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Estudo da variante rs17225178 no gene ARNT2

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Estudo da variante rs17225178 no gene ARNT2

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Este é um estudo de associação genética em todo o genoma (GWAS) que analisa a influência da variante rs17225178 no gene ARNT2 na Síndrome de Asperger.

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Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome

Abstract

Background

Autism Spectrum Conditions (ASC) are neurodevelopmental conditions characterized by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is one subgroup of ASC and differs from classic autism in that in AS there is no language or general cognitive delay. Genetic, epigenetic and environmental factors are implicated in ASC and genes involved in neural connectivity and neurodevelopment are good candidates for studying the susceptibility to ASC. The aryl-hydrocarbon receptor nuclear translocator 2 ( ARNT2) gene encodes a transcription factor involved in neurodevelopmental processes, neuronal connectivity and cellular responses to hypoxia. A mutation in this gene has been identified in individuals with ASC and single nucleotide polymorphisms (SNPs) have been nominally associated with AS and autistic traits in previous studies.

Methods

In this study, we tested 34 SNPs in ARNT2 for association with AS in 118 cases and 412 controls of Caucasian origin. P values were adjusted for multiple comparisons, and linkage disequilibrium (LD) among the SNPs analysed was calculated in our sample. Finally, SNP annotation allowed functional and structural analyses of the genetic variants in ARNT2. We tested the replicability of our result using the genome-wide association studies (GWAS) database of the Psychiatric Genomics Consortium (PGC).

Results

We report statistically significant association of rs17225178 with AS. This SNP modifies transcription factor binding sites and regions that regulate the chromatin state in neural cell lines. It is also included in a LD block in our sample, alongside other genetic variants that alter chromatin regulatory regions in neural cells.

Conclusions

These findings demonstrate that rs17225178 in the ARNT2 gene is associated with AS and support previous studies that pointed out an involvement of this gene in the predisposition to ASC.

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Simona A. Stilo & Robin M. Murray

Kevin M. Antshel & Natalie Russo

Naghmeh Kian, Noosha Samieefar & Nima Rezaei

Background

Autism Spectrum Conditions (ASC) are a group of neurodevelopmental conditions defined by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is a subtype of these conditions, where there is no delay in language or cognitive development [ 1]. The incidence of ASC in the general population is approximately 1 in 100 [ 2]. Male:female sex ratios of 9:1 and 4:1 are observed in individuals with AS [ 3] and classic autism [ 4], respectively, although the true sex ratio in AS may be closer to the sex ratio in classic autism because of under-detection of AS in females [ 5]. ASC are heritable [ 6, 7] and multifactorial conditions, where genetic, epigenetic and environmental factors are involved [ 8, 9].

ASC are neurodevelopmental in origin, and accordingly, genes involved in neural development and connectivity are good candidate genes for studying the genetics of these conditions [ 10, 11]. The aryl-hydrocarbon receptor nuclear translocator 2 ( ARNT2) gene encodes a transcription factor of the basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) family, which is mainly expressed in the kidney and central nervous system [ 12, 13]. ARNT2 dimerizes with the hypoxia-inducible factor-1α (HIF-1α) and regulates cellular responses to hypoxia [ 14]. It also dimerizes with single-minded homolog 1 (SIM1) and participates in the development of paraventricular and supraoptic nuclei in mice [ 15]. Variants in the SIM1 gene have been associated with obesity in humans and have been shown to reduce transcriptional activity of SIM1 combined with ARNT2. Interestingly, 11 individuals carrying these variants have reported atypical behaviours, such as ASC [ 16]. SIM1 controls the expression of methyl CpG binding protein 2 ( MECP2) gene, which is disrupted in individuals with Rett Syndrome who display autistic features [ 17]. Mecp2 mouse models show typical features of Rett Syndrome [ 18, 19], and knockout mice with a deletion of Mecp2 in Sim1-expressing neurons display altered social behaviour [ 20]. Heterozygous Arnt2 mutant mice lack secretory neurons in paraventricular and supraoptic nuclei, implicating the involvement of ARNT2 in neurodevelopmental processes [ 21]. Arnt2 and Sim1 are also involved in long-range axonal guidance and hence lay down the connections between the hypothalamus and the spinal cord in zebrafish [ 22]. ARNT2 is a transcription factor in mouse hippocampus [ 23] and regulates the brain-derived neurotrophic factor ( Bdnf) gene that controls neural activity and is related to several human neurodevelopmental conditions [ 24]. A recent study by Lin and colleagues proposed a role of ARNT2 in the predisposition to ASC. They tested the levels of expression of candidate genes for ASC in neural aggregates in human stem cell models (comparable to a telencephalon in the early stages of development) after heat shock and they observed an alteration of ARNT2 expression [ 25]. One study reported a missense mutation in ARNT2 in individuals with ASC [ 26] and a previous study from our laboratory identified nominal associations between single nucleotide polymorphisms (SNPs) rs3901896 and rs4778599 with AS and autistic traits, respectively [ 27]. A recent study has reported nominally significant association for SNPs in ARNT2 and SIM1 with autistic traits in a general population sample [ 28].

In the current study, we tested 34 SNPs in ARNT2 for association with AS in an independent sample, composed of 118 cases and 412 controls. We found a significant association of rs17225178 with AS after correcting for multiple comparisons and a nominal association of rs3848173 with AS. To test the replicability of this observation, we analysed genome-wide association studies (GWAS) data from the Psychiatric Genomics Consortium (PGC), which tested SNPs for association with ASC in case-control and family-based samples [ 29]. Our study provides further support for a role of ARNT2 in ASC and in particular it points out an involvement of a SNP (that is rs17225178) in the predisposition to AS.

Methods

Participants

Participants were recruited by advertisement from the volunteer database of the Autism Research Centre in Cambridge (United Kingdom). They included both individuals with an AS diagnosis and individuals who were not diagnosed with AS and did not have any close family member with a diagnosis of ASC. All individuals completed the Autism Spectrum Quotient (AQ) online [ 30] to separate cases and controls from the two extreme ends of the AQ continuum. Eighty percent of individuals with an ASC diagnosis score above 32 on the AQ [ 31]. Cases were previously diagnosed by independent clinicians in the United Kingdom for having AS using the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10) [ 32] or the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) [ 1] criteria, at recognized clinics in the United Kingdom, by trained psychiatrists or clinical psychologists. A proportion of these were diagnosed at our clinic in Cambridge (the CLASS Clinic). Individuals without an ASC diagnosis and with an AQ score below or equal to 24 were selected as controls. This ensured that individuals with high autistic traits but without a diagnosis of ASC (the so-called "Broader Autism Phenotype" (BAP) [ 33]) were not included in the study, as this can confound the analysis. The AQ measures autistic traits, has high heritability [ 34] and has a normal distribution in the general population [ 30]. Individuals in the general population show a mean AQ score of 16.4 ± 6.3, while individuals with AS have a mean AQ score of 35.8 ± 6.5 [ 31]. All participants resided in the United Kingdom when the study was conducted and reported Caucasian ancestry for at least three generations. Overall, we tested 530 individuals, among them 118 cases (74 males and 44 females) with a mean AQ score of 35.6 ± 8.9 (males mean: 35.1 ± 8.7, females mean: 36.6 ± 8.8) and 412 controls (185 males and 227 females) with a mean AQ score of 14.9 ± 5.0 (males mean: 16.0 ± 4.4, females mean: 13.9 ± 5.1) (Table 1).

Table 1 Schematic description of the participants enrolled in the current study

SNP selection and genotyping

Thirty-four SNPs in the ARNT2 gene were selected from 80704652 bp to 80889940 bp on chromosome 15 with a mean inter-SNP distance of 7 Kb (GRCH37.p10 Primary Assembly, National Center for Biotechnology Information [NCBI]). We did not have a specific hypothesis about any SNP in particular and tested multiple SNPs within the same gene to identify a possible association of the gene with a given phenotype [ 35]. All chosen SNPs had a minor allele frequency (MAF) > 0.05 in the HapMap CEU (Utah residents with northern and western European ancestry) population (HapMap genome browser, release 27) and were available in the TaqMan SNP Genotyping Assays (Applied Biosystems Inc., Foster City, CA, USA), that was used for genotyping in the current study. rs1446336, rs8034535 and rs7175825 are tag SNPs, as indicated by the HapMap genome browser (release 27). The most upstream SNP is rs1446336 (Chr 15: 80704652) and the most downstream is rs1139650 (Chr 15: 80889940) (see Table 2). Buccal samples were collected by mail from the participants and DNA was extracted and anonymized [ 36]. SNP genotyping was performed following the protocol previously reported [ 27]. Hardy-Weinberg Equilibrium (HWE) was tested in controls using Plink v1.07 [ 37] at α  = 0.05, and all selected SNPs were in HWE.

Table 2 SNPs in ARNT2 analysed in the current study

Statistical analysis

The Chi-Square statistics was applied to test association between 34 SNPs in ARNT2 and AS, using Plink v1.07. We used Bonferroni correction to adjust P values at the α threshold of significance ( α = 0.05). Since Bonferroni correction is conservative when the tests are not independent, we used the SNPSpD web interface [ 38], to account for linkage disequilibrium (LD) patterns between all SNPs in the current sample. The effective number of independent loci was 21 and the subsequent threshold of significance after Bonferroni correction was α = 0.0024. We analysed LD blocks in our sample using Haploview [ 39].

Functional annotation of significant SNPs was conducted using the following softwares: HaploReg v2 [ 40] allows analysis of non-coding genetic variants in terms of their effect on the organization of chromatin regions and regulatory motifs; F-SNP [ 41] is a tool which combines multiple databases for providing the functional roles of genetic variants; the Genetic Association Database [ 42] gives information about genetic studies which previously identified association between the SNPs we analysed and ASC; SNPnexus [ 43] is an online database which was used to define regulatory elements and conserved regions; the University of California Santa Cruz (UCSC) genome browser [ 44] permits to validate functions of the SNPs analysed.

Analysis of replication data

We had access to the summary data of the PGC database and we checked for effect sizes and P values for rs17225178 in the ASC cohort. The PGC analysed genome-wide SNPs for association with ASC in 161 cases, 526 controls, 4,788 trio cases and 4,788 trio pseudocontrols all of Caucasian ancestry [ 29]. Details of methods, ancestry of the participants and analyses for this cohort are provided in reference [ 29].

Consent and ethics

Participants gave their informed consent before participating in this study. The current study had ethics approval from the Cambridge Psychology Research Ethics Committee and the NHS Research Ethics Committee (UK) and was conducted in accordance with the principles of the Declaration of Helsinki.

Results

rs17225178 was significantly associated with AS in our sample with P value = 0.001, and rs3848173 was nominally associated with AS in our sample. Minor alleles of these two SNPs are more prevalent in cases than in controls with odds ratio (OR) > 1 and are associated with an increased risk of AS (Table 3). To replicate our association, we searched for the P value and effect direction of rs17225178 in the PGC dataset. rs17225178 was significant ( P value = 0.0121), and the effect direction was same between the two datasets (OR > 1) (Table 3).

Table 3 Results of SNP association analyses in our sample and in the PGC database

rs17225178 is included in a LD block (25 Kb) in our sample, alongside seven other SNPs (rs3848173, rs8036233, rs3901896, rs17788120, rs3848175, rs895444 and rs12591546) (Figure 1). rs17225178 is located in intron 2 of ARNT2, alongside rs3848175 and rs895444. Intron 1 includes rs8036233, rs3901896, rs3848173 and rs17788120; while rs12591546 is located in intron 3 (Figure 2).

Linkage disequilibrium blocks in the ARNT2 gene calculated in our sample (detail). Block 3 is shown. It includes eight SNPs analysed in the current study. Numbers into the squares indicate D ' values.

Schematic diagram of the ARNT2 gene. White boxes indicate introns. SNP significantly associated with AS in the current study and those in linkage disequilibrium with this genetic variant in our sample are indicated by blue lines.

Discussion

The aim of this study was to investigate the role of common genetic variants in the predisposition to AS, which is a high-functioning form of ASC. The complex genetic nature of ASC has led previous studies to examine multiple genes, mainly involved in neural development and connectivity, sex-hormones signaling and social-emotional pathways [ 10, 11, 27]. In the current study, we tested for a genetic association between 34 SNPs in ARNT2 with AS, and then replicated our significant SNP in a large ASC cohort. Our results show nominal association of rs3848173 with AS and significant association between rs17225178 and AS. We replicated the association of rs17225178 in the PGC ASC cohort, corroborating previous reports that showed an involvement of ARNT2 in ASC. This is consistent with a previous study from our laboratory which indicated nominal association of rs3901896 with AS, and of rs4778599 with autistic traits, measured using the AQ [ 27]. Recent studies have reported a mutation in the ARNT2 gene in ASC [ 26] and common variants associated with autistic traits [ 28].

ARNT2 is located on chromosome 15q24 and includes 20 exons and 19 introns (Figure 2). It spans 193.587 Kb and has eight splice variants. Four transcript variants are protein-coding: one is composed of the first 20 exons and 19 introns; two transcripts include the first 19 exons and 18 introns; one transcript includes the first 6 exons and 5 introns (UCSC genome browser). rs17225178 is located in intron 2 (Figure 2) and modifies transcription factor binding sites (TATA, GATA and TAL1 motifs) and genomic regions that regulate the chromatin state in neural cell lines (hippocampus and brain angular gyrus) (HaploReg v2). SNP annotation did not indicate the presence of miRNA binding sites or other structural variants at the level of this SNP and did not provide information about the effect of rs17225178 on protein structure and function (F-SNP, SNPnexus). We hypothesize that the presence of the risk allele of rs17225178 could influence the binding of different transcription factors or could allow the modification of chromatin states, regulating chromatin accessibility and dynamics. This genetic variant is included in a LD block in our sample, where seven other SNPs (rs8036233, rs3901896, rs3848173, rs17788120, rs3848175, rs895444 and rs12591546) that alter chromatin regulatory regions in neural cell lines are located (HaploReg v2). Two previous studies have reported nominal association of rs3901896 with AS [ 27] and autistic traits (for example, language impairment) [ 28]. This genetic variant is in LD with rs17225178 in our sample (Figure 1). An interesting next step will be to perform molecular studies to analyse possible chromatin modifications and binding of specific transcription factors at the level of regulatory regions of ARNT2, carrying the risk allele or LD region.

This is the first study that shows a statistically significant association of rs17225178 with AS and supports previous findings that indicate an involvement of ARNT2 in ASC. This is consistent with the role of ARNT2 in neural development, as is known from animal models. In particular, ARNT2 is involved in the development of oxytocinergic neurons [ 15]. The oxytocinergic system has been implicated in autism through multiple lines of evidence [ 45- 51]. This result provides further support for the atypical oxytocinergic system in ASC by implicating a gene involved in the development of oxytocinergic neurons. In our study, we find a significant association with rs17225178, which is included in a LD block, alongside rs3901896 that has been nominally associated with AS [ 27] and autistic traits [ 28]. In the previous genetic study carried out in our laboratory [ 27], we did not find association between rs17225178 and AS, as we performed SNP association analysis at the gene level and not for each SNP independently.

The limitation of this study is the small sample size, so further research is required to corroborate these results. Animal models of Rett Syndrome have established a role for Mecp2 [ 18- 20], and it will be important to further test if Arnt2 play an important role in underlying the autistic phenotype. Future work in animals should employ well-designed behavioural assays relevant to autism in Arnt2 knockout mice, such as social-preference paradigms [ 52]. In humans, larger association studies of ARNT2 SNPs with endophenotypes linked to autism will be particularly relevant.

Conclusions

In the current study, we report a statistically significant association between the genetic variant rs17225178 in ARNT2 and AS. Using the PGC ASC dataset, we were able to replicate our results in a larger cohort. The direction of effect was the same in both the cohorts. Our findings provide evidence for the role of ARNT2 in ASC and show the involvement of rs17225178 in this gene in AS, a subgroup of ASC.

Abbreviations

AQ:

Autism Spectrum Quotient

ARNT2:

aryl-hydrocarbon receptor nuclear translocator 2

AS:

Asperger Syndrome

ASC:

Autism Spectrum Conditions

BAP:

broader autism phenotype

BDNF:

brain-derived neurotrophic factor

bHLH-PAS:

basic helix-loop-helix-Per-Arnt-Sim

CEU:

Utah residents with northern and western European ancestry

DSM-IV-TR:

Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision

GWAS:

genome-wide association studies

HIF-1α:

hypoxia-inducible factor-1α

HWE:

Hardy-Weinberg Equilibrium

ICD-10:

International Statistical Classification of Diseases and Related Health Problems, Tenth Revision

LD:

linkage disequilibrium

MAF:

minor allele frequency

MECP2:

methyl CpG binding protein 2

NCBI:

National Center for Biotechnology Information

OR:

odds ratio

PGC:

Psychiatric Genomics Consortium

SIM1:

single-minded homolog 1

SNP:

single nucleotide polymorphism

UCSC:

University of California Santa Cruz

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Acknowledgments

We are grateful to Target Autism Genome, the Nancy Lurie Marks (NLM) Family Foundation, the Autism Research Trust, the Medical Research Council UK, the Max Planck Institute for Psycholinguistics and the Wellcome Trust Sanger Centre. BC was funded by the Medical Research Council UK and VW was funded by the Nehru Trust for Cambridge University and the Cambridge Commonwealth Trust. We thank Robert Plomin, Frank Dudbridge, Lindsey Kent and Ian Craig for relevant discussions, and Jon Breidbord, Allen Chan, Sylvia Lakatosova, Sally Wheelwright, Carrie Allison, Uma Mallya, Alex Politt and Leena Peltonen for support at different points of the project. The study was carried out in collaboration with the NIHR CLAHRC EoE.

Author information

Authors and Affiliations

1. Department of Psychiatry, Autism Research Centre, University of Cambridge, Douglas House, 18B Trumpington Road, Cambridge, CB2 8AH, UK

Agnese Di Napoli, Varun Warrier & Simon Baron-Cohen

2. Cambridgeshire and Peterborough NHS Foundation Trust, CLASS Clinic, Cambridge, UK

Simon Baron-Cohen

3. Centre for Integrative Neuroscience and Neurodynamics, School of Psychology and Clinical Language Sciences, University of Reading, Reading, UK

Bhismadev Chakrabarti

Corresponding authors

Correspondence to Agnese Di Napoli or Simon Baron-Cohen.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BC and SBC designed the study. ADN analysed the data. VW and BC co-supervised data analysis, and SBC received funding for the study. ADN, BC, SBC and VW wrote the paper. All authors read and approved the final manuscript.

Authors' information

ADN and VW are joint first authors; SBC and BC are joint senior authors.

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Di Napoli, A., Warrier, V., Baron-Cohen, S. et al. Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome. Molecular Autism 6, 9 (2015). https://doi.org/10.1186/s13229-015-0009-0
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Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome - Molecular Autism

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Background Autism Spectrum Conditions (ASC) are neurodevelopmental conditions characterized by difficulties in communication and social interaction, alongside unusually repetitive behaviours and narrow interests. Asperger Syndrome (AS) is one subgroup of ASC and differs from classic autism in that in AS there is no language or general cognitive delay.

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PLoS One. 2015; 10(7): e0131202.

Varun Warrier, 1 , ‡ * Bhismadev Chakrabarti, 1 , 2 , ‡ Laura Murphy, 1 Allen Chan, 1 Ian Craig, 3 Uma Mallya, 1 Silvia Lakatošová, 1 Karola Rehnstrom, 4 Leena Peltonen, 4 Sally Wheelwright, 1 Carrie Allison, 1 Simon E. Fisher, 5 , 6 and Simon Baron-Cohen 1 , 7 ,*

Peristera Paschou, Editor

Abstract

Asperger Syndrome (AS) is a neurodevelopmental condition characterized by impairments in social interaction and communication, alongside the presence of unusually repetitive, restricted interests and stereotyped behaviour. Individuals with AS have no delay in cognitive and language development. It is a subset of Autism Spectrum Conditions (ASC), which are highly heritable and has a population prevalence of approximately 1%. Few studies have investigated the genetic basis of AS. To address this gap in the literature, we performed a genome-wide pooled DNA association study to identify candidate loci in 612 individuals (294 cases and 318 controls) of Caucasian ancestry, using the Affymetrix GeneChip Human Mapping version 6.0 array. We identified 11 SNPs that had a p-value below 1x10⁻⁵. These SNPs were independently genotyped in the same sample. Three of the SNPs (rs1268055, rs7785891 and rs2782448) were nominally significant, though none remained significant after Bonferroni correction. Two of our top three SNPs (rs7785891 and rs2782448) lie in loci previously implicated in ASC. However, investigation of the three SNPs in the ASC genome-wide association dataset from the Psychiatric Genomics Consortium indicated that these three SNPs were not significantly associated with ASC. The effect sizes of the variants were modest, indicating that our study was not sufficiently powered to identify causal variants with precision.

Introduction

Asperger Syndrome (AS) is a neurodevelopmental condition and a subset of Autism Spectrum Conditions (ASC) [ 1]. Individuals with ASC have difficulties in social interaction and communication, alongside unusually repetitive and stereotyped behaviour and unusually narrow interests. In AS, language and cognitive development proceed on time. ASC is highly heritable [ 2], with monozygotic twin heritability rate estimated between 73 - 95% [ 3], and has a prevalence of approximately 1% [ 4]. ASC is characterized by high clinical and aetiological heterogeneity. Environmental, epigenetic and genetic factors have been implicated in ASC [ 3, 5- 7]. Currently, more than 660 genes are implicated in ASC ( https://gene.sfari.org/autdb/HG_Home.do), though no single gene or variant accounts for more than 1-2% of cases [ 5, 8]. Additionally, several large copy number variants that duplicate or delete multiple genes have also been identified in association with ASC [ 3, 9].

Though no common variant has been consistently associated with ASC across multiple Genome-wide Association Studies (GWAS) [ 3, 5], it is clear now that they contribute considerably to the variation in ASC [ 10, 11]. Two recent studies have identified that common inherited variation contributes to between 40 - 60% of the variance in ASC [ 10, 11]. However, despite the majority of variance attributable to common inherited variants, as explained earlier, genome-wide association studies have failed to consistently identify causative variations. One explanation for this lack of success is that genome-wide association studies in ASC may be underpowered to detect small effect sizes; the largest ASC GWAS had less than 9000 participants (cases and controls) and, although this seems large, it has been argued that much larger sample sizes are needed (in the range of tens and hundreds of thousands) to successfully identify causative variants [ 9]. An alternative view is that the inability to consistently identify causative common variants is due to the underlying genetic and phenotypic heterogeneity. At a phenotypic level, delay and difficulties in language development is an important source of heterogeneity in ASC. Language delay in individuals with ASC is associated in changes in brain volume in both total grey matter and in specific regions in the brain [ 12]. This different brain architecture points to different biological and genetic networks involved in different forms of ASC. As mentioned earlier, AS is a subset of ASC where individuals have no language delay, suggesting it may have a genetic architecture distinct from the rest of ASC.

Only a few studies have specifically investigated the genetics of AS. In one of the first such studies, we tested for associations between 216 SNPs across 68 candidate genes. We identified nominal associations between SNPs in 14 genes and AS [ 13]. In the current study, we performed a pooled DNA genome wide association in individuals with AS and controls to identify SNPs in a hypothesis-free way. DNA pooling is a rapid, efficient and economical method to identify genetic associations in various conditions [ 14]. We hypothesized that genome-wide DNA pooling would detect the differences in allele frequencies between individuals with AS and controls. SNPs whose p-values were below a pre-defined threshold were then individually genotyped in the same sample using an established approach reported in several previous studies [ 15- 17].

Methods

Participants

612 individuals were genotyped in the pooled genotyping stage. There were 294 cases (males = 254, females = 40, reflecting the male bias in AS [ 5]) and 318 controls (males = 250, females = 68). 607 of these individuals were individually genotyped. 5 (1 case and 4 controls) individuals were not genotyped at this stage due to poor DNA quality. All participants reported Caucasian ancestry for at least 2 generations. All cases were recruited from the Cambridge Autism Research Database (CARD) at www.autismresearchcentre.com, and reported that they had a clinical diagnosis of AS according to DSM IV or ICD-10 criteria. Clinical diagnostic assessment was done by independent clinicians. Control participants were recruited through advertisement and reported that they were free of psychiatric and neurological conditions. Written consent was obtained from all participants. Ethical approval was obtained from the National Health Service Research Ethics Service (NRES).

Pooled DNA Genotyping

DNA from each participant was extracted from buccal swabs and anonymized. DNA was then suspended in Tris-EDTA and quantified using PicoGreen double-stranded DNA quantification reagent (Invitrogen, USA). 100 ng of DNA from each individual was added to their respective pool. The cases were divided into 7 pools with 5 pools for males and 2 pools for females. On average, there were 42 participants in each pool, though the numbers ranged from 12 to 59. Two additional pools with 24 female cases and 44 male cases were genotyped, but were not included in the analysis or taken forward for individual genotyping due to DNA contamination. The controls were divided into 9 pools with an average of 35 participants per pool. The number of participants per control pool ranged from 14 participants to 57.

Genotyping was performed using the Affymetrix GeneChip Human Mapping version 6.0 array (Affymetrix, California, USA) according to the protocol recommended by the company. Cell intensity (.cel) files were generated using GeneChip Scanner 3000 7g. The files generated were converted into relative allele signal scores (RAS) using a custom made script (snpmap.R [ 18]).

To test for differences in allele frequencies for each SNP between the cases and the controls, independent t-tests (equal variance assumed) were performed using the mean RAS scores from the pools. In addition, Levene's test was performed to check for equality of variance. A threshold of significance was chosen a priori at p = 1 x 10 −5. This particular threshold was chosen in order to reduce the risk of false negatives due to the loss of power from DNA pooling [ 19*] and this is a threshold that is typically used in the discovery phase of GWAS studies. All SNPs were screened for quality control. The study design had approximately 38% power to detect variants with an effect of 1.3 for the given threshold of significance after taking power loss due to DNA pooling into consideration. The frequency of both the marker and the effect allele was 0.5 for the power calculation. SNPs were rejected if they had a minor allele frequency (MAF) below 0.01 in the Caucasian population according to the HapMap project, and if the coefficient of variation (calculated as SD/mean) in more than 50% of the pool was greater than 0.2. All SNPs which passed quality control and had a p-value below the threshold of significance were taken forward for individual genotyping to verify the result from the pooled association. Nominally significant SNPs in the individual genotyping stage were further investigated using summary genome-wide association data of the ASC cohort available from the Psychiatric Genomics Consortium (PGC, http://www.med.unc.edu/pgc/). The PGC analysed genome-wide SNPs using DNA from 161 cases, 526 controls, 4788 trio cases and 4788 trio pseudocontrols, all of Caucasian ancestry. A crucial difference in the PGC cohort from our study cohort is that the PGC cohort did not stratify for language delay (and hence includes cases of autistic disorder/childhood autism as well as AS). Additional details of methods, statistical analyses and participant ancestry are provided elsewhere [ 20*].

Individual Genotyping and functional annotation

Individual genotyping was performed by Geneservices UK Ltd using the Sequenom MassARRAY iPLEX platform (Sequenom, San Diego, USA). 5 (1 case and 4 controls) individuals who were genotyped at the pooled DNA analysis stage were not included in this stage due to poor DNA quality. Total genotyping rate was 97%. MAF in the genotyped sample for all the SNPs was above 0.05. Allelic association testing was performed using Plink v1.07 ( http://pngu.mgh.harvard.edu/~purcell/plink/) [ 21]. Functional annotation was performed using Haploreg v2 ( http://www.broadinstitute.org/mammals/haploreg/) [ 22] and SNPnexus ( http://www.snp-nexus.org/) [ 23].

Validation of DNA pooling and replication

To assess the accuracy of DNA pooling in predicting differences in allele frequency, we individually genotyped 12 random SNPs in all the participants. This includes 11 SNPs that did not reach the predefined threshold in the pooled association stage and one SNP, rs7785891, which did reach the threshold. Pearson's correlation coefficient between the mean RAS scores and the allele frequency was calculated at r = 0.65. This correlation is considerably higher than another study that used pooled DNA obtained from cheek swabs on the same platform, though lower than the correlation reported for DNA obtained from blood samples [ 24].

Results

In the DNA-pooling stage, 11 SNPS passed the threshold of significance and quality control. Additionally, 5 SNPs with p-values below 1 x 10 −5 failed quality control at the pooling stage ( Fig 1 ). All the 11 SNPs were individually genotyped and all the SNPs passed quality control in the individual genotyping phase. Three SNPs were nominally significant at p <0.05 in this stage (rs7785891, rs1268055, and rs2782448). None of the SNPs survived correction for multiple testing using Bonferroni correction. None of these three nominally significant SNPs from the individual genotyping stage were significant in the PGC ASC dataset. Results are summarised in Table 1 . A Q-Q plot of the results from the pooling stage is provided in Fig 2 .

Table 1

RSID

Chr

Closest Gene

P-value (pooled genotyping)

P-value (individual genotyping)

Allele 1 (minor allele)

Freq of allele 1

Allele 2 (major allele)

Freq of allele 2

Chi-Sq

Odds Ratio

P-value (PGC cohort)

Discussion

The current study used pooled DNA analysis to identify common variants associated with AS. Using pooled DNA we scanned the genome for SNPs that had a difference in allele frequencies between the case groups and the control groups. A threshold of 1 x 10 −5 was selected in the pooling stage due to the loss of power during DNA pooling and to control for false negatives. SNPs which had p-values below the pre-defined threshold were treated as candidate SNPs and genotyped individually in the same group of individuals to more accurately estimate allele frequencies. Of the 11 SNPs that crossed the threshold of significance in the pooling stage, only three remained nominally significant after the individual genotyping stage. These three SNPs were not significantly associated with ASC in a larger, more heterogeneous ASC cohort from the PGC consortium.

rs778589, the top performing SNP at the pooling stage, is an intronic SNP in DOCK4, a gene previously associated with ASC [ 25, 26]. rs2782448 is an intergenic SNP at 13q21. It is 371 kb from KLHL1 and 7.5 kb from the 3' end of RP11- 459J23.1, a LincRNA identified by the Gencode project. 13q21 has been previously implicated in both autism [ 27, 28] and Specific Language Impairment [ 29]. The third nominally significant SNP, rs1268055, is an intronic SNP in ARMC2, a gene with uncertain function in humans.

The major limitation of this study is power. First, DNA pooling retains only 68% percent of the power [ 19]. Second, even after only selecting for individuals with AS, no SNP remained significant after Bonferroni correction. This indicates that larger sample sizes are required to detect causative alleles of small effect sizes. There is a considerably high correlation between the two stages of analysis, yet of the eleven SNPs selected for individual genotyping, only three remained nominally significant at this stage. The top two associated SNPs that passed quality control in the pooled DNA analysis stage were both nominally significant at the individual genotyping stage. However, they did not remain significant after correction for multiple comparisons. Of the three nominally significant alleles, rs7785891 has an odds ratio above 1, whereas rs1268055 and rs2782448 have odds ratio below 1 (see Table 1 ). Finally, while all our participants reported Caucasian ancestry for at least two generations, population stratification can confound the results and lead to false positives [ 30]. There are currently no known methods to correct for population stratification for pooled DNA association studies, taking into account the polygenicity of the condition.

While the current study tested for association with AS, we also checked to see if the three nominally significant SNPs were significant in an ASC cohort. The direction of effect for all three SNPs was similar to the effect direction in our sample. However, none of the three SNPs were nominally significant in the PGC ASC cohort. This may be due to a) the heterogeneity of the PGC cohort compared to our study cohort, since the former did not stratify for language delay, and/or b) the design of the association study being different (a family based association study based on trios vs a population based study), which may lead to different signal-to-noise ratios. However, it needs to be highlighted that the effect sizes for the SNPs in both the samples were small. This underscores the need for larger sample sizes to effectively identify common variants.

In conclusion, we report the identification three SNPs (rs1268055, rs7785891 and rs2782448) as nominally associated with AS using a genome-wide pooled DNA association study. rs2782448 and rs1268055 lie in genetic loci previously implicated in ASC. None of the SNPs remained significant after Bonferroni correction, underscoring the need for larger sample sizes to uncover alleles with small effect sizes. This is the first genome-wide case-control association study to test common variants for association with AS.

Acknowledgments

We are grateful to Jon Breidbord, Lindsey Kent and Frank Dudbridge for help, advice, and discussions. Professor Peltonen kindly facilitated the collaboration with the Wellcome Trust Sanger Institute, but tragically passed away before the study was completed.

Funding Statement

This work was funded by grants to SB-C from the Nancy Lurie Marks Family Foundation, the Medical Research Council (MRC) UK, Target Autism Genome, and the Autism Research Trust (ART). LM and SEF were supported by the Max Planck Society. VW is funded by St. John's College, Cambridge and the Cambridge Commonwealth Trust.

Data Availability

Summary data has been provided in the manuscript. Due to ethical restrictions related to patient consent, individual-level data are available upon request to Varun Warrier ( ku.ca.mac.lhcsdem@062wv).

References

1. American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders: DSM-IV. 4th edition Washington, DC: American Psychiatric Association; 1994. [ Google Scholar]

12. Lai MC, Lombardo MV, Ecker C, Chakrabarti B, Suckling J, Bullmore ET, et al. Neuroanatomy of Individual Differences in Language in Adult Males with Autism. Cereb Cortex. 2014. [Epub ahead of print]. [ PMC free article] [ PubMed]

Articles from PLOS ONE are provided here courtesy of PLOS

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A Pooled Genome-Wide Association Study of Asperger Syndrome

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PLoS One. 2015; 10(7): e0131202. Varun Warrier, 1 , ‡ * Bhismadev Chakrabarti, 1 , 2 , ‡ Laura Murphy, 1 Allen Chan, 1 Ian Craig, 3 Uma Mallya, 1 Silvia Lakatošová, 1 Karola Rehnstrom, 4 Leena Peltonen, 4 Sally Wheelwright, 1 Carrie Allison, 1 Simon E.

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1. Dificuldade em manter o contacto visual;

2. Hipersensibilidade sensorial;

3. Défice de atenção;

4. Desenvolvimento de habilidades e talentos específicos;

5. Comportamentos repetitivos ou rotineiros (repetição constante de palavras, gestos e expressões);

6. Interesse intenso em áreas específicas;

7. Dificuldade na compreensão das próprias emoções;

8. Descoordenação motora;

9. Problemas de empatia;

10. Dificuldades na comunicação e discurso.

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Artigo sobre fatores genéticos da Síndrome de Asperger

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Artigo sobre fatores genéticos da Síndrome de Asperger

# data[].attributes.attachment_link.title
[Genetic factors in Asperger syndrome] - PubMed

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Asperger syndrome (or Aperger’s disorder) is one of the syndromes of autism spectrum disorders (ASD) (or pervasive developmental disorder). Twin and family studies have indicated that genetic factors play a significant role in the development of ASD, including “classical” autism, in a complicated fa…

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O que é a Perturbação do Espectro do Autismo (PEA)?

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O que é a Perturbação do Espectro do Autismo (PEA)?

# data[].attributes.body
A Perturbação do Espectro do Autismo (PEA) é um distúrbio de neurodesenvolvimento que afeta a comunicação, a aprendizagem, o comportamento e a interação do indivíduo com os outros.
A designação de espectro foi atribuída pela variabilidade dos sintomas, desde as formas mais leves até às formas mais graves.

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Aspetos genéticos da Síndrome de Asperger

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Aspetos genéticos da Síndrome de Asperger

# data[].attributes.headline
Como é que a Síndrome de Asperger (SA) está relacionada com a PEA?

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Como é que a Síndrome de Asperger (SA) está relacionada com a PEA?

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A Síndrome de Asperger (SA) é uma subcategoria da Perturbação do Espectro do Autismo. Esta síndrome afeta sobretudo a interação social e a comunicação. Apesar destas dificuldades, os indivíduos geralmente não apresentam nenhum atraso significativo na linguagem e mostram um nível cognitivo normal ou até mesmo acima da média. Por esta razão, é considerada uma manifestação leve de autismo.

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Da mesma forma, foram reunidos dois vídeos, onde podemos ouvir testemunhos de dois jovens diagnosticados com a síndrome.

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Para podermos obter dados suficientes que nos permitam responder ao problema, é preciso a realização de estudos. Dentro dos artigos que foram analisados, os métodos que se destacavam eram os seguintes:

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Artigo sobre o diagnóstico e o acompanhamento da síndrome

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Artigo sobre o diagnóstico e o acompanhamento da síndrome

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Abstract

Asperger's syndrome (AS), a behavioral disorder that is related to autism, is associated with abnormal social functioning and repetitive behaviors but not with a decrease in intelligence or linguistic functionality. This article reviews the clinical diagnosis of AS and discusses the comorbid disorders that may be present with AS, as well as the efficacy, safety, and tolerability of pharmacotherapies given to AS patients, as reported in preclinical and clinical studies. AS may be present with several comorbid disorders including: attention deficit hyperactivity disorder, anxiety, schizophrenia, bipolar disorder, depression, and Tourette's syndrome. The difficulty in distinguishing AS from autism results in treating the comorbid disorder symptoms, rather than treating the symptoms of AS. Accordingly, there is a great need to further understand the psychobiology of AS and its association with other disorders, which should expand the pharmacological and non-pharmacological therapeutic options and improve the quality of life for AS patients.

Keywords: Asperger's syndrome; autistic spectrum disorders; cognitive behavioral therapy; comorbid disorders; pharmacotherapy; symptoms.

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adrenergic alpha-agonists / therapeutic use

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attention deficit disorder with hyperactivity / complications

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attention deficit disorder with hyperactivity / diagnosis*

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central nervous system stimulants / therapeutic use

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Asperger's syndrome: diagnosis, comorbidity and therapy - PubMed

# data[].attributes.attachment_link.description
Asperger's syndrome (AS), a behavioral disorder that is related to autism, is associated with abnormal social functioning and repetitive behaviors but not with a decrease in intelligence or linguistic functionality. This article reviews the clinical diagnosis of AS and discusses the comorbid disorde ...

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O jovem do vídeo, Dylan Dailor, com apenas 16 anos, fez uma palestra a falar sobre a síndrome. O seu objetivo foi informar e desmistificar algumas ideias que as pessoas têm sobre o autismo.

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Empathy and Asperger’s Syndrome | Dylan Dailor | TEDxAllendaleColumbiaSchool

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Niamh McCann, com apenas 16 anos na época, apresentou esta palestra para abordar os desafios que as raparigas diagnosticadas com SA enfrentam.

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"Copy & Paste’ - Hidden Asperger’s-- Girls with Aspergers | Niamh McCann | TEDxDunLaoghaire

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Este documento contextualiza e explica o que é a Síndrome de Asperger detalhadamente.

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Sindrome de Asperger por Alexandre Fernandes

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Este é um filme francês, baseado em eventos reais, que conta a vida de Hugo, um rapaz diagnosticado com Síndrome de Asperger. O filme acompanha o personagem desde a infância até à idade adulta, retratando as dificuldades procedentes da síndrome.

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O Cérebro de Hugo: Autismo Filme Francês Completo Legendado Português Brasil Psicologia

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Como já havia sido referido anteriormente, a Síndrome de Asperger não é uma doença, pelo que, não existe tratamento. No entanto, existem diferentes tipos de acompanhamento.

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A Terapia Cognitiva Comportamental (TCC) tem como objetivo identificar padrões de comportamento e padrões de pensamento, de modo a encontrar técnicas mais adequadas para alterar essas percepções positivamente.
O profissional de saúde auxilia o indivíduo no controlo das suas emoções e dos seus comportamentos repetitivos.

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Esta terapia tem o objetivo de melhorar a comunicação do indivíduo.

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Em grupo ou individualmente, o terapeuta ensina a criança na interação com os outros e na sua expressão pessoal.

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Não existe nenhuma medicação aprovada pela FDA especificamente para o tratamento da Síndrome de Asperger. No entanto, algumas ajudam no tratamento de outros sintomas, como ansiedade.

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É uma técnica que encoraja comportamentos e a comunicação social positiva do indivíduo. O terapeuta, normalmente, usa reforço positivo para chegar aos resultados.

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Existe "cura" para a Síndrome de Asperger?

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Existe "cura" para a Síndrome de Asperger?

# data[].attributes.body
Um dos erros de abordagem a esta síndrome é a classificação desta como uma doença. Para desmistificar este equívoco, é preciso entender que a Síndrome de Asperger não é considerada uma doença. Portanto, não existe nenhuma cura, mas sim uma série de estratégias e abordagens com o objetivo de melhorar as condições de vida dos indivíduos.

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Qual é a prevalência desta síndrome na nossa sociedade?

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Qual é a prevalência desta síndrome na nossa sociedade?

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Ninguém sabe ao certo quantas pessoas diagnosticadas com a perturbação existem no mundo, no entanto, de acordo com os Centros de Controle e Prevenção de Doenças (CDC), cerca de 1% da população mundial possui algum transtorno do espectro do autismo.

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Pessoas conhecidas com a Síndrome de Asperger

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Pessoas conhecidas com a Síndrome de Asperger

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Por exemplo, a famosa ativista Greta Thunberg foi diagnosticada com a Síndrome de Asperger.

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O diagnóstico da síndrome é difícil de ser realizado, uma vez que os sintomas tem um nível acrescido de variabilidade. Por este motivo, pode acontecer que crianças sejam diagnosticadas tardiamente, quando comparada a outros espectros do autismo. Todavia, algumas dificuldades podem ser identificadas ou diagnosticadas na idade adulta.

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O diagnóstico é feito por um psicólogo, um psiquiatra ou qualquer profissional especializado na área de saúde mental. As orientações para o acompanhamento são adaptadas para cada indivíduo pelo profissional que o atende. O diagnóstico é confirmado, se o indivíduo apresentar dificuldades persistentes na interação social ou/e padrões repetitivos de comportamentos. Estes padrões, geralmente, limitam o funcionamento normal do quotidiano.

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Para iniciar a análise da natureza genética da síndrome, é necessário analisar os fatores de risco.

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O sexo masculino apresenta um maior risco de desenvolver a síndrome em comparação ao sexo feminino. Não se sabe a explicação deste fenómeno, no entanto, alguns estudos sugerem algum tipo de ligação entre níveis de testosterona com a síndrome.

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Como síndrome do X frágil, esclerose tuberosa, epilepsia e síndrome de Tourette.

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A criança ter nascido com pais de uma idade avançada é um fator de risco.

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Os fatores ambientais e genéticos têm pesos diferentes na forma como influenciam o desenvolvimento da síndrome. Como estes requerem uma explicação mais detalhada e minuciosa, vão ser abordados a seguir.

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Através das diferenças observadas entre estudos com gémeos monozigóticos foi possível concluir que o ambiente tem influência no desenvolvimento da síndrome. Todavia, estes fatores sozinhos não são suficientes.
Os fatores ambientais de maior impacto são durante a gravidez.

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Diferentes fatores ambientais durante a gravidez estão associados ao seu desenvolvimento, como, por exemplo:

1. Idade parental avançada;

2. Exposição a poluição do ar;

3. Obesidade, diabetes ou doenças do sistema imunitário;

4. Prematuridade;

5. Complicações que implicaram a falta de oxigénio no cérebro do bebé.

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Embora não tenha sido encontrado nenhum gene específico que contribuísse para o desenvolvimento da síndrome de Asperger, é comum manifestar-se em diferentes gerações familiares.

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Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M. Autism as a strongly genetic disorder: evidence from a British twin study. Psychol

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Estudos com gémeos providenciaram que existe relevância de fatores genéticos no desenvolvimento da síndrome. Neste estudo, observado na imagem, é constatado que a taxa de concordância entre gémeos monozigóticos é proeminentemente superior que em gémeos dizigóticos, o que sugere que a síndrome é resultado sobretudo de fatores genéticos.
Além disso, estes resultados entram em conformidade com o modelo multifatorial, ou seja, a combinação de fatores ambientais com genéticos, uma vez que os gémeos monozigóticos não apresentam 100% de correlação.
Por outro lado, alguns estudos Jorde et al. (1991), Pickles et al. (1995), Bailey et al. (1995) e Bolton et al. (1994) sugeriram a epistase como modelo genético mais adequado. A epistase é um fenómeno genético onde vários genes interagem entre si para formar uma única caraterística.
No entanto, modelo multifatorial é o mais aceite.

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Para além dos estudos de hereditariedade, outros mostraram que pequenas mutações em alguns genes estão associados a esta síndrome. Mais recentemente, métodos de deteção de CNVs (copy number variation) e SNVs (single nucleotide variant) em todo o genoma levaram à identificação de um grande número de variantes raras com penetrância elevada.
A presença destas mutações ou polimorfismos não garantem o desenvolvimento desta perturbação.

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Warrier V, Chakrabarti B, Murphy L, Chan A, Craig I, Mallya U, Lakatošová S, Rehnstrom K, Peltonen L, Wheelwright S, Allison C, Fisher SE, Baron-Cohen S. A Pool

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Genes associados à Perturbação do Espectro do Autismo

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Genes associados à Perturbação do Espectro do Autismo

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Há mais de 1000 genes relevantes no desenvolvimento do autismo, no entanto, 102 deles parecem ter grande relevância. Grande parte destes genes são expressos em linhas neuronais excitatórias e inibitórias, pelo que, podem influenciar as sinapses.
Os genes mais associados à PEA parecem ser expressos em várias zonas do cérebro (pré-natal e momentos iniciais do pós-natal) e afetam um grande número de células do sistema nervoso central.
Esta perturbação tem uma grande heterogeneidade genética, ou seja, pode ser determinada por mutações em mais que um gene, apesar da descoberta de algumas formas monogénicas raras.

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Genes associados apenas à Síndrome de Asperger

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Genes associados apenas à Síndrome de Asperger

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Existem diversos estudos que tentam desvendar quais os genes e as variantes que podem estar associados particularmente a esta síndrome. No entanto, não é possível especificar quais genes têm influencia no processo do seu desenvolvimento.

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O autismo parece partilhar um certo risco com outras doenças neuropsiquiátricas e com outros traços cognitivos. Todavia existem genes mais especificamente associados à PEA do que às perturbações do neurodesenvolvimento em geral.

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Através de GWAS foram identificadas certas perturbações com maior ou menor associação com a PEA. Neste estudo, por exemplo, indivíduos com PEA apresentam um número significativo de haplótipos associados à esquizofrenia.

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Este método tem como objetivo determinar que região do genoma se encontram os genes associados a dada caraterística.

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O estudo de associação genética em todo o genoma (GWAS) é o método mais comum usado para identificar genes candidatos de alguma caraterística complexa.

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Apesar de mostrarem ter importância no desenvolvimento da síndrome, os fatores ambientais são relevantes, se o indivíduo apresentar uma pré-disposição genética para esse desenvolvimento.

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Qual o papel da genética na origem da Síndrome de Asperger?

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Qual o papel da genética na origem da Síndrome de Asperger?

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O desenvolvimento desta síndrome é influenciado por fatores ambientais e genéticos. No entanto, os fatores genéticos são mais determinantes.
Através da analise de diferentes estudos, é possível afirmar também que não existe nenhum gene específico, mas sim vários genes associados à Síndrome de Asperger.

Assim, após a abordar os pontos essenciais sobre a natureza ambiental e genética da Síndrome de Asperger, podemos concluir que esta é uma síndrome multifatorial de neurodesenvolvimento com uma elevada heterogeneidade genética.

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Qual o papel da genética na origem da Síndrome de Asperger?

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Beatriz Caridade, A104981
Ana Rita Lopes, A104894
Alexandre Fernandes, A97030

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1. https://www.cuf.pt/saude-a-z/sindrome-de-asperger

2. https://www.webmd.com/brain/autism/mental-health-aspergers-syndrome

3. https://help.tellmegen.com/is-asperger-syndrome-hereditary/?lang=en

4. Di Napoli, A., Warrier, V., Baron-Cohen, S. et al. Genetic variant rs17225178 in the ARNT2 gene is associated with Asperger Syndrome. Molecular Autism 6, 9 (2015). https://doi.org/10.1186/s13229-015-0009-0

5. Bailey A, Le Couteur A, Gottesman I, Bolton P, Simonoff E, Yuzda E, Rutter M. Autism as a strongly genetic disorder: evidence from a British twin study. Psychol Med. 1995 Jan;25(1):63-77. doi: 10.1017/s0033291700028099. PMID: 7792363.

6. Warrier V, Chakrabarti B, Murphy L, Chan A, Craig I, Mallya U, Lakatošová S, Rehnstrom K, Peltonen L, Wheelwright S, Allison C, Fisher SE, Baron-Cohen S. A Pooled Genome-Wide Association Study of Asperger Syndrome. PLoS One. 2015 Jul 15;10(7):e0131202. doi: 10.1371/journal.pone.0131202. PMID: 26176695; PMCID: PMC4503355.

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Esta síndrome não é uma doença hereditária. Nem todas as doenças genéticas são hereditárias.