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More than 10,000 years ago, the earliest plant breeders were knowingly and unknowingly selecting plants for propagation that had desirable traits, including a shorter growing season, higher yields, and larger seeds or fruits.

Conventional plant breeding is a very long and expensive process that involves crossbreeding two plants, growing new plants from the seeds produced, and painstakingly selecting the offspring with the desired combination of traits—over, and over, and over again. It can take many years to get the right combination of traits, and for new varieties to become available.

What’s a GMO?

GMO stands for genetically modified organism. It’s a term that technically could be applied to almost all plant-based food we eat today, as they’ve all been modified over time, but when people talk about GMOs, they’re most often referring to genetic engineering.

Genetic engineering is a form of biotechnology in which laboratory methods bring together genetic material from multiple sources—a form of genetic recombination. What distinguishes genetic engineering from conventional breeding is the use of recombinant DNA technology.

Here is an example: a gene from another species is added to a crop’s DNA to make it more functional, such as corn that produces a toxin that is lethal to certain crop-killing bugs—meaning that farmers can reduce their insecticide use.

One form of genetic modification can result in “silencing” or turning off specific genes already inside a plant, which can be accomplished through a technique called RNA interference (RNAi). For example, the genes that cause bruising in the Innate® potato, and browning in the Arctic® Apple, have been turned off, which ultimately means that less food is wasted.

Quick fact: apples are one of the most wasted food products on the planet—about 40 per cent are thrown out, much of it because they turn brown once they’re cut, bitten or bruised. Arctic® Apples (a Canadian creation) won’t do that because, with the help of genetic engineering, the enzyme that causes the browning has been turned off.

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There are only 11 GMO crops available today: Genome editing, by comparison, typically involves targeting and changing specific genes that are already there. To edit genes, scientists use a sort of molecular pair of scissors to cut DNA in a desired spot, and then make use of the plant's own natural repair mechanism.

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Giving gene dope to horses

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RESEARCHED AND WRITTEN BY JANE ALLIN

The success of any racehorse is determined by genetics, training and nutrition, which ultimately impact physical traits such as speed, muscular strength and endurance. While training and nutrition no doubt contribute significantly to a racehorse’s performance, inheritance plays an equally if not greater part in influencing performance. At least, that is what the racing industry has based its breeding programs and stud fees on since the birth of horse racing. And of course, doping as a means of performance enhancement, has gone hand in had with these principles in an attempt to manipulate those attributes.

With the development of sophisticated genetic technologies for the genes responsible for these traits comes the potential for novel therapies that will not only benefit treating diseases and musculoskeletal injuries but more insidiously, artificially enhance the performance of the racehorse, otherwise known as gene doping.

Gene doping in human athletics has been on the radar for almost two decades and the race to develop detection methods has been fraught with difficulty, although progress is being made on that front. The World Anti-Doping Agency (WADA) has defined gene doping as the ”transfer of polymers of nucleic acids or nucleic acid analogues, or the use of normal or genetically modified cells that have the capacity to enhance athletic performance”, and has been listed among banned substances and methods since 2003. See What is Prohibited | World Anti-Doping Agency (wada-ama.org).

As gene therapy has shown progress and positive results in recent years, it has made its way into doping regimes in the horse racing world and, just like pharmacological doping agents, it is a threat to both the health of the horse as well as the integrity of the racing industry and a fair wagering environment. Moreover, as with other illegal agents, neither the performance enhancing effects nor adverse side effects of this doping method are well known. Unfortunately, as is observed time and time again, the exploitation of new drugs and other doping agents that can be abused tends to be more rapid than the ability to develop test methods to detect them.

What is gene doping?

Gene doping is an off-shoot of human gene therapy. Gene therapy is used to modify or manipulate the expression of a gene or to alter the properties of biological cells for the purpose of restoring some function related to a damaged or missing gene to treat or cure a disease. Gene therapy aims to fight a disease by replacing, inactivating or repairing a disease‐causing malfunctioning gene.

Therefore, gene doping can be defined as the non-therapeutic use of gene therapy in individuals who do not require it, with the intent of enhancing athletic performance. While this is morally unethical for obvious reasons, more importantly, gene doping could be dangerous, and perhaps even fatal.

In simple terms, gene therapy, and similarly gene doping, involves the introduction of genetic material into cells – essentially the introduction of specific genes that will, in the case of gene therapy, restore proper function to that gene, and in gene doping, enhance athletic performance.

The relationship between cells and genes

The body is composed of trillions of cells, the basic building blocks of all living things. Inside the the nucleus of the cells, there are thousands of genes. These are comprised of a distinct sequence of “nucleotides” that form part of a chromosome. Genes carry the necessary genetic information and instructions for making proteins and enzymes, which help build and maintain the body (e.g. muscles, bones, blood).

BIOLOGY 101 — THE ANATOMY OF EVOLUTION

How are genes introduced into cells?

Gene therapy is not as simple as injecting genes into the bloodstream. Genes cannot enter the target cells on their own, so to put new pieces of DNA into cells in the body, it is typically packaged in a virus. These transport vehicles are called “vectors”. Viruses have a natural ability to deliver genetic material into cells, however, before they can be used to carry the genes into cells they are modified to remove their ability to cause an infectious disease.

Some viruses, such as retroviruses, integrate their genetic material, including the new gene, into a chromosome while, adenoviruses introduce their DNA into the nucleus of the cell, but the not the chromosome. By far the most common class of viruses used today are the viral vectors called “wild-type”adeno-associated viruses (AVV). These are not associated with any pathogenicity unlike some naturally occurring viruses, result in long-term gene expression, and can be engineered to target specific cell or tissue types. See Adeno Associated Virus (AAV) for Cell and Gene Therapy (addgene.org).

The vector containing the gene can be given intravenously directly into the target tissue in the body where it is then taken up by the individual cells (in vivo). Alternatively, the target cells can be removed from the body, the cells cultured in the lab, and genes inserted, then re-introduced to the body (ex vivo).

The process is much more complex than described and there are different types of
delivery vehicles other than viral vectors (e.g. plasmid DNA, bacterial vectors) as well as methods of gene editing (CRISPR) which are all employed as human gene therapy products.  CRISPR technology is a subject all on its own and is both controversial and at the same time at the cutting edge of gene therapy for therapeutic purposes as well as gene doping in human athletes. See https://www.sportsintegrityinitiative.com/scientists-take-step-towards-gene-doping-detection/.

But for the purpose of this discussion, and brevity, will not be dealt with.

Gene doping in sports

Because genes are responsible for the manufacture of proteins and substances that affect cellular structure and function, and if that protein has been shown to be a function of an athlete’s performance, it follows that more of that protein may further enhance performance. With the advent and progression of success in gene therapy treatments comes the inevitable application of its use in the sports world whether that be human or animal. What better sport than horse racing, renowned for its nefarious doping abuse, to experiment on its charges? The future of gene therapy is no doubt exciting and beneficial, but the darker side is its potential for its abuse in sports, despite the potential risks.  And indeed, in the last few years, it has become a growing concern in the horse racing world. See https://tuesdayshorse.wordpress.com/2019/07/04/gene-doping-of-racehorses/.

Equine performance genes for gene doping

In an effort to determine genes associated with racing performance, commercial DNA screening of horses began in 2000 with a racehorse genetic analysis company in the UK called Thoroughbred Genetics Limited. Six years later, the horse genome sequence was finalized, which allowed scientists to identify genome arrays responsible for equine disease and performance based on studies of human genetic pathways attributed to these features. Since this time, a patented genetic test, based on several of these genes, has been used to predict the performance potential of a racehorse over different distances. Genes known to have a favourable effect on a horse’s speed, strength, ability to resist fatigue or mental aptitude are representative options for gene doping. These include; oxygen regulation genes, muscle response genes, energy metabolism genes, and pain and injury repair genes. See https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/full/10.1002/dta.2198.

There are several genes that have been recognized to improve athletic performance, some of which have been implicated in human sporting events. For example, the gene PPAR delta is a gene that was introduced to mice in an experiment (2004) that allowed them to increase their muscle mass that proved to increase their endurance. Insulin‐like growth factor‐1 (IGF‐1) has also been investigated for its role in determining muscle mass and strength. Similarly, expression of the myostatin gene (MSTN) has a strong influence on skeletal muscle mass as do other transforming growth factor (TGF) genes. And then of course there is erythropoietin (EPO), the major regulator of red blood cell production, first made famous by Lance Armstrong. If the gene responsible for EPO production is introduced, this would allow the horse to increase its production or potency of EPO naturally through genetic modification and increase the blood’s oxygen-carrying capacity. These are just a few of the possible mechanisms that could be exploited via genetic manipulation to enhance performance in the race horse.  An in-depth scientific discussion of genes that could potentially be used in equine gene doping is described in the article, “Equine performance genes and the future of doping in horseracing”. See https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/dta.2198.

What about the risks?

While all of this sounds like the doper’s dream, gene therapy has been developed for the treatment of potentially fatal diseases and the repercussions are tolerated when it comes to a matter of life and death, but not so in the healthy athlete. When these techniques are used, human and horse alike, the narrative tells us that the potential side effects can outweigh the pursuit of success on the playing field. Viral vectors used in the transfer of genetic material can trigger a powerful immune response that can lead to multiple organ failure and death. It can also inadvertently create off-target effects where genes not intended for modification are affected and result in the disruption of normal cellular function. Moreover, over- or under-expression of genes encoding proteins can have calamitous effects. For example, the overexpression of EPO can dramatically increase blood viscosity and obstruct regular blood flow, while overexpression of myostatin (MSTN gene) can result in a rapid increase in muscle mass without allowing the heart to adapt, thereby elevating the risk of heart attack. See https://morgansl.com/en/latest/gene-doping-fiction-or-future. Even more sinister, if gene therapy in the horse is used on germ cells, which would alter gametes and be passed onto offspring (versus the somatic cells in tissue), this could lead to unpredicted effects in progenies.

These are just a limited few of the deleterious risks associated with gene therapy designed for therapeutic purposes by reputable sources. Now imagine the use of gene doping products produced and supplied by unregulated and unaccountable clandestine laboratories; the risks are even greater – Frankenstein’s monster re-created?

Detection of gene doping in horses

Over the last several years, the idea that a horse’s genes could be altered to enhance performance has grown from an entirely theoretical perspective to one of reality. Scientific advances in the field of gene therapy have evolved for the purpose of disease and injury intervention, but at the same time, offers a tempting alternative to drugs for improving athletic performance. These developments add to the corruption already present in the racing and breeding industries, and the need to have the capability to detect its presence. To avoid widespread use, tests must be developed to effectively avert the proliferation of gene doping as a means to cheat.

Detection of gene doping also presents an entirely new situation when it comes to routine testing. Unlike other drugs used for doping purposes, which exist as small molecule pharmaceuticals, gene doping vehicles activate cells to produce performance enhancing proteins. These are almost indistinguishable from naturally occurring proteins, which makes detection more difficult.

Recently however, researchers at the University of Pennsylvania School of Veterinary Medicine have developed a new test to precisely detect the local administration of gene doping agents in equine athletes. “The findings from the novel study, supported in part by the Pennsylvania Horse Breeders Association (PHBA) and the Pennsylvania Sate Horse Racing Commission, are a significant breakthrough in the collective fight to advance the welfare and integrity of sport for both horses and humans. ” See https://phys.org/news/2021-03-presence-gene-doping-equines.html.

The study and team of researchers, led by Mary Robinson, Ph.D., VMD, DACVCP – assistant professor of Veterinary Pharmacology and Director of the Equine Pharmacology Laboratory at Penn Vet’s New Bolton Center – developed and validated a quantitative real-time polymerization chain reaction test (PCR) that is able to detect the presence of a gene doping agent in both plasma and synovial fluid after injection into the joint. Not only does it provide a quick, convenient test for pre-race testing, it is also able to detect the agent in blood for up to 28 days. This is significant in that it represents a broad window of opportunity to detect illegal doping both in and outside of competition.

This is a game-changing development:

“The science is closing in on those who seek to use these advancements for wrongful means; the more we learn with each study, the harder it will be for individuals who seek to cheat the system using gene doping strategies.” See https://phys.org/news/2021-03-presence-gene-doping-equines.html.

This research is being conducted as part of a much larger New Bolton project that seeks to expand on its equine “BioBank”. The BioBank is a database consisting of the analysis of numerous types of samples that identify biomarkers in equine athletes. The objective is to eventually create “biological passports” for sport horses that could be used both for predicting pre-existing injuries as well as detecting gene doping agents.

Currently, the PCR test is only capable of detecting a specific gene therapy, but a third study in progress aims to refine the test methodology such that multiple gene doping agents can be identified for even lengthier periods of time.

“ We still have a lot of work to do to better understand the nature of bio-markers and how to fully harness their capabilities, but the science for detecting gene doping is getting there and much more quickly than any of us could have anticipated when we started this research,” added Robinson. “Ideas that once may have seemed unattainable—like a hand-held, stall-side testing device—are now coming into sight as real and tangible possibilities. We just need continued support to help get us there.” See https://phys.org/news/2021-03-presence-gene-doping-equines.html.

Gene doping – today and beyond

With the advent of gene doping, horseracing is facing even more difficult challenges. As in the past, those that exploit illicit means to enhance performance, continue to seek novel methods that are both effective and difficult to detect. Gene doping is a perfect candidate. Over time, gene therapy has advanced in terms of both safety and efficacy, and with it the risk of its misuse as a doping agent in horseracing has become reality. So as to remain a step ahead of drug cheats and to maintain the integrity of the sport, PCR‐based methods offer a viable prospect for detection in the equine athlete.

To date, several genes have been identified as having an effect on equine performance, and are thus potential contenders for gene doping.  Moreover, with time, as genetic technologies in understanding their effects on equine performance evolves, the catalogue of potential doping genes will increase. Implementation of a detection method is therefore paramount to dissuade those contemplating the use of gene doping, as well as protecting the health and welfare of equine athletes.

Featured Image: Adobe Stock

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Grizzly-polar bear hybrids are becoming a reality.

The two bears typically don't occupy the same habitats - polar bears, or Ursus maritimus, like to hang out near or in the sea, while grizzlies, a subspecies of brown bear, or Ursus arctos, prefer forests. But as warmer temperatures hit the Arctic Circle, there have been increasing sightings of crossovers.

Most recently, a bear was shot in northern Canada that didn't quite fit the description of a polar bear.

Its head was the same shape as a grizzly bear and its paws were brown, hunter Didji Ishalook told CBC News.

The reason for the crossover? The bears would rather mate with a different kind of bear than not mate at all, The Washington Post reports. But that's not necessarily great news for the future of the polar bear, which needs arctic ice to thrive.

"I hate to say it, but from a genetic perspective, it's quite likely grizzly bears will eat polar bears up, genetically," University of Alberta professor Andrew Derocher told The Post. The warmer temperatures have led to expanded territories for the grizzlies to roam and run into polar bear mates, which could lead to polar bears as we know them to dissolve into the grizzly population.

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Grizzlies and polar bears are mating for an alarming reason

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Grizzly-polar bear hybrids are becoming a reality. The two bears typically don't occupy the same habitats - polar bears, or Ursus maritimus, like to hang out near or in the sea, while grizzlies, a subspecies of brown bear, or Ursus arctos, prefer forests.

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Scientists are experimenting with "green" microbes in the lab that could someday be used to gobble up oil spills along coastlines without damaging the environment.

© 2011 National Geographic; partially funded by NSF; field producing and videography by Fritz Faerber

RELATED:
Gulf Oil Spill News and Pictures Nature Fighting Back Against Gulf Oil Spill
UNEDITED TRANSCRIPT:

SKIMMING, CONTROLLED BURNS AND DISPERSANTS ARE SOME OF THE MAIN TOOLS USED TO CONTROL BIG OFFSHORE OIL SPILLS LIKE THE APRIL 2010 SPILL IN THE GULF OF MEXICO. BUT ONCE THE OIL GETS ON SHORE OR INTO MARSHLAND, IT POSES A MUCH DIFFERENT SET OF PROBLEMS.

Wetlands soiled by oil can be damaged further when cleaned... SO DEALING WITH OIL ON SHORE IS A KEY AREA OF RESEARCH FOR SCIENTISTS. TULANE UNIVERSITY PROFESSOR KYRIAKOS PAPADOPOULOS AND HIS STUDENTS ARE STUDYING HOW OIL, DISPERSANTS AND BACTERIA MOVE THROUGH POROUS MATERIAL LIKE the SAND and MUD THAT MAKES UP COASTLINES.

SOT: Kyriakos Papadopoulos, Tulane Professor of Chemical and Biomolecular Engineering

"Once the oil reaches sediments and beaches and bayous, how does it transport itself through the porous medium that those beaches and bayous constitute. But the other very important question that we can answer through this is how we can intervene in order for the oil to be removed."

PART OF THAT QUESTION IS WHich PRODUCTS WORK BEST TO REMOVE OIL WHILE DOING THE LEAST HARM TO THE ENVIRONMENT. A COLUMBIA UNIVERSITY TEAM WORKING WITH THE TULANE GROUP IS TESTING AND DEVELOPING something known as REAGENTS. A reagent, in this instance, would be a microorganism able to digest the oil. Ideally, the product created by the microorganism after consuming the oil would be harmless to the environment.

SOT: Som Somasundaran, Columbia University

"The oil that is spilled on marshland you cannot use the techniques that are used on the surface of the ocean. You cannot burn it, you cannot use the booms there because you don't want to disturb the marshland. And so different techniques are required. "

SYNTHETIC BIOLOGY - OR ENLISTING MICROBES TO WORK AS TINY CHEMICAL PLANTS - MAY SOON DELIVER DISPERSANTS AND OTHER PRODUCTS TO HELP CLEAN UP OIL SPILLS. BUT IT'S COMPLICATED - SINCE OIL CAN BE SPILLED under WIDELY DIFFERENT CONDITIONS.

Sot: Somasundaran

"We are looking at all these factors to optimize the window of reagents available for taking care of oil spills. Why window? Because depending on where the spill is - in the Gulf, or in Alaska, the temperature is different, the salinity is different..."

THESE TWO TEAMS, WORKING SOME 13-HUNDRED MILES APART HOPE THEIR RESEARCH WILL YIELD A GREENER CLEANER TECHNIQUE. AND PAPADOPOULOS SAYS SOME OF THE BACTERIA BEHAVIOR HE'S SEEN IN THE LAB COULD MEAN THE NEW MICROBES MAY EVEN move on their own to seek out and CLEAN UP OIL BURIED IN SAND OR SOIL.

Sot: Papadopoulos

"There is a phenomenon we have not published yet, but I hope to publish soon that we call styri-taxis (sp?), meaning bacteria moving to distances they would not have moved otherwise, Just because once they enter the very small, confining spaces of porous medium, they cannot turn back."

A LOT MORE STUDY IS NEEDED, BUT THE SCIENCE COMING OUT OF THESE LABS COULD ONE DAY YIELD A BACTERIUM TO BE SPRINKLED IN THE MARSHES, TO WORK ITS WAY DOWN TO CLEAN UP BURIED OIL.

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Scientists are experimenting with "green" microbes in the lab that could someday be used to gobble up oil spills along coastlines without damaging the environment.

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Kira Peikoff

Kira Peikoff was the editor-in-chief of Leaps.org from 2017 to 2021. As a journalist, her work has appeared in The New York Times, Newsweek, Nautilus, Popular Mechanics, The New York Academy of Sciences, and other outlets. She is also the author of four suspense novels that explore controversial issues arising from scientific innovation: Living Proof, No Time to Die, Die Again Tomorrow, and Mother Knows Best. Peikoff holds a B.A. in Journalism from New York University and an M.S. in Bioethics from Columbia University. She lives in New Jersey with her husband and two young sons. Follow her on Twitter @KiraPeikoff.

Kira Peikoff

Kira Peikoff was the editor-in-chief of Leaps.org from 2017 to 2021. As a journalist, her work has appeared in The New York Times, Newsweek, Nautilus, Popular Mechanics, The New York Academy of Sciences, and other outlets. She is also the author of four suspense novels that explore controversial issues arising from scientific innovation: Living Proof, No Time to Die, Die Again Tomorrow, and Mother Knows Best. Peikoff holds a B.A. in Journalism from New York University and an M.S. in Bioethics from Columbia University. She lives in New Jersey with her husband and two young sons. Follow her on Twitter @KiraPeikoff.

Matt Fuchs is the editor-in-chief of Leaps.org. He is also a contributing reporter to the Washington Post and has written for the New York Times, Time Magazine, WIRED and the Washington Post Magazine, among other outlets. Follow him on Twitter @fuchswriter.

Ron Shinkman

Ron Shinkman is a veteran journalist whose work has appeared in the New England Journal of Medicine publication Catalyst, California Health Report, Fierce Healthcare, and many other publications. He has been a finalist for the prestigious NIHCM Foundation print journalism award twice in the past five years. Shinkman also served as Los Angeles Bureau Chief for Modern Healthcare and as a staff reporter for the Los Angeles Business Journal. He has an M.A. in English from California State University and a B.A. in English from UCLA.

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How Scientists Are Engineering Plastic-Eating Bacteria to Fight the Pollution Crisis

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Kira Peikoff Kira Peikoff was the editor-in-chief of Leaps.org from 2017 to 2021. As a journalist, her work has appeared in The New York Times, Newsweek, Nautilus, Popular Mechanics, The New York Academy of Sciences, and other outlets.

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The responses to the questions provided in this document represent the FDA's view in light of the conclusions and recommendations outlined in the Animal Cloning Risk Assessment, Risk Management Plan, and Guidance for Industry #179. What is cloning, really?
What can go wrong with cloning?
Why is there interest in cloning?
Is it safe to eat food from clones?
What's next?
What is the National Academy of Sciences?
Imagine the perfect dairy cow. For eight years she has gotten pregnant on the first try, given birth easily, and produced gallon upon gallon of the best milk. Even when others in the herd got sick, she stayed healthy. She is ideally suited to the climate in which she lives. The farmer has depended on this cow and her daughters in lean times to carry the farm through, but now she is at the end of her reproductive life.

Although the farmer may have this cow's daughters to carry on the line, he also has another alternative: copying her. Biological copying is referred to as cloning. By cloning his prize cow, breeding the clones, and keeping their offspring, the farmer can introduce the natural positive characteristics into the herd quickly. It would take several more years to achieve these same improvements by conventional breeding.
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Farmers can also clone animals to produce more uniform quality meat. Take, for example, a male swine (boar) that time after time sires offspring that mature quickly and provide lean meat. If a farmer has several of these boars he could quickly produce an entire herd with consistent, high quality meat.

Researchers have been cloning livestock since 1996. When it became apparent that cloning could become a commercial venture in 2001, the Food and Drug Administration's Center for Veterinary Medicine asked that food from clones and their offspring be voluntarily kept out of the food chain. FDA then began an intensive evaluation that included examining the safety of food from these animals.

What is cloning, really?

Cloning is a complex process that lets one exactly copy the genetic, or inherited, traits of an animal (the donor). Livestock species that scientists have successfully cloned are cattle, swine, sheep, and goats. Scientists have also cloned mice, rats, rabbits, cats, mules, horses and one dog. Chickens and other poultry have not been cloned.
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Most people think of livestock breeding taking place through traditional mating, in which males and females physically get together to reproduce. In fact, this is not often the case. Traditional mating is not that efficient, if the goal is to produce as many offspring as possible. For example, a male has enough sperm to produce many more offspring than would be possible by traditional mating. Traditional mating also has certain risks: one or both of the animals may be injured in the process of mating. The female may be hurt by the male because he is often much larger, or an unwilling female may injure the male. There is also a risk of infection or transmission of venereal disease during traditional mating.

Because of these factors, many farmers use assisted reproductive technologies (ARTs) for breeding. These include artificial insemination, embryo transfer, and in vitro fertilization (a process by which egg and sperm are united outside the body). Artificial insemination was first documented in the breeding of horses in the 14th century. The first successful embryo transfer of a cow was in 1951, and the first in vitro fertilization (IVF)-derived animal was a rabbit born in 1959. Livestock production in the United States now uses all these methods regularly. For example, most dairy farms don't have bulls, so more than 70 percent of the Holstein cows bred in the United States are artificially inseminated. The frozen semen can come from a bull many miles, or even many states, away.

Cloning is a more advanced form of these assisted reproductive technologies. Much of the public perception of cloning likely comes from science fiction books and movies. Some people incorrectly believe that clones spring forth fully formed, or are grown in test tubes. This is just not the case.

Clones are born just like other animals. They are similar to identical twins, only born at different times. Just as twins share the same DNA, clones have the same genes as the donor animal. A clone is not a mutant, nor is it a weaker version of the original animal.

In all of the other assisted reproductive technologies, the male and female parents each contribute half of their genes to their offspring. Farmers have worked for years to choose animals with the best traits and breed them together. This increases the chance these good traits will be passed on and become more common in livestock herds. Even though farmers have been able to improve their herds over time, they still can't absolutely predict the characteristics of the offspring, not even their gender. Cloning gives the farmer complete control over the offspring's inherited traits. Thus, a farmer who clones an especially desirable but aging or injured animal knows in advance that the clone will have the genetic potential to be an especially good, younger animal. He can then use that animal to further reproduce by traditional mating or other ARTs

Most cloning today uses a process called somatic cell nuclear transfer (SCNT). Just as with in vitro fertilization, scientists take an immature egg, or oocytes, from a female animal (often from ovaries obtained at the slaughterhouse). But instead of combining it with sperm, they remove the nucleus (which contains the oocytes's genes). This leaves behind the other components necessary for the initial stages of embryo development. Scientists then add the nucleus or cell from the donor animal that has the desirable traits the farmer wishes to copy. After a few other steps, the donor nucleus fuses with the ooplast (the oocytes whose nucleus has been removed), and if all goes well, starts dividing, and an embryo begins to form. The embryo is then implanted in the uterus of a surrogate dam (again the same as with in vitro fertilization), which carries it to term. ("Dam" is a term that livestock breeders use to refer to the female parent of an animal). The clone is delivered just like any other baby animal.

What can go wrong with cloning?

There are no complications that are unique to cloning. The problems seen in clones are also seen in animals born from natural mating or ARTs. They seem to happen more often in clones for a number of reasons that probably have to do with parts of the procedure that occur outside the body. The embryo may fail to develop properly during the in vitro stage or early on after transfer to the surrogate and may be flushed out of the uterus. If it does develop, the embryo may not implant properly into the uterus of the surrogate dam. Alternatively, the placenta may not form properly, and the developing animal won't get the nourishment it needs.

Large Offspring Syndrome (LOS) is seen in pregnancies of cattle and sheep that come from other ARTs and cloning. With LOS, the fetus grows too large in the uterus, making problems for the animal and its surrogate dam. LOS has not been observed in goats and swine.
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Most clones that are normal at birth become as strong and healthy as any other young animals. Calf and lamb clones tend to have more health problems at birth, and may be more likely to die right after birth than conventionally bred animals. Clones born with abnormalities may continue to have health problems for the first few months of life, but by the time clones are young adults, it's not possible to tell them apart from other animals of the same age, even if you conduct a detailed examination. Scientists at FDA and research institutions have looked at extensive health records, the development of clones, and blood work for clones that's similar to what people get when they have physicals. These results show that the clones are perfectly healthy, and walk, wean, grow, mature, and behave just like conventionally bred animals.

Why is there interest in cloning?

The main use of agricultural clones is to produce breeding stock, not food. Clones allow farmers to upgrade the overall quality of their herds by providing more copies of the best animals in the herd. These animals are then used for conventional breeding, and the sexually reproduced offspring become the food producing animals. These animals are not clones-they're just like other sexually reproduced animals. Just as farmers wouldn't use their best conventionally bred breeding animals as sources of food, they are equally unlikely to do so for clones.

Some examples of desirable characteristics in livestock that breeders might want in their herds include the following:

Disease resistance: Sick animals are expensive for farmers. Veterinary bills add up, and unhealthy animals don't produce as much meat or milk. A herd that is resistant to disease is extremely valuable because it doesn't lose any production time to illness, and doesn't cost the farmer extra money for veterinary treatment.

Suitability to Climate: Different types of livestock grow well in different climates. Some of this is natural and some results from selective breeding. For instance, Brahma cattle can cope with the heat and humidity of weather in the southwestern United States, but they often do not produce very high grades of meat. Cloning could allow breeders to select those cattle that can produce high quality meat or milk and thrive in extreme climates and use them to breed more cattle to be used for food production. Similarly, pork production has traditionally been centered in the eastern United States, but is moving to different regions of the United States (e.g., Utah). Cloning could allow breeders to select those swine that naturally do well in the new climate, and use those swine clones to breed more swine to be used for food production.

Quality body type: Farmers naturally want an animal whose body is well suited to its production function. For example, a dairy cow should have a large, well-attached udder so that she can produce lots of milk. She should also be able to carry and deliver calves easily. For animals that produce meat, farmers breed for strong, heavily-muscled, quick-maturing animals that will yield high quality meat in the shortest time possible. The most desirable bulls produce offspring that are relatively small at birth (so that they are easier for the female to carry and deliver) but that grow rapidly and are healthy after birth.

Fertility: Quality dairy cows should be very fertile, as a cow that doesn't get pregnant and bear calves won't produce milk. Male fertility is just as important as that of the female. The more sperm he can produce, the more females a bull can inseminate, and the more animals can be born. Beef cattle or other meat-producing animals such as swine need to have high fertility rates in order to replace animals that are sent to slaughter. Cloning allows farmers and breeders to clone those animals with high fertility rates so that they could bear offspring that would also tend to be very fertile.
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Market preference: Farmers or ranchers may also want to breed livestock to meet the changing tastes of consumers. These include traits like leanness, tenderness, color, size of various cuts, etc. Preferences also vary by culture, and cloning may help tailor products to the preferences of various international markets and ethnic groups.

How does cloning help get these characteristics into the herd more quickly? As we've previously said, cloning allows the breeder to increase the number of breeding animals available to make the actual food production animals. So, if a producer wanted to introduce disease resistance into a herd rapidly, cloning could be used to produce a number of breeding animals that carry the gene for disease resistance, rather than just one. Likewise, if a breeder wants to pass on the genes of a female animal, cloning could result in multiples of that female to breed, rather than just one.
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Is it Safe to Eat Food from Clones?

Yes. Food from cattle, swine, and goat clones is as safe to eat as food from any other cattle, swine, or goat. But it's important to remember that the primary purpose of clones is for breeding, not eating. Dairy, beef, or pork clones make up only a tiny fraction of the total number of food producing animals in the United States. Instead, their offspring would be the animals actually producing meat or milk for the food supply.
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Dairy clones will produce milk after they give birth, and the dairy farmers will want to be able to drink that milk or put it in the food supply. Once clones used for breeding meat-producing animals can no longer reproduce, their breeders may also want to be able to put them into the food supply.

In order to determine whether there would be any risk involved in eating meat or milk from clones or their offspring, in 1999 the FDA asked the National Academy of Sciences (NAS) to identify science-based concerns associated with animal biotechnology, including cloning. The NAS gathered an independent group of top, peer-selected scientists from across the country to conduct this study. The scientists delivered their report in the fall of 2002. That report stated that theoretically there were no concerns for the safety of meat or milk from clones. On the other hand, the report expressed a low level of concern due to a lack of information on the clones at that time, and not for any specific scientific reasons. The report also stated that the meat and milk from the offspring of clones posed no unique food safety concerns.

Meanwhile, FDA itself began the most comprehensive examination of the health of livestock clones that has been conducted. The evaluation has taken over five years. This examination formed the basis of a Draft Risk Assessment to determine whether cloning posed a risk to animal health or to humans eating food from clones or their offspring. FDA conducted a thorough search of the scientific literature on clones, and identified hundreds of peer-reviewed scientific journal articles, which it then reviewed. The agency was also able to obtain health records and blood samples from almost all of the cattle clones that have been produced in the United States and data from clones produced in other countries. FDA compared these health records, and the independently analyzed blood results with similar samples from conventional animals of the same age and breed that were raised on the same farms. FDA received thousands of comments from the public in response to the Draft Risk Assessment. For the final version of the Risk Assessment, FDA conducted an up-to-date review of the literature, added additional information from hundreds of additional references, and made many changes to address some of the public comments.

After reviewing all this information, FDA found that it could not distinguish a healthy clone from a healthy conventionally bred animal. All of the blood values, overall health records, and behaviors were in the same range for clones and conventional animals of the same breed raised on the same farms. FDA also observed that milk from dairy clones does not differ significantly in composition from milk from conventionally bred animals.

In the Risk Assessment, FDA concluded that meat and milk from cattle, swine, and goat clones is as safe as food we eat from those species now. Although we don't have any particular concerns about sheep clones, we did not have enough information to make a decision on the safety of food from sheep clones.

For another study similar to the one conducted on cow clones, the agency also evaluated the health of offspring sexually derived from swine clones, as well as the composition of their meat. After reviewing this very large data set, the agency concluded that all of the blood values, overall health records, and meat composition profiles of the progeny of clones were in the same range as for very closely genetically related conventionally bred swine. Based on these results, other studies from scientific journals, and our understanding of the biological processes involved in cloning, the agency agreed with NAS that food from the sexually reproduced offspring of clones is as safe as food that we eat every day. We reiterate, however, that the sexually reproduced offspring of clones would produce almost all of the food from the overall cloning/breeding process.

What's next?

FDA's Risk Assessment includes data collected or published before mid-2007. The FDA will continue to monitor closely the development of clones and their progeny as a source for food as further data become available.

What is the National Academy of Sciences?

The National Academy of Sciences (NAS) was signed into being by President Abraham Lincoln on March 3, 1863, at the height of the Civil War. As mandated in its Act of Incorporation, the NAS has, since 1863, served to "investigate, examine, experiment, and report upon any subject of science or art" whenever called upon to do so by any department of the government. Scientific issues would become even more contentious and complex in the years following the war. To keep pace with the growing roles that science and technology would play in public life, the institution that was founded in 1863 eventually expanded to include the National Research Council in 1916, the National Academy of Engineering in 1964, and the Institute of Medicine in 1970. Collectively, the four organizations are known as the National Academies. Since 1863, the nation's leaders have often turned to the National Academies for advice on the scientific and technological issues that frequently pervade policy decisions. The Academies and the Institute are honorific societies that elect new members to their ranks each year. The Institute of Medicine also conducts policy studies on health issues, but the bulk of the institution's science policy and technical work is conducted by its operating arm, the National Research Council, created expressly for this purpose. These non-profit organizations provide a public service by working outside the framework of government to ensure independent advice on matters of science, technology, and medicine. They enlist committees of the nation's top scientists, engineers, and other experts--all of whom volunteer their time to study specific concerns. (From the National Academy of Sciences website.)

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A Primer on Cloning and Its Use in Livestock Operations

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The responses to the questions provided in this document represent the FDA's view in light of the conclusions and recommendations outlined in the Animal Cloning Risk Assessment, Risk Management Plan, and Guidance for Industry #179. What is cloning, really? What can go wrong with cloning? Why is there interest in cloning?

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Each person's fingerprints are unique, which is why they have long been used as a way to identify individuals. Surprisingly little is known about the factors that influence a person's fingerprint patterns. Like many other complex traits, studies suggest that both genetic and environmental factors play a role.

A person's fingerprints are based on the patterns of skin ridges (called dermatoglyphs) on the pads of the fingers. These ridges are also present on the toes, the palms of the hands, and the soles of the feet. Although the basic whorl, arch, and loop patterns may be similar, the details of the patterns are specific to each individual.

Dermatoglyphs develop before birth and remain the same throughout life. The ridges begin to develop during the third month of fetal development, and they are fully formed by the sixth month. The function of these ridges is not entirely clear, but they likely increase sensitivity to touch.

The basic size, shape, and spacing of dermatoglyphs appear to be influenced by genetic factors. Studies suggest that multiple genes are involved, so the inheritance pattern is not straightforward. Genes that control the development of the various layers of skin, as well as the muscles, fat, and blood vessels underneath the skin, may all play a role in determining the pattern of ridges. The finer details of the patterns of skin ridges are influenced by other factors during fetal development, including the environment inside the womb. These developmental factors cause each person's dermatoglyphs to be different from everyone else's. Even identical twins, who have the same DNA, have different fingerprints.

Few genes involved in dermatoglyph formation have been identified. Rare diseases characterized by abnormal or absent dermatoglyphs provide some clues as to their genetic basis. For example, a condition known as adermatoglyphia is characterized by an absence of dermatoglyphs, sometimes with other abnormalities of the skin. Adermatoglyphia is caused by mutations in a gene called . Although this gene is clearly important for the formation of dermatoglyphs, its role in their development is unclear.

Scientific journal articles for further reading

Burger B, Fuchs D, Sprecher E, Itin P. The immigration delay disease: adermatoglyphia-inherited absence of epidermal ridges. J Am Acad Dermatol. 2011 May;64(5):974-80. doi: 10.1016/j.jaad.2009.11.013. Epub 2010 Jul 8.PubMed: 20619487.

Nousbeck J, Burger B, Fuchs-Telem D, Pavlovsky M, Fenig S, Sarig O, Itin P, Sprecher E. A mutation in a skin-specific isoform of SMARCAD1 causes autosomal-dominant adermatoglyphia. Am J Hum Genet. 2011 Aug 12;89(2):302-7. doi: 10.1016/j.ajhg.2011.07.004. Epub 2011 Aug 4. PubMed: 21820097. Free full-text available from PubMed Central: PMC3155166.

Warman PH, Ennos A.R. Fingerprints are unlikely to increase the friction of primate fingerpads. J Exp Biol. 2009 Jul;212(Pt 13):2016-22. doi: 10.1242/jeb.028977. PubMed: 19525427.

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Are fingerprints determined by genetics?: MedlinePlus Genetics

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Each person's fingerprints are unique, which is why they have long been used as a way to identify individuals. Surprisingly little is known about the factors that influence a person's fingerprint patterns. Like many other complex traits, studies suggest that both genetic and environmental factors play a role.

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Gene doping in race horses;

https://www.veterinarypracticenews.com/gene-doping-test-supports-health-of-racehorses/

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Eliminating the administration of illicit, gene doping therapies in racehorses and other equine athletes is the driving force behind a new development out of the University of Pennsylvania School of Veterinary Medicine (Penn Vet).

Researchers have created and validated a quantitative test to detect the presence of a potential gene-doping agent in the joint fluid and blood of a horse.

The research, which was supported in part by the Pennsylvania Horse Breeders Association (PHBA) and the Pennsylvania State Horse Racing Commission (PHRA), offers a "significant breakthrough" in the fight to advance the welfare and integrity of sport, both for horses and humans, Penn Vet says.

"As breeders, protecting the health, safety, and wellbeing of our horses is a deeply profound and personal priority for our membership," says PHBA's executive secretary, Brian Sanfratello. "These scientific discoveries get us one step closer to our dream of someday keeping equine sport completely clean."

Gene doping agents trigger cells to produce performance enhancing proteins, according to Penn Vet. These proteins are often elusive due to their similarity to those that naturally occur within the body, making it difficult to determine whether or not an animal or human has had gene therapy administered.

Penn Vet's research team, led by Mary Robinson, PhD, VMD, DACVCP, director of the Equine Pharmacology Laboratory at the college's New Bolton Center, has developed a real-time polymerase chain reaction (PCR) test to detect whether a specific gene therapy agent has been injected into the joint of a horse.

Additionally, the test can perceive the agent in the animal's blood up to 28 days after administration. This window, researchers say, could be useful before races, as well as out of competition testing.

"The ability to detect the presence of these gene doping agents in blood after local administration to joints just magnifies the implications of this game-changing development," says Joanne Haughan, DVM, one of the study's leads. "The science is closing in on those who seek to use these advancements for wrongful means; the more we learn with each study, the harder it will be for individuals who seek to cheat the system using gene doping strategies."

Researchers hope to expand and refine their testing methodology to create screening tests that would successfully identify multiple gene doping agents for even longer periods of time, Penn Vet reports.

"We still have a lot of work to do to better understand the nature of biomarkers and how to fully harness their capabilities, but the science for detecting gene doping is getting there and much more quickly than any of us could have anticipated when we started this research," Dr. Robinson says.

For more, click here.

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Gene-doping test supports health of racehorses

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Eliminating the administration of illicit, gene doping therapies in racehorses and other equine athletes is the driving force behind a new development out of the University of Pennsylvania School of Veterinary Medicine (Penn Vet). Researchers have created and validated a quantitative test to detect the presence of a potential gene-doping agent in the joint fluid and blood of a horse.

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GM is a technology that involves inserting DNA into the genome of an organism. To produce a GM plant, new DNA is transferred into plant cells. Usually, the cells are then grown in tissue culture where they develop into plants. The seeds produced by these plants will inherit the new DNA.

The characteristics of all living organisms are determined by their genetic makeup and its interaction with the environment. The genetic makeup of an organism is its genome, which in all plants and animals is made of DNA. The genome contains genes, regions of DNA that usually carry the instructions for making proteins. It is these proteins that give the plant its characteristics. For example, the colour of flowers is determined by genes that carry the instructions for making proteins involved in producing the pigments that colour petals.

Genetic modification of plants involves adding a specific stretch of DNA into the plant's genome, giving it new or different characteristics. This could include changing the way the plant grows, or making it resistant to a particular disease. The new DNA becomes part of the GM plant's genome which the seeds produced by these plants will contain.

The first stage in making a GM plant requires transfer of DNA into a plant cell. One of the methods used to transfer DNA is to coat the surface of small metal particles with the relevant DNA fragment, and bombard the particles into the plant cells. Another method is to use a bacterium or virus. There are many viruses and bacteria that transfer their DNA into a host cell as a normal part of their life cycle. For GM plants, the bacterium most frequently used is called Agrobacterium tumefaciens. The gene of interest is transferred into the bacterium and the bacterial cells then transfer the new DNA to the genome of the plant cells. The plant cells that have successfully taken up the DNA are then grown to create a new plant. This is possible because individual plant cells have an impressive capacity to generate entire plants. On rare occasions, the process of DNA transfer can happen without deliberate human intervention. For example the sweet potato contains DNA sequences that were transferred thousands of years ago, from Agrobacterium bacteria into the sweet potato genome.

There are other ways to change the genomes of crops, some of which are long established, such as mutational breeding, and others of which are new, such as genome editing, but in this Q&A we are focusing on GM as it is currently usually defined for regulatory purposes in Europe.

See related questions

Page last updated: May 2016
Download all questions and answers (PDF)

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What are GM crops and how is it done? | Royal Society

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GM is a technology that involves inserting DNA into the genome of an organism. To produce a GM plant, new DNA is transferred into plant cells. Usually, the cells are then grown in tissue culture where they develop into plants. The seeds produced by these plants will inherit the new DNA.

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human cloning is a good idea?

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Human reproductive cloning: The curious incident of the dog in the night-time

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Cloning’s fade from the spotlight might provide some insight into public opinion, press behavior, moral panics, and other important aspects about social behavior in the face of startling discoveries in the biosciences.

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Finding out what gene doping is, and if it is safe.

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Azzazy, H. M. E., et al. Doping in the recombinant era: Strategies and counterstrategies. Clinical Biochemistry 38, 959-965 (2005)

Barton, E. R., et al. Muscle-specific expression of insulin-like growth factor 1 counters muscle decline in mdx mice. Journal of Cell Biology 157, 137-148 (2002)

Brownlee, C. Gene doping: Will athletes go for the ultimate high? Science News 166, 280 (2004)

Murray, T. An Olympic tail? Nature Review Genetics 4, 494 (2003) doi:10.1038/nrg1135 ( link to article)

Rivera, V. M., et al. Long-term pharmacologically regulated expression of erythropoietin in patients following AAV-mediated gene transfer. Blood 105, 1424-1430 (2005)

Skipper, M. Gene doping: A new threat for the Olympics? Nature Reviews Genetics 5, 720 (2004) doi:10.1038/nrg1461 ( link to article)

Wang, Y. X., et al. Regulation of muscle fiber type and running endurance by PPAR-δ. PLoS Biology 2, e294 (2004) ( link to article)

World Anti-Doping Agency Home Page. (accessed June 27, 2008).

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gene therapy  igf-1  wada  gene therapy in sports  schwarzenegger mice  gene doping

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Imagine a world in which people look like weight lifters even though they've never set foot in a weight room, where 80-year-old seniors have muscles as strong as those of twentysomething adults, and where Olympic athletes break records in ways that do not seem humanly possible.

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Comfortably seated in the fertility clinic with Vivaldi playing softly in the background, you and your partner are brought coffee and a folder. Inside the folder is an embryo menu. Each embryo has a description, something like this:

Embryo 78 - male
No serious early onset diseases, but a carrier for phenylketonuria (a metabolic malfunction that can cause behavioural and mental disorders. Carriers just have one copy of the gene, so don't get the condition themselves).
Higher than average risk of type 2 diabetes and colon cancer.
Lower than average risk of asthma and autism.
Dark eyes, light brown hair, male pattern baldness.
40% chance of coming in the top half in SAT tests.

There are 200 of these embryos to choose from, all made by in vitro fertilisation (IVF) from you and your partner's eggs and sperm. So, over to you. Which will you choose?

If there's any kind of future for "designer babies", it might look something like this. It's a long way from the image conjured up when artificial conception, and perhaps even artificial gestation, were first mooted as a serious scientific possibility. Inspired by predictions about the future of reproductive technology by the biologists JBS Haldane and Julian Huxley in the 1920s, Huxley's brother Aldous wrote a satirical novel about it.

That book was, of course, Brave New World, published in 1932. Set in the year 2540, it describes a society whose population is grown in vats in an impersonal central hatchery, graded into five tiers of different intelligence by chemical treatment of the embryos. There are no parents as such - families are considered obscene. Instead, the gestating fetuses and babies are tended by workers in white overalls, "their hands gloved with a pale corpse‑coloured rubber", under white, dead lights.

Brave New World has become the inevitable reference point for all media discussion of new advances in reproductive technology. Whether it's Newsweek reporting in 1978 on the birth of Louise Brown, the first "test-tube baby" (the inaccurate phrase speaks volumes) as a "cry round the brave new world", or the New York Times announcing " The brave new world of three-parent IVF " in 2014, the message is that we are heading towards Huxley's hatchery with its racks of tailor-made babies in their "numbered test tubes".

The spectre of a harsh, impersonal and authoritarian dystopia always looms in these discussions of reproductive control and selection. Novelist Kazuo Ishiguro, whose 2005 novel, Never Let Me Go, described children produced and reared as organ donors, last month warned that thanks to advances in gene editing, "we're coming close to the point where we can, objectively in some sense, create people who are superior to others ".

But the prospect of genetic portraits of IVF embryos paints a rather different picture. If it happens at all, the aim will be not to engineer societies but to attract consumers. Should we allow that? Even if we do, would a list of dozens or even hundreds of embryos with diverse yet sketchy genetic endowments be of any use to anyone?

I don't think we are going to see superman or a split in the species any time soon, because we just don't know enough

Henry Greely, bioethicist

The shadow of Frankenstein 's monster haunted the fraught discussion of IVF in the 1970s and 80s, and the misleading term "three-parent baby" to refer to embryos made by the technique of mitochondrial transfer - moving healthy versions of the energy-generating cell compartments called mitochondria from a donor cell to an egg with faulty, potentially fatal versions - insinuates that there must be something "unnatural" about the procedure.

Every new advance puts a fresh spark of life into Huxley's monstrous vision. Ishiguro's dire forecast was spurred by the gene-editing method called Crispr-Cas9, developed in 2012, which uses natural enzymes to target and snip genes with pinpoint accuracy. Thanks to Crispr-Cas9, it seems likely that gene therapies - eliminating mutant genes that cause some severe, mostly very rare diseases - might finally bear fruit, if they can be shown to be safe for human use. Clinical trials are now under way.

But modified babies? Crispr-Cas9 has already been used to genetically modify (nonviable) human embryos in China, to see if it is possible in principle - the results were mixed. And Kathy Niakan of the Francis Crick Institute in the UK has been granted a licence by the Human Fertilisation and Embryology Authority (HFEA) to use Crispr-Cas9 on embryos a few days old to find out more about problems in these early stages of development that can lead to miscarriage and other reproductive problems.

Most countries have not yet legislated on genetic modification in human reproduction, but of those that have, all have banned it. The idea of using Crispr-Cas9 for human reproduction is largely rejected in principle by the medical research community. A team of scientists warned in Nature less than two years ago that genetic manipulation of the germ line (sperm and egg cells) by methods like Crispr-Cas9, even if focused initially on improving health, " could start us down a path towards non-therapeutic genetic enhancement ".

Besides, there seems to be little need for gene editing in reproduction. It would be a difficult, expensive and uncertain way to achieve what can mostly be achieved already in other ways, particularly by just selecting an embryo that has or lacks the gene in question. "Almost everything you can accomplish by gene editing, you can accomplish by embryo selection," says bioethicist Henry Greely of Stanford University in California.

Because of unknown health risks and widespread public distrust of gene editing, bioethicist Ronald Green of Dartmouth College in New Hampshire says he does not foresee widespread use of Crispr-Cas9 in the next two decades, even for the prevention of genetic disease, let alone for designer babies. However, Green does see gene editing appearing on the menu eventually, and perhaps not just for medical therapies. "It is unavoidably in our future," he says, "and I believe that it will become one of the central foci of our social debates later in this century and in the century beyond." He warns that this might be accompanied by "serious errors and health problems as unknown genetic side effects in 'edited' children and populations begin to manifest themselves".

For now, though, if there's going to be anything even vaguely resembling the popular designer-baby fantasy, Greely says it will come from embryo selection, not genetic manipulation. Embryos produced by IVF will be genetically screened - parts or all of their DNA will be read to deduce which gene variants they carry - and the prospective parents will be able to choose which embryos to implant in the hope of achieving a pregnancy. Greely foresees that new methods of harvesting or producing human eggs, along with advances in preimplantation genetic diagnosis (PGD) of IVF embryos, will make selection much more viable and appealing, and thus more common, in 20 years' time.

PGD is already used by couples who know that they carry genes for specific inherited diseases so that they can identify embryos that do not have those genes. The testing, generally on three- to five-day-old embryos, is conducted in around 5% of IVF cycles in the US. In the UK it is performed under licence from the HFEA, which permits screening for around 250 diseases including thalassemia, early-onset Alzheimer's and cystic fibrosis.

As a way of "designing" your baby, PGD is currently unattractive. "Egg harvesting is unpleasant and risky and doesn't give you that many eggs," says Greely, and the success rate for implanted embryos is still typically about one in three. But that will change, he says, thanks to developments that will make human eggs much more abundant and conveniently available, coupled to the possibility of screening their genomes quickly and cheaply.

Carey Mulligan, Keira Knightley and Andrew Garfield in the 2010 film adaptation of Kazuo Ishiguro's Never Let Me Go, in which clones are produced to provide spare organs for their originals. Photograph: 20th Century Fox/Everett/Rex

Advances in methods for reading the genetic code recorded in our chromosomes are going to make it a routine possibility for every one of us - certainly, every newborn child - to have our genes sequenced. "In the next 10 years or so, the chances are that many people in rich countries will have large chunks of their genetic information in their electronic medical records," says Greely.

But using genetic data to predict what kind of person an embryo would become is far more complicated than is often implied. Seeking to justify unquestionably important research on the genetic basis of human health, researchers haven't done much to dispel simplistic ideas about how genes make us. Talk of "IQ genes", "gay genes" and "musical genes" has led to a widespread perception that there is a straightforward one-to-one relationship between our genes and our traits. In general, it's anything but.

There are thousands of mostly rare and nasty genetic diseases that can be pinpointed to a specific gene mutation. Most more common diseases or medical predispositions - for example, diabetes, heart disease or certain types of cancer - are linked to several or even many genes, can't be predicted with any certainty, and depend also on environmental factors such as diet.

When it comes to more complex things like personality and intelligence, we know very little. Even if they are strongly inheritable - it's estimated that up to 80% of intelligence, as measured by IQ, is inherited - we don't know much at all about which genes are involved, and not for want of looking.

At best, Greely says, PGD might tell a prospective parent things like "there's a 60% chance of this child getting in the top half at school, or a 13% chance of being in the top 10%". That's not much use.

We might do better for "cosmetic" traits such as hair or eye colour. Even these "turn out to be more complicated than a lot of people thought," Greely says, but as the number of people whose genomes have been sequenced increases, the predictive ability will improve substantially.

Ewan Birney, director of the European Bioinformatics Institute near Cambridge, points out that, even if other countries don't choose to constrain and regulate PGD in the way the HFEA does in the UK, it will be very far from a crystal ball.

Nearly anything you can measure for humans, he says, can be studied through genetics, and analysing the statistics for huge numbers of people often reveals some genetic component. But that information "is not very predictive on an individual basis," says Birney. "I've had my genome sequenced on the cheap, and it doesn't tell me very much. We've got to get away from the idea that your DNA is your destiny."

If the genetic basis of attributes like intelligence and musicality is too thinly spread and unclear to make selection practical, then tweaking by genetic manipulation certainly seems off the menu too. "I don't think we are going to see superman or a split in the species any time soon," says Greely, "because we just don't know enough and are unlikely to for a long time - or maybe for ever."

If this is all "designer babies" could mean even in principle - freedom from some specific but rare diseases, knowledge of rather trivial aspects of appearance, but only vague, probabilistic information about more general traits like health, attractiveness and intelligence - will people go for it in large enough numbers to sustain an industry?

Greely suspects, even if it is used at first only to avoid serious genetic diseases, we need to start thinking hard about the options we might be faced with. "Choices will be made," he says, "and if informed people do not participate in making those choices, ignorant people will make them."

The Crispr/Cas9 system uses a molecular structure to edit genomes. Photograph: Alamy

Green thinks that technological advances could make "design" increasingly versatile. In the next 40-50 years, he says, "we'll start seeing the use of gene editing and reproductive technologies for enhancement: blond hair and blue eyes, improved athletic abilities, enhanced reading skills or numeracy, and so on."

He's less optimistic about the consequences, saying that we will then see social tensions "as the well-to-do exploit technologies that make them even better off", increasing the relatively worsened health status of the world's poor. As Greely points out, a perfectly feasible 10-20% improvement in health via PGD, added to the comparable advantage that wealth already brings, could lead to a widening of the health gap between rich and poor, both within a society and between nations.

Others doubt that there will be any great demand for embryo selection, especially if genetic forecasts remain sketchy about the most desirable traits. "Where there is a serious problem, such as a deadly condition, or an existing obstacle, such as infertility, I would not be surprised to see people take advantage of technologies such as embryo selection," says law professor and bioethicist R Alta Charo of the University of Wisconsin. "But we already have evidence that people do not flock to technologies when they can conceive without assistance."

The poor take-up of sperm banks offering "superior" sperm, she says, already shows that. For most women, "the emotional significance of reproduction outweighs any notion of 'optimisation'". Charo feels that "our ability to love one another with all our imperfections and foibles outweighs any notion of 'improving' our children through genetics".

All the same, societies are going to face tough choices about how to regulate an industry that offers PGD with an ever-widening scope. "Technologies are very amoral," says Birney. "Societies have to decide how to use them" - and different societies will make different choices.

One of the easiest things to screen for is sex. Gender-specific abortion is formally forbidden in most countries, although it still happens in places such as China and India where there has been a strong cultural preference for boys. But prohibiting selection by gender is another matter. How could it even be implemented and policed? By creating some kind of quota system?

And what would selection against genetic disabilities do to those people who have them? "They have a lot to be worried about here," says Greely. "In terms of whether society thinks I should have been born, but also in terms of how much medical research there is into diseases, how well understood it is for practitioners and how much social support there is."

Once selection beyond avoidance of genetic disease becomes an option - and it does seem likely - the ethical and legal aspects are a minefield. When is it proper for governments to coerce people into, or prohibit them from, particular choices, such as not selecting for a disability? How can one balance individual freedoms and social consequences?

"The most important consideration for me," says Charo, "is to be clear about the distinct roles of personal morality, by which individuals decide whether to seek out technological assistance, versus the role of government, which can prohibit, regulate or promote technology."

She adds: "Too often we discuss these technologies as if personal morality or particular religious views are a sufficient basis for governmental action. But one must ground government action in a stronger set of concerns about promoting the wellbeing of all individuals while permitting the widest range of personal liberty of conscience and choice."

"For better or worse, human beings will not forgo the opportunity to take their evolution into their own hands," says Green. "Will that make our lives happier and better? I'm far from sure."

A scientist at work during an IVF process. Photograph: Ben Birchall/PA

Easy pickings: the future of designer babies

The simplest and surest way to "design" a baby is not to construct its genome by pick'n'mix gene editing but to produce a huge number of embryos and read their genomes to find the one that most closely matches your desires.

Two technological advances are needed for this to happen, says bioethicist Henry Greely of Stanford University in California. The production of embryos for IVF must become easier, more abundant and less unpleasant. And gene sequencing must be fast and cheap enough to reveal the traits an embryo will have. Put them together and you have "Easy PGD" (preimplantation genetic diagnosis): a cheap and painless way of generating large numbers of human embryos and then screening their entire genomes for desired characteristics.

"To get much broader use of PGD, you need a better way to get eggs," Greely says. "The more eggs you can get, the more attractive PGD becomes." One possibility is a one-off medical intervention that extracts a slice of a woman's ovary and freezes it for future ripening and harvesting of eggs. It sounds drastic, but would not be much worse than current egg-extraction and embryo-implantation methods. And it could give access to thousands of eggs for future use.

An even more dramatic approach would be to grow eggs from stem cells - the cells from which all other tissue types can be derived. Some stem cells are present in umbilical blood, which could be harvested at a person's birth and frozen for later use to grow organs - or eggs.

Even mature cells that have advanced beyond the stem-cell stage and become specific tissue types can be returned to a stem-cell-like state by treating them with biological molecules called growth factors. Last October, a team in Japan reported that they had made mouse eggs this way from skin cells, and fertilised them to create apparently healthy and fertile mouse pups.

Thanks to technological advances, the cost of human whole-genome sequencing has plummeted. In 2009 it cost around $50,000; today it is most like $1,500, which is why several private companies can now offer this service. In a few decades it could cost just a few dollars per genome. Then it becomes feasible to think of PGD for hundreds of embryos at a time.

"The science for safe and effective Easy PGD is likely to exist some time in the next 20 to 40 years," says Greely. He thinks it will then become common for children to be conceived through IVF using selected genomes. He forecasts that this will lead to "the coming obsolescence of sex" for procreation.

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Designer babies: an ethical horror waiting to happen?

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Nearly 40 years since the first 'test-tube baby', how close are we to editing out all of our genetic imperfections - and should we even try to do so?

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Finding out if you can really bring back the extinct.

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An extinct rat that once lived on an island in the Indian Ocean may have put the kibosh on scientists’ dreams of resurrecting more famous extinct animals like the woolly mammoth. The Christmas Island rat disappeared just over 100 years ago, but researchers now say even its detailed genome isn’t complete enough to bring it back to life.

The work “shows both how wonderfully close—and yet—how devastatingly far” scientists are from being able to bring back extinct species by genetically transforming a close relative in what’s called “de-extinction,” says Douglas McCauley, an ecologist at the University of California, Santa Barbara, who was not involved with the study.

No species has yet been revived, but de-extinction appeals to many geneticists and futurists. “I know a lot of biologists who think, ‘Can I do this?’” says Karen Wendling, an ethicist at the University of Guelph. Part of the fascination is simply the promise of seeing a vanished species come to life. But putting a key animal back into its original habitat could also help restore ecosystems. The mammoth once kept arctic shrubs and trees under control and fertilized grasses with their manure. Indeed, a company called Colossal Laboratories and Biosciences aims to transform elephants into mammoths, or, at least, mammothlike animals. “There is a lot of hope and hype that de-extinction will save us” from ecosystems failing, McCauley says.

To bring back an extinct species, scientists would first need to sequence its genome, then edit the DNA of a close living relative to match it. Next comes the challenge of making embryos with the revised genome and bringing them to term in a living surrogate mother. So far, scientists have sequenced the genomes of about 20 extinct species, including a cave bear, passenger pigeon, and several types of mammoths and moas. But no one has yet reported re-creating the extinct genome in a living relative.

In the new study, Tom Gilbert, an evolutionary geneticist at the University of Copenhagen, thought it best to start small. “If we want to try something so crazy, why not start with a simple model,” he reasoned. So, he, Jian-Qing Lin, a molecular biologist at Shantou University, and their colleagues, focused on the Christmas Island rat (Rattus macleari), which disappeared by 1908 from that island, located about 1200 kilometers west of Australia. This species “should be a dreamy candidate for de-extinction,” McCauley says, given its close relationship with the Norway rat, a well-studied lab animal with a complete genome sequence that scientists already know how to modify.

Gilbert and Lin extracted DNA from the skins of two preserved Christmas Island rats and sequenced it many times over to get as much of the genome as possible. They achieved more than 60 times’ coverage of it. Old DNA only survives in small fragments, so the team used the genome of the Norway rat as a reference to piece together as much as possible of the vanished rat’s genome. Comparing the two genomes revealed almost 5% of the Christmas Island rat’s genome was still missing, Lin, Gilbert, and their colleagues report today in Current Biology. The lost sequences included bits of about 2500 of the rat’s estimated 34,000 genes. “I was surprised,” Gilbert says. The recovered DNA included the genes for the Christmas Island rat’s characteristic rounded ears, for example, but important immune system and olfaction genes were either missing or incomplete.

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The amount of missing DNA surprised Henry Greely, a bioethicist at Stanford University. “That’s an important issue that I hadn’t focused on.”

The work “really highlights the difficulties, maybe even the ridiculousness, of [de-extinction] efforts,” says Victoria Herridge, an evolutionary biologist at the Natural History Museum in London.

Many of the missing genes will be the ones that make each species unique, Herridge says. She notes that the more time that has passed since an extinct species and its living relative have diverged, the more genes are likely to be missing; the Christmas and Norway rats split 2.6 million years ago, a blink of an eye in evolutionary time; mammoths and the Asian elephants diverged 6 million years ago. “A lot of the talk has made [de-extinction] seem very straightforward, but of course, it’s not,” Herridge says.

McCauley agrees. He says 5% is a big difference—the human genome differs by just 1% from those of chimps and bonobos. “We could make something, but it seems clear it will never be a Christmas Island rat,” he adds. “In which case, what is the point?”

But Andrew Pask, a developmental biologist at the University of Melbourne, Parkville, who is spearheading an effort to bring back an extinct marsupial called the thylacine, or Tasmanian tiger, is unfazed by the new report. “The thylacine is perhaps the best candidate for [de-extinction],” he says. His team sequenced this predator’s genome as well as the genomes of several potential surrogate species, including the dunnart or marsupial mouse. They find that although 5% of the thylacines’ genome is missing, that 5% is primarily repetitive regions that likely won’t affect how the transformed animal looks or behaves, he says.

Moreover, sequencing is “steadily improving” for both modern and extinct DNA, says George Church, the Harvard University geneticist who helped found Colossal. “Many 100% animal genomes will be arriving faster and faster.”

Gilbert now thinks creating an exact replica of a mammoth or a passenger pigeon will be “impossible.” But such efforts might lead to what the International Union for Conservation of Nature calls “proxies,” animals close enough to carry out the same function in the extinct species’ old ecosystem, such as a cold-tolerant “woolly elephant.” And that’s where Church is headed. “The main goal is not making ‘perfect photocopies,’ but making diverse and selective hybrids,” he explains. Right now, his team is focused on defining the mammoth’s cold-tolerance genes for transferring into elephants.

Herridge cautions that in most cases scientists won’t know in advance how the edited genes will affect the animal’s behavior and ability to survive. “Even if we got it genetically perfect, we are still going to have to gestate it in a different species, so why would you think it would be exactly the same?” Wendling adds.

McCauley and others also think de-extinction efforts may take needed resources away from conservation. “You can save eight extant species for [the cost of] one that you make de-extinct,” Wendling points out.

But even those who are not sure that de-extinction could or should happen still fantasize about the possibility. Wendling wants to know what a dodo bird is like. And, Greely says, “Personally, I would love to see a giant ground sloth or an saber-toothed cat!”

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PDF Version: Cunningham

Editor's Note: This article originally appeared in the Volume 17, Number 1&2, Spring/Summer 2010 issue of Dignitas, the Center’s quarterly publication. Subscriptions to Dignitas are available to CBHD Members. To learn more about the benefits of becoming a member click here.

When the great naturalist Joseph Kolreuter painstakingly and methodically cross-pollinated hundreds of plants in the 18th century, he could not have foreseen the 21st century version of hybrids: human-animal (HA) hybrids. HA hybrids confront us with a technology which eludes a readymade ethical conclusion. In “doing bioethics,” particularly with emerging technologies, we find that it takes time to understand, consider and reach an ethical conclusion. An open bioethics conversation among those who share our Judeo-Christian commitments also means that we may be uncertain about preliminary observations as well as ultimate conclusions. HA hybrids is one of those situations. In this column, I’ll address just one aspect of HA hybrids: the insertion of human neurons into animal brains. But first, what are we talking about?

Understand the Biotechnology

In the popular understanding, “hybrids” includes three biotechnologies: chimeras, hybrids, and cybrids. Chimeras are entities created by mixing cells of different animals, usually two different species; each cell retains its original genetic identity. Think of a graft, such as replacing an aging human heart valve with one grown in a pig. Other chimeras are created at a much earlier stage by mixing two embryos, changing the appearance of the new organism. While the centaur is a mythological version, unusual animal hybrids exist, for example, the liger (a combination of a male lion and female tiger), the tigon (offspring of a male tiger and female lion), and the beefalo, a bison/ cattle breed designed for beef production.

Splice, a 2010 summer movie release, tells the dark tale of Dren, a half-human, half-animal lab-created chimera that unpredictably grows and terrorizes people. Actual human chimeras may not raise the same fears. Their new heart valve does not acquire human DNA. Nor does it change their fundamental humanness.

True hybrids are created by integrating some genetic material from one species into an animal of a different species, perhaps by fertilizing the egg of the former with sperm from the latter. Human “cybrids” are hybrids created by a cloning process: human DNA is inserted into a non-human egg that has been enucleated (the animal nucleus has been removed), usually from a cow or rabbit. Cybrids contain more than 99% human DNA; the rabbit or cow mitochondrial DNA in the cytoplasm surrounding the nucleus remains. The United Kingdom, one of few places to permit cybrid research, requires the cybrid embryo to be destroyed after fourteen days. Chinese scientists apparently created a human animal hybrid by inserting human DNA into rabbit eggs for the purpose of extracting the embryonic stem cells.[1]

HA hybrids are produced for a variety of purposes: to observe how transplanted cells differentiate in the host (What kinds of cells do they become?), to test human cells (Are these early cells pluripotent?), to find out what cells will do (Will these become cancerous?), to reveal how these cells are affected by different control systems, to test new drugs for medical treatment, and to grow replacement tissues or organs for xenotransplantation. As the Chinese have claimed, embryonic stem cells might be harvested from cybrid embryos. Their research, which has not been proven elsewhere, would produce human embryos in bulk, to create made-to-order tissues for patients.[2]

Identify Potential Benefits and Risks

HA hybrids might be used to study the causes and development of diseases such as cystic fibrosis, Parkinson’s, AIDS and heart disease, pointing toward new therapies. Genetically engineered mice hybrids with human DNA inserted can generate antibodies to treat cancer that will not be rejected by the human recipient’s body. Researchers may also develop HA hybrids to test new drugs.

Despite their significant research potential, HA hybrids carry some risks. The lessons of history warn of the risk of zoonotic infection. That is, diseases which have been confined to the animal kingdom may cross over to humans. We have witnessed the worldwide calamities triggered by the introduction of HIV, avian virus, and H1N1 influenza (swine flu). A single genetic or protein fragment might be sufficient for crossing the species boundary, causing diseases such as cancer, leukemia, and mad cow disease.

Additional risks include the creation of human diseases and the reality that no one knows how the HA hybrids will develop. When animal viruses cross the species barrier, new strains can emerge which may be carried only by human hosts. Furthermore, while many animal hybrids are sterile, closely related species, such as a mule and a donkey, have been known to reproduce. The “what if” allure of inseminating a primate, such as a chimpanzee, with human sperm may be irresistible. The sensationalized attempt of an early 20th century Soviet scientist to create “humanzees” dramatically illustrates this potential.

Ethical Observations

Ethical inquiry often begins with questions about consequences. As HA experiments proceed, what would be the moral status of these new creatures? Are they protected by animal welfare regulations, or do they deserve human subject research protection? Are there limitations on how much human DNA can be inserted into an animal? How are the risks of zoonotic infection controlled? Would this open the door to using primates to grow donor specific replacement organs, as did the mad scientists in Robin Cook’s Chromosome 6?

While consequences are important considerations, our ultimate concern should involve a deeper level of ethical analysis. Three moral and theological questions immediately come to mind: species boundaries, bodily integrity, and human identity. All of these are aspects of our human dignity. We will explore species boundaries, and touch upon human identity with respect to one specific technology. Although it is easier to state categorically that no human female should be inseminated with animal sperm and vice versa, or that cloning with a rabbit ovum and human nucleus is wrong, other HA possibilities are not clear cases of impermissible mixing of species. One of these is the insertion of human brain cells into animal brains.

It is obviously difficult to study the development of the human central nervous system in human subjects, but it is possible to transplant human brain cells into embryonic, fetal or adult animals, typically mice. Researchers can then track and observe how these cells develop and interact. These HA hybrids are technically chimera, because the human cells do not acquire the mouse DNA; they remain distinct and traceable.

Does this cross a boundary between the human species and the animal? If so, what is the criterion or basis for that boundary? Most people believe that there is a difference, a qualitative difference, between a human being and a tortoise, or even a circus-trained chimpanzee. Christians in the US would describe this as “human dignity.” In the UK, “full moral status” is more commonly used. Both expressions signal that human beings are exceptional, distinct from other creatures.

Human exceptionalism is a difficult problem for biologists who are nominalists. Nominalist theory concludes that only concrete things exist, and that abstract ideas, such as “species,” do not. To illustrate: the nominalist biologist points out that there is no single, universal DNA sequence among human beings; there is no conclusive standard for determining the species of an organism based on its DNA sample. Or, they tout the evolutionary connection of human beings with a common ancestor. According to this perspective, human beings are nothing more than dust and ashes, a particularly clever architecture of molecules and cells.

Science cannot answer why any biological organism is of greater value than another. Admittedly, “species boundary” is a difficult problem, particularly from a biological perspective, but the mere fact of difficulty does not mean that species identities and boundaries do not exist. Think about it: it is hard to define the precise boundary between night and day, but that imprecision does not imply that night and day do not exist. Therefore, while Christian ethicists can and do disagree about where to locate the boundary line in these matters, boundaries do exist, and it is an important part of ethical reflection to strive to discern them.

Matter Causes Brain Causes Mind?

The nominalist presupposes that the human mind derives only from the brain, which is composed solely of matter, and that the mind is the basis for possessing human dignity. The nominalist would be concerned about a change in the structure of the brain that might cause a change in its function. When an undefined threshold is reached, that is, a critical number of neurons have been inserted and have integrated themselves into the mouse brain, the mouse brain might demonstrate human-like cognitive characteristics. For the nominalist, this is the threshold of unease, if not outright certainty, that a species or moral barrier has been breached.

If unease is based on “humanlike” cognition alone, it appears to condition species membership and our concomitant moral worth on mental abilities. If we stop to consider a radical, logical extension of this ethical position, the ramifications are chilling. Those with significantly impaired cognitive function—the uncle in a persistent vegetative state, the daughter with serious developmental delays, the elderly grandmother with dementia, the anencephalic newborn boy—are judged to have less moral worth than the Rhodes scholar or moral philosopher. What is respected here is not humandignity, but human cognition. While cognitive capacity is one evidence of our singularity, it is not the basis of the ontological reality, the truth about our status as creatures made in the image of God. There are many other capacities which are distinctively human, for example, humor, preservation of history, artistic creativity, imagination, self-awareness. No single human being fully expresses all mental capacities, and some humans lack one or more entirely. Yet, it would be wrong to conclude they are not human beings with dignity.

Some advocates of human dignity are concerned about transplanting human neurons into animal brains on different grounds. Because of the brain’s intimate connection with personal identity, on this view, brain transplantation—a theoretical possibility for now—would be clearly immoral. The insertion of human cells into an animal brain could be problematic in two ways. The first concerns the origin of the cells: bone marrow stem cells might not be problematic, but neuronal progenitor cells, which raise the “possibility of humanlike connections between the neurons,” are troubling.[3] The second concern has to do with the potential of the inserted cells to change the architecture of the brain, that is, its weight, shape, and size. It is not clear what percentage of human neurons constitutes a “significant” alteration. Thus, prudence counsels that we not engage in a procedure which potentially alters identity.

It is important to note that not all HA hybrid research may violate species boundaries. Inserting a small fragment of human DNA into a mouse to develop a cancer-fighting drug, for example, might not implicate human dignity. Growing a human-tolerant pig valve for a heart patient is therapeutic, not threatening.

Thus, we have two different arguments against integrating human DNA into animal brains, one based on nominalist grounds, and the other on dignitarian grounds. A whole host of ethical concerns remain. What about concerns for animal welfare? The host animal and its offspring may suffer terribly. Could “human dignity” apply to HA hybrids? Are they human, or actually something else? In addition to neurons, are there other types of cells that raise specific concerns, such as gametes, or organs, such as the uterus? Does it matter at what stage of biological development the species mixing occurs? It could be at fertilization, at the embryonic stage, or somewhat later. How is this relevant?

Humanzees or Dren from Splice may be fanciful creations. The mass production of human-animal embryos may not. Somewhere in between we may find the highest and best purposes of research, those therapeutic goals that do not violate ethical standards. We still lack a Christian consensus on all aspects of the HA hybrid question, but we must persevere and continue the difficult work of thinking through ethical issues, principles, and their application. A premature conclusion may initially satisfy, but ultimately prove to be a barrier to both encouraging ethical research and respecting human dignity in all its stages, ages, and variations.

References

[2] David Derbyshire, “Experiments fail: Controversial human-animal hybrid embryos ‘will not deliver medical benefit.’” http://www.dailymail.co.uk/ sciencetech/article-1134483/Experiments-fail-Controversial-human-animalhybrid- embryos-deliver-medical-benefit.html (accessed September 22, 2010).

[3] Tara L. Seyfer, “An Overview of Chimeras and Hybrids,” The National Catholic Bioethics Quarterly (Spring 2006): 37-49, 47.

Cite as: Paige C. Cunningham, "Ligers, Tigons, and Splice: Human-Animal Hybrids,” Dignitas 17, no. 1&2 (2010): 14–16.

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Ligers, Tigons, and Splice: Human-Animal Hybrids

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When the great naturalist Joseph Kolreuter painstakingly and methodically cross-pollinated hundreds of plants in the 18th century, he could not have foreseen the 21st century version of hybrids: human-animal (HA) hybrids. HA hybrids confront us with a technology which eludes a readymade ethical conc…

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how human organ farming could save 20 lives everyday.

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In a new paper published in Stem Cell Reports, Bhanu Telugu and co-inventor Chi-Hun Park of the University of Maryland (UMD) Department of Animal and Avian Sciences show for the first time that newly established stem cells from pigs, when injected into embryos, contributed to the development of only the organ of interest (the embryonic gut and liver), laying the groundwork for stem cell therapeutics and organ transplantation. Telugu's start-up company, Renovate Biosciences Inc. (RBI), was founded with the goal of leveraging the potential of stem cells to treat terminal diseases that would otherwise require organ transplants, either by avoiding the need for transplants altogether or creating a new pipeline for growing transplantable human organs. With the number of people who suffer from organ failures and the 20 deaths per day in the U.S. alone purely from a lack of available organs for transplant, finding a new way to provide organs and therapeutic options to transplant patients is a critical need. In this paper, Telugu and his team are sharing their first steps towards growing fully transplantable human organs in a pig host.

"This paper is really about using the stem cells from pigs for the first time and showing that they actually can be injected into embryos and only go to the endodermal target organs like the liver, which is very important for delivering safe therapeutic solutions going forward," says Telugu. "This is an important milestone. It's a pipe dream in a way because a lot of things need to work out between here and full organ transplantation, but this paper sets the stage for all our future research. We can't really just go and start working with humans in work like this, so we started with pig-to-pig transfer in this paper, working with the stem cells and putting them back into other pigs to track the process to make sure it is safe for liver production as proof-of-concept."

Telugu and his team pitched this work at UMD Bioscience Day on behalf of his company, RBI, and received the Inventor Pitch Award and the UMD Invention of the Year Award in 2018. In order to protect the intellectual property, Telugu worked with the UMD Office of Technology Commercialization (OTC) to secure patents and open the work up for additional fundraising to carry this technology through the preclinical and clinical stages. The Maryland Stem Cell Foundation provided some funding to advance this work, and Telugu is thankful that Maryland funds technologies in the human stem cell space.

"There are many terminal cases where people need some sort of an organ replacement, like organ failure and degenerative diseases that cannot be cured by drugs," explains Telugu. "The traditional paradigm is to find a donor organ, but as of today there are still thousands of patients waiting for transplants, and there is no keeping up with the demand. Researchers have thought for a long time that stem cells could help solve this problem, and these stem cells have the ability to go into a specific organ as opposed to those that go into any lineage. In this case, you can differentiate the cells and place them where they are needed to help rescue a diseased organ, eliminating the need for transplant or at least buying the patient some time. Just making the human liver and collecting them early from a neonatal piglet, the hepatocyte [liver] cells alone are a $3 billion opportunity per year. And in the future, we can move into organ transplantation, first with the liver, and then looking at other organs of interest like the pancreas and lungs."

According to Telugu, this has distinct advantages over other methods that researchers are currently using to create donor organs in pigs, since the organs Telugu and his team are working with are actually of human origin and are therefore more likely to be accepted when transplanted. "Transplant rejections are pretty common even between humans and humans," says Telugu, "and if it is such a problem normally, you can imagine how an organ from a pig could be difficult to accept and may not essentially perform the same functions. Pig proteins may not function the same, so that remains a huge barrier for other methods that are not actually growing fully human organs like ours."

This work has the potential to solve a major problem in the treatment of organ failure and other degenerative diseases, which is what Telugu and his work is all about. "Being a veterinarian by training, we always look at the problem and try to find solutions to them," says Telugu. "Most animal scientists operate by looking for solutions, so integrating research and entrepreneurship to get this to the market where it is needed is essential. We are one of the few groups on the planet that are working in this space, and we have a great team of embryologists here at Maryland to do this work. We are uniquely positioned to accomplish this with both genome editing and stem cell biology expertise, and being able to prove the concept with this paper is a great first step towards our goals."

Journal Reference:

Chi-Hun Park, Young-Hee Jeoung, Kyung-Jun Uh, Ki-Eun Park, Jessica Bridge, Anne Powell, Jie Li, Laramie Pence, Luhui Zhang, Tianbin Liu, Hai-Xi Sun, Ying Gu, Yue Shen, Jun Wu, Juan-Carlos Izpisua Belmonte, Bhanu P. Telugu. Extraembryonic Endoderm (XEN) Cells Capable of Contributing to Embryonic Chimeras Established from Pig Embryos. Stem Cell Reports, 2020; DOI: 10.1016/j.stemcr.2020.11.011

Cite This Page:

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stem cells; liver disease; prostate cancer; skin cancer; nanotechnology; organic chemistry; thermodynamics; biochemistry

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Growing human organs for transplantation with new proof-of-concept

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With the number of people who suffer from organ failures and the growing need for available organs for transplant, finding a new way to provide organs and therapeutic options to transplant patients is a critical need.

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The advancement is Science has enabled to produce first ever genetically designed Baby named Adam Nash.

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When Adam Nash was still an embryo, living in a dish in the lab, scientists tested his DNA to make sure it was free of Fanconi anemia, the rare inherited blood disease from which his sister Molly suffered. They also checked his DNA for a marker that would reveal whether he shared the same tissue type. Molly needed a donor match for stem cell therapy, and her parents were determined to find one. Adam was conceived so the stem cells in his umbilical cord could be the lifesaving treatment for his sister.

Adam Nash is considered to be the first designer baby, born in 2000 using in vitro fertilizaton with pre-implantation genetic diagnosis, a technique used to choose desired characteristics. The media covered the story with empathy for the parents' motives but not without reminding the reader that "eye color, athletic ability, beauty, intelligence, height, stopping a propensity towards obesity, guaranteeing freedom from certain mental and physical illnesses, all of these could in the future be available to parents deciding to have a designer baby. "

The designer babies have thus been called the "future-we-should-not-want" for each new reproductive technology or intervention. But the babies never came and are nowhere close. I am not surprised.

I study the prediction of complex diseases and human traits that result from interactions between multiple genes and lifestyle factors. This research shows that geneticists cannot read the genetic code and know who will be above average in intelligence and athleticism. Such traits and diseases that result from multiple genes and lifestyle factors cannot be predicted using just DNA, and cannot be designed. Not now. And very unlikely ever.

Designer babies are next

The inevitable rise of designer babies was proclaimed in 1978 after the birth of Louise Brown, the first IVF baby, as the next step toward "a brave world where parents can select their child's gender and traits." The same situation occurred in 1994 when a 59-year-old British woman stretched the limits of nature by giving birth to twins using donated eggs that were implanted in her womb at a fertility clinic in Italy.

The response was the same in 1999, when a fertility clinic in Fairfax, Virginia, offered sex selection of embryos to screen against diseases that only happen in boys. In 2013, when 23andMe was granted a patent for a tool that predicts the likelihood of traits in babies based on DNA of two parents, the question of patenting designer babies was raised. In 2016, when the U.K. permitted a woman to donate her healthy mitochondria to a couple using IVF to conceive a child, raising the number of parents to three, fears of unnatural children rose again. Last month, when Genomic Prediction, a New Jersey company, announced its DNA screening panel for embryos would also assess the risk for complex diseases such as Type 2 diabetes and heart disease that are caused by multiple genes, fears of engineering babies with high IQ or athletic prowess emerged.

The same issues arose on Nov. 26 when He Jiankui reported at the Second International Summit on Human Genome Editing in Hong Kong that he had successfully edited the DNA of twin baby girls born last month.

The designer baby doom scenarios have not evolved with the technology. It's been the same story for decades. It's the same "desirable" traits and the same assumption that parents want to select these traits if technology allowed. But no one seems to be questioning whether these traits are solely a product of our genes such that they can be selected or edited in embryos.

Wondering about designer babies was understandable in the early days, but repetition of these supposed fears now suggests lack of understanding of how DNA, and the genes they encode, work.

Designing favorable traits in babies is not simple

Although there are exceptions, DNA generally differs between people in two ways: There are DNA mutations and DNA variations.

Mutations cause rare diseases like Huntington's disease and cystic fibrosis, which are caused by a single gene. Mutations in the BRCA genes substantially increase the risk of breast and ovarian cancer. Selecting embryos that do not have these mutations removes the entire or main cause of disease - women who don't have BRCA mutations can still develop breast cancer through other causes, like all women.

Variations are changes in the genetic code that are more common than mutations and associated with common traits and diseases. DNA variants increase the likelihood that you may have a trait or develop a disease but do not determine or cause it. Association means that in several large study populations, a DNA variant was more frequent among people with the trait than those without, often only slightly more frequent.

These variants don't determine a trait, but increase its likelihood by interacting with other DNA variants and nongenetic influences such as upbringing, lifestyle and environment. To design such traits in embryos would require multiple DNA changes in multiple genes and orchestrating or controlling relevant environment and lifestyle influences too.

Let's compare it to driving a car. DNA mutations are like the flat tires and the failing brakes: technical problems that make driving problematic, no matter where you drive. DNA variations are like the color and the type of car, or other features of the car that may affect the driving experience and even might create problems over time. For example, a convertible is a delight when driving on Hollywood's Sunset Boulevard on a breezy summer evening, but cruel when crossing a high mountain pass in midwinter. Whether features of the car are an asset or a liability depends on the context and that context might change - they are never ideal all the time.

Another hurdle

Most DNA mutations do nothing else other than cause the disease, but DNA variations may play a role in many diseases and traits. Take variations in the "red hair" gene, which not only increases the chance that your child will have red hair, but also increases their risk of skin cancer. Or variations in the and "eye color" genes that are also associated with the risk of various cancers, Parkinson's and Alzheimer's disease. To be sure, these are statistical associations, reported in the scientific literature, some may be confirmed; others may not. But the message is clear: Editing DNA variations for "desirable" traits may have adverse consequences, including many that scientists don't know about yet.

We can see this in the analysis of He Jiankui's gene-edited babies. By trying to make the babies resistant to HIV, He might have greatly increased susceptibility to infections by West Nile virus or influenza.

To be sure, even though complex traits such as intelligence, athletics and musicality cannot be selected or designed, there will be opportunists who will try to offer these traits, even if totally premature and unsupported by science. Like Stephen Hsu, the co-founder of Genomic Prediction who said about his offer to test embryos for polygenic risk, the risk of a disease based on multiple genes, "I think people are going to demand that. If we don't do it, some other company will." And also He said: "There will be someone, somewhere, who is doing this. If it's not me, it's someone else." People need to be protected against this irresponsible and unethical use of DNA testing and editing.

Science brought incredible progress in reproductive technology, but didn't bring designer babies one step closer. The creation of designer babies is not limited by technology, but by biology: The origins of common traits and diseases are too complex and intertwined to modify the DNA without introducing unwanted effects.

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Those designer babies everyone is freaking out about - it's not likely to happen

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When Adam Nash was still an embryo, living in a dish in the lab, scientists tested his DNA to make sure it was free of Fanconi anemia, the rare inherited blood disease from which his sister Molly suffered. They also checked his DNA for a marker that would reveal whether he shared the same tissue type.

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https://www.who.int/news/item/12-07-2021-who-issues-new-recommendations-on-human-genome-editing-for-the-advancement-of-public-health

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Two new companion reports released today by the World Health Organization (WHO) provide the first global recommendations to help establish human genome editing as a tool for public health, with an emphasis on safety, effectiveness and ethics.

The forward-looking new reports result from the first broad, global consultation looking at somatic, germline and heritable human genome editing. The consultation, which spanned over two years, involved hundreds of participants representing diverse perspectives from around the world, including scientists and researchers, patient groups, faith leaders and indigenous peoples.

"Human genome editing has the potential to advance our ability to treat and cure disease, but the full impact will only be realized if we deploy it for the benefit of all people, instead of fueling more health inequity between and within countries," said Dr Tedros Adhanom Ghebreyesus, WHO Director-General.

Potential benefits of human genome editing include faster and more accurate diagnosis, more targeted treatments and prevention of genetic disorders. Somatic gene therapies, which involve modifying a patient's DNA to treat or cure a disease, have been successfully used to address HIV, sickle-cell disease and transthyretin amyloidosis. The technique could also vastly improve treatment for a variety of cancers.

However, some risks exist, for example, with germline and heritable human genome editing, which alter the genome of human embryos and could be passed on to subsequent generations, modifying descendants' traits.

The reports published today deliver recommendations on the governance and oversight of human genome editing in nine discrete areas, including human genome editing registries; international research and medical travel; illegal, unregistered, unethical or unsafe research; intellectual property; and education, engagement and empowerment. The recommendations focus on systems-level improvements needed to build capacity in all countries to ensure that human genome editing is used safely, effectively, and ethically.

The reports also provide a new governance framework that identifies specific tools, institutions and scenarios to illustrate practical challenges in implementing, regulating and overseeing research into the human genome. The governance framework offers concrete recommendations for dealing with specific scenarios such as:

A hypothetical clinical trial of somatic human genome editing for sickle cell disease proposed to take place in West Africa

Proposed use of somatic or epigenetic genome editing to enhance athletic performance

An imaginary clinic based in a country with minimal oversight of heritable human genome editing that offers these services to international clients following in vitro fertilization and preimplantation genetic diagnosis

"These new reports from WHO's Expert Advisory Committee represent a leap forward for this area of rapidly emerging science," said WHO's Chief Scientist, Dr Soumya Swaminathan. "As global research delves deeper into the human genome, we must minimize risks and leverage ways that science can drive better health for everyone, everywhere."

What's next
WHO will:

Convene a small expert committee to consider next steps for the Registry, including how to better monitor clinical trials using human genome editing technologies of concern

Convene multisector stakeholders to develop an accessible mechanism for confidential reporting of concerns about possibly illegal, unregistered, unethical and unsafe human genome editing research and other activities

As part of a commitment to increase 'education, engagement and empowerment', lead regional webinars focusing on regional/local needs. Work within the Science Division to consider how to build an inclusive global dialogue on frontier technologies, including cross-UN working and the creation of web-based resources for reliable information on frontier technologies, including human genome editing.

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WHO issues new recommendations on human genome editing for the advancement of public health

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Two new companion reports released today by the World Health Organization (WHO) provide the first global recommendations to help establish human genome editing as a tool for public health, with an emphasis on safety, effectiveness and ethics. The forward-looking new reports result from the first broad, global consultation looking at somatic, germline and heritable human genome editing.

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870449/

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Abstract

Purpose

As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos.

Methods

3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos.

Results

By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5 Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations.

Conclusions

This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

Electronic supplementary material

The online version of this article (doi:10.1007/s10815-016-0710-8) contains supplementary material, which is available to authorized users.

CRISPR/Cas9, Genetic modification, Keywords: CCR5, Human 3PN embryos

Introduction

Sequencing the genomes of numerous species, including humans, has uncovered the genetic blueprints encoded in DNA that direct the development and progression of organisms on Earth, which are constantly challenged by natural selection [ 1, 2]. The ability to make random or targeted modifications to genomic sequences allows us to study the functions of genetic components and engineer organisms for research and practical applications. The recent development of site-specific DNA endonuclease technologies, such as zinc finger nuclease (ZFN) [ 3], transcription activator-like effector nuclease (TALEN) [ 4], and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) nuclease system (CRISPR/Cas) [ 5], has greatly accelerated genome engineering applications and broadened the spectrum of species that is applicable to such methods. When these systems bind to a target DNA sequence in the genome, they create a DNA double strand break (DSB), the repair of which leads to specific DNA sequence modifications.

By introducing the CRISPR/Cas system into a zygote, methods have been established for the one-step generation of mice carrying mutations in single and multiple genes and of mice carrying reporter and conditional alleles [ 6, 7]. Using a similar approach, two groups have demonstrated that precise genetic modifications could be achieved in early mouse embryos to correct disease-causing mutations, leading to the production of healthy adult animals [ 8, 9]. These studies highlighted that the correction of genetic mutations in germline cells or early embryos could serve as a permanent solution for genetic diseases. Moreover, Niu et al. and Liu et al. recently showed that the injection of zygotes with CRISPR/Cas and TALEN systems could generate targeted genetic mutations in non-human primates [ 10, 11]. More recently, Liang et al. reported that the CRISPR/Cas9 system can mediate genetic modifications in human tripronuclear zygotes [ 12]. In the present study, we successfully introduced the naturally occurring CCR5Δ32 allele into early human tripronuclear (3PN) embryos by CRISPR/Cas-mediated genome editing. However, the efficiency of homology directed repair (HDR) of the CCR5 Δ32 allele was low, and the edited embryos were mosaic. Our study serves two purposes: first, to evaluate technologies and establish principles for the introduction of precise genetic modifications in early human embryos and to identify any significant technical hurdles; and, second, to raise awareness of the potential legal and ethical challenges that our society will face from the increasing accessibility of genome engineering technologies.

Materials and methods

Source of tripronuclear human zygotes

From April 2014 to September 2014, a total of 213 3PN human zygotes from 87 patients with a visible second polar body were obtained 18-20 h post-insemination from the Third Affiliated Hospital of Guangzhou Medical University.

Tripronuclear human zygote injection

The 3PN zygotes were transferred into HTF medium (Quinn's Advantage ™ Medium with HEPES). They were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo injections, donor oligo-1 (90 bp) or oligo-2 (90 bp) (100 ng/μl) or dsDonor (1 kb) (100 ng/μl) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos. The injected embryos were cultured in G1.5 media until day three. All embryos were maintained in a humidified incubator at 37 °C, 5 % CO ₂, and 20 % oxygen.

Production of Cas9 mRNA, gRNAs, and DNA donors

In vitro transcription (IVT) of Cas9 mRNA was performed as previously described [ 13]. Briefly, T7 promoter was added to Cas9 coding region by PCR amplification, using px330 vector [ 14] as template. The PCR product was gel-purified and used as a template for IVT using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). For gRNA synthesis, the IVT templates were generated by PCR amplification using primers listed in Table S1. The T7-gRNA PCR product was purified and used as the template for IVT using a MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the gRNAs were purified using a MEGAclear kit (Life Technologies). All single-stranded oligos were ordered from Invitrogen. The 1 kb dsDNA donor was ordered as gBlock from Integrated DNA Technologies.

Whole-genome amplification

Whole-genome amplification (WGA) was performed using the REPLI-g Single Cell Kit (no. 150345, Qiagen) based on MDA technologies according to the manufacturers' recommended protocols. Following completion of the WGA reaction, the products were purified using the HP PCR Product Purification Kit (no. 11732668001, Roche). Purified DNA (1 μl) was run on agarose gels to confirm successful amplification, and the remaining material was kept at −20 °C until processing for PCR.

DNA sequencing analysis

For embryos injected with Cas9 mRNA and gRNAs, with or without single-stranded oligodeoxynucleotides (ssODNs), the 324 bp region flanking the CCR5 target site was amplified by PCR with specific primers (CCR5 geno F and R) from the WGA product of each embryo. For embryos injected with Cas9 mRNA, gRNAs, and 1 kb dsDNA donor, the target region was amplified using primer outside the 1 kb donor sequence. First, the PCR products were sequenced to identify the presence of mutation peaks in the sequencing chromatography. Subsequently, the PCR products from each embryo were cloned into a pMD18T TA cloning vector (Takara) and transformed into competent Escherichia coli cells. The DNA plasmids from 10 to 50 randomly picked colonies were sequenced using M13 universal primers. The sequences of all the primers used are listed in Table S2.

Off-target analysis

Potential off-targets were predicted by searching the human genome (hg19) for matches to the 20 bp CCR5 gRNA1 and gRNA2 sequences, allowing for up to four mismatches followed by NGG PAM sequence. Matches were ranked first by the ascending number of mismatches and then by the ascending distance from the PAM sequence. According to the output of the scan, potential off-targets with less than four mismatches in the seed sequence were selected for PCR amplification using diluted WGA DNA as templates. The PCR products of the candidates were sequenced, and the primers for amplifying the candidate sites are listed in Table S1.

Results

Study design

To address the ethical and public concerns regarding how the CRISPR/Cas9 system could be used to engineer viable human embryos with the intention to produce a living human, we limited our study to using 3PN embryos. The 3PN embryos used in this experiment do not live beyond 3 days and are incapable of in vivo development. As one major type of polyspermy, 3PN zygotes with three pronuclei (often two sperm nuclei and one oocyte nucleus) are commonly found during the in vitro fertilization process. Although polyspermy is considered invariably pathological and the early embryo either fails to develop or develops abnormally [ 15, 16], 3PN zygotes are capable of substantial in vitro development [ 17] and therefore serve as a good system for investigating the general principles of genome engineering in early human embryos.

We chose the C-C chemokine receptor type 5 ( CCR5) gene, which encodes the major co-receptor used by HIV-1 to infect human cells, as an exemplary target for our studies. The null CCR5Δ32 allele is present at high frequencies in Scandinavian and Northern European populations and is absent in non-European populations [ 18]. Individuals who are heterozygous or homozygous for this allele are healthy and have a slower progression or resistance to HIV infections [ 19, 20]. Multiple studies have demonstrated that site-specific nuclease-mediated disruption of CCR5 function occurred in CD34+ hematopoietic stem/progenitor cells (HSPCs) and CD4+ T cells [ 21- 23] and in both human embryonic stem cells and iPS cells [ 24- 26]. We wanted to test whether we could introduce this naturally occurring beneficial CCR5 Δ32 allele into early human 3PN embryos.

Generation of CCR5 mutant alleles with high efficiency

We designed two gRNAs, each targeting at one end of the boundary of the desired 32 bp deletion within CCR5 exon 4 (Fig. 1a ). When the CRISPR/Cas-mediated DSBs are repaired through the non-homologous end-joining (NHEJ) pathway, small insertions or deletions (indels) can occur and lead to CCR5 mutant alleles.

First, we sought to evaluate the in vitro developmental potential of 3PN zygotes injected with the CRISPR/Cas system. Cas9 mRNA and gRNA1 or gRNA2 were delivered into 3PN zygotes by cytoplasmic injection. Injected zygotes were cultured in G1.5 media (Vitrolife Sweden AB) for 3 days to reach an 8-16-cell stage (Fig. 1b ). Approximately 72 % of the 3PN embryos injected with water (control group) developed into an 8-16-cell stage. In comparison, 64 and 62 % of the zygotes injected with Cas9 mRNA, in combination with gRNA1 and gRNA2, respectively, developed to an 8-16-cell stage, suggesting a minimally detrimental effect.

Each injected embryo that developed to 8-cell stage and beyond was collected independently, and the genomic DNA was amplified using a REPLI-g Single Cell Kit (Qiagen). The targeted region was amplified by PCR and characterized by Sanger sequencing. The PCR products from identified mutant samples were subcloned into plasmids, and each allele was identified by sequencing (Fig. 1c ). The efficiency of genetic modification was above 50 % using either gRNA1 or gRNA2, indicating that potentially beneficial genetic modifications could be introduced into early human embryos with high efficiency (Table ). Because all of the 3PN embryo donors were from non-European populations, we observed a complete absence of the naturally occurring CCR5 Δ32 allele in both the control embryos and the injected embryos, as expected.

Table 1

Groupsa

Injected 3PN zygotes

Development (%)

Genetic modification

Cleavage

8-16 cell

NHEJ mutations (percent)

Δ32 allele (%)

Introduction of CCR5Δ32 allele

Next, we tested whether we could introduce the Δ32 allele precisely at the CCR5 locus through the HDR pathway by using ssODNs as donors. Two 90-nt-long ssODNs were designed from the sense strand (ssODN1) and the anti-sense strand (ssODN2) of the CCR5 locus, each containing 45 nt sequences homologous to the sequence from each side of the 32 nt intended deletion (Table S2). We delivered the Cas9 mRNA, gRNA1, and ssODN1 using both cytoplasmic and pronuclear injection methods. Cytoplasmic injection performed better both for embryo development and targeted mutation efficiency (Table ), although the difference was not statistically significant due to the small sample size. We were able to produce the CCR5Δ32 allele in one of the 20 total embryos by cytoplasmic injection (Fig. 1d , Table ). We sought to improve this efficiency by providing a double stranded donor with 1 kb total homology but did not see significant improvement (Table ).

Because NHEJ repair is usually more efficient and because two DSBs can be precisely joined through the NHEJ pathway, we co-injected Cas9 mRNA along with both gRNAs targeting the boundaries of the 32 bp region without providing any donor DNA template. Using this strategy, we were able to generate embryos containing the CCR5Δ32 allele in four of the 26 total samples (Tables 1 and ). All of the alleles identified from the embryos containing the CCR5Δ32 allele are shown in Fig. 2 , and the PCR subcloning results are summarized in Tables 2 and Fig. S1. In the embryos containing the CCR5 Δ32 allele, the other alleles at the same locus either remained wild type or contained different indel mutations that were not predefined.

Table 2

Experiment

WT allele/total (%)

Other mutation/total (%)

Δ32 allele/total (%)

Because the majority of 3PN embryos have triploid chromosome complements or mosaic arrangements [ 16], it is very difficult to assess the mosaicism arising from genome editing. However, the presence of three to four unique alleles in embryos suggests that the mosaicism of genetic modification could occur (Fig. 2 ) because mosaicism is well documented in similar studies in animal models [ 7, 27]. In addition, the number of alleles identified from each embryo might be an underestimation due to limitations in the PCR genotyping approach.

To address the potential off-target effects, we predicted the top 11 and 17 potential off-targets for gRNA1 and gRNA2, respectively, based on published findings [ 7, 28], and we genotyped all 28 loci in three embryos containing CCR5Δ32 allele (Table ). None of these loci contained indel mutations at the predicted off-targeted regions (Table S2).

Discussion

In this study, we demonstrated that a naturally occurring beneficial allele could be introduced into early human 3PN embryos through zygote injection of the CRISPR/Cas system. By testing different strategies, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. Similar to the results obtained in other species, NHEJ-mediated indel mutations could be obtained with high efficiency, whereas the HDR-mediated precise modifications had a lower efficiency. Because of the scarcity of human embryos, we had a relatively small number of samples for each group. Therefore, the differences in development and mutation rates among groups are not statistically significant, and we cannot make any absolute conclusions. We would like to highlight the fact that even in the embryos in which we successfully introduced the CCR5Δ32 allele, the other alleles at the same locus either were wild type or contained indel mutations. Liang et al. showed that the efficiency of HDR of the β-globin gene was 4.7 % (per injected zygote) and that the modified embryos displayed mosaicism in which wild-type cells and genetically modified cells coexisted [ 12]. Our result is consistent with the study by Liang et al. Although mosaicism may be avoided by a more careful consideration of the timing and method of microinjection, Long et al. demonstrated that the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction [ 9]. Therefore, significant technical hurdles will need to be overcome to gain control of the genotype of both alleles in a viable embryo.

Off-target effects are a major concern for the CRISPR/Cas system. Several studies have reported that the CRISPR/Cas system induced heritable off-target mutations in both mice and humans [ 13, 29]. In this study, we did not detect off-site targeting as predicted for a total of 28 potential off-target sites. Recent studies used various strategies to experimentally map off-target mutations of the CRISPR/Cas system and found that computational prediction based on sequence similarity to target sites failed to predict many off-target mutations [ 30- 32]. Therefore, the embryos likely carried off-target mutations undetected by our genotyping, and the off-target effects thus merit further inquiry.

Liang et al. reported that off-target mutations were detected in modified 3PN embryos by the T7E1 assay and whole-exome sequencing [ 12]. However, considering that the off-target effect is site dependent and that more specific strategies using more sophisticated enzymes and meticulous design of the guiding molecule have already been established [ 33- 35], off-target mutagenesis may be minimized by optimizing the procedure [ 36]. Furthermore, small molecules that enhance CRISPR-mediated HDR have also been reported [ 37], suggesting that the CRISPR/Cas system could be a reliable genome editing tool for humans. We intend to investigate the effects of these molecules on CRISPR-mediated HDR in human 3PN embryos in our future studies.

Because we used 3PN embryos from donors with no known genetic disorders, we chose to introduce this naturally occurring allele as a test case for generating a precise genetic modification. Compared with 2PN embryos, 3PN embryos only have one more sperm nucleus and are capable of substantial in vitro development. Thus, our results suggest that similar techniques could be applied to correct disease-causing mutations in human early embryos. It is likely that other programmable endonucleases can also be applied in a similar manner, although with potentially different efficiencies and specificities.

These results call for immediate attention being paid to the regulation of the application of genome engineering technologies in humans for genetic modification in early embryos and related work, such as the genetic modification of human germline cells. Because human in vitro fertilization methods are well established and site-specific nuclease technologies are readily available, it is foreseeable that a genetically modified human could be generated. We believe that any attempt to generate genetically modified humans through the modification of early embryos needs to be strictly prohibited until we can resolve both ethical and scientific issues. Several major scientific issues remain. First, for any germline genetic modification in humans, we believe that the resulting allele needs to be precisely predefined. This requires the further improvement of precise modification efficiency as well as assays to select the embryos containing the desired allele. Second, genetic mosaicism, likely due to nuclease action after the one-cell stage, needs to be resolved. Genome engineering of germline stem cells might be one potential solution [ 38]. Third, the specificity of the technologies needs to be further investigated and improved to ensure that no off-target mutations will be introduced. Fourth, for any introduced allele, even a naturally occurring allele in the human population, the effect of its introduction into a different genetic background needs to be carefully evaluated. Without a much better understanding of the genetic network at a systematic level, we cannot predict the genome-wide effects of changing even a single allele.

Genome engineering technologies for germline editing need to be significantly improved to solve scientific issues that include, but are not limited to, the ones presented above before they may be considered for application in humans. Preimplantation genetic diagnosis (PGD) has already been used clinically in some countries to screen out human embryos with mutations responsible for genetic conditions such as thalassemia and spinal muscular atrophy. The clinical use of PGD appears to justify germline genome editing research because only embryos that contain no suspected mutations, but have undergone the physical intervention of embryonic cell biopsy for genetic testing, are used for embryo transfer. Even with a perfected technology, we believe that genome engineering in the human germline should only be considered if no less invasive alternative approach is available.

A thorough discussion regarding the related ethical issues is beyond the scope of our study, and the topic needs to be addressed comprehensively by the broad international community. However, to start the discussion, we would like to raise several important questions. One of the most important questions is, "how do we define the norm of human and human genome?" With the rapid development of human genomics, a large number of human alleles associated with diseases and human physiology are being discovered. Once we understand the underlying biology, would it be acceptable to introduce alleles that improve human health and life span or to eliminate those imposing serious disease risks (e.g., Huntington's disease)?

Despite the significant scientific and ethical issues involved, however, we believe that it is necessary to keep developing and improving the technologies for precise genetic modifications in humans by using animal models and in vitro systems such as human 3PN embryos and germline stem cells. The availability and improvement of genome engineering technologies in human early embryos, the proof-of-concept that we and Liang et al. [ 12] provide, may provide solutions for genetic diseases, improve human health, or play an essential role in surviving future disruptive selection pressures. Recently, a prominent ad hoc group of scientists called for a moratorium on clinical applications of germline gene editing [ 39]. We also advocate for preventing any application of genome editing in the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig S1(192K, gif)

Acknowledgments

We thank Dr. Haoyi Wang from Institute of Zoology in Chinese Academy of Sciences for helpful advice. This work was supported in part by the National Natural Science Foundation of China (81370766, 81570101, 81370670), Guangdong Province Higher Education Funding (2013KJCX0149, Yq2013135), and Guangdong Province Science and Technology Project (2014A02011029, 2015B020227002). Y. F. was supported by the State 863 project 2015AA020307.

Authors' contributions

Y.F. designed the study. Y.F., X.K., and Y.H. recruited the patients and signed the informed consent. Y.F. performed embryo injection. X.K. and Y.H. collected embryos. W.H. and Q.Y. performed the WGA and PCR genotyping and off-target prediction. Y.C., X.G., and X.S. contributed to off-target sites genotyping. Y.F. analyzed the data and wrote the paper.

Compliance with ethical standards

This study was approved by the ethics committee of the Third Affiliated Hospital of Guangzhou Medical University (LLSC2014018). The methods used in the present study closely followed the guidelines legislated and posted by the Ministry of Health of the People's Republic of China. The patients involved in this study knew about and understood the usage of polyspermic zygotes and voluntarily donated them after providing informed consent.

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Capsule As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species.

Xiangjin Kang, Wenyin He and Yuling Huang contributed equally to this work.
Electronic supplementary material
The online version of this article (doi:10.1007/s10815-016-0710-8) contains supplementary material, which is available to authorized users.

An erratum to this article is available at http://dx.doi.org/10.1007/s10815-017-0946-y.

References

13. Liu P, Chen S, Li X, Qin L, Huang K, Wang L et al. Low immunogenicity of neural progenitor cells differentiated from induced pluripotent stem cells derived from less immunogenic somatic cells. PLoS One. 8(7):e69617. [ PMC free article] [ PubMed]

14. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 339(6121):819-23. [ PMC free article] [ PubMed]

29. Hong SG, Winkler T, Wu C, Guo V, Pittaluga S, Nicolae A et al. Path to the clinic: assessment of iPSC-based cell therapies in vivo in a nonhuman primate model. Cell Rep. 7(4):1298-309. [ PMC free article] [ PubMed]

Articles from Journal of Assisted Reproduction and Genetics are provided here courtesy of Springer Science+Business Media, LLC

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Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing

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As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise ...

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For better or worse, genetic testing of embryos offers a potential gateway into a new era of human control over reproduction. Couples at risk of having a child with a severe or life-limiting disease such as cystic fibrosis or Duchenne muscular dystrophy have used preimplantation genetic testing (PGT) for decades to select among embryos created through in vitro fertilization (IVF) for those that do not carry the disease-causing gene. But what new iteration of genetic testing could tempt healthy, fertile couples to reject our traditional time-tested and wildly popular process of baby making in favor of hormone shots, egg extractions and DNA analysis?

A California-based start-up called Orchid Biosciences claims it has an answer to that question. The company offers prospective parents genetic testing prior to conception to calculate risk scores estimating their own likelihood of confronting common illnesses such as heart disease, diabetes, and schizophrenia and the likelihood that they will pass such risks along to a future child. Parents-to-be can then use IVF, along with Orchid's upcoming embryo screening package, to identify the healthiest of their embryos for a pregnancy.

Orchid aims to use PGT and IVF to expand what is already a thriving marketplace in screening tests for prospective parents. Initially, the only people offered tests to prevent genetic disease in the next generation were those whose ancestry put them at higher risk for a specific condition, such as Tay-Sachs disease in the Ashkenazi Jewish population. The first genetic screen intended for universal use and covering a wide range of diseases was introduced by Counsyl (now part of Myriad Genetics) in 2010. Today carrier screening is a $1.7-billion industry. These tests search for genetic problems that otherwise come to light only after the birth of an affected child. But diseases caused by a single gene are rare. Most children are born healthy, and most couples who do carrier screening come away reassured.

By contrast, Orchid's risk assessment includes common diseases, ensuring that a high percentage of prospective parents who do this version of preconception testing will find something to worry about. Those who choose to act on their concerns will soon have the option of paying for IVF plus Orchid's embryo-testing package. According to its promotional materials, the company will provide a scorecard intended to identify, among various embryos, the future children least likely to develop heart disease, breast cancer, prostate cancer, type 1 or 2 diabetes, and five other conditions that make up Orchid's current common disease risk portfolio.

With a marketing strategy that encourages routine use of IVF for those who can afford it, Orchid breaks new ground in introducing the first-but likely not the last-consumer-driven model of human reproduction. The ambitions of this new Silicon Valley venture into health care are backed by the imprimatur of health-tech luminaries, including 23andMe co-founder and Orchid investor Anne Wojcicki. Orchid's first product on the market is its "Couple Report," at a cost of $1,100. Phase two, scheduled for launch later this year, examines embryos conceived by IVF, allowing the couple to pick and choose among potential children in a process that Orchid CEO Noor Siddiqui, speaking in an interview on the podcast Mendelspod in April, referred to as "embryo prioritization." Siddiqui is a former Thiel Foundation Fellow whose interests lie in the use of technology in medicine. She did not respond to repeated requests for an interview from Scientific American.

Geneticists have greeted Orchid's launch with skepticism, in large part because of objections to the company's use of a technique called polygenic risk scores to assess an embryo's lifetime risk of common diseases. Heart disease runs in families just like musical ability or height, but only in exceptional cases can the inherited risk be traced to a single gene. Hundreds or even thousands of genes each contribute in a small way. Polygenic risk scores attempt to sum up the overall likelihood of a particular outcome-such as getting a disease-by simply observing which patterns of variation in a genome are associated with a higher or lower probability of having the condition. In other words, this method gives us information about who might be more or less likely to get sick without explaining why. The statistical association is real but hardly definitive, and it tracks population-level trends that may not be relevant for the individual in question.

Researchers who work with polygenic risk scores are concerned about their use in this context. "We don't know what these variants are doing biologically," says Peter Kraft, a professor of epidemiology and biostatistics at the Harvard T. H. Chan School of Public Health. "Something that's associated with a decreased risk of breast cancer could be associated with all other kinds of things, some of which might actually increase your risk of something else. We just don't know enough yet."

Some version of prenatal planning as envisioned by Orchid may be possible eventually, but few experts seem to share their optimism that today is that day. A July 1 special report in the New England Journal of Medicine pointed out the inherent weakness of using polygenic risk scores to distinguish among sibling embryos-which, unlike random individuals in a population, will be identical in 50 percent of the genetic variation that is examined to generate a score.The report concluded with recommendations on how to convey any purported benefits from polygenic scores in embryo selection responsibly-and the need to emphasize the underlying uncertainties in the data. "Any one of the issues discussed in this article would be difficult to communicate accurately-even to other scientists and clinicians," the authors noted. "Collectively, these issues constitute a formidable challenge for [companies selling these services], which must ensure that their customers understand what they are doing." The report also called for the Federal Trade Commission to look carefully at claims made by any company using polygenic scoring to pick embryos.

Current polygenic risk scores have limited predictive strength and reflect the shortcomings of genetic databases, which are overwhelmingly Eurocentric. Alicia Martin, an instructor at Massachusetts General Hospital and the Broad Institute of the Massachusetts Institute of Technology and Harvard University, says her research examining polygenic risk scores suggests "they don't transfer well to other populations that have been understudied." In fact, the National Institutes of Health announced in mid-June that it will be giving out $38 million in grants over five years to find ways to enhance disease prediction in diverse populations using polygenic risk scores. Speaking of Orchid, Martin says, "I think it is premature to try to roll this out."

In an interview about embryo screening and ethics featured on the company's Web site, Jonathan Anomaly, a University of Pennsylvania bioethicist, suggested the current biases are a problem to be solved by getting customers and doing the testing. "As I understand it," he said, "Orchid is actively building statistical models to improve ancestry adaptation and adjustments for genetic risk scores, which will increase accessibility of the product to all individuals."

Still, better data sets will not allay all concerns about embryo selection. The combined expense of testing and IVF means that unequal access to these technologies will continue to be an issue. In her Mendelspod interview, Siddiqui insisted, "We think that everyone who wants to have a baby should be able to, and we want our technology to be as accessible to everyone who wants it," adding that the lack of insurance coverage for IVF is a major problem that needs to be addressed in the U.S.

But should insurance companies pay for fertile couples to embryo-shop? This issue is complicated, especially in light of the fact that polygenic risk scores can generate predictions for more than just heart disease and cancer. They can be devised for any trait with a heritable component, and existing models offer predictions for educational attainment, neuroticism and same-sex sexual behavior, all with the same caveats and limitations as Orchid's current tests for major diseases. To be clear, tests for these behavioral traits are not part of Orchid's current genetic panel. But when talking about tests the company does offer, Siddiqui suggested that the ultimate decision makers should be the parents-to-be. "I think at the end of the day, you have to respect patient autonomy," she said.

Despite Orchid's hard lean into parental free choice, bioethicists such as Gabriel Lázaro-Muñoz of the Center for Medical Ethics and Health Policy at Baylor College of Medicine worry that Orchid's system of ranking embryos may unduly influence prospective parents and replace a very necessary broader societal debate on what qualifies as a good life. It is problematic for that reason, according to Lázaro-Muñoz, to have these companies "bias the conversation."

Lurking in the background of every discussion on embryo selection and ethics is the specter of eugenics. "I think we have to be very aware of our history," Lázaro-Muñoz says, "in terms of sterilization and state-mandated programs in the past that were aimed at ... exterminating individuals with some of these conditions."

Clearly Orchid anticipates pushback. The company's promotional materials include guides to fertility planning and the genetics of irritable bowel disease but also a set of talking points for concerned relatives described as " How to respond to your family skeptics -playing God, designer babies, and genetic enhancement."

"Yes, we're going there," the guide says. Ethics? Bring it on. This is not a company in a defensive crouch. The "Our Principles" section of its Web site positions genetic testing as a human right. "From a reproductive freedom perspective, we stand for a couple's right to have access to information that enables them to mitigate disease risk for their future child," it says. Like other Silicon Valley health care technology pioneers, Orchid presents itself less as a product than as a social justice movement with a little commercial venture on the side, like a gift shop.

Orchid dismisses suggestions from detractors that its marketing oversells what polygenic risk scores have to offer in the context of screening embryos. "Parents are asking for this information and deserve to know it," Siddiqui told Mendelspod, warning that those who stand in the way are "frankly being a little bit paternalistic." And if prospective parents are not asking, Siddiqui suggested, perhaps they should be. When it comes to the next generation, "we've been sort of just rolling the dice," Siddiqui said, while "the ability to actually stack the odds against disease is ... sort of a new capability that humanity has just gotten online."

The suggestion that embryo selection is not only something people can do but something they should do raises perhaps one of the thorniest ethical issues of all. In the Mendelspod interview, Siddiqui drew a contrast between "earned" and "unearned" bad luck. "You can get hit by a car, right? That's totally out of your control. But what is earned bad luck?" she asked before answering her own question. "I mean, that's the idea of ... you're going base jumping constantly, and then you break your leg.... You kind of exposed yourself to higher risk there."

Ultimately, if technology allows Orchid to offer a product that meaningfully reduces the risk of disease susceptibility in the next generation, does that mean that anyone who can't or won't use it deserves their bad luck? If the basic, no-frills version of human reproduction comes to be seen as a form of careless parenting, it invites a callous parsing of who does and does not deserve their fate-and, by extension, who does and does not deserve resources and support.

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A New Era of Designer Babies May Be Based on Overhyped Science

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For better or worse, genetic testing of embryos offers a potential gateway into a new era of human control over reproduction. Couples at risk of having a child with a severe or life-limiting disease such as cystic fibrosis or Duchenne muscular dystrophy have used preimplantation genetic testing (PGT) for decades to select among embryos created through in vitro fertilization (IVF) for those that do not carry the disease-causing gene.

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https://www.future-science.com/doi/10.2144/btn-2019-0056

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Cloning in Bacteria is fine, but cloning in human life is unethical

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The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone. Researchers have cloned a wide range of biological materials, including genes, cells, tissues and even entire organisms, such as a sheep.

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The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone.

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Designer babies are a horror waiting to happen and here's why https://www.theguardian.com/science/2017/jan/08/designer-babies-ethical-horror-waiting-to-happen

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Comfortably seated in the fertility clinic with Vivaldi playing softly in the background, you and your partner are brought coffee and a folder. Inside the folder is an embryo menu. Each embryo has a description, something like this:

Embryo 78 - male
No serious early onset diseases, but a carrier for phenylketonuria (a metabolic malfunction that can cause behavioural and mental disorders. Carriers just have one copy of the gene, so don't get the condition themselves).
Higher than average risk of type 2 diabetes and colon cancer.
Lower than average risk of asthma and autism.
Dark eyes, light brown hair, male pattern baldness.
40% chance of coming in the top half in SAT tests.

There are 200 of these embryos to choose from, all made by in vitro fertilisation (IVF) from you and your partner's eggs and sperm. So, over to you. Which will you choose?

If there's any kind of future for "designer babies", it might look something like this. It's a long way from the image conjured up when artificial conception, and perhaps even artificial gestation, were first mooted as a serious scientific possibility. Inspired by predictions about the future of reproductive technology by the biologists JBS Haldane and Julian Huxley in the 1920s, Huxley's brother Aldous wrote a satirical novel about it.

That book was, of course, Brave New World, published in 1932. Set in the year 2540, it describes a society whose population is grown in vats in an impersonal central hatchery, graded into five tiers of different intelligence by chemical treatment of the embryos. There are no parents as such - families are considered obscene. Instead, the gestating fetuses and babies are tended by workers in white overalls, "their hands gloved with a pale corpse‑coloured rubber", under white, dead lights.

Brave New World has become the inevitable reference point for all media discussion of new advances in reproductive technology. Whether it's Newsweek reporting in 1978 on the birth of Louise Brown, the first "test-tube baby" (the inaccurate phrase speaks volumes) as a "cry round the brave new world", or the New York Times announcing " The brave new world of three-parent IVF " in 2014, the message is that we are heading towards Huxley's hatchery with its racks of tailor-made babies in their "numbered test tubes".

The spectre of a harsh, impersonal and authoritarian dystopia always looms in these discussions of reproductive control and selection. Novelist Kazuo Ishiguro, whose 2005 novel, Never Let Me Go, described children produced and reared as organ donors, last month warned that thanks to advances in gene editing, "we're coming close to the point where we can, objectively in some sense, create people who are superior to others ".

But the prospect of genetic portraits of IVF embryos paints a rather different picture. If it happens at all, the aim will be not to engineer societies but to attract consumers. Should we allow that? Even if we do, would a list of dozens or even hundreds of embryos with diverse yet sketchy genetic endowments be of any use to anyone?

I don't think we are going to see superman or a split in the species any time soon, because we just don't know enough

Henry Greely, bioethicist

The shadow of Frankenstein 's monster haunted the fraught discussion of IVF in the 1970s and 80s, and the misleading term "three-parent baby" to refer to embryos made by the technique of mitochondrial transfer - moving healthy versions of the energy-generating cell compartments called mitochondria from a donor cell to an egg with faulty, potentially fatal versions - insinuates that there must be something "unnatural" about the procedure.

Every new advance puts a fresh spark of life into Huxley's monstrous vision. Ishiguro's dire forecast was spurred by the gene-editing method called Crispr-Cas9, developed in 2012, which uses natural enzymes to target and snip genes with pinpoint accuracy. Thanks to Crispr-Cas9, it seems likely that gene therapies - eliminating mutant genes that cause some severe, mostly very rare diseases - might finally bear fruit, if they can be shown to be safe for human use. Clinical trials are now under way.

But modified babies? Crispr-Cas9 has already been used to genetically modify (nonviable) human embryos in China, to see if it is possible in principle - the results were mixed. And Kathy Niakan of the Francis Crick Institute in the UK has been granted a licence by the Human Fertilisation and Embryology Authority (HFEA) to use Crispr-Cas9 on embryos a few days old to find out more about problems in these early stages of development that can lead to miscarriage and other reproductive problems.

Most countries have not yet legislated on genetic modification in human reproduction, but of those that have, all have banned it. The idea of using Crispr-Cas9 for human reproduction is largely rejected in principle by the medical research community. A team of scientists warned in Nature less than two years ago that genetic manipulation of the germ line (sperm and egg cells) by methods like Crispr-Cas9, even if focused initially on improving health, " could start us down a path towards non-therapeutic genetic enhancement ".

Besides, there seems to be little need for gene editing in reproduction. It would be a difficult, expensive and uncertain way to achieve what can mostly be achieved already in other ways, particularly by just selecting an embryo that has or lacks the gene in question. "Almost everything you can accomplish by gene editing, you can accomplish by embryo selection," says bioethicist Henry Greely of Stanford University in California.

Because of unknown health risks and widespread public distrust of gene editing, bioethicist Ronald Green of Dartmouth College in New Hampshire says he does not foresee widespread use of Crispr-Cas9 in the next two decades, even for the prevention of genetic disease, let alone for designer babies. However, Green does see gene editing appearing on the menu eventually, and perhaps not just for medical therapies. "It is unavoidably in our future," he says, "and I believe that it will become one of the central foci of our social debates later in this century and in the century beyond." He warns that this might be accompanied by "serious errors and health problems as unknown genetic side effects in 'edited' children and populations begin to manifest themselves".

For now, though, if there's going to be anything even vaguely resembling the popular designer-baby fantasy, Greely says it will come from embryo selection, not genetic manipulation. Embryos produced by IVF will be genetically screened - parts or all of their DNA will be read to deduce which gene variants they carry - and the prospective parents will be able to choose which embryos to implant in the hope of achieving a pregnancy. Greely foresees that new methods of harvesting or producing human eggs, along with advances in preimplantation genetic diagnosis (PGD) of IVF embryos, will make selection much more viable and appealing, and thus more common, in 20 years' time.

PGD is already used by couples who know that they carry genes for specific inherited diseases so that they can identify embryos that do not have those genes. The testing, generally on three- to five-day-old embryos, is conducted in around 5% of IVF cycles in the US. In the UK it is performed under licence from the HFEA, which permits screening for around 250 diseases including thalassemia, early-onset Alzheimer's and cystic fibrosis.

As a way of "designing" your baby, PGD is currently unattractive. "Egg harvesting is unpleasant and risky and doesn't give you that many eggs," says Greely, and the success rate for implanted embryos is still typically about one in three. But that will change, he says, thanks to developments that will make human eggs much more abundant and conveniently available, coupled to the possibility of screening their genomes quickly and cheaply.

Carey Mulligan, Keira Knightley and Andrew Garfield in the 2010 film adaptation of Kazuo Ishiguro's Never Let Me Go, in which clones are produced to provide spare organs for their originals. Photograph: 20th Century Fox/Everett/Rex

Advances in methods for reading the genetic code recorded in our chromosomes are going to make it a routine possibility for every one of us - certainly, every newborn child - to have our genes sequenced. "In the next 10 years or so, the chances are that many people in rich countries will have large chunks of their genetic information in their electronic medical records," says Greely.

But using genetic data to predict what kind of person an embryo would become is far more complicated than is often implied. Seeking to justify unquestionably important research on the genetic basis of human health, researchers haven't done much to dispel simplistic ideas about how genes make us. Talk of "IQ genes", "gay genes" and "musical genes" has led to a widespread perception that there is a straightforward one-to-one relationship between our genes and our traits. In general, it's anything but.

There are thousands of mostly rare and nasty genetic diseases that can be pinpointed to a specific gene mutation. Most more common diseases or medical predispositions - for example, diabetes, heart disease or certain types of cancer - are linked to several or even many genes, can't be predicted with any certainty, and depend also on environmental factors such as diet.

When it comes to more complex things like personality and intelligence, we know very little. Even if they are strongly inheritable - it's estimated that up to 80% of intelligence, as measured by IQ, is inherited - we don't know much at all about which genes are involved, and not for want of looking.

At best, Greely says, PGD might tell a prospective parent things like "there's a 60% chance of this child getting in the top half at school, or a 13% chance of being in the top 10%". That's not much use.

We might do better for "cosmetic" traits such as hair or eye colour. Even these "turn out to be more complicated than a lot of people thought," Greely says, but as the number of people whose genomes have been sequenced increases, the predictive ability will improve substantially.

Ewan Birney, director of the European Bioinformatics Institute near Cambridge, points out that, even if other countries don't choose to constrain and regulate PGD in the way the HFEA does in the UK, it will be very far from a crystal ball.

Nearly anything you can measure for humans, he says, can be studied through genetics, and analysing the statistics for huge numbers of people often reveals some genetic component. But that information "is not very predictive on an individual basis," says Birney. "I've had my genome sequenced on the cheap, and it doesn't tell me very much. We've got to get away from the idea that your DNA is your destiny."

If the genetic basis of attributes like intelligence and musicality is too thinly spread and unclear to make selection practical, then tweaking by genetic manipulation certainly seems off the menu too. "I don't think we are going to see superman or a split in the species any time soon," says Greely, "because we just don't know enough and are unlikely to for a long time - or maybe for ever."

If this is all "designer babies" could mean even in principle - freedom from some specific but rare diseases, knowledge of rather trivial aspects of appearance, but only vague, probabilistic information about more general traits like health, attractiveness and intelligence - will people go for it in large enough numbers to sustain an industry?

Greely suspects, even if it is used at first only to avoid serious genetic diseases, we need to start thinking hard about the options we might be faced with. "Choices will be made," he says, "and if informed people do not participate in making those choices, ignorant people will make them."

The Crispr/Cas9 system uses a molecular structure to edit genomes. Photograph: Alamy

Green thinks that technological advances could make "design" increasingly versatile. In the next 40-50 years, he says, "we'll start seeing the use of gene editing and reproductive technologies for enhancement: blond hair and blue eyes, improved athletic abilities, enhanced reading skills or numeracy, and so on."

He's less optimistic about the consequences, saying that we will then see social tensions "as the well-to-do exploit technologies that make them even better off", increasing the relatively worsened health status of the world's poor. As Greely points out, a perfectly feasible 10-20% improvement in health via PGD, added to the comparable advantage that wealth already brings, could lead to a widening of the health gap between rich and poor, both within a society and between nations.

Others doubt that there will be any great demand for embryo selection, especially if genetic forecasts remain sketchy about the most desirable traits. "Where there is a serious problem, such as a deadly condition, or an existing obstacle, such as infertility, I would not be surprised to see people take advantage of technologies such as embryo selection," says law professor and bioethicist R Alta Charo of the University of Wisconsin. "But we already have evidence that people do not flock to technologies when they can conceive without assistance."

The poor take-up of sperm banks offering "superior" sperm, she says, already shows that. For most women, "the emotional significance of reproduction outweighs any notion of 'optimisation'". Charo feels that "our ability to love one another with all our imperfections and foibles outweighs any notion of 'improving' our children through genetics".

All the same, societies are going to face tough choices about how to regulate an industry that offers PGD with an ever-widening scope. "Technologies are very amoral," says Birney. "Societies have to decide how to use them" - and different societies will make different choices.

One of the easiest things to screen for is sex. Gender-specific abortion is formally forbidden in most countries, although it still happens in places such as China and India where there has been a strong cultural preference for boys. But prohibiting selection by gender is another matter. How could it even be implemented and policed? By creating some kind of quota system?

And what would selection against genetic disabilities do to those people who have them? "They have a lot to be worried about here," says Greely. "In terms of whether society thinks I should have been born, but also in terms of how much medical research there is into diseases, how well understood it is for practitioners and how much social support there is."

Once selection beyond avoidance of genetic disease becomes an option - and it does seem likely - the ethical and legal aspects are a minefield. When is it proper for governments to coerce people into, or prohibit them from, particular choices, such as not selecting for a disability? How can one balance individual freedoms and social consequences?

"The most important consideration for me," says Charo, "is to be clear about the distinct roles of personal morality, by which individuals decide whether to seek out technological assistance, versus the role of government, which can prohibit, regulate or promote technology."

She adds: "Too often we discuss these technologies as if personal morality or particular religious views are a sufficient basis for governmental action. But one must ground government action in a stronger set of concerns about promoting the wellbeing of all individuals while permitting the widest range of personal liberty of conscience and choice."

"For better or worse, human beings will not forgo the opportunity to take their evolution into their own hands," says Green. "Will that make our lives happier and better? I'm far from sure."

A scientist at work during an IVF process. Photograph: Ben Birchall/PA

Easy pickings: the future of designer babies

The simplest and surest way to "design" a baby is not to construct its genome by pick'n'mix gene editing but to produce a huge number of embryos and read their genomes to find the one that most closely matches your desires.

Two technological advances are needed for this to happen, says bioethicist Henry Greely of Stanford University in California. The production of embryos for IVF must become easier, more abundant and less unpleasant. And gene sequencing must be fast and cheap enough to reveal the traits an embryo will have. Put them together and you have "Easy PGD" (preimplantation genetic diagnosis): a cheap and painless way of generating large numbers of human embryos and then screening their entire genomes for desired characteristics.

"To get much broader use of PGD, you need a better way to get eggs," Greely says. "The more eggs you can get, the more attractive PGD becomes." One possibility is a one-off medical intervention that extracts a slice of a woman's ovary and freezes it for future ripening and harvesting of eggs. It sounds drastic, but would not be much worse than current egg-extraction and embryo-implantation methods. And it could give access to thousands of eggs for future use.

An even more dramatic approach would be to grow eggs from stem cells - the cells from which all other tissue types can be derived. Some stem cells are present in umbilical blood, which could be harvested at a person's birth and frozen for later use to grow organs - or eggs.

Even mature cells that have advanced beyond the stem-cell stage and become specific tissue types can be returned to a stem-cell-like state by treating them with biological molecules called growth factors. Last October, a team in Japan reported that they had made mouse eggs this way from skin cells, and fertilised them to create apparently healthy and fertile mouse pups.

Thanks to technological advances, the cost of human whole-genome sequencing has plummeted. In 2009 it cost around $50,000; today it is most like $1,500, which is why several private companies can now offer this service. In a few decades it could cost just a few dollars per genome. Then it becomes feasible to think of PGD for hundreds of embryos at a time.

"The science for safe and effective Easy PGD is likely to exist some time in the next 20 to 40 years," says Greely. He thinks it will then become common for children to be conceived through IVF using selected genomes. He forecasts that this will lead to "the coming obsolescence of sex" for procreation.

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Designer babies: an ethical horror waiting to happen?

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Nearly 40 years since the first 'test-tube baby', how close are we to editing out all of our genetic imperfections - and should we even try to do so?

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https://www.nature.com/articles/s41380-018-0070-0

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Abstract

Decades of research show that genes play an vital role in the etiology of attention deficit hyperactivity disorder (ADHD) and its comorbidity with other disorders. Family, twin, and adoption studies show that ADHD runs in families. ADHD’s high heritability of 74% motivated the search for ADHD susceptibility genes. Genetic linkage studies show that the effects of DNA risk variants on ADHD must, individually, be very small. Genome-wide association studies (GWAS) have implicated several genetic loci at the genome-wide level of statistical significance. These studies also show that about a third of ADHD’s heritability is due to a polygenic component comprising many common variants each having small effects. From studies of copy number variants we have also learned that the rare insertions or deletions account for part of ADHD’s heritability. These findings have implicated new biological pathways that may eventually have implications for treatment development.

Attention deficit hyperactivity disorder (ADHD) is a childhood-onset condition with impairing symptoms of inattention, impulsivity, and hyperactivity. Decades of research have documented and replicated key facts about the disorder (for a review, see ref. [1]). It occurs in about 5% of children with little geographic or cross-cultural variation in prevalence and often co-occurs with other conditions, including mood, anxiety, conduct, learning, and substance use disorders. Longitudinal studies show that two-thirds of ADHD youth will continue to have impairing symptoms of ADHD in adulthood. People with ADHD are at risk for a wide range of functional impairments: school failure, peer rejection, injuries due to accidents, criminal behavior, occupational failure, divorce, suicide, and premature death. Although many details of ADHD’s pathophysiology are unknown, neuropsychological and neuroimaging studies implicate brain circuits regulating executive functioning, reward processing, timing, and temporal information processing.

This article reviews data about the role that genes play in the etiology of ADHD from two perspectives. Family, twin, and adoption studies provide a firm foundation for asserting that genes are involved in the etiology of ADHD. The view from molecular genetics provides a basis for understanding mechanisms whereby genes affect biological pathways that lead to ADHD.

Family, twin and adoption studies of ADHD

Evidence for heritability from family, adoption, and twin studies

A study of 894 ADHD probands and 1135 of their siblings aged 5–17 years old found a ninefold increased risk of ADHD in siblings of ADHD probands compared with siblings of controls [2]. Adoption studies suggest that the familial factors of ADHD are attributable to genetic factors rather than shared environmental factors [3, 4] with the most recent one reporting rates of ADHD to be greater among biological relatives of non-adopted ADHD children than adoptive relatives of adopted ADHD children. The adoptive relatives had a risk for ADHD like the risk in relatives of control children [4].

Twin studies rely on the difference between the within-pair similarities of monozygotic (MZ) twin pairs, who are genetically identical, and dizygotic (DZ) twin pairs, who share, on average, 50% of their segregating genes. The mean heritability across 37 twin studies of ADHD or measures of inattentiveness and hyperactivity is 74% (Fig. 1). A similar heritability estimate of around 80% was seen in a study of MZ and DZ twins, full siblings, and maternal and paternal half-siblings [5]. The heritability is similar in males and females and for the inattentive and hyperactive-impulsive components of ADHD [6,7,8].

Fig. 1

Heritability of ADHD from twin studies of ADHD diagnoses or symptom counts [153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173]

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Only a few of the twin studies in Fig. 1 used categorical measures of ADHD [9,10,11,12]. Their heritability estimates range from 77 to 88%, which is consistent with the larger number of studies using symptom count measures of ADHD. Twin studies have explored whether ADHD is best viewed as a categorical disorder or as an extreme of a continuous trait. A study of 16,366 Swedish twins found a strong genetic link between the extreme and the sub-threshold variation of DSM-IV ADHD symptoms [13]. This study confirmed an early study of 583 same-sexed twin pairs using ADHD-III-R symptoms [14]. Both studies suggest that the diagnosis of ADHD is the extreme of a continuous distribution of ADHD symptoms in the population and that the etiologic factors involved in the disorder also account for the full range of symptoms. These data are consistent with clinical studies showing the clinical implications of subthreshold ADHD [15].

ADHD’s clinical features and course

Reporter effects

Parent and teacher ratings of ADHD symptoms result in high heritability estimates (70–80%) [6]. In contrast, studies using self-ratings in adolescence and adulthood show lower heritabilities (<50%) [16,17,18,19]. Two twin studies examined these rater effects [20, 21]. They showed that self-ratings, as well as different-parent and different-teacher ratings within twin pairs, were associated with lower heritability estimates (~30–40%) compared with heritabilities based on same-parent and same-teacher ratings (~70–80%) [20,21,22]. Low reliability of self-reports may explain why heritability estimates are lower in studies of self-rated ADHD symptoms. Using different informants for ADHD symptom ratings of each twin in a pair introduces rater effects (i.e., each rater experiences and reports different ADHD symptoms) or rater bias (i.e., a rater consistently over- or underestimates ADHD symptoms or similarities between twins). These effects could explain why heritability estimates are lower in studies relying on different informants for each twin in a pair compared with studies using the same raters [23].

Developmental effects

The first twin studies of ADHD in adults used self-reports and estimated heritability at 30–40% (Fig. 2), (e.g. [24]), which is substantially lower than the heritability among children and adolescents. In contrast, one study estimated heritability to be 80% after combining self and parent ratings into a composite index of ADHD. Another study found the heritability of clinically diagnosed ADHD in adults to be 72% [25]. These findings (Fig. 2) suggest that the heritability of ADHD is stable during the transition from childhood into adulthood. They explain previous reports of low heritability for ADHD symptoms in adults as due to measurement error from rater effects. The higher heritabilities for clinically diagnosed adult ADHD confirm family studies suggesting that persistent ADHD is highly familial [5, 26, 27].

Fig. 2

Heritability of ADHD in adults depends on method of diagnosis

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Twin studies show that both stable and dynamic genetic risk factors influence ADHD over the course of the development from childhood to early adulthood [7, 28,29,30]. These study findings explain the developmental structure of genetic risk factors for ADHD with both stable and dynamic processes. The stable component of the genetic risk suggests that persistent ADHD and its pediatric form are genetically linked. The dynamic component suggests that the set of genetic variants accounting for the onset of ADHD differs from those accounting for the persistence and remission of the disorder. For a review of the genetics of adult ADHD, see Franke et al. [26].

Psychiatric comorbidity

Multivariate twin and sibling studies have found a general genetic factor that influences ADHD and a broad spectrum of neuropsychiatric conditions [31, 32]. These studies have shown that a latent shared genetic factor accounts for up to 45% of co-variance across childhood externalizing, internalizing, and phobia symptoms [31, 33] and 31% of co-variance in childhood neurodevelopmental symptoms [34]. Similar results have been reported for register-based clinical diagnoses, with one study showing that a general genetic factor explained 10–36% of disorder liability across several psychiatric diagnoses [32]. Two studies have assessed the contribution of measured genetic variants for a general psychopathology dimension. One study estimated the SNP-heritability as 18% for maternal ratings of total problems on the Child Behavior Checklist, which measures internalizing, externalizing, and attention problems [35]. Similarly, another study estimated the SNP heritability as 38% for a general psychopathology factor derived from childhood psychopathology symptoms assessed by multiple raters [36]. These studies also support spectrum-specific genetic factors, such as genetic factors that load specifically on externalizing disorders [31]. The finding of externalizing-specific genetic factors for ADHD is consistent with a large number of twin and family studies demonstrating genetic overlaps of ADHD with oppositional-defiant disorder symptoms [37], conduct disorder [38], antisocial behavior [39], and substance use problems [40,41,42].

Twin studies have tested for genetic overlap between ADHD and autism spectrum disorders (ASD) [43, 44], which often co-occur [45]. Studies of community samples of youth, from the United States of America [46], the United Kingdom [47], and Sweden [11, 48] show that genetic factors influence this comorbidity. Ronald et al. [47]. found genetic correlations between ADHD and ASD above 0.50. Similar results have been found in adult twin studies [49]. A register-based study in Sweden found that individuals with ASD and their relatives were at increased risk of ADHD. The pattern of association across relatives supported the existence of a genetic overlap between clinically ascertained ASD and ADHD [50]. Some features of ASD are differentially linked to either the inattentive or the hyperactive-impulsive components of ADHD [51, 52]. For instance, Polderman et al. [51]. found that the symptoms reflecting the repetitive and restricted aspects of ASD showed the strongest genetic association with ADHD and a Swedish twin study found that the subcomponents of ADHD and ASD are influenced by specific genetic factors [48].

Fewer studies have explored how genetic factors contribute to the co-occurrence between ADHD and internalizing disorders. A large family study found an increased risk of attempted and completed suicide in first- and second-degree relatives of ADHD probands [53]. The pattern of familial risks across different levels of relatedness suggests that shared genetic factors are important for these associations [53]. Family studies that studied the association between ADHD and depression suggest that the co-occurrence is influenced by shared familial factors [54, 55]. Twin studies of this issue suggest that shared genetic factors explain the overlap of ADHD with depression, anxiety, and internalizing symptoms [56,57,58,59,60]. For example, Cole et al. [59]. found that shared genetic factors explained most of the association between traits of ADHD and depression. Similar results were found by Spatola et al. [60]., who used a multivariate twin analysis to study the overlap between different subscales of the Child Behavior Check List (CBCL), such as affective problems, anxiety problems, and attention-deficit/hyperactivity problems.

In contrast to the wealth of information about the familial co-transmission of ADHD and many other disorders, very little is known about ADHD’s familial links to intellectual disability (ID). A meta-analysis reported that the intelligence quotient (IQ) of youth with ADHD is nine points lower than typically developing peers [61] and much evidence suggests it is valid to diagnose ADHD in the context of ID [62]. Faraone et al. [63] studied the genetic association of ADHD and ID in Swedish medical registry data. Individuals with ID were at increased risk for ADHD and relatives of ID cases had an increased risk for ADHD compared with relatives of those without ID. Model fitting analyses attributed 91% of the correlation between the liabilities of ADHD and ID to genetic factors. This work attributes nearly all the comorbidity between ADHD and ID to genetic factors. Only a few twin and family studies have explored how genetic factors contribute to non-psychiatric comorbidity. The literature suggests novel etiologic links with asthma [64], obesity [65], and epilepsy [66].

The search for common genetic variants

Genetic linkage studies

Genetic linkage was the first genome-wide method applied to ADHD. This method searches the genome for evidence that a segment of DNA is transmitted with a disorder within families. A review of the linkage literature found substantial disagreement about which chromosomal regions are linked to ADHD [67]. Although there is some overlap in “suggestive” findings, no finding met genome-wide significance [68]. To make sense of these results, Zhou et al. [69] applied Genome Scan Meta-Analysis. They found genome-wide significant linkage for a region on chromosome 16 between 64 Mb and 83 Mb. Because the linkage method only detects genetic variants that have large effects, the paucity of significant findings for other loci suggests that common DNA variants having a large effect on ADHD are unlikely to exist. Nearly all ADHD linkage studies have selected either sibling pairs or small families from outbred populations. Another approach is to assess for linkage in multigenerational population isolates. Arcos-Burgos et al. [70] used this strategy to study 16 multi-generational families from Colombia. In some of these families, they found evidence supporting linkage to chromosomes 4q13.2, 5q33.3, 8q11.23, 11q22, and 17p11. one region implicated LPHN3. For a review of supporting evidence, see ref. [71].

Candidate gene association studies

Early molecular genetic studies of ADHD sought to associate ADHD with genes that had some a priori plausibility as being involved in its etiology. Because the drugs that treat ADHD target dopaminergic or noradrenergic transmission, many studies examined “candidate genes” in these pathways. Results were frequently contradictory [26, 67]. In the meta-analyses of Gizer et al. [72], eight candidate DNA variants showed a statistically significant association with ADHD across multiple studies. These variants implicated six genes: the serotonin transporter gene (5HTT), the dopamine transporter gene (DAT1), the D4 dopamine receptor gene (DRD4), the D5 dopamine receptor gene (DRD5), the serotonin 1B receptor gene (HTR1B) and a gene coding for a synaptic vesicle regulating protein known as SNAP25. A meta-analysis covering all genetic association studies of adults with ADHD reported a significant association between adult ADHD and BAIAP2 (brain-specific angiogenesis inhibitor 1-associated protein 2). BAIAP2 is involved in neuronal proliferation, survival, and maturation and dendritic spine morphogenesis and may affect neuronal growth-cone guidance. These findings were significant even after Bonferroni correction [73]. For both the child and adult meta-analyses, the strength of each association, as measured by the odds ratio, is small, less than 1.5.

Many studies examined the dopamine transporter gene (SLC6A3), especially a 40-base pair variable number of tandem repeats regulatory polymorphism located in the 3′-untranslated region of the gene. This variant produces two common alleles with 9- and 10-repeats (9R and 10R). In humans, the 10R allele of this polymorphism has been associated with ADHD in youth [67] while the 9R allele is associated with ADHD in adults [74]. A meta-analysis showed that the 9R allele is associated with increased DAT activity in human adults as measured by positron emission tomography [75].

Genome-wide significant common variants

Genome-wide association studies (GWAS) scan the entire genome to detect common DNA variants having very small etiologic effects. By “common” we mean greater than 1% of the population. To do this, GWAS assay hundreds of thousands or even millions of single nucleotide polymorphisms (SNPs). Doing so has a statistical cost: to assert genome-wide statistical significance, an observed association must have a p value less than 0.00000005. This stringent p value needs very large samples.

The initial GWAS of ADHD [76,77,78,79,80,81,82,83,84,85,86] did not discover any DNA variants that achieved genome-wide significance, even when most of these samples were combined in meta-analysis having a sample size of 2064 trios (two parents and an ADHD child), 896 ADHD patients, and 2455 controls [87]. That study did find statistical significance for a group of candidate genes previously nominated by members of the International Multisite ADHD Genetics (IMAGE) project [88]. For a review of early GWAS studies, see Franke et al. [89]. Examination of the “molecular landscape” derived from the top findings from these initial GWAS studies along with other data concluded that genes regulating directed neurite outgrowth were strongly implicated in the etiology of ADHD [90]. Pathway and gene set analyses of GWAS data implicated pathways involved in the regulation of neurotransmitter release, neurite outgrowth and axon guidance as contributors to the etiology of ADHD [91,92,93].

A consortium of ADHD researchers completed a GWAS meta-analysis of 12 studies comprising 20,183 people with ADHD and 35,191 controls. For methodologic details about the studies contributing data to this meta-analysis, see Demontis et al. [94]. Twelve loci achieved genome-wide significance. None of the genome-wide significant SNPs showed significant heterogeneity between studies. Among the implicated genes, FOXP2 is especially notable because prior work had implicated it in adult ADHD (Ribases, 2012 #26445) and in speech and language disorders [95]. A FOXP2 knockout mouse study found that the gene regulates dopamine in ADHD-associated brain regions [96].

As described by Demontis et al. [94], other genes implicated by the genome-wide significant loci have relevant biological roles. DUSP6 regulates neurotransmitter homeostasis by affecting dopamine levels in the synapses. SEMA6D is expressed in the brain. It regulates neuronal wiring during embryonic development. ST3GAL3 harbors missense mutations associated with ID. LINC00461 is expressed in brain and includes variants associated with educational attainment. Another gene implicated at that locus is MEF2C, which has been associated with ID and several psychiatric disorders.

The consortium conducted several gene set analyses including three sets of genes regulated by FOXP2: (1) genes enriched in wild-type versus control FOXP2 knockout mouse brains; (2) genes showing differential expression in wild-type versus FOXP2 knockout mouse brains; and (3) genes enriched in basal ganglia or inferior frontal cortex from human fetal brain samples. None of these sets were associated with ADHD. Also, non-significant was a set of candidate genes for ADHD previously proposed by a panel of ADHD experts [88]. Among these, only SLC9A9 showed a weak association with ADHD. No Gene Ontology gene sets attained statistical significance but a set of genes showing high intolerance to loss of function did associate with ADHD.

Common variant ADHD as a polygenic disorder

The GWAS analyses also showed that much of ADHD’s heritability is due to the polygenic effects of many common variants each having very small effects. The SNP heritability was 0.22, which is about one-third of ADHD’s heritability computed from twin studies [97]. The polygenic architecture for ADHD was confirmed by estimating polygenic risk scores in one subset of the sample and showing that it predicted ADHD, in a dose-dependent manner, in a validation subset. As seen for other psychiatric disorders [98], the variance explained by these risk scores was low (5.5%).

Further evidence for the validity of the ADHD’s polygenic background comes from analyses showing that the relevant SNPs were enriched for annotations implicating conserved regions of the genome (which are known to have biological significance) and for regulatory elements specific to the central nervous system. The discovery of a polygenic susceptibility to ADHD does not show which DNA variants comprise the susceptibility. It does, however, support the idea that more genome-wide significant variants will be discovered in larger samples.

Martin et al. [99] showed that ADHD’s polygenic liability derived from a clinical sample predicted ASD traits in a population sample, which confirms twin study data [48, 51] and gene set analyses [100] showing genetic overlap between ADHD and ASDs. The polygenic liability score derived from Martin et al.’s ADHD case-control clinical sample also predicted both inattention and hyperactivity in the general population. This latter finding was replicated by Groen-Blokhuis et al. [101] who found that ADHD polygenic risk scores significantly predicted both parent and teacher ratings of attention in preschool- and school-aged children in the population. Likewise, Stergiakouli et al. [102] showed that the polygenic liability for ADHD traits in a population sample predicted ADHD clinical diagnoses in a case-control study. These results confirmed conclusions from twin studies that the liability for clinically defined ADHD is the extreme of a trait that varies continuously in the population [13].

Other polygenic score studies are confirming cross-disorder genetic associations previously predicted by family and twin studies. We have long known that ADHD co-occurs with conduct disorder. Both family and twin studies have implicated shared genes in this association [38, 103,104,105]. Consistent with this prior work, Hamshere et al. [106] reported a high polygenic risk for ADHD among children with comorbid conduct problems. In a large population study, Larsson et al. [107] reported that the relatives of ADHD individuals had an increased risk for schizophrenia and bipolar disorder. Consistent with that report, the polygenic risk score derived from a large GWAS of schizophrenia significantly discriminated ADHD cases from controls [108]. This discrimination was strongest for alleles that were risk alleles for both adult schizophrenia and adult bipolar disorder, which confirms prior family and twin data suggesting a genetic link between ADHD and bipolar disorder [109]. Moreover, a joint GWAS of ADHD and bipolar disorder reported a significant correlation between the polygenic scores of ADHD and bipolar disorder and also identified genome-wide significant loci for the two disorders [110]. Similarly, prior reports of familial co-transmission of ADHD and depression [54] have been extended by showing shared SNP heritability between the two disorders [98]. Using a novel drug challenge paradigm, Hart et al. [111] found that the polygenic scores for both schizophrenia and ADHD were associated with the euphoric response to amphetamine, which suggests that the genetic association between these disorders may be due to variants in the neural systems regulating the euphoric response to amphetamine.

Using GWAS results from many studies, it is possible to compute genetic correlations that indicate the degree to which the polygenic architectures of two disorders or traits overlap. When Demontis et al. [94] correlated ADHD’s polygenic risk with 220 disorders and traits, many highly significant correlations emerged. Figure 3 shows some of the most significant of these correlations (each passing the Bonferroni significance threshold). Some of these genetic correlations fit with prior expectations (e.g., with neuroticism, depression and the cross disorder GWAS). Others are consistent with the clinical epidemiology of ADHD (e.g., with obesity, IQ, smoking and school achievement). In some cases, these significant correlations offer new directions for understanding comorbidity. For example, some have interpreted the comorbidity between ADHD and obesity, which has been confirmed via meta-analysis [112], as being caused by the impulsivity associated with ADHD. The genetic correlation data suggest that shared genetic risk factors, and an underlying shared pathophysiology, account for this comorbidity.

Fig. 3

Genetic correlations of ADHD with other traits based on LD score regression

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Some of the genetic correlations in Fig. 3 are entirely novel. These include ADHD’s genetic correlations with medical outcomes (lung cancer, coronary artery disease, parents’ age at death) and with demographics (number of children in the family, age first child born). There are, however, some consistent findings in the prior literature, which suggest that people with ADHD are more likely to have larger families [113] and more likely to die prematurely [114].

The search for rare genetic variants

Initially, information about rare DNA variants (<1% of the population) came from reports of syndromic chromosomal anomalies associated with multiple medical and psychiatric problems along with ADHD. Examples are velo-cardio facial syndrome fragile-X syndrome, Turner syndrome, tuberous sclerosis, neurofibromatosis, Klinefelter syndrome, and Williams syndrome (Fig. 4). In a single family, a peri-centric inversion of chromosome 3 co-segregating with ADHD symptoms [115, 116] implicated SLC9A9. Mutations of that gene lead to an animal model of ADHD [117, 118] and have been associated with both autism [119, 120] and ADHD [121].

Fig. 4

Prevalence of ADHD in rare genetic syndromes

Full size image

The common variant genotyping arrays used in GWAS studies can detect large copy number variants (CNVs). Because CNVs often delete or duplicate a large genomic segment spanning part of a gene or even entire genes, they often have clear implications for gene functioning. Studies of CNVs in ADHD assessed ADHD youth and controls for the presence of large (>500 kb), rare CNVs [77, 122,123,124,125,126,127]. Each study, except one, found an odds ratio greater than one, indicating a greater burden of large, rare CNVs among ADHD patients compared with controls. The discrepant study used a different definition of burden [125]. Only three studies found a statistically significant burden among ADHD patients for large CNVs (for a summary, see Thapar et al. [128]). As their review shows, deletions and duplications are equally over-represented in ADHD samples although statistical significance emerged only for duplications. Thapar et al. also found enrichment for duplications (but not deletions) previously implicated in schizophrenia and, to a lesser extent, ASDs. The top biological pathways implicated by these CNV studies were: respiratory electron transport, organonitrogen compound catabolic process, transmembrane transporter activity, carbohydrate derivative catabolic process, ligand-gated ion channel activity, methyltransferase activity, transmembrane transport and ion gated channel activity.

A study of 489 ADHD patients and 1285 controls found rare CNVs in the parkinson protein two gene (PARK2) [123]. The result was significant after empirical correction for genome-wide testing. PARK2 regulates the cell’s ubiquitin-proteasome system which helps dispose damaged, misshapen, and excess proteins. Two other genes involved in this pathway (FBXO33 and RNF122) had been implicated in other studies [84, 129]. A study of adult ADHD did not find a significant effect for large CNVs, but did find a significant effect for small CNVs [126].

The CNV studies have implicated several biological pathways. Williams et al. [127] found that ADHD patients harbored duplications in the alpha-7 nicotinic acetylcholine receptor gene (CHRNA7) and showed that the finding replicated in four independent cohorts from the United Kingdom, the United States, and Canada. Another replication was reported in an Italian sample [130]. The implication of the nicotinic system is particularly interesting given that nicotinic neurons modulate dopaminergic neurons, ADHD patients have a high rate of smoking [131] and nicotine administration reduces ADHD symptoms [132]. In a sample of 99 children and adolescents with severe ADHD. Lesch et al. [124] found several CNVs, including a 3 Mb duplication on chromosome 7p15.2-15.3 harboring neuropeptide Y (NPY). Investigation of other family members yielded an association of this duplication with increased NPY plasma concentrations and functional magnetic imaging assessed brain abnormalities.

Thapar et al. [128] reported biological pathway studies of ADHD CNV data pooled from five studies. These CNV data were enriched for genes previously implicated in schizophrenia, Fragile X intellectual disability and, to a lesser degree, autism. Several biological pathways were significantly enriched in the ADHD CNV findings, most notably ion channel pathways, which had been implicated in cross-disorder analyses of ADHD, autism, schizophrenia, bipolar disorder, and depression [133]. The CNV analyses also pointed to pathways regulating immune functioning and oxidative stress. These pathways had previously been implicated in ADHD by non-genetic studies, (e.g. refs. [134,135,136]).

Elia et al. [122] showed that CNVs impacting metabotropic glutamate receptor genes were significantly enriched across multiple cohorts of patients. Supporting evidence came from Akutagava-Martins et al. [137] who reported that CNVs in glutamatergic genes were associated with the cognitive and clinical impairments of ADHD. In a pharmacogenomics GWAS, an SNP in glutamate receptor gene GRM7 was one of the most significant findings [138]. Glutamatergic defects have been observed in a rat model of ADHD [139, 140] and magnetic resonance spectroscopy in humans shows dysregulation of glutamate and glutamate/glutamine concentrations in ADHD patients(e.g. ref. [141]).

An exome sequencing study of ADHD [121] reported results for 123 adults with persistent ADHD and 82 healthy controls. Significantly more cases than controls had a rare missense or disruptive variant in a set of ADHD candidate genes. In an exome sequencing study of ADHD patients without a family history of ADHD, Kim et al. [142] reported six de novo missense SNVs in brain-expressed genes: TBC1D9, DAGLA, QARS, CSMD2, TRPM2, and WDR83. They also sequenced 26 genes implicated in ID and ASDs but found only one potentially deleterious variant. In an exome chip study, Zayats et al. [143] assayed a sample of 1846 cases and 7519 controls to search for rare genetic variants. They detected four study-wide significant loci that implicated four genes known to be expressed in the brain during prenatal stages of development: NT5DC1, SEC23IP, PSD, and ZCCHC4. Hawi et al. [144] found novel rare variants in the BDNF gene by sequencing 117 genes in 152 youth with ADHD and 188 controls.

Pharmacogenetics of ADHD

Several studies have clarified the genetics of the metabolism of ADHD patients. Some patients are slow metbolizers of atomoxetine due to variants in the cytochrome P450 isoenzyme 2D6, which is regulated by the CYP2D6 gene. As a result, the half-life of atomoxetine ranges from 5.2 h in rapid metabolizers to 21.6 h in slow metabolizers [145]. Some work has looked into CES1 variants regarding the regulation of methylphenidate metabolism and CYP2D6/CYP3A4 variants and the metabolism of ADHD, but the evidence base has not generated consistent results for either children [146] or adults [73].

Myer et al. [147] used meta-analysis to evaluate pharmacogenetic studies of the efficacy response to methylphenidate for the treatment of ADHD. They found 36 studies comprising 3647 ADHD youth treated with methylphenidate. Statistically significant effects were found for: rs1800544 in ADRA2A (odds ratio (OR): 1.69; confidence interval (CI): 1.12−2.55), rs4680 COMT (OR: 1.40; CI: 1.04−1.87), rs5569 SLC6A2 (OR: 1.73; CI: 1.26−2.37), and rs28386840 SLC6A2 (OR: 2.93; CI: 1.76−4.90), and, repeat variants VNTR 4 DRD4 (OR: 1.66; CI: 1.16−2.37) and VNTR 10 SLC6A3 (OR: 0.74; CI: 0.60−0.90). The following variants did not reach statistical significance: rs1947274 LPHN3 (OR: 0.95; CI: 0.71−1.26), rs5661665 LPHN3 (OR: 1.07; CI: 0.84−1.37) and VNTR 7 DRD4 (OR: 0.68; confidence interval: 0.47−1.00). The significant findings were not due to publication biases. Although the odds ratios are small, these findings suggest that a personalized medicine approach to ADHD is a reasonable goal of future research.

Conclusions and future directions

There can be no doubt that DNA variants in genes or regulatory regions increase the risk for ADHD. In rare cases, a single genetic defect may lead to ADHD in the absence of other DNA variants. We do not know how many of these rare variants exist or if such variants require environmental triggers for ADHD to emerge. It is equally clear that no common DNA variants are necessary and sufficient causes of ADHD. Genome-wide association studies show that a genetic susceptibility to ADHD comprised of many common DNA variants accounts for about one-third of the twin study estimates of ADHD’s heritability. We do not know yet which variants or how many of them make up the polygenic component. The heritability that cannot be explained by main effects of rare or common variants is likely due to gene−gene interactions, gene−environment interactions or gene−environment correlations.

The convincing evidence for genes as risk factors for ADHD does not exclude the environment as a source of etiology. The fact that twin estimates of heritability are less than 100% asserts quite strongly that environmental factors must be involved. ADHD’s heritability is high, and that estimate encompasses gene by environment interaction. Thus, it is possible that such interactions will account for much of ADHD’s etiology. Environmental risk factors likely work through epigenetic mechanisms, which have barely been studied in ADHD [148]. The importance of the environment can also be seen in the fact that, as for other complex genetic disorders, much of ADHD’s heritability is explained by SNPs in regulatory regions rather than coding regions [149].

Another hypothesis for future research to explore is the possibility that ADHD is an omnigenic disorder. The omnigenic model of Boyle et al. [150] posits the existence of a small number of “core genes” having “biologically interpretable roles in disease” along with a much greater quantity of “peripheral genes” regulating the core genes. Because there are many more peripheral genes, they account for a greater proportion of the variability in heritability than do the core genes. Because core genes are more likely than peripheral genes to be relevant for developing biomarkers and treatment targets, separating these two classes from one another will require more research.

Gene discovery for ADHD has succeeded but has left us with unexpected results. None of the genome-wide significant findings had been predicted a priori and a set of ADHD candidate genes, implicated primarily by the disorder’s neuropharmacology, did not reach statistical significance. These findings challenge the idea that the core of ADHD’s pathophysiology rests within the machinery of catecholaminergic transmission. Instead, it is possible that the catecholaminergic dysregulation believed to underlie ADHD is a secondary compensation to ADHD’s primary etiology (see discussion by Hess et al. [151]).

In the years to come, we can expect breakthroughs in the genetics of ADHD to come from several fields of study. Our knowledge of rare variants should increase dramatically as we learn more about CNVs and as reports from exome, full genome and targeted sequencing studies unfold. With the discovery of genome-wide significant common variants, we look forward to studies that discover the functional variants responsible for these findings. With the discovery of these functional variants, we will learn more about the mechanisms whereby genetic risk variants increase the risk for ADHD.

Accumulating evidence from family, twin, and molecular genetic studies suggests that the disorder we know as ADHD is the extreme of a dimensional trait in the population. The dimensional nature of ADHD has wide-ranging implications. If we view ADHD as analogous to cholesterol levels, then diagnostic approaches should focus on defining the full continuum of “ADHD-traits” along with clinically meaningful thresholds for defining who does and does not need treatment and who has clinically subthreshold traits that call for careful monitoring. The dimensional nature of ADHD should also shift the debate about the increases in ADHD’s prevalence in recent years. Instead of assuming that misdiagnoses are the main explanation for the increased prevalence, perhaps researchers should explore to what extent the threshold for diagnosis has decreased over time and whether changes in the threshold are clinically sensible or not. A shift from categorical to dimensional constructs harmonizes with the Research Domain Criteria (RDoC) initiative of the National Institute of Mental Health [152]. RDoC seeks to define and validate dimensional constructs mediating psychopathology along with the neurobiological underpinnings of these constructs.

Unraveling the genetics of ADHD will be challenging. Technological advances are moving at a rapid pace. The next decade of work should give us more accurate measures of brain structure and function along with much more genomic, transcriptomic and epigenomic data. These advances will set the stage for breakthroughs in our understanding of the etiology of ADHD and in our ability to diagnose and treat the disorder.

References

1. Faraone SV, Asherson P, Banaschewski T, Biederman J, Buitelaar JK, Ramos-Quiroga JA, et al. Attention-deficit/hyperactivity disorder. Nat Rev Dis Prim. 2015;1:15020.

PubMed  Article  Google Scholar

2. Chen W, Zhou K, Sham P, Franke B, Kuntsi J, Campbell D, et al. DSM-IV combined type ADHD shows familial association with sibling trait scores: a sampling strategy for QTL linkage. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:1450–60.

CAS  PubMed  Article  Google Scholar

3. Alberts-Corush J, Firestone P, Goodman JT. Attention and impulsivity characteristics of the biological and adoptive parents of hyperactive and normal control children. Am J Orthopsychiatry. 1986;56:413–23.

PubMed  Article  Google Scholar

4. Sprich S, Biederman J, Crawford MH, Mundy E, Faraone SV. Adoptive and biological families of children and adolescents with ADHD. J Am Acad Child Adolesc Psychiatry. 2000;39:1432–7.

CAS  PubMed  Article  Google Scholar

5. Chen Q, Brikell I, Lichtenstein P, Serlachius E, Kuja-Halkola R, Sandin S, et al. Familial aggregation of attention-deficit/hyperactivity disorder. J Child Psychol Psychiatry. 2017;58:231–9.

PubMed  Article  Google Scholar

6. Nikolas MA, Burt SA. Genetic and environmental influences on ADHD symptom dimensions of inattention and hyperactivity: a meta-analysis. J Abnorm Psychol. 2010;119:1–17.

PubMed  Article  Google Scholar

7. Larsson H, Lichtenstein P, Larsson JO. Genetic contributions to the development of ADHD subtypes from childhood to adolescence. J Am Acad Child Adolesc Psychiatry. 2006;45:973–81.

PubMed  Article  Google Scholar

8. McLoughlin G, Ronald A, Kuntsi J, Asherson P, Plomin R. Genetic support for the dual nature of attention deficit hyperactivity disorder: substantial genetic overlap between the inattentive and hyperactive-impulsive components. J Abnorm Child Psychol. 2007;35:999–1008.

PubMed  PubMed Central  Article  Google Scholar

9. Sherman D, Iacono W, McGue M. Attention deficit hyperactivity disorder dimensions: a twin study of inattention and impulsivity hyperactivity. J Am Acad Child Adolesc Psychiatry. 1997;36:745–53.

CAS  PubMed  Article  Google Scholar

10. Langner I, Garbe E, Banaschewski T, Mikolajczyk RT. Twin and sibling studies using health insurance data: the example of attention deficit/hyperactivity disorder (ADHD). PLoS ONE. 2013;8:e62177.

CAS  PubMed  PubMed Central  Article  Google Scholar

11. Lichtenstein P, Carlstrom E, Rastam M, Gillberg C, Anckarsater H. The genetics of autism spectrum disorders and related neuropsychiatric disorders in childhood. Am J Psychiatry. 2010;167:1357–63.

PubMed  Article  Google Scholar

12. Thapar A, Harrington R, Ross K, McGuffin P. Does the definition of ADHD affect heritability? J Am Acad Child Adolesc Psychiatry. 2000;39:1528–36.

CAS  PubMed  Article  Google Scholar

13. Larsson H, Anckarsater H, Rastam M, Chang Z, Lichtenstein P. Childhood attention-deficit hyperactivity disorder as an extreme of a continuous trait: a quantitative genetic study of 8,500 twin pairs. J Child Psychol Psychiatry. 2012;53:73–80.

PubMed  Article  Google Scholar

14. Levy F, Hay D, McStephen M, Wood C, Waldman I. Attention-deficit hyperactivity disorder: a category or a continuum? Genetic analysis of a large-scale twin study. J Am Acad Child Adolesc Psychiatry. 1997;36:737–44.

CAS  PubMed  Article  Google Scholar

15. Faraone SV, Biederman J, Spencer T, Mick E, Murray K, Petty C, et al. Diagnosing adult attention deficit hyperactivity disorder: are late onset and subthreshold diagnoses valid? Am J Psychiatry. 2006;163:1720–9. quiz 859

PubMed  Article  Google Scholar

16. Haberstick BC, Timberlake D, Hopfer CJ, Lessem JM, Ehringer MA, Hewitt JK. Genetic and environmental contributions to retrospectively reported DSM-IV childhood attention deficit hyperactivity disorder. Psychol Med. 2008;38:1057–66.

CAS  PubMed  Article  Google Scholar

17. Boomsma DI, Saviouk V, Hottenga JJ, Distel MA, de Moor MH, Vink JM, et al. Genetic epidemiology of attention deficit hyperactivity disorder (ADHD index) in adults. PLoS ONE. 2010;5:e10621.

PubMed  PubMed Central  Article  CAS  Google Scholar

18. Brikell I, Kuja-Halkola R, Larsson H. Heritability of attention-deficit hyperactivity disorder in adults. Am J Med Genet B Neuropsychiatr Genet. 2015;168:406–13.

PubMed  Article  Google Scholar

19. Larsson H, Asherson P, Chang Z, Ljung T, Friedrichs B, Larsson JO, et al. Genetic and environmental influences on adult attention deficit hyperactivity disorder symptoms: a large Swedish population-based study of twins. Psychol Med. 2013;43:197–207.

CAS  PubMed  Article  Google Scholar

20. Kan KJ, van Beijsterveldt CE, Bartels M, Boomsma DI. Assessing genetic influences on behavior: informant and context dependency as illustrated by the analysis of attention problems. Behav Genet. 2014;44:326–36.

PubMed  Article  Google Scholar

21. Merwood A, Greven CU, Price TS, Rijsdijk F, Kuntsi J, McLoughlin G, et al. Different heritabilities but shared etiological influences for parent, teacher and self-ratings of ADHD symptoms: an adolescent twin study. Psychol Med. 2013;43:1973–84.

CAS  PubMed  Article  Google Scholar

22. Kan KJ, Dolan CV, Nivard MG, Middeldorp CM, van Beijsterveldt CE, Willemsen G, et al. Genetic and environmental stability in attention problems across the lifespan: evidence from the Netherlands twin register. J Am Acad Child Adolesc Psychiatry. 2013;52:12–25.

PubMed  Article  Google Scholar

23. Freitag CM, Rohde LA, Lempp T, Romanos M. Phenotypic and measurement influences on heritability estimates in childhood ADHD. Eur Child Adolesc Psychiatry. 2010;19:311–23.

PubMed  Article  Google Scholar

24. Schultz MR, Rabi K, Faraone SV, Kremen W, Lyons MJ. Efficacy of retrospective recall of attention-deficit hyperactivity disorder symptoms: a twin study. Twin Res Hum Genet. 2006;9:220–32.

PubMed  Article  Google Scholar

25. Larsson H, Chang Z, D'Onofrio BM, Lichtenstein P. The heritability of clinically diagnosed attention deficit hyperactivity disorder across the lifespan. Psychol Med. 2014;44:2223–9.

CAS  PubMed  Article  Google Scholar

26. Franke B, Faraone SV, Asherson P, Buitelaar J, Bau CH, Ramos-Quiroga JA, et al. The genetics of attention deficit/hyperactivity disorder in adults, a review. Mol Psychiatry. 2011;17:960–87.

PubMed  PubMed Central  Article  CAS  Google Scholar

27. Faraone SV. Genetics of adult attention-deficit/hyperactivity disorder. Psychiatr Clin North Am. 2004;27:303–21.

PubMed  Article  Google Scholar

28. Chang Z, Lichtenstein P, Asherson PJ, Larsson H. Developmental twin study of attention problems high heritabilities throughout development. JAMA Psychiatry. 2013;70:311–8.

PubMed  Article  Google Scholar

29. Rietveld MJ, Hudziak JJ, Bartels M, van Beijsterveldt CE, Boomsma DI. Heritability of attention problems in children: longitudinal results from a study of twins, age 3 to 12. J Child Psychol Psychiatry. 2004;45:577–88.

CAS  PubMed  Article  Google Scholar

30. Kuntsi J, Rijsdijk F, Ronald A, Asherson P, Plomin R. Genetic influences on the stability of attention-deficit/hyperactivity disorder symptoms from early to middle childhood. Biol Psychiatry. 2005;57:647–54.

PubMed  Article  Google Scholar

31. Lahey BB, Van Hulle CA, Singh AL, Waldman ID, Rathouz PJ. Higher-order genetic and environmental structure of prevalent forms of child and adolescent psychopathology. Arch Gen Psychiatry. 2011;68:181–9.

PubMed  PubMed Central  Article  Google Scholar

32. Pettersson E, Larsson H, Lichtenstein P. Common psychiatric disorders share the same genetic origin: a multivariate sibling study of the Swedish population. Mol Psychiatry. 2016;21:717–21.

CAS  PubMed  Article  Google Scholar

33. Waldman ID, Poore HE, van Hulle C, Rathouz PJ, Lahey BB. External validity of a hierarchical dimensional model of child and adolescent psychopathology: Tests using confirmatory factor analyses and multivariate behavior genetic analyses. J Abnorm Psychol. 2016;125:1053–66.

PubMed  Article  PubMed Central  Google Scholar

34. Pettersson E, Anckarsater H, Gillberg C, Lichtenstein P. Different neurodevelopmental symptoms have a common genetic etiology. J Child Psychol Psychiatry. 2013;54:1356–65.

PubMed  Article  Google Scholar

35. Pappa I, Fedko IO, Mileva-Seitz VR, Hottenga JJ, Bakermans-Kranenburg MJ, Bartels M, et al. Single nucleotide polymorphism heritability of behavior problems in childhood: genome-wide complex trait analysis. J Am Acad Child Adolesc Psychiatry. 2015;54:737–44.

PubMed  Article  Google Scholar

36. Neumann A, Pappa I, Lahey BB, Verhulst FC, Medina-Gomez C, Jaddoe VW, et al. Single nucleotide polymorphism heritability of a general psychopathology factor in children. J Am Acad Child Adolesc Psychiatry. 2016;55:1038–45 e4..

PubMed  Article  Google Scholar

37. Nadder TS, Rutter M, Silberg J, Maes H, Eaves L. Genetic effects on the variatio and covariation of attention deficit-hyperactivity disorder (ADHD) and oppositional-defiant disorder/conduct disorder (ODD/CD) symptomatologies across informant and occasion of measurement. Psychol Med. 2002;32:39–53.

CAS  PubMed  Article  Google Scholar

38. Faraone SV, Biederman J, Monuteaux MC. Attention-deficit disorder and conduct disorder in girls: evidence for a familial subtype. Biol Psychiatry. 2000;48:21–9.

CAS  PubMed  Article  Google Scholar

39. Kuja-Halkola R, Lichtenstein P, D’Onofrio BM, Larsson H. Codevelopment of ADHD and externalizing behavior from childhood to adulthood. J Child Psychol Psychiatry. 2015;56:640–7.

PubMed  Article  Google Scholar

40. Chang Z, Lichtenstein P, Larsson H. The effects of childhood ADHD symptoms on early-onset substance use: a Swedish twin study. J Abnorm Child Psychol. 2012;40:425–35.

PubMed  Article  Google Scholar

41. Capusan AJ, Bendtsen P, Marteinsdottir I, Kuja-Halkola R, Larsson H. Genetic and environmental contributions to the association between attention deficit hyperactivity disorder and alcohol dependence in adulthood: a large population-based twin study. Am J Med Genet B Neuropsychiatr Genet. 2015;168:414–22.

PubMed  Article  Google Scholar

42. Skoglund C, Chen Q, Franck J, Lichtenstein P, Larsson H. Attention-deficit/hyperactivity disorder and risk for substance use disorders in relatives. Biol Psychiatry. 2015;77:880–6.

PubMed  Article  Google Scholar

43. Rommelse NN, Geurts HM, Franke B, Buitelaar JK, Hartman CA. A review on cognitive and brain endophenotypes that may be common in autism spectrum disorder and attention-deficit/hyperactivity disorder and facilitate the search for pleiotropic genes. Neurosci Biobehav Rev. 2011;35:1363–96.

CAS  PubMed  Article  Google Scholar

44. Rommelse NN, Franke B, Geurts HM, Hartman CA, Buitelaar JK. Shared heritability of attention-deficit/hyperactivity disorder and autism spectrum disorder. Eur Child Adolesc Psychiatry. 2010;19:281–95.

PubMed  PubMed Central  Article  Google Scholar

45. Antshel KM, Zhang-James Y, Faraone SV. The comorbidity of ADHD and autism spectrum disorder. Expert Rev Neurother. 2013;13:1117–28.

CAS  PubMed  Article  Google Scholar

46. Ronald A, Edelson LR, Asherson P, Saudino KJ. Exploring the relationship between autistic-like traits and ADHD behaviors in early childhood: findings from a community twin study of 2-year-olds. J Abnorm Child Psychol. 2010;38:185–96.

PubMed  PubMed Central  Article  Google Scholar

47. Ronald A, Simonoff E, Kuntsi J, Asherson P, Plomin R. Evidence for overlapping genetic influences on autistic and ADHD behaviours in a community twin sample. J Child Psychol Psychiatry. 2008;49:535–42.

PubMed  Article  Google Scholar

48. Ronald A, Larsson H, Anckarsater H, Lichtenstein P. Symptoms of autism and ADHD: a Swedish twin study examining their overlap. J Abnorm Psychol. 2014;123:440–51.

PubMed  Article  Google Scholar

49. Lundstrom S, Chang Z, Kerekes N, Gumpert CH, Rastam M, Gillberg C, et al. Autistic-like traits and their association with mental health problems in two nationwide twin cohorts of children and adults. Psychol Med. 2011;41:2423–33.

CAS  PubMed  Article  Google Scholar

50. Ghirardi L, Brikell I, Kuja-Halkola R, Freitag CM, Franke B, Asherson P, et al. The familial co-aggregation of ASD and ADHD: a register-based cohort study. Mol Psychiatry. 2017;23:257–62.

PubMed  PubMed Central  Article  CAS  Google Scholar

51. Polderman TJ, Hoekstra RA, Posthuma D, Larsson H. The co-occurrence of autistic and ADHD dimensions in adults: an etiological study in 17,770 twins. Transl Psychiatry. 2014;4:e435.

CAS  PubMed  PubMed Central  Article  Google Scholar

52. Polderman TJ, Hoekstra RA, Vinkhuyzen AA, Sullivan PF, van der Sluis S, Posthuma D. Attentional switching forms a genetic link between attention problems and autistic traits in adults. Psychol Med. 2013;43:1985–96.

CAS  PubMed  Article  Google Scholar

53. Ljung T, Chen Q, Lichtenstein P, Larsson H. Common etiological factors of attention-deficit/hyperactivity disorder and suicidal behavior: a population-based study in Sweden. JAMA Psychiatry. 2014;71:958–64.

PubMed  Article  Google Scholar

54. Faraone SV, Biederman J. Do attention deficit hyperactivity disorder and major depression share familial risk factors? J Nerv Ment Dis. 1997;185:533–41.

CAS  PubMed  Article  Google Scholar

55. Faraone SV, Biederman J. Depression: a family affair. Lancet. 1998;351:158.

CAS  PubMed  Article  Google Scholar

56. Rydell M, Taylor MJ, Larsson H Genetic and environmental contributions to the association between ADHD and affective problems in early childhood-A Swedish population-based twin study. Am J Med Genet B Neuropsychiatr Genet. 2017;174:538–46.

PubMed  Article  Google Scholar

57. Segenreich D, Paez MS, Regalla MA, Fortes D, Faraone SV, Sergeant J, et al. Multilevel analysis of ADHD, anxiety and depression symptoms aggregation in families. Eur Child Adolesc Psychiatry. 2015;24:525–36.

PubMed  Article  Google Scholar

58. Chen TJ, Ji CY, Wang SS, Lichtenstein P, Larsson H, Chang Z. Genetic and environmental influences on the relationship between ADHD symptoms and internalizing problems: A Chinese twin study. Am J Med Genet B Neuropsychiatr Genet. 2016;171:931–7.

PubMed  Article  Google Scholar

59. Cole J, Ball HA, Martin NC, Scourfield J, McGuffin P. Genetic overlap between measures of hyperactivity/inattention and mood in children and adolescents. J Am Acad Child Adolesc Psychiatry. 2009;48:1094–101.

PubMed  Article  Google Scholar

60. Spatola CA, Fagnani C, Pesenti-Gritti P, Ogliari A, Stazi MA, Battaglia M. A general population twin study of the CBCL/6-18 DSM-oriented scales. J Am Acad Child Adolesc Psychiatry. 2007;46:619–27.

PubMed  Article  Google Scholar

61. Frazier TW, Demaree HA, Youngstrom EA. Meta-analysis of intellectual and neuropsychological test performance in attention-deficit/hyperactivity disorder. Neuropsychology. 2004;18:543–55.

PubMed  Article  Google Scholar

62. Antshel KM, Phillips MH, Gordon M, Barkley R, Faraone SV. Is ADHD a valid disorder in children with intellectual delays? Clin Psychol Rev. 2006;26:555–72.

PubMed  Article  Google Scholar

63. Faraone SV, Ghirardi L, Kuja-Halkola R, Lichtenstein P, Larsson H. The familial co-aggregation of attention-deficit/hyperactivity disorder and intellectual disability: a register-based family study. J Am Acad Child Adolesc Psychiatry. 2017;56:167–74 e1.

PubMed  Article  Google Scholar

64. Mogensen N, Larsson H, Lundholm C, Almqvist C. Association between childhood asthma and ADHD symptoms in adolescence—a prospective population-based twin study. Allergy. 2011;66:1224–30.

CAS  PubMed  Article  Google Scholar

65. Chen Q, Kuja-Halkola R, Sjolander A, Serlachius E, Cortese S, Faraone SV, et al. Shared familial risk factors between attention-deficit/hyperactivity disorder and overweight/obesity—a population-based familial coaggregation study in Sweden. J Child Psychol Psychiatry. 2017;58:711–8.

PubMed  Article  Google Scholar

66. Brikell I, Ghirardi L, D’Onofrio BM, Dunn DW, Almqvist C, Dalsgaard S, et al. Familial liability to epilepsy and attention-deficit/hyperactivity disorder: a nationwide cohort study. Biol Psychiatry. 2018;83:173–80.

PubMed  Article  Google Scholar

67. Faraone SV, Mick E. Molecular genetics of attention deficit hyperactivity disorder. Psychiatr Clin North Am. 2010;33:159–80.

PubMed  PubMed Central  Article  Google Scholar

68. Lander E, Kruglyak L. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat Genet. 1995;11:241–7.

CAS  PubMed  Article  Google Scholar

69. Zhou K, Dempfle A, Arcos-Burgos M, Bakker SC, Banaschewski T, Biederman J, et al. Meta-analysis of genome-wide linkage scans of attention deficit hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:1392–8.

CAS  PubMed  PubMed Central  Article  Google Scholar

70. Arcos-Burgos M, Castellanos FX, Pineda D, Lopera F, David Palacio J, Guillermo Palacio L, et al. Attention-deficit/hyperactivity disorder in a population isolate: linkage to Loci at 4q13.2, 5q33.3, 11q22, and 17p11. Am J Hum Genet. 2004;75:998–1014.

CAS  PubMed  PubMed Central  Article  Google Scholar

71. Arcos-Burgos M, Muenke M. Toward a better understanding of ADHD: LPHN3 gene variants and the susceptibility to develop ADHD. Atten Defic Hyperact Disord. 2010;2:139–47.

PubMed  PubMed Central  Article  Google Scholar

72. Gizer IR, Ficks C, Waldman ID. Candidate gene studies of ADHD: a meta-analytic review. Hum Genet. 2009;126:51–90.

CAS  PubMed  Article  Google Scholar

73. Bonvicini C, Faraone SV, Scassellati C. Attention-deficit hyperactivity disorder in adults: a systematic review and meta-analysis of genetic, pharmacogenetic and biochemical studies. Mol Psychiatry. 2016;21:1643.

CAS  PubMed  PubMed Central  Article  Google Scholar

74. Franke B, Vasquez AA, Johansson S, Hoogman M, Romanos J, Boreatti-Hummer A, et al. Multicenter analysis of the SLC6A3/DAT1 VNTR haplotype in persistent ADHD suggests differential involvement of the gene in childhood and persistent ADHD. Neuropsychopharmacology. 2010;35:656–64.

CAS  PubMed  Article  Google Scholar

75. Faraone SV, Spencer TJ, Madras BK, Zhang-James Y, Biederman J. Functional effects of dopamine transporter gene genotypes on in vivo dopamine transporter functioning: a meta-analysis. Mol Psychiatry. 2014;19:880–9.

CAS  PubMed  Article  Google Scholar

76. Neale BM, Medland S, Ripke S, Anney RJ, Asherson P, Buitelaar J, et al. Case-control genome-wide association study of attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry. 2010;49:906–20.

PubMed  PubMed Central  Article  Google Scholar

77. Yang L, Neale BM, Liu L, Lee SH, Wray NR, Ji N, et al. Polygenic transmission and complex neuro developmental network for attention deficit hyperactivity disorder: genome-wide association study of both common and rare variants. Am J Med Genet B Neuropsychiatr Genet. 2013;162:419–30.

CAS  Article  Google Scholar

78. Ebejer JL, Duffy DL, van der Werf J, Wright MJ, Montgomery G, Gillespie NA, et al. Genome-wide association study of inattention and hyperactivity-impulsivity measured as quantitative traits. Twin Res Hum Genet. 2013;16:560–74.

PubMed  Article  Google Scholar

79. Fliers EA, Vasquez AA, Poelmans G, Rommelse N, Altink M, Buschgens C, et al. Genome-wide association study of motor coordination problems in ADHD identifies genes for brain and muscle function. World J Biol Psychiatry. 2012;13:211–22.

PubMed  Article  Google Scholar

80. Hinney A, Scherag A, Jarick I, Albayrak O, Putter C, Pechlivanis S, et al. Genome-wide association study in German patients with attention deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2011;156B:888–97.

PubMed  Article  Google Scholar

81. Lasky-Su J, Anney RJ, Neale BM, Franke B, Zhou K, Maller JB, et al. Genome-wide association scan of the time to onset of attention deficit hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:1355–8.

PubMed  PubMed Central  Article  Google Scholar

82. Lasky-Su J, Neale BM, Franke B, Anney RJ, Zhou K, Maller JB, et al. Genome-wide association scan of quantitative traits for attention deficit hyperactivity disorder identifies novel associations and confirms candidate gene associations. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:1345–54.

CAS  PubMed  Article  Google Scholar

83. Mick E, Todorov A, Smalley S, Hu X, Loo S, Todd RD, et al. Family-based genome-wide association scan of attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry. 2010;49:898–905 e3.

PubMed  PubMed Central  Article  Google Scholar

84. Sanchez-Mora C, Ramos-Quiroga JA, Bosch R, Corrales M, Garcia-Martinez I, Nogueira M, et al. Case-control genome-wide association study of persistent attention-deficit hyperactivity disorder identifies FBXO33 as a novel susceptibility gene for the disorder. Neuropsychopharmacology. 2015;40:915–26.

CAS  PubMed  Article  Google Scholar

85. Lesch KP, Timmesfeld N, Renner TJ, Halperin R, Roser C, Nguyen TT, et al. Molecular genetics of adult ADHD: converging evidence from genome-wide association and extended pedigree linkage studies. J Neural Transm. 2008;115:1573–85.

CAS  PubMed  Article  Google Scholar

86. Zayats T, Athanasiu L, Sonderby I, Djurovic S, Westlye LT, Tamnes CK, et al. Genome-wide analysis of attention deficit hyperactivity disorder in Norway. PLoS ONE. 2015;10:e0122501.

PubMed  PubMed Central  Article  CAS  Google Scholar

87. Neale BM, Medland SE, Ripke S, Asherson P, Franke B, Lesch KP, et al. Meta-analysis of genome-wide association studies of attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry. 2010;49:884–97.

PubMed  PubMed Central  Article  Google Scholar

88. Brookes K, Xu X, Chen W, Zhou K, Neale B, Lowe N, et al. The analysis of 51 genes in DSM-IV combined type attention deficit hyperactivity disorder: association signals in DRD4, DAT1 and 16 other genes. Mol Psychiatry. 2006;11:934–53.

CAS  PubMed  Article  Google Scholar

89. Franke B, Neale BM, Faraone SV. Genome-wide association studies in ADHD. Hum Genet. 2009;126:13–50.

CAS  PubMed  PubMed Central  Article  Google Scholar

90. Poelmans G, Pauls DL, Buitelaar JK, Franke B. Integrated genome-wide association study findings: identification of a neurodevelopmental network for attention deficit hyperactivity disorder. Am J Psychiatry. 2011;168:365–77.

PubMed  Article  Google Scholar

91. Mooney MA, McWeeney SK, Faraone SV, Hinney A, Hebebrand J, Consortium I, et al. Pathway analysis in attention deficit hyperactivity disorder: an ensemble approach. Am J Med Genet B Neuropsychiatr Genet. 2016;171:815–26.

CAS  PubMed  PubMed Central  Article  Google Scholar

92. Aebi M, van Donkelaar MM, Poelmans G, Buitelaar JK, Sonuga-Barke EJ, Stringaris A, et al. Gene-set and multivariate genome-wide association analysis of oppositional defiant behavior subtypes in attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2016;171:573–88.

CAS  PubMed  Article  Google Scholar

93. Groenman AP, Greven CU, van Donkelaar MM, Schellekens A, van Hulzen KJ, Rommelse N, et al. Dopamine and serotonin genetic risk scores predicting substance and nicotine use in attention deficit/hyperactivity disorder. Addict Biol. 2016;21:915–23.

CAS  PubMed  Article  Google Scholar

94. Demontis D, Walters RK, Martin J, Mattheisen M, Als TD, Agerbo E, et al. Discovery of the first genome-wide significant risk loci for ADHD. Submitted for publication, bioRxiv. 2017;14558:1–43

Google Scholar

95. Lai CS, Gerrelli D, Monaco AP, Fisher SE, Copp AJ. FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder. Brain. 2003;126(Pt 11):2455–62.

PubMed  Article  Google Scholar

96. Enard W, Gehre S, Hammerschmidt K, Holter SM, Blass T, Somel M, et al. A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell . 2009;137:961–71.

CAS  PubMed  Article  Google Scholar

97. Faraone SV, Perlis RH, Doyle AE, Smoller JW, Goralnick JJ, Holmgren MA, et al. Molecular genetics of attention-deficit/hyperactivity disorder. Biol Psychiatry. 2005;57:1313–23.

CAS  Article  PubMed  Google Scholar

98. Lee SH, Ripke S, Neale BM, Faraone SV, Purcell SM, Perlis RH, et al. Genetic relationship between five psychiatric disorders estimated from genome-wide SNPs. Nat Genet. 2013;45:984–94.

CAS  PubMed  Article  Google Scholar

99. Martin J, Hamshere ML, Stergiakouli E, O’Donovan MC, Thapar A. Genetic risk for attention-deficit/hyperactivity disorder contributes to neurodevelopmental traits in the general population. Biol Psychiatry. 2014;76:664–71.

PubMed  PubMed Central  Article  Google Scholar

100. Bralten J, van Hulzen KJ, Martens MB, Galesloot TE, Arias Vasquez A, Kiemeney LA, et al. Autism spectrum disorders and autistic traits share genetics and biology. Mol Psychiatry. 2017;00:1–8.

101. Groen-Blokhuis MM, Middeldorp CM, Kan KJ, Abdellaoui A, van Beijsterveldt CE, Ehli EA, et al. Attention-deficit/hyperactivity disorder polygenic risk scores predict attention problems in a population-based sample of children. J Am Acad Child Adolesc Psychiatry. 2014;53:1123–9 e6.

PubMed  Article  Google Scholar

102. Stergiakouli E, Martin J, Hamshere ML, Langley K, Evans DM, St Pourcain B, et al. Shared genetic influences between attention-deficit/hyperactivity disorder (ADHD) traits in children and clinical ADHD. J Am Acad Child Adolesc Psychiatry. 2015;54:322–7.

PubMed  PubMed Central  Article  Google Scholar

103. Faraone S, Biederman J, Garcia Jetton J, Tsuang M. Attention deficit disorder and conduct disorder: longitudinal evidence for a familial subtype. Psychol Med. 1997;27:291–300.

CAS  PubMed  Article  Google Scholar

104. Faraone SV, Biederman J, Keenan K, Tsuang MT. Separation of DSM-III attention deficit disorder and conduct disorder: evidence from a family-genetic study of American child psychiatric patients. Psychol Med. 1991;21:109–21.

CAS  PubMed  Article  Google Scholar

105. Thapar A, Harrington R, McGuffin P. Examining the comorbidity of ADHD-related behaviours and conduct problems using a twin study design. Br J Psychiatry. 2001;179:224–9.

CAS  PubMed  Article  Google Scholar

106. Hamshere ML, Langley K, Martin J, Agha SS, Stergiakouli E, Anney RJ, et al. High loading of polygenic risk for ADHD in children with comorbid aggression. Am J Psychiatry. 2013;170:909–16.

PubMed  PubMed Central  Article  Google Scholar

107. Larsson H, Ryden E, Boman M, Langstrom N, Lichtenstein P, Landen M. Risk of bipolar disorder and schizophrenia in relatives of people with attention-deficit hyperactivity disorder. Br J Psychiatry. 2013;203:103–6.

PubMed  PubMed Central  Article  Google Scholar

108. Hamshere ML, Stergiakouli E, Langley K, Martin J, Holmans P, Kent L, et al. Shared polygenic contribution between childhood attention-deficit hyperactivity disorder and adult schizophrenia. Br J Psychiatry. 2013;203:107–11.

PubMed  PubMed Central  Article  Google Scholar

109. Faraone SV, Biederman J, Wozniak J. Examining the comorbidity between attention deficit hyperactivity disorder and bipolar I disorder: a meta-analysis of family genetic studies. Am J Psychiatry. 2012;169:1256–66.

PubMed  Article  Google Scholar

110. van Hulzen KJE, Scholz CJ, Franke B, Ripke S, Klein M, McQuillin A, et al. Genetic overlap between attention-deficit/hyperactivity disorder and bipolar disorder: evidence from genome-wide association study meta-analysis. Biol Psychiatry. 2017;82:634–41.

PubMed  Article  Google Scholar

111. Hart AB, Gamazon ER, Engelhardt BE, Sklar P, Kahler AK, Hultman CM, et al. Genetic variation associated with euphorigenic effects of d-amphetamine is associated with diminished risk for schizophrenia and attention deficit hyperactivity disorder. Proc Natl Acad Sci USA. 2014;111:5968–73.

CAS  PubMed  Article  PubMed Central  Google Scholar

112. Cortese S, Moreira-Maia CR, St Fleur D, Morcillo-Penalver C, Rohde LA, Faraone SV. Association between ADHD and obesity: a systematic review and meta-analysis. Am J Psychiatry. 2015;173:34–43. appiajp201515020266

PubMed  Article  Google Scholar

113. Barkley RA, Murphy KR, Fischer M. ADHD in Adults: What the Science Says. New York, NY: Guilford Press; 2010.

Google Scholar

114. Dalsgaard S, Ostergaard SD, Leckman JF, Mortensen PB, Pedersen MG. Mortality in children, adolescents, and adults with attention deficit hyperactivity disorder: a nationwide cohort study. Lancet. 2015;385:2190–6.

PubMed  Article  Google Scholar

115. Lo-Castro A, D’Agati E, Curatolo P. ADHD and genetic syndromes. Brain Dev. 2010;33:456–61.

PubMed  Article  Google Scholar

116. de Silva MG, Elliott K, Dahl HH, Fitzpatrick E, Wilcox S, Delatycki M, et al. Disruption of a novel member of a sodium/hydrogen exchanger family and DOCK3 is associated with an attention deficit hyperactivity disorder-like phenotype. J Med Genet. 2003;40:733–40.

PubMed  PubMed Central  Article  Google Scholar

117. Zhang-James Y, Middleton FA, Sagvolden T, Faraone SV. Differential expression of SLC9A9 and interacting molecules in the hippocampus of rat models for attention deficit/hyperactivity disorder. Dev Neurosci. 2012;34:218–27.

CAS  PubMed  Article  Google Scholar

118. Zhang-James Y, Dasbanerjee T, Sagvolden T, Middleton FA, Faraone SV. SLC9A9 mutations, gene expression, and protein−protein interactions in rat models of attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2011;156:835–43.

CAS  PubMed Central  Article  Google Scholar

119. Patak J, Hess JL, Zhang-James Y, Glatt SJ, Faraone SV. SLC9A9 Co-expression modules in autism-associated brain regions. Autism Res. 2016;10:414–29

Google Scholar

120. Patak J, Zhang-James Y, Faraone SV. Endosomal system genetics and autism spectrum disorders: a literature review. Neurosci Biobehav Rev. 2016;65:95–112.

CAS  PubMed  PubMed Central  Article  Google Scholar

121. Demontis D, Lescai F, Borglum A, Glerup S, Ostergaard SD, Mors O, et al. Whole-exome sequencing reveals increased burden of rare functional and disruptive variants in candidate risk genes in individuals with persistent attention-deficit/hyperactivity disorder. J Am Acad Child Adolesc Psychiatry. 2016;55:521–3.

Article  PubMed  Google Scholar

122. Elia J, Glessner JT, Wang K, Takahashi N, Shtir CJ, Hadley D, et al. Genome-wide copy number variation study associates metabotropic glutamate receptor gene networks with attention deficit hyperactivity disorder. Nat Genet. 2012;44:78–84.

CAS  Article  Google Scholar

123. Jarick I, Volckmar AL, Putter C, Pechlivanis S, Nguyen TT, Dauvermann MR, et al. Genome-wide analysis of rare copy number variations reveals PARK2 as a candidate gene for attention-deficit/hyperactivity disorder. Mol Psychiatry. 2012;19:115–21.

PubMed  PubMed Central  Article  CAS  Google Scholar

124. Lesch KP, Selch S, Renner TJ, Jacob C, Nguyen TT, Hahn T, et al. Genome-wide copy number variation analysis in attention-deficit/hyperactivity disorder: association with neuropeptide Y gene dosage in an extended pedigree. Mol Psychiatry. 2011;16:491–503.

CAS  PubMed  Article  Google Scholar

125. Lionel AC, Crosbie J, Barbosa N, Goodale T, Thiruvahindrapuram B, Rickaby J, et al. Rare copy number variation discovery and cross-disorder comparisons identify risk genes for ADHD. Sci Transl Med. 2011;3:95ra75.

CAS  PubMed  Article  Google Scholar

126. Ramos-Quiroga JA, Sanchez-Mora C, Casas M, Garcia-Martinez I, Bosch R, Nogueira M, et al. Genome-wide copy number variation analysis in adult attention-deficit and hyperactivity disorder. J Psychiatr Res. 2014;49:60–7.

PubMed  Article  Google Scholar

127. Williams NM, Franke B, Mick E, Anney RJ, Freitag CM, Gill M, et al. Genome-wide analysis of copy number variants in attention deficit/hyperactivity disorder confirms the role of rare variants and implicates duplications at 15q13.3. Am J Psychiatry. 2012;169:195–204.

PubMed  PubMed Central  Article  Google Scholar

128. Thapar A, Martin J, Mick E, Arias Vasquez A, Langley K, Scherer SW, et al. Psychiatric gene discoveries shape evidence on ADHD’s biology. Mol Psychiatry. 2015;21:1202–7.

PubMed  PubMed Central  Article  CAS  Google Scholar

129. Garcia-Martinez I, Sanchez-Mora C, Soler Artigas M, Rovira P, Pagerols M, Corrales M, et al. Gene-wide association study reveals RNF122 ubiquitin ligase as a novel susceptibility gene for attention deficit hyperactivity disorder. Sci Rep. 2017;7:5407.

PubMed  PubMed Central  Article  CAS  Google Scholar

130. Valbonesi S, Magri C, Traversa M, Faraone SV, Cattaneo A, Milanesi E, et al. Copy number variants in attention-deficit hyperactive disorder: identification of the 15q13 deletion and its functional role. Psychiatr Genet. 2015;25:59–70.

CAS  PubMed  Article  Google Scholar

131. Kollins SH, McClernon FJ, Fuemmeler BF. Association between smoking and attention-deficit/hyperactivity disorder symptoms in a population-based sample of young adults. Arch Gen Psychiatry. 2005;62:1142–7.

PubMed  Article  Google Scholar

132. Levin ED, Conners CK, Silva D, Hinton SC, Meck WH, March J, et al. Transdermal nicotine effects on attention. Psychopharmacol (Berl). 1998;140:135–41.

CAS  Article  Google Scholar

133. Cross-Disorder Group of the Psychiatric Genomics Consortium. Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis. Lancet. 2013;381:1371–9.

PubMed Central  Article  CAS  Google Scholar

134. Buske-Kirschbaum A, Schmitt J, Plessow F, Romanos M, Weidinger S, Roessner V. Psychoendocrine and psychoneuroimmunological mechanisms in the comorbidity of atopic eczema and attention deficit/hyperactivity disorder. Psychoneuroendocrinology. 2013;38:12–23.

CAS  PubMed  Article  Google Scholar

135. Joseph N, Zhang-James Y, Perl A, Faraone SV. Oxidative stress and attention deficit hyperactivity disorder: a meta-analysis. J Atten Disord. 2015;19:915–24.

PubMed  Article  Google Scholar

136. Chen MH, Su TP, Chen YS, Hsu JW, Huang KL, Chang WH, et al. Comorbidity of allergic and autoimmune diseases among patients with ADHD: a nationwide population-based study. J Atten Disord. 2013;21:219-27.

Article  Google Scholar

137. Akutagava-Martins GC, Salatino-Oliveira A, Genro JP, Contini V, Polanczyk G, Zeni C, et al. Glutamatergic copy number variants and their role in attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2014;165:502–9.

CAS  Article  Google Scholar

138. Mick E, Neale B, Middleton FA, McGough JJ, Faraone SV. Genome-wide association study of response to methylphenidate in 187 children with attention-deficit/hyperactivity disorder. Am J Med Genet B Neuropsychiatr Genet. 2008;147B:1412–8.

CAS  PubMed  Article  Google Scholar

139. Russell VA. Dopamine hypofunction possibly results from a defect in glutamate-stimulated release of dopamine in the nucleus accumbens shell of a rat model for attention deficit hyperactivity disorder--the spontaneously hypertensive rat. Neurosci Biobehav Rev. 2003;27:671–82.

CAS  PubMed  Article  Google Scholar

140. Jensen V, Rinholm JE, Johansen TJ, Medin T, Storm-Mathisen J, Sagvolden T, et al. N-methyl-d-aspartate receptor subunit dysfunction at hippocampal glutamatergic synapses in an animal model of attention-deficit/hyperactivity disorder. Neuroscience. 2009;158:353–64.

CAS  PubMed  Article  Google Scholar

141. Carrey NJ, MacMaster FP, Gaudet L, Schmidt MH. Striatal creatine and glutamate/glutamine in attention-deficit/hyperactivity disorder. J Child Adolesc Psychopharmacol. 2007;17:11–7.

PubMed  Article  Google Scholar

142. Kim DS, Burt AA, Ranchalis JE, Wilmot B, Smith JD, Patterson KE, et al. Sequencing of sporadic Attention-Deficit Hyperactivity Disorder (ADHD) identifies novel and potentially pathogenic de novo variants and excludes overlap with genes associated with autism spectrum disorder. Am J Med Genet B Neuropsychiatr Genet. 2017;174:381–9.

CAS  PubMed  PubMed Central  Article  Google Scholar

143. Zayats T, Jacobsen KK, Kleppe R, Jacob CP, Kittel-Schneider S, Ribases M, et al. Exome chip analyses in adult attention deficit hyperactivity disorder. Transl Psychiatry. 2016;6:e923.

CAS  PubMed  PubMed Central  Article  Google Scholar

144. Hawi Z, Cummins TD, Tong J, Arcos-Burgos M, Zhao Q, Matthews N, et al. Rare DNA variants in the brain-derived neurotrophic factor gene increase risk for attention-deficit hyperactivity disorder: a next-generation sequencing study. Mol Psychiatry. 2017;22:580–4.

CAS  PubMed  Article  Google Scholar

145. Trzepacz PT, Williams DW, Feldman PD, Wrishko RE, Witcher JW, Buitelaar JK. CYP2D6 metabolizer status and atomoxetine dosing in children and adolescents with ADHD. Eur Neuropsychopharmacol. 2008;18:79–86.

CAS  PubMed  Article  Google Scholar

146. Froehlich TE, McGough JJ, Stein MA. Progress and promise of attention-deficit hyperactivity disorder pharmacogenetics. CNS Drugs. 2010;24:99–117.

CAS  PubMed  PubMed Central  Article  Google Scholar

147. Myer NM, Boland JR, Faraone SV. Pharmacogenetics predictors of methylphenidate efficacy in childhood ADHD. Mol Psychiatry. 2017;00:1–8.

148. Walton E, Pingault JB, Cecil CA, Gaunt TR, Relton CL, Mill J, et al. Epigenetic profiling of ADHD symptoms trajectories: a prospective, methylome-wide study. Mol Psychiatry. 2016;22:250–6.

PubMed  PubMed Central  Article  CAS  Google Scholar

149. Gusev A, Lee SH, Trynka G, Finucane H, Vilhjalmsson BJ, Xu H, et al. Partitioning heritability of regulatory and cell-type-specific variants across 11 common diseases. Am J Hum Genet. 2014;95:535–52.

CAS  PubMed  PubMed Central  Article  Google Scholar

150. Boyle EA, Li YI, Pritchard JK. An expanded view of complex traits: from polygenic to omnigenic. Cell . 2017;169:1177–86.

CAS  PubMed  PubMed Central  Article  Google Scholar

151. Hess JL, Akutagava-Martins GC, Patak JD, Glatt SJ, Faraone SV. Why is there selective subcortical vulnerability in ADHD? Clues from postmortem brain gene expression data. Molecular Psychiatry, 00:1–7.

152. Sanislow CA, Pine DS, Quinn KJ, Kozak MJ, Garvey MA, Heinssen RK, et al. Developing constructs for psychopathology research: research domain criteria. J Abnorm Psychol. 2010;119:631–9.

PubMed  Article  Google Scholar

153. Chang Z, Lichtenstein P, Asherson PJ, Larsson H. Developmental twin study of attention problems: high heritabilities throughout development. JAMA Psychiatry. 2013;70:311–8.

PubMed  Article  Google Scholar

154. Polderman TJ, Huizink AC, Verhulst FC, van Beijsterveldt CE, Boomsma DI, Bartels M. A genetic study on attention problems and academic skills: results of a longitudinal study in twins. J Can Acad Child Adolesc Psychiatry. 2011;20:22–34.

PubMed  PubMed Central  Google Scholar

155. Greven CU, Rijsdijk FV, Plomin R. A twin study of ADHD symptoms in early adolescence: hyperactivity-impulsivity and inattentiveness show substantial genetic overlap but also genetic specificity. J Abnorm Child Psychol. 2011;39:265–75.

PubMed  Article  Google Scholar

156. Ilott N, Saudino KJ, Wood A, Asherson P. A genetic study of ADHD and activity level in infancy. Genes Brain Behav. 2010;9:296–304.

CAS  PubMed  Article  Google Scholar

157. Bornovalova MA, Hicks BM, Iacono WG, McGue M. Familial transmission and heritability of childhood disruptive disorders. Am J Psychiatry. 2010;167:1066–74.

PubMed  PubMed Central  Article  Google Scholar

158. Tuvblad C, Zheng M, Raine A, Baker LA. A common genetic factor explains the covariation among ADHD ODD and CD symptoms in 9−10 year old boys and girls. J Abnorm Child Psychol. 2009;37:153–67.

PubMed  PubMed Central  Article  Google Scholar

159. Polderman TJ, Derks EM, Hudziak JJ, Verhulst FC, Posthuma D, Boomsma DI. Across the continuum of attention skills: a twin study of the SWAN ADHD rating scale. J Child Psychol Psychiatry. 2007;48:1080–7.

PubMed  Article  Google Scholar

160. Derks EM, Dolan CV, Hudziak JJ, Neale MC, Boomsma DI. Assessment and etiology of attention deficit hyperactivity disorder and oppositional defiant disorder in boys and girls. Behav Genet. 2007;37:559–66.

PubMed  PubMed Central  Article  Google Scholar

161. Hudziak JJ, Derks EM, Althoff RR, Rettew DC, Boomsma DI. The genetic and environmental contributions to attention deficit hyperactivity disorder as measured by the conners’ rating scales—revised. Am J Psychiatry. 2005;162:1614–20.

PubMed  Article  Google Scholar

162. Dick DM, Viken RJ, Kaprio J, Pulkkinen L, Rose RJ. Understanding the covariation among childhood externalizing symptoms: genetic and environmental influences on conduct disorder, attention deficit hyperactivity disorder, and oppositional defiant disorder symptoms. J Abnorm Child Psychol. 2005;33:219–29.

PubMed  Article  Google Scholar

163. Larsson JO, Larsson H, Lichtenstein P. Genetic and environmental contributions to stability and change of ADHD symptoms between 8 and 13 years of age: a longitudinal twin study. J Am Acad Child Adolesc Psychiatry. 2004;43:1265–75.

Article  Google Scholar

164. Martin N, Scourfield J, McGuffin P. Observer effects and heritability of childhood attention-deficit hyperactivity disorder symptoms. Br J Psychiatry. 2002;180:260–5.

PubMed  Article  Google Scholar

165. Kuntsi J, Stevenson J. Psychological mechanisms in hyperactivity: II The role of genetic factors. J Child Psychol Psychiatry. 2001;42:211–9.

CAS  PubMed  Article  Google Scholar

166. Coolidge FL, Thede LL, Young SE. Heritability and the comorbidity of attention deficit hyperactivity disorder with behavioral disorders and executive function deficits: a preliminary investigation. Dev Neuropsychol. 2000;17:273–87.

CAS  PubMed  Article  Google Scholar

167. Schmitz S, Fulker DW, Mrazek DA. Problem behavior in early and middle childhood: an initial behavior genetic analysis. J Child Psychol Psychiatry. 1995;36:1443–58.

CAS  PubMed  Article  Google Scholar

168. Stevenson J. Evidence for a genetic etiology in hyperactivity in children. Behav Genet. 1992;22:337–44.

CAS  PubMed  Article  Google Scholar

169. Edelbrock C, Rende R, Plomin R, Thompson LA. A twin study of competence and problem behavior in childhood and early adolescence. J Child Psychol Psychiat. 1995;36:775–85.

CAS  PubMed  Article  Google Scholar

170. Gillis JJ, Gilger JW, Pennington BF, DeFries JC. Attention deficit disorder in reading-disabled twins: evidence for a genetic etiology. J Abnorm Child Psychol. 1992;20:303–15.

CAS  PubMed  Article  Google Scholar

171. Goodman R. Genetic factors in hyperactivity: account for about half of the explainable variance. Br Med J. 1989;298:1407–8.

CAS  Article  Google Scholar

172. Willerman L. Activity level and hyperactivity in twins. Child Dev. 1973;44:288–93.

CAS  PubMed  Article  Google Scholar

173. Matheny AP Jr., Brown AM. Activity, motor coordination and attention: individual differences in twins. Percept Mot Skills. 1971;32:151–8.

PubMed  Article  Google Scholar

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Acknowledgements

Dr. Faraone is supported by the K.G. Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Bergen, Norway, the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 602805, the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 667302 and NIMH grant 5R01MH101519. Dr. Larsson is supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 667302 and the Swedish Research Council (2013-2280; 2014-3831).

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1. Departments of Psychiatry and of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA

Stephen V. Faraone

2. School of Medical Sciences, Örebro University, Örebro, Sweden

Henrik Larsson

3. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden

Henrik Larsson

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1. Stephen V. Faraone

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In the past year, SVF received income, potential income, travel expenses continuing education support and/or research support from Lundbeck, KenPharm, Rhodes, Arbor, Ironshore, Shire, Akili Interactive Labs, CogCubed, Alcobra, VAYA, Sunovion, Genomind, and NeuroLifeSciences. With his institution, he has US patent US20130217707 A1 for the use of sodium−hydrogen exchange inhibitors in the treatment of ADHD. HL has served as a speaker for Eli-Lilly and Shire and has received research grants from Shire.

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Faraone, S.V., Larsson, H. Genetics of attention deficit hyperactivity disorder. Mol Psychiatry 24, 562–575 (2019). https://doi.org/10.1038/s41380-018-0070-0

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Received: 29 August 2017

Revised: 31 January 2018

Accepted: 19 February 2018

Published: 11 June 2018

Issue Date: April 2019

DOI: https://doi.org/10.1038/s41380-018-0070-0

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Genetics of attention deficit hyperactivity disorder - Molecular Psychiatry

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Molecular Psychiatry - Genetics of attention deficit hyperactivity disorder

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How does temperature effect the evolution of neuronal gene expression in common fruit flies.

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Abstract

Neuronal activity is temperature sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet, we do not know enough about the evolutionary response of neuronal phenotypes in new temperature environments. Here, we use long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes. Here, we demonstrate a direct relationship between thermal selective pressure and the evolution of neuronally expressed molecular and behavioral phenotypes. Several essential neuronal genes evolve lower expression at high temperatures and higher expression at low temperatures, with dopaminergic neurons standing out by displaying the most consistent expression change across independent replicates. We functionally validate the link between evolved gene expression and behavioral changes by pharmacological intervention in the experimentally evolved D. simulans populations as well as by genetically triggered expression changes of key genes in D. melanogaster. As natural temperature clines confirm our results for Drosophila and Anopheles populations, we conclude that neuronal dopamine evolution is a key factor for temperature adaptation.

Introduction

In the light of the ongoing climatic change, the mechanisms for temperature adaptation are of particular interest ( Hoffmann and Sgro 2011; Lister and Garcia 2018). Many essential biological processes are affected by temperature and neuronal activity is one of them. Changes in neuronal phenotypes within a single generation (neuronal plasticity) induced by thermal stress are well documented ( Milburn et al. 1995; Sigrist et al. 2003; Zhong and Wu 2004; Peng et al. 2007; Yang and Zheng 2014). However, whether such changes are adaptive and favored by natural selection across multiple generations is unclear. For instance, temperature stress increases arborization (synaptic connectivity with other neurons) and excitability of neurons in individual Drosophila flies ( Sigrist et al. 2003). Constitutive neuronal hyperconnectivity and hyperexcitability is, however, deleterious ( Dong et al. 2009; Hillary and Grafman 2017). It is therefore not clear whether temperature stress persisting for multiple generations triggers genetic change for neuronal traits. The central role of neuronal tissue in regulating essential organismal processes ( Sokolowski 2001) and its temperature sensitivity, makes this an important research question-particularly in the light of the most recent global climate change.

Although adaptation to local environments over multiple generations is common, adaptive phenotypes are difficult to study in the wild. To avoid the lack of replication and the challenge of many, poorly characterized confounding factors in natural populations, experimental evolution provides a powerful alternative to study adaptive phenotypes ( Kawecki et al. 2012; Schlötterer et al. 2015; Barret et al. 2019). Here, we explored the phenotypic consequences of adaptation to novel temperatures using experimental evolution and a combination of RNA-seq with genetic and pharmacological manipulations to characterize and validate the putatively selected phenotypes. We identified a concordant response to selection in neuronally expressed dopamine signaling genes, with lower expression of genes for dopamine synthesis, recycling, and transsynaptic signaling, consistent with the changes in expression of core synaptic signaling genes. This key neuronal adaptation resulted in enhanced locomotor activity in hot environments, a phenotype that could be confirmed by pharmacological intervention in the evolved populations, and replicated by genetic manipulation of gene expression in D. melanogaster. Our study provides direct evidence that environmental temperature drives evolutionary changes specific to neuronal tissues.

Results

Temperature Induces Evolution of Neuronal Gene Expression

We studied temperature adaptation in replicate D. simulans populations. For half a day, we shifted the environmental temperature from physiologically benign and preferred 18-20 °C ( Liu et al. 2003; Kwon et al. 2008) to stressfully high 28 °C or low 10 °C (12/12 h circadian cycle, hot regime: 28/18 °C; cold regime: 20/10 °C; supplementary fig. S1 and , Supplementary Material online; David et al. 2004). In flies, prolonged exposure to the extreme thermal limits (28 °C or 10 °C) is detrimental for viability and reproduction ( Hoffmann 2010). This imposed a specific and controlled thermal selective pressure for up to 100 generations in highly replicated, outbred D. simulans populations. We tracked the phenotypic outcome of thermal selection in ten hot-evolved, five cold-evolved, and five ancestral populations. Prior to measuring concordant gene expression changes across replicates with RNA-seq, we maintained all replicates (ancestral, hot-, and cold-evolved) for two generations at the high-temperature regime. The phenotypic analysis focused on the high-temperature regime because we were interested in adaptation to the most stressful environment. This avoided an arbitrary temperature choice, because the ancestral temperature regime is unknown. This design also assured that observed differences in gene expression are caused by genetic differences and not by environmental perturbations or evolution of plasticity.

Relative to the ancestral populations, 2,813 genes (29% of all expressed genes, N = 9,283) changed their expression significantly (FDR < 0.05) in the evolved population. The statistical test determining the significance of change relies on the consistency across replicates and significant gene expression changes indicate that selection rather than drift is the most likely driver of the gene expression differences ( supplementary fig. S2, Supplementary Material online). About 15% of these genes changed expression in the same direction in hot- and cold-evolved flies ( supplementary fig. S3 and tables S1 and S2, Supplementary Material online). We attributed this to adaptation to culturing conditions unrelated to temperature and did not consider these genes further. About 11% (300) of the genes changed gene expression only in hot-evolved replicates but remained unchanged in cold-evolved replicates (hot adaptive gene expression, fig. 1A and supplementary fig. S3 and , Supplementary Material online). Most genes (74%) changed their expression in the hot- and cold-evolved populations in opposite direction (compared with the ancestral state). We interpret this antagonistic evolution in the two temperature regimes as strong evidence for selection driving widespread temperature-specific gene expression (temperature adaptive gene expression, fig. 1A and supplementary fig. S3 and , Supplementary Material online).

As a first step toward functional characterization of temperature adaptation, we pooled all genes that were either significantly up- or downregulated in hot-evolved replicates. Upregulated genes were only enriched for several (23) sperm motility-related gene ontology (GO) categories, whereas downregulated genes showed enrichment for >200 GO categories ( supplementary table S3, Supplementary Material online). Of special interest were the categories related to neuronal function and development, because the same set of genes was also enriched for expression in head and brain tissues ( supplementary table S4, Supplementary Material online), specific neuronal anatomical categories ( supplementary table S5, Supplementary Material online), and neuronal activity-regulated genes, ARGs (Fisher's exact test P = 0.005, fig. 1B and supplementary table S6, Supplementary Material online; Chen et al. 2016). We further characterized the evolution of the neuronal system by asking whether genes with a well-documented role in neuronal activity, presynaptic active zone, and synaptic vesicle release consistently evolved their expression in opposite directions in hot- and cold-evolved flies ( fig. 1D). Consistent with the enrichment analyses, populations in hot temperature regimes evolved significantly lower expression of genes with functionally characterized roles in neuronal activity and synaptic transmission ( Harris and Littleton 2015; Gramates et al. 2017), whereas cold-evolved population showed higher expression of the same group of genes. ( fig. 1D and E; supplementary table S2, Supplementary Material online). Thus, surprisingly, independent D. simulans populations consistently responded to opposing temperature regimes by evolving neuronal gene expression in different directions.

Neuronal Evolution Is Consistent with Evolution of Dopaminergic Signaling

Similar to several single-cell RNA-seq studies ( Croset et al. 2018; Davie et al. 2018), we looked for changes in the expression of vesicular neurotransmitter-specific transporters to identify the type of neuronal populations underlying the observed evolved gene expression changes. Only the vesicular monoamine transporter ( VMAT) evolved an expression change related to temperature (FDR = 0.05), restricting the candidates to monoaminergic neurons using dopamine, serotonin, octopamine, and tyramine as neurotransmitters ( fig. 1C). Although gene expression in serotonergic, octopaminergic, and tyraminergic neurons did not have consistent patterns of temperature-specific, evolved expression changes ( supplementary fig. S4, Supplementary Material online), dopaminergic neurons (DANs), and their synaptic targets, the mushroom body neurons (MBNs) ("Calyx of adult mushroom body," FDR = 0.05; supplementary table S5, Supplementary Material online) showed clear anatomy ontology enrichments. This implicates DANs and MBNs as the likely cellular neuronal substrates for the evolved gene expression changes. Consequently, we specifically tested the evolved expression of genes dedicated to dopamine biosynthesis and its neuronal recycling. The key dopamine synthesis genes ( ple and ddc, encoding for tyrosine hydroxylase and dopa-decarboxylase) and the dopamine transporter gene ( DAT) were among the downregulated genes in hot-evolved flies (FDR = 0.01, FDR = 0.01, FDR = 9.12×10 −05; fig. 1C and supplementary table S2, Supplementary Material online). One of the most pronounced evolutionary changes (FDR = 1.07×10 −06, fig. 1D supplementary table S1, Supplementary Material online) was seen for Hr38, a nuclear hormone receptor ( Palanker et al. 2006). Among a range of other regulatory functions, Hr38 encodes a transcription factor that regulates the expression of ple and ddc ( Sekine et al. 2011) and is known to be the most strongly induced ARG in DANs and other neurons ( Sekine et al. 2011; Chen et al. 2016; Croset et al. 2018). The dopamine receptor genes ( Dop1R1, Dop2R, Dop1R2, and DopEcR) are primarily expressed in the MBNs and showed the same evolutionary trend. Due to their low expression level, all but DopEcR ( P value = 0.12, FDR = 0.26) could not be tested for statistical significance ( supplementary fig. S4, Supplementary Material online). Although many types of neurons may have contributed to thermal adaptation, our data suggest that the strongest and most consistent evolutionary signal is related to dopamine biology.

Cuticular Dopamine Traits Do Not Respond to Thermal Selection

Dopamine also serves an essential role in other processes unrelated to neuronal signaling, such as cuticular pigmentation ( Takahashi 2013). Cuticular pigmentation is related to temperature ( Watt 1969) and UV-radiation ( Bastide et al. 2014), both mediated by exposure to sunlight. Even though sunlight was absent in our experiment, we studied pigmentation to rule out that selection is operating on other dopamine-related processes in the body, rather than in the brain. We found a significant, but mild decrease of whole-body melanin content in hot-evolved adults compared with the ancestral population ( fig. 2A and supplementary table S7, Supplementary Material online). The same trend was seen for evolved flies propagated at constant 25 °C, rather than a temperature regime fluctuating between 18 and 28 °C ( supplementary fig. S5 and , Supplementary Material online). Interestingly, despite the lack of sunlight in the experiment, this result is consistent with the studies of pigmentation along latitudinal and altitudinal clines ( Bastide et al. 2014). On the other hand, focusing on thoracic cuticle pigmentation rather than melanin content, no significant differences between ancestral and evolved flies were detected ( supplementary fig. S6 and , Supplementary Material online). The lack of temperature-specific expression changes of genes used for converting dopamine to melanin (i.e., genes supporting a nonbrain-specific function) is fully consistent with the similar pigmentation of evolved and ancestral populations ( supplementary figs. S7 and S8 and , Supplementary Material online). In contrast, genes expressed only in neurons ( DAT, VMAT, and dopamine receptors) evolved temperature-specific changes in expression. Hence, we conclude that thermal selection operates primarily on a neuronal dopamine trait rather than on pigmentation.

Evolved Differences in Locomotor Activity at High Temperatures

The evolution of neuronal dopamine signaling can be demonstrated by the analysis of behavioral traits which are controlled by dopamine, such as spontaneous locomotor activity ( Van Swinderen and Andretic 2011). Consistent with altered dopamine levels, we observed a significantly higher spontaneous locomotor activity of hot-evolved flies compared with ancestral and cold-evolved flies at 28 °C ( fig. 2B and supplementary table S8, Supplementary Material online). The difference was exacerbated in a sensitized assay based on a temperature-ramping regime, with extreme temperatures up to 35 °C (see Materials and Methods; fig. 2C and D and supplementary table S8, Supplementary Material online). Because locomotion is not exclusively regulated by dopamine, this assay did not provide us with sufficient evidence to confirm the change in activity was due to evolution of dopamine levels. However, changes in locomotion specific to dopamine levels can be assayed across diverse genetic backgrounds with the help of targeted pharmacological perturbation of dopamine.

Pharmacological Intervention Confirms the Evolution of Dopamine Signaling

We tested if the evolved gene expression changes altered synaptic dopamine levels in hot- and cold-evolved populations in opposite directions. We modulated synaptic dopamine levels in adult flies by pharmacological intervention with cocaine and 3IY. Due to short-lived effects of cocaine ( McClung and Hirsh 1998), we tested the impact of the drugs using startle response, rather than spontaneous locomotor activity. 3IY acts antagonistically to tyrosine, the rate-limiting substrate for dopamine synthesis enzyme tyrosine hydroxylase ( ple), whereas cocaine binds specifically to dopamine transporter ( DAT) and blocks the reuptake of dopamine from the synaptic cleft, effectively increasing dopamine neurotransmission ( fig. 2A). Both drugs induce a disbalance of dopamine levels in the DANs and synaptic cleft which results in impaired locomotor ability. Importantly, the impact of both drugs is expected to be different for flies with high and low dopamine levels ( fig. 3A). Cocaine is expected to have a stronger effect on flies with high gene expression dedicated to neuronal dopamine signaling (cold-evolved), whereas the effect of 3IY should be more pronounced in flies with low dopamine levels (hot-evolved: fig. 3A). As expected, in the startle response assay, hot-evolved replicates were highly sensitive to 3IY, whereas cold-evolved replicates were highly sensitive to cocaine ( fig. 3B and supplementary table S9, Supplementary Material online). Hence, pharmacological intervention confirms that hot- and cold-evolved replicates evolved distinct dopamine levels, as suggested by our gene expression analysis. Furthermore, we show that distinct levels of synaptic dopamine lead to pronounced behavioral differences in the hot- versus cold-evolved populations.

Knockdown of Neuronal Dopamine Genes at High Temperatures Recapitulates Hot-Evolved Phenotype

We aimed to genetically recapitulate the evolved gene expression changes in D. simulans populations evolving in hot temperature using its closely related sister species, D. melanogaster, which allows diverse genetic interventions and behavioral analysis. As genetic null mutations in tyrosine hydroxylase and dopa-decarboxylase encoded by pale ( ple) and ddc are lethal, we used tissue-specific genetic constructs to evaluate the neuronal function of ple. The homozygous lethal mutant ple ² can be rescued with either a wild-type rescue construct, or a construct specifically lacking neuronal ple transcripts (brain dopamine-deficient strain; Cichewicz et al. 2017). We measured the spontaneous locomotor activity of both ple ² rescued strains in the same temperature-sensitized assays that were used for the evolved D. simulans populations. Contrary to our expectation, the locomotor activity of flies deficient for dopamine in the brain was severely impaired at all temperatures when compared with the ple ² wild-type rescue ( supplementary fig. S12, Supplementary Material online). However, the magnitude of dopamine level perturbation determines the direction of the effects ( Yamamoto and Seto 2014). Full knockouts generate severe phenotypes that are unlikely to be maintained in natural populations. In line with this, we find mild, yet significant changes in gene expression in the evolved populations. Although the phenotype of brain dopamine-deficient strain is consistent with previously reported knockouts at benign temperatures, it also suggests that simple gene knockouts, which could be easily achieved in D. simulans, will not be informative for the evolutionary processes in our experiment. Hence, we turned to GAL4-RNAi toolkit to reduce ple and ddc expression. We used several RNAi lines with different knockdown efficiencies and genetic backgrounds. Similar to ple ² mutants, strong knockdowns using short hairpin RNAi ( Ni et al. 2011) for ple and ddc with elav-GAL4 drivers also resulted in flies with strongly impaired locomotor activity independent of temperature ( supplementary fig. S12, Supplementary Material online). This reaffirmed our expectation that the natural, evolved D. simulans behavioral phenotypes are unlikely to rely on strong depletion of neuronal dopamine synthesis. Conversely, neuronal knockdowns using less efficient, long hairpins that have been reported to achieve a milder knockdown ( Ni et al. 2011), were more suitable for testing the effect of adaptive, rather than fully disruptive changes in gene expression. Knockdown against ple and ddc in conjunction with elav-GAL4 driving expression of long hairpin probes yielded adult flies with increased activity in stressfully hot temperatures, essentially recapitulating the observed behavior of hot-evolved D. simulans ( fig. 4A and B). We expanded our RNAi knockdown test to genes dedicated to neuronal dopamine synthesis, recycling, and transsynaptic signaling in all postmitotic neurons ( elav-GAL4; fig. 4A and B and supplementary fig. S9, Supplementary Material online), DANs ( ddc-GAL4; fig. 4B and supplementary fig. S10, Supplementary Material online), and MBNs (247-GAL4, mef2; fig. 4B and supplementary fig. S11, Supplementary Material online). The effect of gene knockdown on locomotor activity was evaluated using the sensitized temperature-ramping assay. Two different genetic backgrounds were used for each knockdown GAL4>RNAi combination to avoid false-positive results (in total 54 independent types of GAL4>RNAi crosses and nine types of GAL4>control crosses; fig. 4B and supplementary tables S10 and S11, Supplementary Material online). Knockdown of neuronal dopamine genes significantly increased locomotor activity at high temperatures compared with control knockdown crosses ( fig. 4A and B and supplementary figs. S9-S11 and , Supplementary Material online), again emulating the evolved neuronal phenotypes we find in D. simulans hot-evolved populations.

Targeting RNAi in DANs and MBNs separately revealed the temperature-dependent phenotype of ple, ddc, VMAT, DAT in DANs, and Dop1R1 in MBNs ( fig. 4B). We found no changes in activity after knocking down DAN-expressed genes ( ple, ddc, Vmat) in nondopaminergic, MBNs, which confirmed that the GAL4 drivers appropriately induce RNAi expression in the different tissues. More importantly, this experiment demonstrates that a genetic assay targeting a specific neuronal circuit faithfully recovers the same behavioral phenotype in D. melanogaster that was observed in thermally adapted D. simulans. Remarkably, we also show that the same set of genes is contributing to the phenotype in both species.

Beyond this, we also find the same behavioral phenotype in RNAi knockdowns of dopamine receptors in DANs ( fig. 4B). This suggests potential autoregulatory roles for Dop1R1, Dop2R, and DopEcR in DANs. Consistent with this hypothesis, dopamine receptor expression in DANs has previously been observed in scRNA-seq ( Croset et al. 2018). Furthermore, RNAi against Dop2R in DANs, indicated an autoregulatory function in anesthesia-resistant memory formation ( Scholz-Kornehl and Schwärzel 2016). We propose that dopamine autoregulation in DANs could be an interesting topic for future studies.

We also used RNAi to demonstrate that genes for synaptic dopamine recycling ( VMAT and DAT) are not required for dopamine synthesis in the thoracic cuticle. pannier-GAL4-driven RNAi of ple and ddc expression during thoracic cuticle formation showed that dopamine synthesis is required for thoracic cuticular pigmentation ( fig. 4C). However, pannier-GAL4-driven RNAi of VMAT and DAT did not affect pigmentation ( fig. 4C), providing further support that neuronal dopamine signaling evolved in our experiments, but pigmentation did not.

Overall, the neuronal RNAi knockdowns in D. melanogaster demonstrated that experimentally reducing the dopamine-dedicated gene expression by RNAi elicits temperature-dependent locomotor behavior that closely mimics the evolved phenotype in hot-evolved D. simulans, where expression of these genes was lowered through natural laboratory selection. It is, however, noteworthy to address a couple of potential caveats. First, based on previous reports and guidelines provided by stock centers that supplied the RNAi lines, we assumed the long hairpin RNAi achieves a milder gene expression knockdown than short hairpin RNAi or null mutants. We used two independent RNAi hairpins for each knockdown in different genetic backgrounds and in combination with four GAL4 drivers (three neuronal, one cuticular) to ensure the expression of RNAi is appropriate and results are valid. We note, however, that we did not explicitly test the efficiency of each knockdown using RT-PCR. Second, unlike the assay in which we directly pharmacologically perturbed dopamine levels in experimentally evolved populations ( fig. 3), the RNAi-mediated knockdown exercise in D. melanogaster only provides indirect evidence for the temperature-dependent evolution of dopamine signaling. Nevertheless, this recapitulation of the hot-evolved phenotype clearly upholds the general if not adaptive role of dopamine signaling at high temperature.

Dopamine Signaling Is Clinal in Natural Populations

Our results indicate that the evolution of dopamine signaling is a very strong response to ambient temperature in experimental D. simulans populations. With hot- and cold-evolved populations responding in opposite direction, this evolutionary response cannot be explained by adaptation to laboratory conditions or genetic drift. But how do these results from controlled, laboratory evolution relate to natural populations? The contrast of local populations along a geographic cline is frequently employed to identify clinal polymorphisms, a pattern that can be related to temperature adaptation. By mining clinal polymorphism data for geographic clines of natural Drosophila and Anopheles populations ( Cheng et al. 2012; Fabian et al. 2012; Reinhardt et al. 2014; Machado et al. 2016; Sedghifar et al. 2016), we identified latitudinal allele frequency differences for genes encoding neuronally expressed dopamine receptors ( Dop1R1, Dop2R, DopEcR) and transporters ( DAT and VMAT) ( supplementary table S12, Supplementary Material online). Although not explicitly noted in the original publications due to the vast number of clinal loci, this observation strongly argues that the evolution of temperature-dependent neuronal dopamine signaling is not restricted to laboratory experiments, but most likely reflects a general response of insects to thermal selection in nature.

Discussion

The impact of temperature changes on the biology of a fly is highly complex. Using a combination of experimental evolution, gene expression analysis, pharmacological intervention, and RNAi-mediated gene knockdown, we revealed that neuronal dopamine signaling evolved in response to a novel, hot temperature environment. However, it is still unclear how this altered signaling improves fitness in a new temperature regime beyond enhancing locomotor ability. The correlation of locomotor activity with mating success could be a simple explanation behind evolution of dopamine-dependent locomotion ( Cobb et al. 1987). Furthermore, with different individual DAN circuits being the basis of neuronal dopamine pleiotropy, many different behaviors including sleep, arousal, aggression, courtship, and valence to sensory perceptions in learning and memory ( Yamamoto and Seto 2014) are affected and may also be potential important targets of selection.

Somehow counterintuitive was the observation that a reduced expression of dopamine-related genes results in a higher locomotor activity at elevated temperatures. We propose that a DAN hyperactivity of ancestral flies in hot temperatures may exhaust the signaling system, resulting in a decreased locomotor ability and consequently reduced fitness. In support of this hypothesis, we found an enrichment for downregulation of neuronal activity-regulated genes and downregulation of neuronal K⁺ ion channels in hot-evolved populations. Moreover, consistent with a fitness cost of elevated dopamine signaling at high temperatures, low-dopamine D. melanogaster prefers higher temperatures and vice versa ( Ueno et al. 2012). Based on our results, it is not possible to decide whether the altered dopamine signaling is the outcome of regulatory evolution or reflects allometric changes in neuron composition in the brain. Single-cell sequencing of hot-evolved flies may answer this question.

Using two opposing temperature regimes, this study distinguished between laboratory adaptation, where gene expression changed in the same direction and temperature-driven changes. Because the environment in natural laboratory selection is considerably less complex than in natural populations, it is easier to interpret the observed evolutionary changes. Only in combination with results from the natural laboratory selection, the signal of latitudinal clinal dopamine signaling could be recognized in the blurry signal of many clinal variants that would have otherwise remained unrecognized. Nevertheless, it is also important to recognize that our study only identified one mechanism of temperature adaptation, which resulted in a clear signal. We anticipate that other adaptive mechanisms can be detected in natural laboratory selection starting from populations with a different genetic composition. With several such studies, it should be possible to disentangle adaptive processes in natural populations that can be related to temperature from those arising from other evolutionary forces.

Materials and Methods

Evolution Experiment

About 202 isofemale lines were combined 15 times to seed each of the 15 replicated populations. Of those, ten replicates were maintained at the high-temperature regime 12 h/12 h 28/18 °C and 12 h/12 h light/dark circadian cycle for 100 generations (hot-evolved replicates; as described in Barghi et al. [2019]), whereas five replicates (cold-evolved replicates) were maintained at the low-temperature regime 12 h/12 h 20/10 °C and 12 h/12 h light/dark for over 50 generations ( supplementary fig. S1, Supplementary Material online). Population size was maintained each generation between 1,000 and 1,250 adult individuals per replicate. The isofemale lines used to seed the selection lines were maintained in the isofemale state (overlapping generations at ∼18 °C and 12 h/12 light/dark) to enable the reconstitution of the ancestral population at the end of the evolution experiments ( Nouhaud et al. 2016).

RNA-Seq Common Garden Experiment

RNA-seq common garden was performed after 103 generations at high-temperature regime (or 52 generations in low-temperature regime, supplementary fig. S1, Supplementary Material online). We pooled ∼180 founder isofemale lines (22 were lost over the years) five times to seed five replicates of the reconstituted ancestral population. Both evolved and ancestral replicates (ten hot-evolved, five cold-evolved, and five ancestral replicates) were reared for at least two generations in the same conditions (28/18 °C, 12 h/12 h light/dark cycle, 400 eggs per bottle) to account for maternal effects. Whole fifty 5-day-old males were flash-frozen in liquid nitrogen and stored at −80 °C until RNA extraction.

RNA-Extraction and Library Preparation

Flies were homogenized in Qiazol (Qiagen, Hilden, Germany) and total RNA was extracted from the homogenate using the Qiagen RNeasy Universal Plus Mini kit with DNase I treatment. Barcoded and stranded mRNA libraries were prepared using the TruSeq-stranded mRNA Library Prep Kit for Neoprep (Illumina, San Diego, CA). Neoprep runs were performed using software version 1.1.0.8 and protocol version 1.1.7.6 starting with 100 ng of total RNA. All samples were randomized across library cards, and library cards with the same lot number were used for all Neoprep runs to avoid batch effects. Barcoded libraries were quantified using the Qubit DNA HS assay kit and pooled in equimolar amounts. Reads with a length of 50 bp were sequenced on an Illumina HiSeq 2500 platform.

Differential Expression Analysis

All RNA-seq reads were trimmed based on quality (-min_quality 20) with ReadTools ( Gomez-Sanchez and Schlötterer 2018) and mapped to the D. simulans genome ( Palmieri et al. 2015) using GSNAP ( Wu and Nacu 2010) with enabled split read mapping (-N 1) and relaxed mismatch parameter (-m 0.08) to account for polymorphisms. Reads were counted based on the D. simulans annotation ( Palmieri et al. 2015) using RSubread ( Liao et al. 2013). Differential expression analysis between the evolved and ancestral replicates was performed in the EdgeR framework ( Robinson et al. 2010) after filtering 30% of genes with the lowest expression. To test for differential gene expression, we modeled evolution type ("ancestral," "hot-evolved," "cold-evolved") and anchored ancestral expression as the intercept (counts ∼ 0 + evolution). Different evolution types were assigned based on criteria in supplementary table S1, Supplementary Material online. All P values were FDR corrected for multiple testing with the significance cutoff FDR < 0.05.

Enrichment Tests

GO enrichment was performed using Gorilla ( Eden et al. 2009) with all expressed genes ( N = 9,283, supplementary table S1, Supplementary Material online) as the background set. Tissue and anatomy ontology enrichments were calculated using Fisher's exact test and were based on FlyAtlas2 expression data (with tissue biased expression defined as tissue: carcass ratio >1; Leader et al. 2018) and the Flybase anatomy ontology database from FlyBase Consortium ( Gramates et al. 2017) (obtained from Victor Strelets), respectively. Where required, significance values were adjusted for multiple testing.

Locomotor Activity Assays

Spontaneous locomotor activity was measured for ancestral and evolved replicates (hot-evolved generation F140 and cold-evolved generation F70; supplementary fig. S1, Supplementary Material online) using Drosophila Activity Monitor System (DAMS, TriKinetics, USA). After two generations, at common garden conditions in the high-temperature regime (28/18 °C), activity of male flies was recorded either under the native high-temperature regime assay (28/18 °C) over 6 days or at a sensitized high-temperature-ramping assay with gradual temperature increase from 18 to 35 °C over 7 h (as indicated in fig. 2C). Maximal temperature (35 °C) was chosen as the highest temperature at which flies would not die after ramping has completed. To test for consistency across independently evolving replicates, we compared MODEL1 = β + Zμ + ε and MODEL2 = Zμ + ε fits within an ANOVA framework, where y is summed activity for each individual ( i) in each replicate ( r) of a given evolution type ( E) in a particular measurement day ( m) using lmer function in lme4 R package (fixed and random effects are indicated in upper and lower cases, also in the following models). Pairwise differences between evolved and ancestral populations were tested using Tukey's post hoc test with the TukeyHSD function in lsmeans R package ( supplementary table S7, Supplementary Material online).

Drug Sensitivity

To measure sensitivity to perturbation of dopamine signaling, we employed the startle response assay after pharmacological treatment with cocaine (Cocaine, COC-156-FB, Lipomed AG, Austria) or 3IY (3-iodo-l-tyrosine, I8250, Sigma Aldrich, USA). Flies were reared for two generations in the high-temperature regime common garden experiment ( supplementary fig. S1, Supplementary Material online). We treated 50 male flies with 2 µg of cocaine dissolved in ethanol and vaporized using a cocaine vaporizer (1-min exposure) constructed as described in McClung and Hirsh (1998). For 3IY treatment, 50 male flies were treated for 3 days with 2 mg 3IY dissolved in 2% yeast and 5% sucrose solution (soaked filter paper) or with yeast-sucrose solution only. Startle-induced locomotion was assayed using modified method from Liao et al. (2014) with finer resolution of measurement. Flies were transferred from vials with food to a 100-ml glass graduated cylinder after 6 h in the 28 °C part of the high-temperature cycle. After a short (∼30 s) acclimatization period, flies were forced to the bottom of the cylinder by firmly tapping it on the bench. Control and treated flies in each replicate were assayed at the same time. The startle response was recorded with a Samsung Galaxy S6 camera (high-definition resolution, 60 fps capture, white balance 38,000, and auto-focus) and measured as the average attained height of the 50 flies 30 s after being startled. Height was assessed for each fly using the volume marks on the cylinder. The sensitivity to chemicals was measured as the difference between startle response of the drug-treated flies and the untreated flies reared under the same conditions (28/18 °C). The interaction between evolution type and drug effect was assessed with the AIC goodness of fit of MODEL1 = β + Zμ + ε compared with reduced MODEL2 = Zμ + ε, where y is the attained height 30 s after startle response for an individual fly i of a given replicate ( r) and measurement ( m) of evolution type ( E) in a control or drug treatment ( D). The differences in sensitivity to the drug between evolution types were estimated based on the interaction between evolution type ( E) and drug ( D) ( supplementary table S8, Supplementary Material online).

Pigmentation and Melanin Content

To assay pigmentation and melanin changes, flies were reared at constant 25 °C common garden and very low egg density (150 eggs/bottle) or 28/18 °C and low egg density (400 eggs/bottle; supplementary fig. S1, Supplementary Material online). Around ten flies from each replicate in the 25 °C common garden were mounted on a cover slide submerged in 70% ethanol and thorax images were taken the same day using the same imaging parameters and illumination. Pixel color was quantified for images of ancestral and evolved replicates, as well as "yellow" (melanin-deficient) and "ebony" (highly pigmented) mutant D. simulans strains as extreme pigmentation reference points ( supplementary table S9, Supplementary Material online). Between-group analysis with subsequent leave-one-out predictive analysis and data projection ( Culhane et al. 2002) was used to quantify pigmentation change in evolved replicates. To determine relative melanin content, 200 males from 28/18 °C and 25 °C common garden (ancestral, evolved, and yellow mutants) were vacuum-dried at 80 °C for 72 h and pulverized with a porcelain mortar into fine powder (∼5-7 mg of powder per sample was obtained) and homogenates were analyzed by infrared (IR) spectroscopy. To obtain IR spectra of a thin uniform layer, 3-5 mg of homogenized powder was pressured on a diamond ATR plate using self-leveling sapphire anvil of a Cary 660 Fourier transform infrared (FTIR) spectrometer (Agilent Technologies) coupled with Golden Gate single-reflection diamond Attenuated Total Reflectance (ATR) accessory (Specac). Absorption spectra were recorded in a midinfrared region (4,000-400 cm −1) at a nominal resolution of 4 cm −1 (at RT; 23 ± 1 °C). Two replicate spectra (50 scans/spectrum) of each sample were recorded using different aliquots. Molecular vibrations observed in IR spectra were assigned using electronic spectral libraries (NIST, Agilent Technologies, IMB Jena Image Library of Biological Macromolecules) and published spectral data (see supplementary methods, Supplementary Material online). Relative melanin content was determined from the IR absorbance for determined melanin-specific wavenumbers with melanin absorption index (MAI) >1 (MAI maxima at 2,925, 2,853, 1,743, 1,153, 1,077, and 1,023 cm −1; see supplementary methods, Supplementary Material online). Full and reduced general mixed models were fitted to the melanin absorbance data (lme4 R package; MODEL1 = β + Zμ, + ε and MODEL2 y= Zμ, + ε, where y is absorbance value for each individual i, in each replicate r, measurement m, of evolution type E) to test for replicate consistency ( supplementary table S6, Supplementary Material online). The best model fit was chosen based on AIC score within an ANOVA framework. Differences between evolution types were tested using Tukey's post hoc test (lsmeans R package).

GAL4-UAS Knockdown Assays

The D. melanogaster strains used for genetic knockdown are given in supplementary table S9, Supplementary Material online. Mini- white⁺ GAL4 transgenes and white genetic backgrounds have been suggested to alter several behavioral traits in males and to affect dopamine levels ( Borycz et al. 2008; Krstic et al. 2013; Qiu and Xiao 2018). Therefore, we crossed RNAi line virgins to GAL4 line males, making the assayed F1 males exhibit wild-type eye colors for Bloomington and VDRC/KK RNAi lines, and very dark eye color for VDRC/GD lines. To avoid genetic background effect that may occur in GAL4/+ and UAS/+ controls, we crossed a UAS-mCherry RNAi line with the same genetic background as Bloomington lines to the tested GAL4 line. Similarly, we used VDRC and KK control lines (same genetic background as UAS-RNAi line but with empty landing sites for the RNAi construct) crossed to GAL4 as controls for knockdown crosses with VDRC and KK genetic backgrounds.

Flies were raised at 25 °C in three replicates for the activity assay after neuronal knockdown. Locomotion was assayed for 20-40 males per line. Flies were aged 3-6 days and assayed in a sensitized high-temperature assay (see Locomotor Activity Assays) at 25 °C during the dark part of the cycle. All crosses were assayed in parallel, including controls, in multiple measurements. We used Dunnet adjusted pairwise comparison between knockdown crosses and appropriate controls to account for comparisons of each knockdown cross with the same control. Comparisons were performed using mean activity estimated after fitting a model = β + Zμ + ε, where y is summed activity measured at a temperature above a threshold (see fig. 3A) for individual fly i from GAL4xUAS-RNAi line cross L, replicate r, and measurement m ( supplementary table S2, Supplementary Material online).

A single cross was produced for the cuticular knockdown with pnr-GAL4. Flies were mounted and imaged following the same protocol as for the pigmentation assay. Due to the severity of the effects on pigmentation, it was possible to assess cuticle discoloration visually by assigning a binary value ("changed" or "unchanged").

Supplementary Material

Supplementary data are available at Molecular Biology and Evolution online.

Acknowledgments

We thank the Birman, Knoblich, and Keleman Labs, and VDRC and BDSC for D. melanogaster lines; the FlyBase Consortium for the anatomy ontology file; Miljenko Jakšić, Ivan Tomić, Marko Jakšić, and Jay Hirsh for the advice and equipment needed to construct the "fly crack pipe"; Rupert Mazzucco and Marlies Dolezal for advice on the statistical analysis; and Graham Tebb, Andy Clark, and Yasir Ahmed-Braimah for comments on the article. This study was funded by European Research Council (ERC) grant ArchAdapt, Austrian Science Fund (FWF) grant FWF W1225-B20 , and Marie Skłodowska-Curie Actions.

All raw RNA-seq data are uploaded to ENA database (accession number PRJEB35504) and available upon publication. All raw data and full code used to produce results (raw RNA-seq count tables, raw locomotor activity data, startle response data, pigmentation images, and raw IR spectra) will be uploaded and available for download on DataDryad upon publication.

References

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Neuronal Function and Dopamine Signaling Evolve at High Temperature in Drosophila

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Neuronal activity is temperature sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet, we do not know enough about the evolutionary response of neuronal phenotypes in new temperature environments. Here, we use long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes.

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Designer baby's are already here and are the future of genetics.

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Designer babies aren’t futuristic. They’re already here.

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Are we designing inequality into our genes?

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Pre-natal testing for genetic differences--comparing cultural ideas

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Were Jaguar Child Sculpture

With the discovery of the earliest identified case of Down syndrome ever, it furthers the view that Down syndrome has always been a part of us.

During the COVID-19 pandemic, researchers published findings of an ancient tomb on the Western Coast of Ireland. Prior to the Irish tomb, other teams of researchers had speculated based on various factors on what was the earliest case of Down syndrome.

Prior discoveries of Down syndrome

The picture at the top of this post is of a sculpture from the Olmec civilization from Latin America. The Olmecs existed between 1,500 BC and 300 AD. Based on this and several other figures, with their distinctive facial features and, for some, the flexibility of the poses, scholars theorized that not only was Down syndrome present in Olmec culture, but that it was venerated. The Olmecs worshiped the Jaguar and many of these figures are considered depictions of Were Jaguars, children spawned from the deified Jaguar. According to these scholars, the Olmecs considered Down syndrome a condition closer to being god-like.

One of the most viewed posts on this website is about a portrait from the Renaissance that is considered the earliest depiction of Down syndrome in western civilization. Similar to the Olmec depictions, those with Down syndrome are portrayed as cherubs and shepherds at the adoration of the birth of Jesus Christ.

In doing research for this post, I learned of a far earlier suspected case of Down syndrome. Archaelogists analyzed the remains from a Native American burial ground on Santa Rosa Island, part of the Channel Islands off the coast of California. Though their analysis was not definitive, their examination of the remains led them to conclude the individual likely had Down syndrome. The remains dated to 5,200 BC. An article from teh Journal of Contemporary Anthropology details these cases and all previous known cases extensively and is available at this link.

The oldest known diagnosed case of Down syndrome

The report on the Irish Tomb contains the oldest, most definitive diagnosis of Down syndrome.

The report itself is behind a paywall and requires a subscription. The focus of the report was not on the remains of a child with Down syndrome. Rather, the anthropologists were analyzing the burial tomb for the hierarchy of this ancient civilization.

Fortunately, NPR chose to feature the earliest known diagnosis. And “diagnosis” is correctly used in this instance. Unlike the theorizing based on physical depictions of Olmec sculptures or analysis of skeletal remains of Native Americans, these researchers extracted DNA from the skeletal remains and actually diagnosed a child having Down syndrome … 7,500 years ago, circa 5,500 BC!

To put in context how ancient that is: it is over 5 millennia before the Roman Empire; it is over 2 and a half millennia from the establishment of the first Jewish Temple; it coincides with the first known use of the wheel.

Though sadly, the child is estimated to have died at just six months old, the researchers note something of comfort. The site was a monumental tomb, with many children and adults buried there. And, this child, too, was included in the tomb.

Has human civilization evolved in its care for those with Down syndrome?

The comprehensive Journal of Contemporary Anthropology article has a thought-provoking conclusion after reviewing all then-known instances of Down syndrome in ancient human history:

On the basis of both material culture and skeletal remains exhibiting Down syndrome like phenotypes, and basic logic, it is likely that Down syndrome has been present in mankind for as long as chromosome 21 has existed.

(citations omitted). Beyond Down syndrome likely being a part of our human history for all of time, the Olmec sculptures, the Renaissance portrait, and the Irish tomb further demonstrate that, at least in those cultures, Down syndrome was included, even venerated and adored.

However, for every positive treatment of Down syndrome, the human record has off-setting negative examples. In many cultures, it was commonplace to leave disabled members of the tribe abandoned, to die of exposure or be killed by the animals. Within the lifetime of some of those living with us today, we know of programs here in the United States to sterilize, warehouse, and segregate individuals with Down syndrome and other intellectual and developmental disabilities. These policies formed the beginnings of the Holocaust, with it starting with the systematic killing of individuals with intellectual disabilities.

By comparison to those negative examples, at least in first world countries, life for individuals with Down syndrome is tremendously better. Fewer are institutionalized, with the norm being welcoming the child into a loving home, most often that of the birth parents, but also the homes of foster and adoptive parents. In the social rituals of American culture, very often individuals with Down syndrome are adored, selected as prom kings and queens, and being congratulated for long years of service at their place of employment.

It is also not uncommon for individuals with Down syndrome to be depicted in pop culture movies and t.v. shows as being imbued with a greater wisdom and spirituality, making them individuals to be venerated akin to the Olmec culture sans the chimera quality of were jaguars.

Ancient acceptance

While the quality of life, on balance, has gotten better in how human society treats those with Down syndrome, there remains a lesson from the Irish tomb, the Olmec civilization, and the Native American burial ground: one of simple acceptance.

As detailed at length on this blog, the technology that allowed for the definitive diagnosis of the oldest case of Down syndrome–genetic testing–has been implemented to prevent the births of children of Down syndrome.

Obviously, such a means was not available to the ancients. Instead, nature would take its course, the mother would become pregnant, give birth, and then the ancient tribes would have to decide how to treat this new member who had more obvious needs. In the NPR report, it shares how a chemical analysis of the child’s bones show that he was breastfed, just like every other child born into that tribe. And, when he died, he joined his ancestors in the tomb.

Perhaps if our society began with this default setting of accepting these individuals that have been worshiped, adored, and included for as long as there has been human history our evolution would remain truer to our nature.

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Down syndrome has always been a part of us

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Were Jaguar Child Sculpture With the discovery of the earliest identified case of Down syndrome ever, it furthers the view that Down syndrome has always been a part of us.

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Make research benefit everyone--need for diversity in genetics research

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PROVIDENCE, R.I. [Brown University] - By using new methods for analyzing DNA data and medical records, researchers from Brown University are helping improve the understanding of complex traits that will make more discoveries relevant to non-white, non-European ancestry groups.

In a study published in the May 5 issue of the American Journal of Human Genetics, the researchers illustrated examples of the robust associations of trait determinants, or patterns of similarity, while studying 25 traits in over 600,000 individuals from seven diverse human ancestries. With these similarities, discoveries about the nature of diseases or illnesses and their responses to potential treatments become more relevant to larger groups of people - including populations that had previously been ignored or understudied.

Genome-wide association (GWA) datasets, which are commonly used by geneticists, are based on the assumption that individual genetic mutations underwrite the genetic basis of traits, explained study author Sohini Ramachandran, a professor of biology and computer science who directs both the Center for Computational Molecular Biology and the Data Science Initiative at Brown. The idea is that a discovery about those mutations will be relevant to all people across a range of diverse ancestry groups, so that if the finding is used to develop treatments for genetic conditions, it will be applicable for all people with that condition.

However, recent studies have shown that GWA results estimated from self-identified European individuals are not transferable to non-European individuals. Because of this, the insights from the datasets are largely biased toward sampling individuals with European ancestry. The statistical hypothesis underlying the GWA framework is unfairly restrictive, Ramachandran said.

Thus the researchers used a new "enrichment analysis" methodology, previously developed in a collaboration between Ramachandran and Brown assistant professor of biostatistics Lorin Crawford, to address bias and underrepresentation.

"In this paper, which involved very careful analysis of a ream of data across multiple biobanks, we show that data viewed only through a very specific GWA lens may look disparate and irreconcilable," Ramachandran said. "Yet, viewed in a more equitable way, with a more expansive methodology, it becomes biologically unified, interpretable, and, importantly, actionable."

Ramachandran's interest in the topic started when she learned about a study showing that children with acute lymphoblastic leukemia had different responses to the standard treatment regimen depending upon their ancestry group - for example, non-white children were more likely to relapse and had a worse prognosis. As an evolutionary biologist and population geneticist, Ramachandran started thinking about the increasing reliance on GWA studies in the development of "personalized" treatments for diseases and conditions.

"There wasn't a lot of discussion around the extent to which the results from these studies were going to be directly applicable to all ancestries," she said. "And based on population genetics and theory, it just seemed really unlikely that this was going to pan out in a way that was equitable."

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Researchers highlight more equitable way to analyze DNA data from understudied groups

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PROVIDENCE, R.I. [Brown University] - By using new methods for analyzing DNA data and medical records, researchers from Brown University are helping improve the understanding of complex traits that will make more discoveries relevant to non-white, non-European ancestry groups.

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What are the details behind how sex is determined in humans?Compare today with First Nation's two-spirit ideals.

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Sex Redefined: The Idea of 2 Sexes Is Overly Simplistic

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Biologists now think there is a larger spectrum than just binary female and male