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1. Nombre de las especies en que se desarrolla el estudio de caso

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1. Nombre de las especies en que se desarrolla el estudio de caso

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Homo sapiens

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2. A nivel global, ¿En qué países tiene relevancia el tema tratado en su estudio de caso?

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2. A nivel global, ¿En qué países tiene relevancia el tema tratado en su estudio de caso?

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El tema de la intolerancia a la lactosa es relevante en diversos países de Europa y África, ya que en estos lugares se encuentra el mayor número de estudios realizados (Figura).

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3. ¿Creen que el tema de su estudio tiene relevancia en Ecuador? ¿Por qué?

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3. ¿Creen que el tema de su estudio tiene relevancia en Ecuador? ¿Por qué?

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Se sabe que la intolerancia a la lactosa cuenta con molestos síntomas gastrointestinales, que al no ser diagnosticados correctamente, pueden ser prolongados. De manera que informarse sobre el tema puede fomentar la realización de estas pruebas para la total confirmación de la intolerancia. Si es necesaria la eliminación de la lactosa de la dieta, implementar otro tipo de fuente de calcio, ya que la falta de calcio puede llevar a problemas como la osteoporosis. Por lo que un conocimiento sobre este tema en Ecuador podría ser importante para evitar molestias gastrointestinales como el consumo de otras fuentes de calcio para evitar enfermedades por deficiencia de calcio. Sin mencionar el hecho de la falta de estudios del tipo en el país.

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4. ¿A qué área corresponde la aplicación de su estudio de caso?

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4. ¿A qué área corresponde la aplicación de su estudio de caso?

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El área corresponde a la genética poblacional

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5. Aspectos éticos y legales del estudio de caso 

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5. Aspectos éticos y legales del estudio de caso 

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Aspectos éticos:

Se deben tomar en cuenta los siguientes aspectos:

-El valor, en cuanto a que la investigación debe buscar   mejorar la salud o el conocimiento.
- La validez científica: la investigación debe ser metodológicamente sensata, de manera que los participantes de la investigación no pierden su tiempo con investigaciones que deben repetirse.
-La selección de seres humanos o sujetos debe ser justa: los participantes en las investigaciones deben ser seleccionados en forma justa y equitativa y sin prejuicios personales o preferencias.
- Proporción favorable de riesgo/ beneficio: los riesgos a los participantes de la investigación deben ser mínimos y los beneficios potenciales deben ser aumentados, los beneficios potenciales para los individuos y los conocimientos ganados para la sociedad deben sobrepasar los riesgos.
- Consentimiento informado: los individuos deben ser informados acerca de la investigación y dar su consentimiento voluntario antes de convertirse en participantes de la investigación.
-Respeto para los seres humanos participantes: Los participantes en la investigación deben mantener protegida su privacidad, tener la opción de dejar la investigación y tener un monitoreo de su bienestar.

Aspectos legales:

En el aspecto legal se debe tomar en cuenta un consentimiento informado, reservando los derechos del individuo.
Dentro del presente estudio se debe tener en consideración que los datos e información recogida de los participantes debe ser tratada bajo el principio de confidencialidad, es así que el participante una vez aceptado el consentimiento informado, sabe que trato le darán a sus datos. Si el estudio se realiza en el territorio ecuatoriano, el uso de los datos obtenidos debe ir acorde a la manifestado en Ley Orgánica de Protección de Datos Personales, misma que tiene por objeto garantizar el derecho a la protección de datos personales, que incluye el acceso y decisión sobre la información y datos de este carácter, así como su correspondiente protección.

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ARTÍCULOS DE INVESTIGACIÓN

Diagnóstico de intolerancia a la lactosa en adultos: rendimiento comparativo de la clínica, test de hidrógeno espirado y test genético
Comparative performance of symptoms questionnaire, hydrogen test and genetic test for lactose intolerance
Antonio Rollán¹, Cecilia Vial², Soledad Quesada1,a, Karena Espinoza², Mary Hatton², Alonso Puga3,b, Gabriela Repetto²

¹ Unidad de Gastroenterología, Departamento de Medicina, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo, Santiago de Chile, Chile.

² Centro de Genética Humana, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo, Santiago de Chile, Chile.

³ Centro de Genómica y Bioinformática. Instituto de Biotecnología. Universidad Mayor, Santiago de Chile, Chile.

Background: Genetically programmed adult-type hypolactasia affects 56% of Chilean population. Ideally, diagnosis should be confirmed. Aim: To compare diagnostic yield of genetic test, hydrogen (H₂) expiratory test and a validated symptomatic structured survey (SS). Material and Methods: Patients submitted to H₂ test answered a historic (anamnestic) and current SS (after the ingestion of 25 g of lactose). A blood sample was obtained for determination of genetic polymorphisms C/T_13910, C/G_13907 and G/A_22018 by polymerase chain reaction. The gold standard for diagnosis of lactose intolerance (LI) was the agreement of at least two of three tests. Results: Sixty-one participants aged 39 ± 12 years (21 males), were studied. Anamnestic SS was diagnostic of LI in all cases (score > 7), while current SS detected LI in 27/61 (46%). H₂ test (an increase > 15 ppm after ingestion of 25 g of lactose) showed LI in 31/61 (51%). The locus C/G_13907 showed no polymorphism and locus G/A_22018 was in complete linkage disequilibrium with C/T_13910. Genotype C/C_13910, associated to hypolactasia, was present in 30/58 (52%). According to the gold-standard, 32/61 (52.5%) patients were diagnosed as LI. Sensitivity and specificity were, respectively, 79% and 69% for current SS, 93% and 93% for H2 test and 97% and 93% for the genetic test. The last two showed a positive likelihood ratio (LR) > 10 and a negative LR < 0.1, figures within the range considered clinically useful. Conclusions: Genotype C/C_13910 is responsible for hypolactasia in this population. Anamnestic report of symptoms after milk ingestion and symptoms after lactose ingestion, are not accurate enough. H₂ and genetic tests are simple and similarly accurate to diagnose lactose intolerance in adults.

Key words: Genetic testing; lactose intolerance; lactose tolerance test; questionnaires.

Muchos pacientes relacionan sus síntomas digestivos con la ingesta de lácteos. Para diagnosticar intolerancia a la lactosa (IL) debe demostrarse su mala absorción (MAL) en el intestino delgado ¹, la que habitualmente se debe a la disminución de la actividad de lactasa (lactasa phlorizin hidrolasa) en el ribete en cepillo de los enterocitos (hipolactasia). Este déficit enzimático puede ser adquirido (secundario) o genético (primario), siendo este último mucho más frecuente.

Los niños (como los recién nacidos de cualquier mamífero) expresan alta actividad de lactasa. En una proporción variable de la población, luego del destete se produce una reducción genéticamente programada en su síntesis, que determina hipolactasia del adulto ². La no persistencia de lactasa (NPL) condiciona que la lactosa alcance el colon donde, al ser metabolizada por las bacterias colónicas, puede generar dolor abdominal, diarrea y meteorismo, aunque la mayoría de los sujetos NPL son asintomáticos. La persistencia de actividad de lactasa (PL) en el adulto es una característica hereditaria dominante, adquirida evolutivamente ³. Estos conceptos se resumen en la Tabla.1 ⁴.

Tabla 1. Definiciones y métodos diagnósticos en intolerancia a lactosa

Recientemente se han identificado los polimorfismos genéticos determinantes del fenotipo PL o NPL. Tres polimorfismos de nucleótido único (SNP)(C/T_13910, G/A_22018 y C/G_13907) ubicados en regiones intrónicas del gen MCM6, localizado en la región cromosómica 2q21, regulan la expresión del gen de la lactasa (LCT) ⁵⁻⁷. La variante C/T_13910 es la principal determinante de PL/NPL en Europa y poblaciones nómades de África. El genotipo C/C_13910 determina fenotipo NPL y C/T o T/T_13910, fenotipo PL ⁶. El polimorfismo G/A_22018 se ubica en el intrón 9 del MCM6 y el alelo A se asocia a PL. Este polimorfismo se encuentra en fuerte desequilibrio de ligamiento con el anterior ⁷. Otras variantes, como el polimorfismo C/G_13907, parecen ser importantes en algunas poblaciones de África ⁸.

La frecuencia poblacional de NPL es variablenativas de América presentan las mayores tasas reportadas 9,4. En el norte de Europa alcanza 2-8% ⁹. En Chile, un estudio reciente demostró NPL (C/C _13910) en 56% de población hispana (mestiza) y en 88% de mapuches ¹⁴. La mayoría de los sujetos NPL son asintomáticos o toleran dosis diarias de lactosa equivalentes a una taza de leche (12 g) y la mayoría de los que reportan intolerancia a lácteos no tienen realmente MAL ⁹. ¹, aunque pudiera aumentar debido a la inmigracion ¹⁰. En Polonia alcanza a 30% ¹¹, en Brasil 67% (caucásicos y mulatos) ¹² y en afro-americanos en Estados Unidos de Norteamérica hasta 75% ¹³. Poblaciones

Los síntomas luego de ingerir leche admiten diversas interpretaciones. La IL (NPL) y los trastornos digestivos funcionales (TDF) comparten el mismo complejo sintomático y pueden coexistir. Comparados con población sana, la IL es más frecuente en sujetos con TDF, mientras que la frecuencia de NPL es la misma. Existe poca correlación entre los síntomas y el resultado de métodos diagnósticos objetivos ¹⁵. Los principales métodos diagnósticos para detectar IL se resumen en la Tabla.1.

El objetivo principal de este estudio fue evaluar prospectivamente el rendimiento diagnóstico de diversos polimorfismos del gen MCM6 en adultos chilenos con síntomas sugerentes de IL, correlacionarlos con MAL y con la presencia de síntomas post ingesta de leche o lactosa.

Enviados para test de H ₂ espirado post lactosa por sospecha clínica de IL. Se obtuvo una muestra de sangre y se aplicó un cuestionario de síntomas. Todos participaron voluntariamente y firmaron consentimiento informado específico. El estudio fue aprobado por el Comité de Ética de nuestra institución.

Se registró el diagnóstico y los síntomas principales. Los pacientes respondieron un cuestionario de síntomas de 5 ítems, tanto anamnéstico, recordando las molestias presentadas al ingerir leche (ESE anamnéstica) como luego de ingerir 25 g de lactosa (ESE actual). Las molestias se califican en una escala visual análoga (EVA 0-10) y el puntaje total (0-50), se obtiene sumando los 5 ítems. De acuerdo a Casellas y col, utilizando 50 g de lactosa y test espiratorio como 67% para MAL gold standard, un puntaje > 6,5 tiene sensibilidad de 75% y especificidad de ¹⁶.

Luego de 48 h de dieta baja en fibra, sin lactosa, sin tabaco y ayuno de 12 h se realizó una determinación de H ₂ y CH ₄ basal en aire espirado mediante analizador Quintron (Quintron Instrument Company, Milwaukee, WI, USA), en el Laboratorio de Fisiología Digestiva de Clinica Alemana. Después de ingerir 25 g de lactosa en 250 ml de agua, se repitieron las determinaciones cada 30 min durante 3 h. Se consideró MAL un aumento > 15 partes por millón (ppm) de H ₂ respecto al basal en cualquier determinación ¹⁷. Evaluamos además el rendimiento diagnóstico de la determinación conjunta de H ₂ y CH ₄.

Se extrajo ADN genómico de sangre periférica mediante ensayo comercial (Axygen CA, USA). Los polimorfismos se estudiaron mediante reacción en cadena de la polimerasa, seguida de digestión por enzimas de restricción y análisis de polimorfismos de longitud de fragmentos (PCR -RFLP) según protocolo descrito previamente ². Las determinaciones se realizaron en el Laboratorio de Genética de la Facultad de Medicina Clinica Alemana-Universidad del Desarrollo. El genotipo se determina a partir del tamaño de los fragmentos, analizados en gel de agarosa. Los partidores y enzimas fueron los siguientes:

Polimorfismo C/T-13910: Partidor 1: GCTGGCAATACAGATAAGATAATGGA, Partidor 2: CTGCTTTGGTTGAAGCGAAGAT, Enzima: HinfI (New England Biolabs, MA, USA).

Polimorfismo C/G-13907: Partidor 1: GGATCTCCTTTTGGACTTTCC, Partidor 2: TGGACCTAAACCAATAATGATGAA, Enzima: Hpy8I (Fermentas, MD, USA).

Polimorfismo G/A-22018: Partidor 1: TAA AAATGGGTTTTCGCCATG, Enzima: HhaI (Fermentas, MD, USA).

Las variables numéricas fueron descritas mediante promedios y desviación estándar y comparadas mediante test t de Student. Las variables categóricas se describen mediante números y porcentajes y comparadas mediante test de chi-cuadrado. Las frecuencias genotípicas observadas del locus C/T_13910 se compararon con las esperadas según el equilibrio de Hardy-Weinberg (p ² + 2pq + q ²; p = Frecuencia alelo T; q = Frecuencia alelo C). Utilizando como gold standard la concordancia de dos de tres test utilizados, calculamos sensibilidad, especificidad y razones de verosimilitud (likelihood ratio (LR)) positivas y negativas para cada uno de los test diagnósticos. Se consideró significativo p < 0,05.

Se incluyeron 61 pacientes, cuyos datos demográficos, sintomatología y patología asociada se resumen en la Tabla.2.

Tabla 2. Demografía, sintomatología y patología asociada (n = 61)

Cincuenta y ocho pacientes (95%) ingerían habitualmente leche y/o derivados, aunque 52 (85%) referían molestias digestivas luego de consumirla y 11 (18%) sólo consumían leche sin lactosa. Diecisiete pacientes (28%) reportaron familiares directos con IL.

La ESE anamnéstica y actual se resumen en la Figuras.1 y 2. El meteorismo y el borborigmo fueron los síntomas más relevantes, mientras vómitos o diarrea fueron excepcionales en la ESE actual. El puntaje total promedio en la ESE anamnéstica fue significativamente mayor que en la ESE actual (19,4 vs 9,5; p < 0,05) y todos los pacientes (menos uno que no respondió) cumplían el criterio para diagnosticar IL (puntaje > 7), mientras sólo 27 pacientes (46%) tuvieron puntaje > 7 en la ESE actual (p < 0,05) y 11 pacientes (19%) no refirieron molestia alguna. La ESE anamnéstica tuvo una sensibilidad de 93,1%, pero una especificidad de 3,5%. El rendimiento diagnóstico de la ESE actual se resume en la Tabla.4.

Figura 1. Box-plot encuesta sintomática anamnéstica (Histórica). La caja muestra la mediana (línea central) y los percentiles 25 y 75 (primer y tercer cuartil respectivamente). Las extensiones verticales ("whiskers") comprenden hasta 1,5 veces el rango intercuartil. Los círculos muestran datos fuera de este rango ("outliers").

Figura 2. Box-plot encuesta sintomática actual (post ingesta de 25 g de lactosa). La caja muestra la mediana (línea central) y los percentiles 25 y 75 (primer y tercer cuartil respectivamente). Las extensiones verticales ("whiskers") comprenden hasta 1,5 veces el rango intercuartil. Los círculos muestran datos fuera de este rango ("outliers").

Tabla 4. Rendimiento de los métodos diagnósticos para Intolerancia a lactosa*

El test de H ₂ espirado post 25 g de lactosa demostró MAL en 31 pacientes (51%). La suma de H ₂ y CH ₄ > 16 ppm o de H ₂ y 2x CH ₄ > 17 ppm ¹⁸ no mejoró el rendimiento diagnóstico en comparación con la determinación exclusiva de H ₂, (datos no mostrados). El rendimiento diagnóstico se muestra en la Tabla.4.

Evaluamos 58 pacientes (en 3 pacientes no fue posible realizar la determinación). La observadas y las esperadas de acuerdo a la ley de Hardy-Weinberg. Las frecuencias de C/C (51,7%), C/T (27,6%) y T/T (20,7%) se encuentran en equilibrio de Hardy-Weinberg en esta población (χ Figura.3 muestra un gel representativo de los diferentes genotipos. El locus C/G_13907 no mostró polimorfismo en esta población. El locus A/G_22018 mostró un completo desequilibrio de ligamiento con el locus C/T_13910. Los análisis posteriores se efectuaron considerando sólo el locus C/T_13910. Se demostró genotipo C/C_13910, en 30/58 pacientes (52%). La ² = 4,15; p = 0,12). El rendimiento diagnóstico del test genético se muestra en la Tabla.4. Tabla.3 muestra las frecuencias genotípicas

Figura 3. Polimorfismos gen MCM6 (PCR-RFLP). LC-003, LC-004 y LC-005: Identificación de tres pacientes distintos. C. ADN, C.Dig: Controles internos.

Tabla 3. Frecuencia genotípica locus MCM6 C/T13910 (n = 58)

El test espiratorio y el test genético coincidieron en 52/58 pacientes (90%). En 4/6 pacientes discordantes el diagnóstico final (determinado por el gold standard) coincidió con el test genético y en uno de los 2 restantes el test espiratorio mostró elevación significativa de CH ₄, de modo que eventualmente 5 de los 6 discordantes habrían sido correctamente diagnosticados por el test genético.

De acuerdo al gold standard, 32 pacientes (52,5%) fueron diagnosticados como IL. En los 3 pacientes en que no se contó con el test genético los otros dos tests fueron coincidentes (2 tolerantes y 1 intolerante), de modo que fueron incluidos en el análisis.

La Tabla.4 muestra el rendimiento diagnóstico global. Tanto el test espiratorio como el test genético muestran un rendimiento muy elevado y comparable. El rendimiento de la ESE actual fue menor y los LR no alcanzan los niveles requeridos para recomendar su uso clínico, aunque no es posible demostrar diferencias significativas entre los tres tests (ver IC 95% en Tabla.4), probablemente por el reducido tamaño muestral.

La frecuencia de IL en esta muestra de pacientes sintomáticos (52,5%) resultó muy similar al 56% esperado para población hispánica (mestiza) chilena ¹⁴. Si bien no puede descartarse que la población de origen tenga una menor prevalencia de NPL, el que los genotipos se encuentren en equilibrio de Hardy-Weinberg sugiere que la muestra es similar a la población original. La historia de síntomas post ingesta de leche tiene escasa utilidad para seleccionar pacientes con NPL y MAL, como ha sido repetidamente reportado en la literatura 15,18,19.

En la práctica clínica habitual los síntomas asociados a la ingesta de leche pueden atribuirse a NPL, a TDF o a una mezcla de ambos. Lo recomendable pareciera ser evaluar la respuesta a la supresión de lactosa: si los síntomas desaparecen no es clara la necesidad de una mayor precisión diagnóstica, dado el bajo riesgo de una patología orgánica significativa. Sin embargo, la supresión de lactosa es difícil de sostener en sujetos con alta apetencia por la leche y la respuesta clínica es a veces incompleta o inconsistente. La compleja relación entre síntomas y dieta en pacientes con TDF puede originar una innecesaria supresión de lácteos en una alta proporción de la población. Un reciente consenso del National Institute of Health (NIH, USA) advierte sobre el riesgo de un inadecuado consumo de calcio y vitamina D en estos pacientes ⁹. Esta advertencia parece aun más relevante en países más pobres y con alta frecuencia de NPL, sugiriendo la conveniencia de confirmar IL, al menos en los casos en que la respuesta clínica no sea bien clara, lo que implica la necesidad de validar métodos diagnósticos objetivos.

Como ya fue señalado ( Tabla.1), los métodos disponibles evalúan aspectos diferentes del problema, lo que impide contar con un gold standard objetivo. Al igual que en otros estudios similares, escogimos la concordancia entre los métodos para calificar el estado final de nuestros pacientes, lo que constituye una aproximación aceptada para evaluar el rendimiento de diversos métodos diagnósticos cuando no existe un gold standard aceptado ²¹.

La Encuesta Sintomática Estructurada constituye un instrumento atractivo por su simplicidad, bajo costo y facilidad de aplicación. Casellas ¹⁶, utilizando 50 g de lactosa y test espiratorio como gold standard, demostró sensibilidad de 75% y especificidad de 67%. Si bien nosotros utilizamos sólo 25 g de lactosa obtuvimos resultados muy similares (sensibilidad 79% y especificidad 69%). Aunque el rendimiento diagnóstico fue muy superior a la ESE anamnéstica, los LR de la ESE post lactosa actual ( Tabla.4) no alcanzan los niveles aceptados para recomendar su uso clínico (LR (+) > 10 y LR (-) < 0,1) ²¹.

El test espiratorio y el test genético tuvieron un rendimiento diagnóstico elevado y similar en este estudio. Ambos métodos muestran valores de LR (+) y (-) en rangos considerados clínicamente útiles. Si bien el test espiratorio no está bien estandarizado y existen importante diferencias en su aplicación, incluyendo la dosis de lactosa, el considerar o no el CH ₄, los puntos de corte a utilizar y además múltiples aspectos, tales como las característica de la flora intestinal, la dieta previa y el tabaco, pueden influir en su rendimiento, diversos estudios coinciden en reportar un excelente rendimiento diagnóstico, por lo que se ha constituido en el método preferido en la mayor parte de los centros ¹⁸. Un reciente consenso ha sugerido recomendaciones para estandarizar su aplicación, incluyendo considerar sólo los niveles de H ₂ y no los de CH ₄, lo que coincide con nuestros resultados ¹⁷. Las principales ventajas del test espiratorio son su naturaleza no invasiva y la ausencia de riesgos, mientras su limitación más importante es la necesidad de restricciones dietéticas previas y la duración del test (hasta 4 h) ¹⁷ .

Coincidiendo con un estudio previo en Chile ¹⁴, nuestros resultados confirman que la mutación europea (C/T _13910) es la principal determinante de PL/NPL en nuestra población. La determinación genética presenta ventajas de simplicidad (sólo requiere una muestra de ADN, sin restricción dietética alguna) y de interpretación (el resultado es claramente positivo o negativo) que la hacen atractiva para su uso clínico. Potenciales desventajas son, sin embargo, que el genotipo C/C_13910 no necesariamente implica MAL, ya que el "switch off" del gen pudiera ocurrir a edades variables y la posibilidad de que existan otros genotipos asociados al fenotipo MAL. La excelente correlación que observamos entre ambos métodos (genético y respiratorio) y el óptimo rendimiento del test en esta muestra sugieren que estas objeciones son teóricas, al menos en el adulto. En niños con genotipo C/C_13910, un estudio reciente en India mostró que la supresión de la lactasa se inicia entre los 3 y 5 años y que a los 8 años de edad, todos los niños mostraban una actividad muy reducida ²². Otro estudio en niños africanos y finlandeses sugiere que la disminución de la actividad enzimática pudiera ser más precoz en niños africanos ²³. Estos datos deben ser corroborados en otras poblaciones y en nuestro centro estamos iniciando un protocolo al respecto. La principal desventaja del método genético es su escasa disponibilidad, la necesidad de laboratorios especializados y su alto costo. La posibilidad de obtener ADN de muestra de saliva o de un frotis de mucosa oral permitiría mejorar aún más su aceptación y eventualmente promover una eventual disminución de costos.

En resumen, este estudio de pacientes con síntomas sugerentes de IL confirma que aun en poblaciones con alta frecuencia de hipolactasia el antecedente anamnéstico tiene escaso valor diagnóstico y que la cuantificación de síntomas inmediatamente después de ingerir lactosa tampoco alcanza un rendimiento diagnóstico suficiente. Confirmamos que la alta frecuencia de NPL en nuestra población se asocia al polimorfismo C/T_13910 del gen MCM6 y demostramos un alto rendimiento diagnóstico del test genético, comparable o superior al test espiratorio y con claras ventajas de administración e interpretación.

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16. Casellas F, Varela E, Aparici A, Casaus M, Rodríguez P. Development, validation, and applicability of a symptoms questionnaire for lactose malabsorption screening. Dig Dis Sci 2009; 54 (5): 1059-65. [ Links ]

17. Gasbarrini A, Corazza GR, Gasbarrini G, Montalto M, Di Stefano M, Basilisco G, et al. Methodology and indications of H2-breath testing in gastrointestinal diseases: the Rome Consensus Conference. Aliment Pharmacol Ther 2009; 29 Suppl 1:1-49. [ Links ]

21. Stengel D, Bauwens K, Sehouli J, Ekkernkamp A, Porzsolt F. A likelihood ratio approach to meta-analysis of diagnostic studies. J Med Screen 2003; 10 (1): 47-51. [ Links ]

22. Kuchay RA, Thapa BR, Mahmood A, Mahmood S. Effect of C/T -13910 cis-acting regulatory variant on expression and activity of lactase in Indian children and its implication for early genetic screening of adult-type hypolactasia. Clin Chim Acta 2011; 412 (21-22): 1924-30. [ Links ]

23. Rasinperä H, Savilahti E, Enattah NS, Kuokkanen M, Tötterman N, Lindahl H, et al. A genetic test which can be used to diagnose adult-type hypolactasia in children. Gut 2004; 53 (11): 1571-6. [ Links ]

Recibido el 24 de enero de 2012, aceptado el 23 de mayo de 2012.

Correspondencia:

Dr. Antonio Rollán

Fax: 5866032

Manquehue 1410. Piso 3, Vitacura.

E-mail: arollan@alemana.cl

Conflictos de Intereses:

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Diagnóstico de intolerancia a la lactosa en adultos: rendimiento comparativo de la clínica, test de hidrógeno espirado y test genético

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ARTÍCULOS DE INVESTIGACIÓN Diagnóstico de intolerancia a la lactosa en adultos: rendimiento comparativo de la clínica, test de hidrógeno espirado y test genético Comparative performance of symptoms questionnaire, hydrogen test and genetic test for lactose intolerance Antonio Rollán1, Cecilia Vial2, Soledad Quesada1,a, Karena Espinoza2, Mary Hatton2, Alonso Puga3,b, Gabriela Repetto2 1 Unidad de Gastroenterología, Departamento de Medicina, Facultad de Medicina Clínica Alemana-Universidad del Desarrollo, Santiago de Chile, Chile.

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Importancia de la intolerancia a la lactosa en individuos con síntomas gastrointestinales

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La intolerancia a la lactosa es un padecimiento con elevada prevalencia a nivel mundial principalmente en países hispanoamericanos, asiáticos y africa…

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Elián Alvarado
Liz Escandón
Natalia Vergara

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Situación actual de la intolerancia a la lactosa en la infancia

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FACTORES DE RIESGO Y BAJA MASA ÓSEA en la Ciudad de MÉrida

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FACTORES DE RIESGO Y BAJA MASA ÓSEA en la Ciudad de MÉrida

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Servicios Personalizados

Similares en SciELO

Revista de La Facultad de Medicina, Volumen 24 Número 2, 2001 (181-184)

FACTORES DE RIESGO Y BAJA MASA ÓSEA en la Ciudad de MÉrida

F Chakal2, B Castelli3, J Durán2 y G Medina1.

Residente de Post-grado de Ortopedia y Traumatología, Hospital Vargas de Caracas, Venezuela.

Residente de Post-grado, Hospital Vargas de Caracas, Venezuela.

Jefe del Servicio de Traumatología y Ortopedia, Hospital Vargas de Caracas, Venezuela.

RESUMEN:

El propósito de nuestro trabajo fue determinar la presencia de factores de riesgo de osteoporosis y la disminución de la masa ósea, en mujeres mayores de 49 años en la ciudad de Mérida. Se tomaron ciento sesenta y tres (163) fichas de mujeres mayores de 49 años que viven en esta ciudad, de las cuales 39 de ellas se realizaron estudios de densitometría ósea, a este grupo se le determinaron los factores de riesgo y se analizaron estadísticamente. Al realizar el análisis entre factores de riesgo y disminución de la masa ósea, ninguno de ellos representó tener significancia estadística. Dentro de los factores estudiados, la lactancia, el sedentarismo y la intolerancia a los lácteos, son los de mayor peso relativo pero no estadísticamente significativo.

Palabras Clave: Factores de riesgo, Baja masa ósea, Densitometría ósea (DE).

ABSTRACT:

The purpose of our work was to determine the presence of factors of osteoporosis risk factors and the decrease of the bone mass, in women older than 49 years in the city of Merida. They reviewed a hundred sixty three (163) women's records in which they were older than 49 years and lived in this city, in which 39 of them were carried out studies of bone densitometry, to this group were determined the risk factors and they were statiscally analyzed. When carrying out the analysis between risk factors and decrease of the bone mass, none of them had statistical significance. Among the studied factors, the nursing, the sedentarism and the intolerance to the milky products, they were those of more relative but not statistically significant.

Key Words: Risk factors, Low bone mass, Bone densitometry (BMD).

__________________

INTRODUCCIÓN

Entendiendo como osteoporosis, una enfermedad metabólica ósea caracterizada por baja masa ósea y una relación mineral/colágeno normal con deterioro de la micro- arquitectura del tejido óseo, que inducen mayor fragilidad y el consiguiente aumento del riesgo de fractura.

Expertos de la Organización Mundial de la Salud (OMS) han propuesto que las mujeres con valores de densidad ósea inferiores a más de 2,5 DE (desviaciones estándar) del valor medio del adulto joven sean consideradas osteoporóticas. Si presentan una o más fracturas por fragilidad, se las clasificaría como casos de osteoporosis grave o establecida. Las mujeres con valores entre 1 y 2,5 DE por debajo de los valores medios del adulto joven serían clasificadas como osteopénicas.

Estudios epidemiológicos retrospectivos han identificado una serie de factores que predisponen a la adquisición de un bajo pico de masa ósea y que la pérdida de esta masa sea más rápida. Dichos factores de riesgo corresponden a categorías tales como: edad o relacionados con la edad, genéticos como variables antropométricas, nutricionales como la baja ingesta de calcio, la inactividad física, hábitos tabaquitos y alcohólicos en exceso, hormonas endógenas, enfermedades crónicas, y características físicas del hueso.

Se ha señalado que la ingestión de ciertos medicamentos, la menarquia tardía, la menopausia precoz, fracturas previas, intolerancia a la lactosa, lactancia materna por períodos prolongados, la multiparidad y la caída de los dientes pudiera acelerar la pérdida de la masa ósea (Tabla 1).

Tabla 1: Factores de riesgo de osteoporosis.

Fuente: Departamento de Medicina Universidad de Hawaii.

MATERIALES Y MÉTODOS

De un trabajo referente a calcio iónico realizado por el grupo de investigaciones del metabolismo óseo (GIMO) en el período 1990-1993 se tomaron doscientas fichas de mujeres mayores de 49 años que viven en la ciudad de Mérida, Venezuela, y de ellas solo se pudieron contactar 163 vía telefónica para un estudio retrospectivo tipo corte transversal. De esas 163 mujeres, a 39 se les había realizado una determinación de masa ósea por densitometría de tipo DEXA.

A este grupo se le determinó edad cronológica, edad de aparición de la menarquia y la menopausia, estatura, paridad, lactancia, actividad física, ingesta de lácteos, hábitos tabáquicos y alcohólicos, consumo de medicamentos y fracturas. Todas las densitometrías se realizaron con un equipo NORLAND CX 36 valoradas por un solo observador (Tabla 2).

Tabla 2: Densitometría mineral ósea. Criterios de la Organización mundial de la Salud para el diagnóstico de osteoporosis.

DMO: Densidad mineral ósea; DE: desviación estándar.
* The Who Study Group. Assessment of fracture risk and its application to screening for Postmenopausal osteoporosis. Ginebra; Organización Mundial de la Salud; 1994.

RESULTADOS

Los resultados obtenidos se analizaron desde el punto de vista estadístico para determinar su significancia.

De las 163 mujeres encuestadas, el 23,9% se habían realizado una densitometría tipo DEXA (Tabla 3 y Tabla 4).

Al realizar el análisis de regresión lineal múltiple de la relación entre factores de riesgo y osteoporosis se puede concluir que ninguno de ellos son estadísticamente significativos para originar alteraciones esqueléticas tanto en cuello femoral como en columna lumbar L2-L4.

Tabla 3: Mujeres con baja masa ósea (osteopénicas)

Fuente:Formatos de recolección.

Tabla 4: Mujeres osteoporóticas.

Fuente: Formatos de recolección.

DISCUSIÓN

La osteoporosis se define como una enfermedad esquelética en la que hay reducción de la masa ósea, acompañada de alteraciones de la micro arquitectura del esqueleto, de manera que hay aumento significativo del riesgo de fractura. En la mayor parte de las circunstancias, la prevención de la osteoporosis está orientada hacia la prevención de las fracturas, en consecuencia, la prioridad es la evaluación del riesgo para intentar una intervención oportuna. Se han incriminado a un gran número de factores en la patogenia de la osteoporosis, algunos modifican claramente el comienzo, la duración o la velocidad de pérdida ósea de los individuos. Otros son bastantes raros en la población, de modo que su utilidad como factores de riesgo es modesta. Por lo tanto, se puede llevar a cabo una evaluación clínica grosera del riesgo, determinando comparativamente escasos factores de riesgo, la mayoría de los cuales pueden ser investigados durante una anamnesis adecuada. Los factores generales están representados por el sexo femenino, raza blanca o asiática, y posmenopausia (probablemente con mayor riesgo en caso de menopausia precoz quirúrgica o natural). Para los que cumplen estos criterios, los antecedentes personales de fractura antes de los 45 años o los de fractura por osteoporosis en un familiar directo, el bajo peso corporal, el consumo de cigarrillos, la ingesta alcohólica excesiva, ingesta diaria de cafeína se suman al riesgo (y cada uno confiere riesgo adicional). Factores como enfermedad crónica, sedentarismo y fármacos (esteroides, diuréticos, hormona tiroidea), se sobreagregan a estas características simples. Otros son suficientemente endémicos en la población para que casi puedan ser asumidos, hábitos nutricionales, ingesta de calcio y actividad física inadecuada. La existencia de estos factores se pueden utilizar de dos maneras, primero para sensibilizar al paciente e inducir en ellos conciencia sobre la probabilidad de padecer esta enfermedad, segundo se deben discutir con el paciente los que son pasibles de ser eliminados o modificados. Por ende, es necesario realizar una cuidadosa historia clínica en pacientes ambulatorios ya que la revisión de éstos factores de riesgo representa un enfoque inicial útil de prevención, evolución y de posterior tratamiento de ser necesario. En este estudio tipo corte transversal dichos factores se describieron desde el punto de vista epidemiológico, se trataron de asociar a cada paciente en particular, pero desde el punto de vista estadístico no fueron significativos, generando controversias en cuanto a la asociación de factores de riesgo y osteoporosis en esta comunidad, obligando a los autores a profundizar en los mismos.

CONCLUSIONES

Este corte transversal evidencia que amamantar a los hijos, la intolerancia a la leche, el sedentarismo son los factores de mayor peso relativo pero no estadísticamente significativo.

En este estudio tanto en las mujeres con baja masa ósea como en las osteoporóticas el escore T siempre es más bajo a nivel del cuello del fémur que en la columna lumbar L2-L4.

Se han descrito numerosos factores de riesgo, desde el punto de vista epidemiológico, en este trabajo se trataron de asociar para cada paciente en particular. Pero los resultados desde el punto de vista estadístico al ser no significativos generan controversias y este hecho obliga a los autores a profundizar en el estudio de la asociación de factores de riesgo y osteoporosis en esta comunidad.

REFERENCIAS BIBLIOGRÁFICAS

1. Melton LJ. Epidemiology of Vertebral fractures in Women. Am J Epidemiol. 1989; 129: 1000-1011. [ Links ]

2. Wasnich R. Bone mass measurement: prediction of risk: Am J Med. 1993; 95: 65-105 [ Links ]

3. Kanis JA, Melton LJ III, Christiansen C, Johnston CC y Khaltaev N. The diagnosis of osteoporosis. J Bone miner Res. 1994; 9: 1137-1141. [ Links ]

4. Greenfield EM, Goldberg VM. Genetic determination of bone density. Lancet 1997; 350: 1263-4. [ Links ]

5. Reid IR. Steroid- induced osteoporosis. Osteoporosis Int 1997; 137: 209-17. [ Links ]

6. The WHO Study Group. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis. Ginebra: Organización Mundial de la Salud; 1994. [ Links ]

7. Blumsohn A, Eastell R. Pathophysiology of osteoporosis: age- related factors. En: Riggs Bl, Meltón LJ III, eds. Osteoporosis, etiology, diagnosis and management. Filadelfia: Lippicncott-Raven; 1995: 161-182. [ Links ]

8. Mckane Wr, Khosla, S Egan Ks, Robins SP, Burritt MF y Riggs BL. Role of calcium intake in modulation age-related increases in parathyroid function and bone resorption. 1996; 81: 1699-1703. [ Links ]

9. Arlot Me, Sornay-Rendu E, Garnero P, Marty B y Delmas PD. Apparent pre-and postmenopausal bone loss evaluated by DXA at different skeletal sites in women: the OFELY cohort. J Bone Res Miner. 1997; 12: 683-690. [ Links ]

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Factores de Riesgo y Baja Masa Ósea en la Ciudad de Mérida

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Similares en SciELO Revista de La Facultad de Medicina, Volumen 24 Número 2, 2001 (181-184) FACTORES DE RIESGO Y BAJA MASA ÓSEA en la Ciudad de MÉrida F Chakal2, B Castelli3, J Durán2 y G Medina1. Residente de Post-grado de Ortopedia y Traumatología, Hospital Vargas de Caracas, Venezuela.

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Estudio cuantitativo del contenido mineral óseo en niños con intolerancia a la lactosa

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Estudio cuantitativo del contenido mineral óseo en niños con intolerancia a la lactosa

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Se presenta un trabajo de investigación de carácter prospectivo, planteado con el objetivo de establecer si existe alguna diferencia en la densidad mineral ósea (BMD) en los niños malabsorbedores de lactosa respecto a un grupo control sano establecido previamente y como se modificaría esta masa ósea según incluyan o no lactosa en su dieta en el último año.

Para confirmar el diagnóstico de malabsorción de lactosa e indicar el tratamiento dietético a seguir por los mismos hemos utilizado el test de hidrógeno espirado realizado con la fórmula láctea habitual y tras la adición de lactasa comercial.

Para el cálculo de la BMD hemos utilizado la Dualfotoabsorciometría mediante un densitómetro óseo Hologic QDR-1000 que nos da el resultado en gramos de hidroxiapatita de calcio por centímetro cuadrado referentes a las vértebras L2, L3 y L4.

La población de nuestro estudio está formada por 380 niños de edades comprendidas entre 1 y 14 años dividido en dos grupos. Un grupo control de 291 niños y otro de 89 pacientes que presentaban clínica compatible con intolerancia a la lactosa. Este último grupo a su vez se han dividido en dos subgrupos B1 y B2, el primero de ellos, grupo B1, formado por niños que han realizado un tratamiento dietético con una alimentación carente en lactosa por un periodo superior o igual a un año; y el segundo, grupo B2, formado por 24 niños que estaban tomando fórmulas lácteas o bien leche de vaca y derivados, adicionándoles previamente lactasa comercial (KERULAC, Artu Biologicals N.V.). Este grupo a su vez lo podemos dividir en dos en función del tiempo que habían realizado este tratamiento: un subgrupo B2A formado por aquellos que lo habían realizado durante un periodo de tiempo superior al año y un subgrupo B2B por aquellos que la habían estado tomando durante un periodo de tiempo superior a los 6 meses y previamente fórmula sin lactosa por un periodo superior al año.

Del estudio de los resultados hemos encontrado los siguientes hallazgos con significación estadística: existe una correlación lineal entre el valor de la BMD y la edad, peso, altura y superficie corporal con un coeficiente de correlación muy significativo, tanto cuando se consideró toda la población estudiada, como en cada uno de los grupos en los que se dividió. La BMD se puede considerar que viene dada en función de un polinomio de tercer grado en el que participan el peso, la altura y el sexo de los niños.

Existen diferencias estadísticamente significativas entre la BMD del Grupo B1 con respecto a la BMD del Grupo A, B2 y B2A; y no existen diferencias estadísticamente significativas entre la BMD del Grupo A o grupo control y la BMD del Grupo B2 de niños que consumen Kerulac; ni entre la BMD del Grupo B2B y la BMD de los Grupos B2A ni B1. Estas diferencias entre los grupos no se han apreciado en los niños menores de los dos años de edad.

El test de hidrógeno espirado utilizado con la fórmula láctea habitual y posteriormente en los casos patológicos con la adicción de lactasa constituye un método muy útil y sensible para el diagnóstico y control de niños con sospecha de intolerancia a la lactosa, así como referencia terapéutica para el seguimiento de estos niños.

La lactosa en un disacárido de gran importancia en la alimentación humana destacando su participación como agente facilitador en la absorción de calcio, fósforo, magnesio y otros oligoelementos básicos para una adecuada mineralización ósea. Este proceso es fundamental en los primeros años de la vida, cuando la masa ósea se desarrolla y aumenta, hasta adquirir su pico máximo para posteriormente empezar a disminuir.

Esto pone de manifiesto la importancia de alcanzar un adecuado desarrollo del esqueleto durante los primeros años de la vida que podría proteger de la osteoporosis del adulto. El periodo de almacenamiento mineral óseo llega según diferentes autores hasta los 20-35 años siendo durante la infancia más intenso este proceso.

La malabsorción intestinal de la lactosa es una patología muy frecuente en Pediatría aunque su diagnóstico no siempre es fácil de realizar por precisar la disponibilidad de un procedimiento sofisticado lo cual conduce a prescribir de forma empírica dietas carenciales excluyendo este nutriente en algunas ocasiones.

Actualmente disponemos de un procedimiento inocuo, fácil, cómodo, fiable y no invasivo que es el test de hidrógeno espirado, para realizar el diagnóstico de la malabsorción intestinal de los carbohidratos en general, y de la lactosa en particular. Además existe una alternativa terapéutica que facilita el tratamiento de estos enfermos sin excluir este disacárido de la dieta, evitando los perjuicios económicos y nutricionales que ello puede conllevar.

También es posible estudiar el contenido mineral óseo mediante una técnica no invasiva, fiable y de costo aceptable con el fin de valorar las alteraciones que se pueden producir en la masa ósea de los niños que presenta una historia clínica con dieta exenta de lactosa y enjuiciar las ventajas que puede aportar la administración sustitutiva de lactasa en el tratamiento de estos enfermos. A partir de estos datos plantearemos el siguiente OBJETIVO:

Establecer si existe alguna diferencia en la densidad mineral ósea en niños intolerantes a la lactosa respecto a un grupo control sano establecido previamente y como se modificaría esta masa ósea según que la pauta dietética seguida en el último año por estos niños afectos incluyera o no lactosa.

CONCLUSIONES:

1. Es obligación ineludible del pediatra prevenir en el niño problemas de salud de la edad adulta.

2. La osteopenia puede ser una consecuencia inmediata o tardía de una dieta inadecuada, exenta en lactosa.

3. La intolerancia a la lactosa, problema frecuente en pediatría, requiere tratamiento dietético específico que puede causar iatrogenia, por lo que no debería aplicarse sin un diagnóstico correcto.

4. El test de hidrógeno espirado constituye un método muy útil, sensible, económico e incruento para el diagnóstico y control de niños con sospecha de intolerancia a la lactosa y nos puede servir como referencia terapéutica para la introducción o no de lactasa comercial como tratamiento enzimático sustitutivo.

5. El empleo de lactasa comercial adicionada previamente a la fórmula láctea habitual constituye una alternativa terapéutica eficaz en la intolerancia a la lactosa que aporta ventajas nutricionales, económicas y de administración.

6. La fotodensitometría dual constituye una nueva técnica no invasiva para el cálculo de la masa ósea. Su elevada precisión, su tasación rápida, exacta y altamente reproducible de la densidad de masa ósea y la posibilidad de poder realizar estudios repetidos con una mínima dosis de irradiación, hacen que sea de gran utilidad en el diagnóstico precoz y el seguimiento de la patología metabólica ósea.

7. Es preciso contar con patrones de normalidad de densitometrías óseas en cada medio geográfico y para cada sexo y edad, a ser posible con carácter evolutivo para conocer mejor las alteraciones existentes en las distintas afecciones pediátricas.

8. La densitometría dual fotónica aplicada al estudio de niños que incluyen o excluyen la lactosa permite establecer diferencias estadísticamente significativas entre ambos grupos. De forma que la ausencia de lactosa en la dieta condiciona una diminución de la masa mineral ósea respecto a un grupo control.

9. El tratamiento de los niños intolerantes a la lactosa con fórmulas adaptadas que contienen lactosa o leche de vaca adicionadas con lactasa contribuye a la consecución de una masa ósea en estos niños superponible a un grupo control y supone una remisión parcial de la osteopenia de los mismos a los 6 meses de tratamiento.

10. Hasta la edad de los dos años no comienzan a observarse diferencias estadísticamente significativas en la BMD de los niños intolerantes a la lactosa con respecto al grupo control, debido quizás a una influencia genética en estas primeras etapas de la vida para posteriormente descender cuando esta depende del aporte exógeno de minerales exclusivamente.

11. Sería conveniente la realización de un control densitométrico evolutivo a estos niños con intolerancia a la lactosa para comprobar si la osteopenia demostrada se mantiene, corrige o incrementa.

# data[].attributes.attachment_link.keywords[]
estudio cuantitativo del contenido mineral óseo en niños con intolerancia a la lactosa

# data[].attributes.attachment_link.title
Estudio cuantitativo del contenido mineral óseo en niños con intolerancia a la lactosa

# data[].attributes.attachment_link.description
Se presenta un trabajo de investigación de carácter prospectivo, planteado con el objetivo de establecer si existe alguna diferencia en la densidad mineral ósea (BMD) en los niños malabsorbedores de lactosa respecto a un grupo control sano establecido previamente y como se modificaría esta masa ósea según incluyan o no lactosa en su dieta en el último año.

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Deficiencia de lactasa, intolerancia a la lactosa y pico de masa ósea en adultos jóvenes colombianos 

# data[].attributes.subject
Deficiencia de lactasa, intolerancia a la lactosa y pico de masa ósea en adultos jóvenes colombianos 

# data[].attributes.attachment_link.title
Deficiencia de lactasa, intolerancia a la lactosa y pico de masa ósea en adultos jóvenes colombianos

# data[].attributes.attachment_link.description
Deficiencia de lactasa, intolerancia a la lactosa y pico de masa ósea en adultos jóvenes colombianos Lactase deficiency, lactose intolerance, and peak bone mass in colombian young adults Luis Alberto Ángel Arango 1, Enrique Calvo Ayala 2, Yesid Muñoz 3, Bernard Messing 4 1 Profesor Titular, Unidad de Gastroenterología, Departamento de Medicina Interna, Facultad de Medicina, Universidad Nacional de Colombia.

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Identificación de las variantes c/t-13910 y g/a-22018 asociadas con hipolactasia primaria tipo adulto (HPTA) en caucásicos, en una población afrodescendiente de la Costa Caribe colombiana

# data[].attributes.subject
Identificación de las variantes c/t-13910 y g/a-22018 asociadas con hipolactasia primaria tipo adulto (HPTA) en caucásicos, en una población afrodescendiente de la Costa Caribe colombiana

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Resumen

La Hipolactasia primaria tipo adulto (HPTA), es una condición determinada genéticamente, de herencia autosómica recesiva, afecta a 2/3 partes de la población mundial, se caracteriza por una declinación filológica en la actividad de la lactasa, en células intestinales, después del destete. La prevalencia de la HPTA depende del origen étnico, asiáticos cerca del 100%, africanos 70% y caucásicos 30%, en Colombia mayor del 50%. La HPTA se asocia a dos genotipos, C/C-13910 y G/G-22018, situados a 14kb y 22kb corriente arriba del gen de la lactasa. En el presente trabajo se muestran las frecuencias alélicas y genotípicas de estos SNPs en una población afrocolombiana. Para tal propósito, se extrajo ADN a 130 afrodescendientes y se identificaron estos polimorfismos mediante la técnica PCR/RFLP. Las frecuencias alélicas y genotípicas y el Equilibrio de Hardy Weinberg (HWE), se determinaron con el software TFPGA1.3. Las frecuencias genotípicas para el C/T-13910: CC(90%), CT (10%) y TT(0%). Las frecuencias alélicas para *C(95%) y *T(5%). Las frecuencias genotípicas esperadas por HWE: CC(90%), CT(9,5%), TT(0.3%) y muestra concordancia con el HWE (p>0.05). Para el G/A-22018 las frecuencias genotípicas observadas: AA (42%), GA (58%) y GG (0%). Las frecuencias alélicas para *A (71%) y *G(29%). Las frecuencias genotípicas esperadas, por HWE: AA (51%), GA (41%), GG (8%) y no muestra concordancia con el HWE (p<0.05). El C/T-13910 podría utilizarse como marcador genético de Hipolactasia en la población de estudio, pero G/A-22018 no parece estar ligado a Hipolactasia, por lo cual no sería un buen predictor de esta condición.

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Identificación de las variantes c/t-13910 y g/a-22018 asociadas con hipolactasia primaria tipo adulto (HPTA) en caucásicos, en una población afrodescendiente de la Costa Caribe colombiana

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La Hipolactasia primaria tipo adulto (HPTA), es una condición determinada genéticamente, de herencia autosómica recesiva, afecta a 2/3 partes de la población mundial, se caracteriza por una declinación filológica en la actividad de la lactasa, en células intestinales, después del destete.

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Genetic diversity of lactase persistence in East African populations

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Genetic diversity of lactase persistence in East African populations

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https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-015-1833-1

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Abstract

Background

The expression of lactase which digests lactose from milk in humans is generally lost after weaning, but selected mutations influencing the promoter of the lactase gene have spread into the human populations. This is considered a classical example of gene-culture co-evolution, and several studies suggested that the lactase gene has been under strong directional evolutionary selective pressure in the past 5000 to 10,000 years.

Results

In the present study we investigated the distribution of three gene variants leading to lactase persistence in 12 different East African populations as well as one European population. Our results show that with the exception of Copts and Nilotic populations who are fully lactose non-persistent, the majority of populations of East Africa show at least partly lactose persistence, with both ethnic and socio-economic aspects playing an important role in the distribution of genetic variants. In this study, the variants C/G-13907 and T/G-13915, which are the major variants among the nomadic Arabs in the Arabia and Beja of East Africa, showed remarkable frequencies in Sudanese populations, especially those of pastoralists, in line with the historical links and bidirectional migration of nomadic populations between Arabia and East Africa. The C/T-13910 variant, generally associated with European populations is uniquely present among the Fulani.

Conclusions

These data indicate that a combination of socio-economic, ethnic and evolutionary factors converged to shape the genetic structure of lactase persistence in East African populations.

Background

The milk sugar lactose requires hydrolysis in the intestinal tract into the monosaccharides glucose and galactose in order to facilitate their absorption by the enterocytes. The breakdown of lactose in the small intestine is catalyzed by the enzyme lactase, a glycoside hydrolase, which is encoded in humans by the LCT gene [ 1]. Lactase persistence represents the continued ability to break down lactose in adults, and is a dominant Mendelian polymorphic trait identifiable through its phenotypic effect lactose tolerance. Globally, the percentage of lactase persistence haplotype is approximately 35 %, with the highest frequency being found among European and Americans of European origin and its lowest frequencies present in sub-Saharan Africa and southeast Asia [ 2- 4].

The ability of adult humans to digest lactose sugar and the nutritional benefit conferred by this trait (by using milk as an additional energy source) is considered as a classical example of gene-culture co-evolution [ 5, 6]. Many studies suggested that the LCT gene has been under strong directional evolutionary selective pressure resulting from shared cultural traits, animal domestication and adult milk consumption within the past 5000-10,000 years [ 7- 10]. The phenotype of lactase persistence is due to the presence of a haplotype extending more than 2 M nucleotide base pairs on chromosome 2. To date, five major variants (C/T-13910, C/G-13907, T/G-13915, T/G-14009 and G/C-14010) that originated on different haplotype backgrounds in intron 13 of the MCM6 gene are known to regulate the LCT enhancer and are associated with lactase persistence in humans: the C/T-13910 SNP found mainly in Europeans, and C/G-13907, T/G-13915 and G/C-14010 SNPs found in African and Middle Eastern populations, while T/G-14009 is found in Ethiopia [ 2, 10- 13]. The association of these genotypes with the phenotype of lactose tolerance has recently been confirmed for African populations [ 13, 14].

The link between domestication of cattle and the inhabitants of the Nile Valley has a long history [ 15]. Archeological excavations revealed that people of Neolithic and Kerma who lived in the present day northern Sudan and southern Egypt some 5000 years ago raised livestock from which around 80 % were cattle [ 16, 17]. Moreover, historical and archeological findings show evidence of milk fermentation by the later communities as well. Interestingly, drawings attributed to people of the ancient Kingdom of Meroe (690 BC to AD 323) are dominated by cattle. Also the classical Greek writer Strabo (7 BC) mentioned that the Meroites, which are called Ethiopians by classical writers, used fermented milk in their foods. This information indicates that cattle were the major livestock of ancient kingdoms of the Nile Valley [ 18, 19]. Today, consumption of fresh and fermented milk is widespread in East African populations, including pastoral and agriculturalist communities in the Sudan, South Sudan and Ethiopia. Milk and its products play an important role in the traditional diet of the people, and serve as a major source of cash income for most families. Cattle are the primary source of milk in the region, although nomadic populations also raise sheep, goats and camels [ 17, 20].

The aim of the present study was to assess the presence of three MCM6 variants modulating LCT. Variation in these SNPs lead to the tolerance to lactose in different populations from East Africa living along the Nile Valley in the current territory of the Sudan, South Sudan and Ethiopia. We correlated this information with the occupational characteristics of each of the populations, as well as with ethnic and historical aspects, to increase our understanding of population history in this ancient space of human civilizations.

Methods

To investigate the genetic diversity of lactase persistence alleles among East African populations, a total of 488 saliva samples of unrelated individuals from 12 different East-African ethnic groups from the Sudan, South Sudan and Ethiopia were collected from different geographical area in the Sudan prior to separation.

The samples used in the present study were collected and studied with ethical approval and informed consent (ref. SUM/2010/7). All protocols were approved by the IRB of the Sudan Medical and Scientific Research Institute (SUMASRI) at the University of Medical Sciences and Technology in Khartoum, and were carried out in accordance with the approved guidelines.

Appropriate informed consent and questionnaires were obtained from all participants before sample collection; and samples were grouped according to their self-declared ethnic background of parents and grandparents, and languages. Sample sizes, geographic origin, and linguistic affiliation for each population are reported in Table 1. Saliva samples were collected using OrageneTM collection kits (DNA Genotek, Ontario, Canada). The DNA isolation, purification, and genotyping were performed at Radboud University Medical Center, Nijmegen, The Netherlands, following the manufacturer's standard protocols.

Table 1 Geographic locations, sample size, socio-economic activities and linguistic affiliation of populations analyzed for lactase haplotypes in this study

All samples were genotyped for three SNPs in the MCM6 gene, which have previously been shown to regulate transcriptional activity of the LCT promoter and therefore confer tolerance to lactose (C/G-13907 (rs41525747), C/T-13910 (rs4988235) and T/G-13915 (rs41380347)) [ 10]. SNPs were genotyped using Sanger sequencing, and for C/T-13910 we additionally performed TaqMan genotyping to confirm the Sanger sequencing results. In order to compare the African samples with samples from European origin, we genotyped the MCM6 SNPs in DNA isolated from blood samples collected from 46 healthy Dutch volunteers. Isolation was performed using the Gentra Pure Gene Blood kit (Qiagen, Venlo, The Netherlands) following the manufacturer's protocol. Data analysis was performed using Arlequin v.3 and Genetic Data Analysis v.1.0 programs [ 21, 22]. The data set supporting the results of this article summarized in the Additional file 1 of this article.

Results

The genotype and haplotype frequencies of the SNPs conferring lactase persistence among the selected populations are shown in Fig. 1 and Table 2 as well as Additional file 1: Table S1. The variants T/G-13915, C/G-13907 and C/T-13910 are found among East African populations with frequencies of 15.5, 5.5 and 4 % respectively. The C/T-13910 allele, which is the most common in European populations, was identified in high frequency among Dutch (80 %), Fulani (46 %) and Shokrya (2.5 %). The variants C/G-13907 and T/G-13915, which were found previously in Arabia and among Beja of East Africa [ 1, 10- 12, 23]. were found in high frequencies among Beja, Arabs, Nubians and Ethiopians. None of the three genotyped variants was observed in Copts of the Sudan or Nilotes of South Sudan.

Distribution of MCM6 haplotypes among different East African populations: a Pie charts representing the proportion of each haplotype among the selected populations in this study. The arrows show the approximate locations of the populations. The Fulani is a nomadic group and has no specific geographical location. b We additionally added a European (Dutch) cohort to compare the different groups. c The location of the Sudan, South Sudan and Ethiopia and the neighboring countries in Africa. d The key and distribution of LP haplotypes, columns of bases represent the genotypes of T/G-13915, C/T-13910 and C/G-13907 respectively. Modified from d-maps.com

Table 2 Frequencies of haplotype combinations of the three LP variants, T/G-13915, C/T-13910 and C/G-13907 respectively among the populations genotyped in this study

The SNPs in the various populations were tested for Hardy-Weinberg Equilibrium and pairwise disequilibrium using the three variants C/G-13907, C/T-13910 and T/G-13915. The Copts and Nilotes were excluded from the analysis due to the absence of any variants among the genotyped population. The values of observed and expected heterozygosity and fixation indices and their significance are shown in Table 3. No locus of the three polymorphic loci showed a significant departure from HWE with one exception of the variant T/G-13915 in Nuba ( P = 0.01). In addition, there was no significant pairwise disequilibrium between loci, with the only exception found between C/T-13910 and T/G-13915 ( P = 0.01), and between C/G-13907 and T/G-13915 ( P = 0.01) in Nuba.

Table 3 Frequencies of expected and observed heterozygosity and fixation index for the LP variants C/G-13907, C/T-13910 and T/G-13915 among ten ethnic groups of East Africa and Dutch from Europe

Discussion

In the present study variable frequencies in the distribution of MCM6 genetic variants that influence expression of LCT gene were identified in selected East African ethnic groups living in the present territory of the Sudan, South Sudan and Ethiopia (see Table 1). Shokrya, Fulani and Beja-Bani Amir of the Sudan show the highest genetic diversity of LP haplotypes. These three groups are pastoralist, and milk consumption is an important part of their diet. We can thus assume that genotyping data support the hypothesis of a significant selective advantage of lactose absorption. Interestingly, the European haplotype C/T-13910 is uniquely present in considerable frequencies among the Fulani; which may suggest the possible gene flow between Fulani and European populations, or a relatively recent back migration of this group to Africa [ 24- 26]. Remarkably, a notable frequency of the "T" allele was shown in the Arab nomadic group Shokrya, which may be a sign of gene flow from Fulani into this group as a result of their wide distribution in the West and East of the Sudan, which is part of Africa's Sahel.

Both Nilotes and Copts show the absence of lactase persistence alleles. Nilotes are inhabited in the South Sudan and are agro-pastoralists who dwell in the savannas region. Nilotes as well as Nuba breed cattle, but animals are kept not so much for the production of meat and milk, but rather as symbols of wealth and social standing (cows are used for paying marriage dowry) [ 28].

Nilotes are of unusually large stature, and live in the savannas of the Upper Nile region. They comprise Dinka, Nuer, Shilluk, Anyuak, Baria and a number of small ethnic groups. Nilotes speak languages within the Nilo-Saharan family. Farming and hunting are the major nutritional sources in Dinka and Nuer, populations that breed cattle and lead a semi-nomadic life, while Shilluk, Baria and Anyuak are agriculturalists [ 27, 28].

The Copts (ancient Egyptians) descend from longstanding cultures of city-states and empires along the Nile, the socio-activities of this group being farming and trade. These different socio-cultural aspects of Copts, Nilotes and Nuba, most likely combined with the serendipitous lack of arising of novel LCT mutations, explain the absence/low frequencies of LP alleles among these ancient sedentary populations of the Nile Valley.

The other residential groups of the Nile (Shwaiga, Gaalien, Mahas and Halfawien) are agriculturists. Despite the fact that their economy is mainly agricultural, they exhibit moderate allele frequencies of LP. This is intriguing in the sense that one would expect sedentary populations to be more able to admix and receive genes from their nomadic counterparts, as the area surrounding the Nile is economically attractive. This assumption is supported by the negative values of the fixation indices of these groups, which may indicate the increase of heterozygosity. Arabs, Nubians and Beja of the Sudan, and Ethiopians show high frequencies of the allele T/G-13915, which is the major LP variant among the nomadic Arabs in Arabian Peninsula [ 23]. This supports the historical link and bidirectional migration of nomadic populations between Arabia and East Africa. And the significant frequencies of this allele of the nomadic Arabs among Nubians of the Sudan may be due to the gene flow form Afro-Asiatic into this Nilo-Saharan group. Nubians reside at the entering port of the Sudan along the Nile at the border with Egypt, where they were influenced by Arabs as a direct result of the penetration of large numbers of Arabs from Arabia and Egypt into the Sudan over a long period of time following the arrival of Islam around 651 AD [ 29].

Ethiopians genotyped in this study consist of three major groups, Oromo, Amhara and Tigray, they practice farming and animal breeding. Ethiopians display a genetic similarity with Beja of the Sudan; both of them have significant frequencies of lactase persistence haplotypes.

Darfurians is a collective term for a number of residential agriculturalist groups living in arid areas in west of the Sudan (Darfur). Most of the groups in the area practice seasonal cultivation of crops during rainy seasons. They do not rely on fresh milk which is likely the reason why they have low proportion of lactase persistence alleles.

Fresh milk is commonly used in pastoralist communities, and although agriculturalists of the Sudan, South Sudan and Ethiopia show lower frequencies of lactase persistence compared to pastoralists, the milk products are widely consumed by all socio-economic classes of East African agriculturalists. Both pastoralists and non-pastoralists exhibit different cultures of fermented milk products, including Rob (sour yogurt) in the Sudan and South Sudan, white cheese, Mish and Gariss in the Sudan, and Ergo in Ethiopia. Rob and Ergo are naturally fermented cow's or goat's milk products [ 20, 30, 31]. They are the most common traditional dairy fermented food in many areas in the Sudan and Ethiopia. The bulk of these products are produced by the nomadic and semi-nomadic communities during the rainy seasons when fresh milk is available in abundance. The milk is fermented using traditional procedures to obtain sour milk and butter. The white cheese and Mish are known in urban communities, and they were probably introduced into the Sudan from Egypt during the Turkish rule in the 19th Century. The major forms of milk fermentation give a good example of the integration of Arab and African cultures in the Sudan [ 17]. Gariss is the special fermented milk of Arab camel herders in east and west of the Sudan. The camel herders spend sometimes months sustained on Gariss alone [ 32]. Acetic acid bacteria and yeasts are involved in the fermentation of fresh milk. Starter culture of fermented milk contains lactose and phosphorylation enzymes produced by lactic acid bacteria to hydrolyze lactose and to initiate the lactic acid glucose phosphate energy cycles. Lactose is hydrolyzed to the monosaccharides, glucose and galactose. The galactose is converted in the milk to glucose, which may facilitate the absorption of it in the small intestine among the lactose intolerant people with no known symptoms of malabsorption of milk [ 32].

In conclusion, in the present study we have investigated the distribution of LCT gene variants that are associated with LP in twelve different East African populations. With the exception of Copts and Nilotes who are fully lactose intolerant, the majority of populations of East Africa have lactase persistence at moderate frequency. The genetic cause of lactose tolerance is however variable, with both ethnic and socio-economic aspects playing an important role in the distribution of genetic variants. The allele T/G 13915, the major LP variant among the nomadic Arabs in Arabian Peninsula, shows high frequencies in Sudanese populations, in line with the historical links and bidirectional migration of nomadic populations between Arabia and East Africa.

Conclusion

These data tally with the previous data of the distribution of lactase persistence in East Africa, although future studies are warranted to investigate the distribution of more LP variants among the populations studied here. In conclusion, our study indicates that a combination of life style/culture with evolutionary pressure played a crucial role in shaping genetic structure of LP in African populations.

Abbreviations

LP:

lactase persistence

SNP:

single nucleotide polymorphism

References

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Authors' contributions

HH, MO and HT performed sample collection and processing. AvE, and MJ carried out the genetic determination of the different cohorts. HH, AvE, MJ, LJ, HT and MN drafted the manuscript. MN, LJ and HH participated in the design of the study. HH and HT performed the statistical analysis. HH, MJ and AvE prepared the figures and tables. All authors read and approved the final manuscript.

Acknowledgements

We would like to express our gratitude to Prof. Abdalla Elkhawad from the University of Medical Sciences and Technology in Khartoum, Mr. Abdalla Teia Juma and Mr. Waleed Hussein for their help in sample collection. Most of all, we thank all of the participants who generously donated saliva samples for DNA genotyping. M.J. and M.G.N. were supported by an ERC Consolidator Grant (#310372 to M.G.N).

Competing interests

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

1. Banoon ART and Cytogenetics Centre, Bahrain Defence Force Hospital, Manama, Kingdom of Bahrain

Hisham Y. Hassan

2. Department of Internal Medicine and Radboudumc Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands

Anke van Erp, Martin Jaeger, Marije Oosting & Leo A. B. Joosten

3. Sudan Medical and Scientific Research Institute, University of Medical Sciences and Technology, Khartoum, Sudan

Hanan Tahir

4. Department of Internal Medicine (463), Radboud University Medical Center, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands

Mihai G. Netea

Corresponding author

Correspondence to Mihai G. Netea.

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Hassan, H.Y., van Erp, A., Jaeger, M. et al. Genetic diversity of lactase persistence in East African populations. BMC Res Notes 9, 8 (2016). https://doi.org/10.1186/s13104-015-1833-1
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DOI: https://doi.org/10.1186/s13104-015-1833-1

Keywords

Lactase

East Africa

Sudan

Nilotes

# data[].attributes.attachment_link.title
Genetic diversity of lactase persistence in East African populations - BMC Research Notes

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Background The expression of lactase which digests lactose from milk in humans is generally lost after weaning, but selected mutations influencing the promoter of the lactase gene have spread into the human populations. This is considered a classical example of gene-culture co-evolution, and several studies suggested that the lactase gene has been under strong directional evolutionary selective pressure in the past 5000 to 10,000 years.

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https://fohs.bgu.ac.il/develop/mbmls/generegeukhandout.pdf

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Worldwide Interactive map on Lactase Persistence (LP) Phenotype Frequencies

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Worldwide Interactive map on Lactase Persistence (LP) Phenotype Frequencies

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http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software

# data[].attributes.attachment_link.readable_text
With this interactive map we present and summarize all the LP frequency data reported in the scientific literature until July 2020. This tool incorporates detailed information on the geographical location, ethnicity, number of individuals analyzed, reported frequency and reference for each included study. Interactive mapping involves zooming in and out, panning around, identifying specific features, and querying underlying data. Therefore, our tool allows the generation of detailed reports and other means of using or visualizing information in a map. This allows a more attractive way of presenting large sets of data that would not be enough-well exploited in static plots or large tables.

For optimal use, we recommend zooming the map until reaching the desired geographical location or country, and then investigate all reported frequencies. If there is more than one study available in a particular location, data will be displayed in the form of an interactive grey cluster. The number of studies included in each cluster is indicated with a number within the circle. The geographical location mapped for all the studies of a cluster can be visualized as a blue area when moving the mouse over the circle.

The genetic basis for population variation in lactase production as a dominant trait is well-described although not yet complete, with cis-element mutations responsible for LP identified in the transcriptional enhancer MCM6

Among identified variants, the -13910:C>T (rs4988235) and -13495:C>T (rs4954490) have almost reached fixation in some parts of Europe, while others such as -13907:C>G (rs41525747), -13915:T>G (rs41380347), -14009:T>G (rs869051967) and -14010:G>C (rs145946881) are found at variable frequencies in the Middle East and Africa. Besides highlighting as the most widespread and strongly associated LP variants, these six SNPs have been reported as functional markers according to a great range both in vitro transfection assays and in vivo studies.

For them, we created an interactive map following the same format than the one previously-presented for LP phenotype data.

Again, for optimal use, we recommend zooming the map until reaching the desired geographical location or country, and then investigate all reported frequencies. If there is more than one study available in a particular location, data will be displayed in the form of an interactive grey cluster. The number of studies included in each cluster is indicated with a number within the circle. The geographical location mapped for all the studies of a cluster can be visualized as a blue area when moving the mouse over the circle.

References:

Anguita-Ruiz A, Aguilera CM, Gil Á. Genetics of Lactose Intolerance: An Updated Review and Online Interactive World Maps of Phenotype and Genotype Frequencies. Nutrients. 2020;12(9):E2689. Published 2020 Sep 3. doi:10.3390/nu12092689

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bioquímica de la nutrición. implicaciones terapéuticas

# data[].attributes.attachment_link.description
With this interactive map we present and summarize all the LP frequency data reported in the scientific literature until July 2020. This tool incorporates detailed information on the geographical location, ethnicity, number of individuals analyzed, reported frequency and reference for each included study.

# data[].attributes.headline
Genetics of Lactose Intolerance: An Updated Review and Online Interactive World Maps of Phenotype and Genotype Frequencies

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Genetics of Lactose Intolerance: An Updated Review and Online Interactive World Maps of Phenotype and Genotype Frequencies

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551416/

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1. Introduction

Lactose is the main carbohydrate present in milk and one of the main sources of energy during the nursing period in mammals. To take advantage of milk lactose, mammals have to first hydrolyze it into glucose and galactose, monosaccharides that can be easily absorbed by the intestinal tract [ 1]. In humans, the ability to digest milk lactose is conferred by a β-galactosidase enzyme called lactase-phlorizin hydrolase (LPH) [ 2, 3]. The LPH enzyme is encoded by the lactase (LCT) gene, located on the chromosome 2q21. Exclusively expressed in the small intestine, in the apical part of microvilli within the brush border membrane of enterocytes, the LPH enzyme reaches the highest levels of activity during the nursing period [ 4]. After the weaning phase, however, in the majority of humans, the activity of LPH declines rapidly because of a decrease in the levels of the enzyme, and this trait is known as lactase non-persistence (LNP) [ 4, 5, 6]. As a consequence of LNP, the majority of humans are unable to digest lactose during adulthood, and some suffer clinical complications when they consume it. It has been estimated that approximately two-thirds of humans are LNP worldwide [ 7]. In the remaining third, however, there are individuals with the ability to digest milk and other lactose-rich dairy products during adulthood. This trait is known as lactase persistence (LP), and is particularly common in descendants from populations that have traditionally practiced cattle domestication [ 7, 8]. Today, there is good functional evidence that the trait of LP has a genetic basis and follows a dominant pattern of inheritance [ 9]. Notably, it has been postulated that LP could be independently caused by, at least, five or more single nucleotide polymorphisms (SNPs) in a regulatory region (a transcriptional enhancer) called MCM6 (minichromosome maintenance complex component 6), which is located upstream of the LCT gene [ 9, 10, 11, 12, 13, 14, 15, 16, 17].

The frequency of LP is high in northern European populations, decreases across Southern Europe and the Middle East, and is low in non-pastoralist Asian and African communities [ 17, 18, 19, 20]. Notably, LP is also common in pastoralist populations from Africa. It is hypothesized that natural selection has elicited a prime role in determining the current frequencies of LP in different human communities since the development of cattle domestication in the Middle East and North Africa around ~7500-9000 years before present (BP). Although LP and LNP frequencies have been widely studied worldwide, there is a lack of a work collecting all newly available information published to date [ 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32]; especially the new frequency data reported for American populations. Given the great interest of this research topic, an online graphic resource gathering all reported lactase frequency data (both at the phenotypic and genetics levels) would be of much utility for the scientific community. With this purpose, interactive maps and other geographic information system (GIS) tools have been extensively employed with great success in other evolutionary biology areas [ 33]. Interactive mapping involves using maps that allow zooming in and out, panning around, identifying specific features, and querying underlying data, such as by topic or an accurate indicator (e.g., the allele frequency of a certain LP variant in a particular sub-population or ethnicity), generating reports, and other means of using or visualizing information in a map. This allows a more attractive way of presenting large sets of data that would not be sufficiently well exploited in static plots or large tables. Moreover, the online format of these resources allows easy access for researchers and constant update.

On the other hand, although great efforts have been devoted to elucidating the exact molecular mechanisms responsible for the natural decline in LPH activity after weaning (LNP), some gaps remain to be fulfilled [ 34]. Neither is there a unique, accepted theory about the evolutionary origin of LP and what was the advantage that it conferred to carriers, and that favored such a transmission across generations.

Taking all this into consideration, the aims of the present review were: (1) to gather and summarize all available information on LNP and LP genetic mechanisms and evolutionary adaptation theories, and (2) to create online interactive world maps, including all LP phenotype and genotype frequency data reported to date. As far as we are aware, this work constitutes the first interactive, and most updated, online resource for studying and exploring the phenotypic and genetic data on lactase phenotypes. This review is intended for general clinicians, nutritionists, dietitians, paediatricians, evolutionary biology researchers, geneticists, and any researcher working on the topic.

3. Results and Discussion

3.1. Lactose: A Unique Sugar

The main carbohydrate in milk is the disaccharide lactose (β-d-Galactopyranosyl-(1→4)-d-glucose), which requires hydrolysis in the intestinal tract so that its component monosaccharides, galactose and glucose, can be taken up into the enterocytes and used later as a source of energy or structural elements. Lactose is found exclusively in mammal's milk, although in a variable concentration, depending on the species. While humans have a mean composition of about 7 g of lactose per 100 mL of milk, other mammals present lower concentrations (e.g., 4.8, 4.1, and 4.8 g per 100 mL in cow, goat, and sheep milk, respectively) [ 37].

The hydrolysis of milk lactose in the intestine is catalyzed by the enzyme lactase-phlorizin hydrolase (LPH) (EC 3.2.1.108-EC 3.2.1.62), a β-galactosidase located in the brush border membrane of small-intestinal enterocytes. In humans, LPH is expressed only in the small intestine and is confined to absorptive enterocytes on the villi and not the proliferating cells of the crypts. Within the small intestine, LPH further demonstrates positional regulation, as exhibited by a tightly controlled pattern of expression along the proximal to the distal axis in both animals and humans, with high levels in the mid-intestine (mid-jejunum), and reduced levels in the duodenum and distal ileum [ 38, 39, 40]. This pattern of expression closely parallels that of another digestive hydrolase, i.e., sucrase-isomaltase [ 41]. However, lactase is expressed only at low levels throughout fetal life, whereas sucrase-isomaltase is expressed at high levels in the small intestine of early fetuses, and also transiently in the fetal colon [ 42].

3.2. The Lactase-Phlorizin Hydrolase Enzyme

In humans, the LPH enzyme is a 160 kDa transmembrane glycoprotein with a C-terminus (of 26 amino acids), which is intracellular, and an N-terminus, found at the luminal surface of the lipid bilayer of the microvillus membrane of enterocytes [ 1, 3]. The transmembrane-spanning region is a short sequence of hydrophobic amino acids (19 residues). LPH is a multifunctional enzyme with several substrate specificities. Therefore, it can hydrolyze, in addition to lactose, lactosylceramide, cellobiose, cellotriose, flavonoid glucosides, and phlorizin. The primary sequence of the LPH protein in humans, rabbits, and rats reveals a four-fold internal homology (designated domains I-IV), most likely due to two independent duplication events during evolution [ 43]. It has been shown that domains I and II together serve to regulate protein folding in the endoplasmic reticulum; they are not glycosylated and they lack enzymatic activity. Domains III and IV are heavily glycosylated, are inserted into the microvillus membrane, and contain the two active sites in which each nucleophile is a glutamate residue. The amino acid sequence around the active site glutamic acid (E) in domain III is PIYITENG, whereas that of domain IV is PIYVTENG. First, nascent LPH (195 kDa) is synthesized in the endoplasmic reticulum and undergoes cotranslational, dolichol-dependent, high-mannose glycosylation, yielding a molecular mass of 215 kDa. Complex glycosylation of domains III and IV occurs in the Golgi, generating a structure of 220 kDa (O-glycosylations in serines and threonines as well as N-glycosylations in asparagine), and this glycosylation probably affects enzymatic activity as well as folding and intracellular transport [ 44]. Subsequently, there is a cleavage of a small N-terminal pro-enzyme, and of domains I and II that serve as a chaperone for the remaining molecule, which is inserted into the microvillus membrane. Finally, an extracellular cleavage event (perhaps carried out by pancreatic proteases) removes a small residue from the N-terminus, producing the final, mature LPH enzyme [ 45]. The role of the extra-cellular cleavage product remains unknown [ 3].

The human LCT gene, located on chromosome 2q21, comprises 17 exons and covers approximately 49 kb, giving rise to a messenger RNA of slightly more than 6 kb (). From the initiation codon to the stop codon, the human LCT mRNA encodes 1927 amino acids, forming the complete translation product. Initial analyses of the gene identified several genetic variants within both the coding region and the 5′ -flanking region [ 43], although none were considered to have functional significance. Studies in transgenic mice have indicated that approximately 1 kb of the 5′ -flanking sequence of LCT in the pig, and 2 kb in the rat, are sufficient to direct appropriate tissue, cell, and villus expression, as well as a developmental decline after weaning [ 46, 47, 48]. Therefore, the majority of LCT regulatory elements may be located within, or surrounding, these regions. The first 100 bp of the proximal LCT promoter of the mammals analyzed to date (rat, mouse, pig, and human) are virtually identical, and appear to be similarly regulated. Interestingly, binding sites for caudal type homeobox 2 (Cdx-2), hepatocyte nuclear factor 1α (HNF1-α), and GATA transcription factors are in similar positions relative to the transcriptional start site [ 49, 50, 51, 52]. In addition, a number of other transcription factors have been found to interact with the LCT 5′ -flanking sequence, albeit some in more distal loci, including HOXC11 (homeobox C11), HNF-3, C/EBP (CCAAT/enhancer binding protein), and FREAC-2/3 (forkhead box F2) [ 53, 54, 55, 56], and have also been found to activate promoter-reporter constructs in transfection assays. In contrast, the transcription factor Pdx-1 (pancreatic and duodenal homeobox 1) appears to reduce the LCT expression in vitro [ 57]. As HNF1-α and GATA factors are found together in the intestine and colon, their interactions in the activation of LCT gene function have been extensively studied. In mouse intestine, the expression patterns of HNF1-α and GATA-4 closely correlate with LCT expression [ 51], being a physical interaction between these factors, necessary for the cooperative activation of the LCT promoter [ 52]. In contrast to the other mammals analyzed, the first 1000 bp of the 5′ -flanking region of the human LCT gene contains two Alu sequences of approximately 300 bp each, whereas the more distal region contains additional repetitive DNA sequences, making it difficult to directly compare this more distal regulatory region to those of other mammals. Whether inserted repetitive DNA segments affect LCT expression is still unknown. On the other hand, exon 17 of MCM6, a cell cycle regulatory gene ending 3.5 kb from the start site of the human LCT gene, seems to play a role as a regulatory enhancer of LCT [ 58]. The transcriptional start site of the MCM6 gene lies approximately 39 kb 5′ of the LCT transcriptional start site. These two genes are close together, but the available evidence indicates that their regulation is independent [ 58].

3.3. Lactase Phenotypes and Their Genetic Origin

Lactase enzyme activity is high and vital during infancy, when milk is the main food. In most mammals, however, lactase activity declines after the weaning phase (to <10% of the neonatal values); with reduced activity maintained throughout adult life, and this is also the case in the majority of humans throughout the world who are described as LNP. This down-regulation is genetically programmed and seems to affect 70% of the world adult population. In other healthy humans (approximately a third of all humans) [ 59], especially people of Northern European ascendance and a few other population groups around the world, lactase activity persists at high levels throughout adult life, and this trait is known as LP. People who are LP can usually hydrolyze large amounts of lactose during adulthood and can thus consume large quantities of fresh milk without complication. People with LNP (also referred to as adult-type hypolactasia or lactase restriction) have a much lower lactose digestion capacity (LDC) than those with LP, and thus often, but not always, show symptoms after consumption of fresh milk [ 4]. When referring to LNP it is important to differentiate the term from others such as congenital lactase deficiency and secondary hypolactasia. Congenital lactase deficiency is extremely rare and appears at the onset of nursing, caused by severe mutations on both alleles of the LCT gene [ 60]. Secondary hypolactasia otherwise is caused by direct damage to mucosal lactase (e.g., celiac, giardiasis, enteritis), and does not have a genetic basis [ 61]. In this review, we will focus exclusively on the LNP and LP phenotypes, leaving behind any other type of hypolactasia. Other common terms usually employed concerning lactase phenotypes are lactose malabsorption and lactose intolerance, both referring to the consequences derived from an LNP status [ 62]. Lactose malabsorption refers to lowered blood glucose rise or increased breath hydrogen rise after an oral lactose load, and lactose intolerance describes the symptoms that normally accompany lactose malabsorption (e.g., diarrhea, farting, stomach pain, etc.).

About the genetics underlying LNP and LP, although considerable research efforts have been devoted, to date, some gaps remain unfulfilled. Concerning LNP, it seems that it is the direct action of transcriptional repressors, around the age of 5 years, which elicits the natural decline in lactase levels [ 46, 47, 48]. According to promoter studies, one plausible candidate could be the transcription factor Pdx-1, which has shown to directly downregulate LCT expression in vitro [ 57]. The reduction in the rate of LPH transcription would then elicit a consequent decrease in LPH-protein synthesis and enzyme activity along the proximal to the distal gradient, as well as the virtual extinction of expression in the duodenum and distal ileum. Besides the action of transcription factors, some epigenetics mechanisms (mainly DNA methylation) could also be involved in LNP regulation, as demonstrated by Labrie et al. (2016) [ 63]. Likewise, there have been described interpersonal differences in terminal glycosylation of the carbohydrate side chains of lactase, due to polymorphisms in Lewis (FUT3) and Secretor (FUT2) genes, corresponding to secretion of Lewis ABO (H) histo-blood group antigen CA19-9 [ 64]. Since these variants exert a direct influence on protein stability [ 44], not only DNA-binding elements, but also post-transcriptional factors, might contribute to the natural decline of intestinal lactase levels [ 5]. On the other hand, the persistence of LPH activity throughout adulthood (LP) seems to be a genetic trait following a dominant inheritance pattern, and involving the action of cis-element mutations. Particularly, it has been described that the effect of several SNPs leads to an alternative path for LCT expression that is not downregulated, as the original pathway is [ 4]. The molecular mechanism for these SNPs is the creation of new binding sites for specific transcription factors, such as the octamer-binding protein 1 (Oct-1) [ 65]. Interestingly, identified causal mutations seem not to be at LCT itself but 14 kb chromosomally upstream of LCT, on the same haplotype, in an intron of the previously introduced MCM6 locus. As we have already mentioned, the MCM6 has been described as a regulatory enhancer that modulates LCT expression, and hence protein levels [ 4, 16, 66]. A graphical summary of all the plausible genetics mechanisms involved in the development of lactase phenotypes can be found in .

The identification of lactase phenotypes in humans (LP and LNP) can be done directly by assay of the lactase activity from a small intestine biopsy, or indirectly by lactose-tolerance tests [ 67]. Although the direct determination of lactase activity in intestinal samples is always better than lactose-tolerance tests, it constitutes an invasive and impractical technique for all situations. Among alternative lactose-tolerance tests, it highlights the measurement of an increase in blood glucose after giving a lactose load of 50 g, so that people with high lactase activity will present a significant rise in blood glucose concentration within 15 to 45 min, following lactose administration. Another lactose-tolerance test, and preferable over the blood glucose test, involves testing breath hydrogen after a lactose load or, ideally, after the ingestion of one serving (250 mL) of pasteurized or sterilized milk [ 68]. In LNP people, undigested lactose reaches the colon, where it is fermented, leading to the production of fatty acids and gases, including hydrogen, which is excreted in the breath. According to the genetic basis of LP, several genetic tests have also become available as alternatives to lactose tolerance tests and intestinal biopsies. Although these genetic tests constitute non-invasive and useful diagnostic tools, depending on the genetic variants analyzed, they are not always appropriate for all populations.

3.4. Lactase Phenotypes and Their Genetics Origin

By means of either of these diagnostic tools, an impressive number of studies since the 1960s have investigated the distribution of lactase phenotypes worldwide, reporting sharply different LP frequencies among distinct human populations, and raising a provocative question in relation to evolutionary genetics. With their reviews, Itan et al. (2010) and Storhaug et al. (2017) gathered all available LP frequency phenotype data until 2016, becoming a reference for the study of LP phenotype worldwide distributions. Nonetheless, no review study has been recently published with updated data. Taking this into consideration, in the present work, we have collected all LP frequency data available till 2020 (also including the data from Itan et al. (2010), and Storhaug et al. (2017)) ( Supplementary Table S1), and generated an interactive world map. Our interactive map constitutes the most comprehensive and updated resource for exploring LP frequencies worldwide. Besides including all available literature reports on LP frequencies till 20 July 2020, our map further offers a detailed description for each included study (incorporating information related to geographical location, ethnicity, number of individuals analyzed, reported frequency, and literature references). One of the greatest virtues of our interactive map is its ability to organize large amounts of data in a single shot, which otherwise would be hosted in unpleasantly big tables. As a fully extendable and interactive graphic, our tool further constitutes an upgrade over previously published static world maps, allowing users a personalized data exploration at the same time as accessing complete reports by population. For the construction of our database and interactive map, LP frequency data obtained either by intestinal biopsy or any type of presented lactose-tolerance and genetic tests have been considered. Although the interactive map is hosted at ( http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software), in the present article we show a static capture of the map (showing mean LP frequency values per country) ().

As can be observed, the frequency of LP varies greatly among populations, ranging from 0% to almost 100%, with the highest rates found in people of northern European descent and some populations from West Africa, East Africa, and the Middle East ( http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software). The frequency decreases as one moves to the south and east of the map (with 30% of LP reported in Italy). This descending gradient is disrupted in some European countries with a strong cultural admixture tradition, such is the case of Spain (with reported frequencies ranging from 47% to 91%). Interestingly, LP is particularly frequent in some milk consuming nomads and pastoralist communities of the Afro-Arabian area in comparison to their neighboring populations (e.g., 86% in Beduin Saudi, 88% in Ben-Amir, 80% in Haddendoa, and 70% in Fulani). There is a very low level of LP in the Asiatic countries (15% in China, and 0% in South Korea, Vietnam, and Cambodia). Regarding the USA and Mexico, although they present a relatively low mean LP frequency (48 and 52% respectively), highly variable LP sub-frequencies, according to ethnicity, have also been reported. For example, while LP is present in 83% to 93% of white Americans, with European or Scandinavian extraction, it shows a frequency of around 30% in Mexicans from rural areas, and 12-40% in African Americans. Conversely, LNP is found in 83% of Ashkenazi Jews in Israel, and in 81-92% of pure-bred American Indians. Low LP frequencies have also been found generally in South America (with 20% in Colombia, 6% in Peru, 38% in Chile, 37% in Brazil, and 30% in Uruguay), except for some white-ancestry Mennonite sub-populations in Brazil.

Although LP has been investigated in an impressive number of individuals, some geographical areas remain phenotypically understudied, notably in North Africa (e.g., Tunisia, Libya, Mali, or Mauritania), West Africa, and specific locations around the Caucasus. The situation should also be clarified in the many Caribbean and South American countries, such as Argentina, Bolivia, Ecuador, Uruguay, Venezuela, Dominican Republic, Panamá, Guatemala, or Cuba, for whom we lack LP phenotype data entirely.

3.5. Current Status of Genetics Aetiology of Lactase Persistence

The genetic basis for population variation in lactase production as a dominant trait is well-described, although not yet complete, with cis-element mutations responsible for LP identified in the transcriptional enhancer MCM6 [ 9, 10, 11, 12, 13, 14, 15, 16, 17]. Although LP has been known for almost a century, it was not until 2002 that the first LP mutation was discovered [ 69]. Such a delayed discovery was probably caused by the location of the LP alleles, which map 14 kb upstream of the LCT gene and not within, or immediately upstream, of it. Among identified variants, the −13910:C>T (rs4988235) [ 69] has almost reached fixation in some parts of Europe, while others such as −13907:C>G (rs41525747), −13915:T>G (rs41380347), −14009:T>G (rs869051967), and −14010:G>C (rs145946881) are found at variable frequencies in the Middle East and Africa [ 10, 12, 14]. Besides being highlighted as the most widespread and strongly associated LP variants, these five SNPs have been reported as functional markers according to a vast range, of both in vitro transfection assays, and in vivo studies [ 10, 12, 13, 16, 39, 65, 66, 70, 71, 72, 73, 74].

In addition to these five genetic markers, up till now, eighteen new SNPs mapping the MCM6 have also been associated with LP in specific populations, thereby making a total of twenty-three known SNPs that currently underlie the genetic etiology of LP (). Interestingly, these variants seem to have arisen during the same time period, but independently in different human populations, and this is the reason why LP has become a textbook example of convergent regulatory evolution, and gene-culture co-evolution. In the presented , we display the known identifiers for mentioned variants as well as any evidence of functional control on LCT expression according to the literature. Analysis of an 80 kb haplotype covering the region of LCT and the upstream MCM6 enhancer has further confirmed a tight association of these LP variants with particular haplotypes [ 10, 12, 14], and shows that haplotype diversity also differs between populations, with the least diversity observed in Northern Europeans [ 9].

Table 1

Of the twenty-three genetic variants included in , there are ten with a rare frequency (q < 5%) in the populations where they were discovered, as well as another SNP in complete linkage disequilibrium (LD) with the well-known −13.910:C>T. For the remaining twelve frequent and widely studied SNPs, we have created a database including all available information for minor allele worldwide frequencies reported till July 2020 in the literature ( Supplementary Table S2). For the most genotyped variants among these twelve markers (those SNPs that have been studied in more than ten different populations around the world), we have further created an interactive map following the same format as the one previously presented for LP phenotype data. The six genetic variants represented in the interactive map comprise −13910:C>T (rs4988235), −13495:C>T (rs4954490), −14009:T>G (rs869051967), −13907:C>G (rs41525747), −13915:T>G (rs41380347), and −14010:G>C (rs145946881)), for which we further include information related to ethnicity, number of genotyped individuals, reported minor allele frequencies, and literature references. This interactive map is hosted at ( http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software) and a static capture is presented in . For the construction of such a database and interactive map, genotype data available in the work from Liebert et al. (2017) [ 9] as well as in any additional genetic study published from 2016 to 2020 have been considered. Owing to that, our map constitutes the first resource presenting distribution data for LP associated alleles in American populations.

As can be observed in the map, highly variable SNP frequencies have been described worldwide, differing not only between countries but also between different sub-populations or ethnicities within the same geographical location. By far, the −13.910:C>T (rs4988235) is the more widely extended and studied variant, with a presence in lots of ethnicities and cultures. First identified in 2002, in a study of Finnish families by Enattah et al. (2002) [ 69], this variant has been further confirmed as a cis-acting enhancer of the LCT promoter in both in vitro and in vivo studies [ 13, 66, 75]. The molecular mechanism through which the T allele of this variant increases LCT expression consists of the creation of a new binding site for the Oct-1, a transcription factor that interacts with human HNF1-α to bind to the LCT promoter [ 65]. Therefore, this allele leads to an alternative path for LCT expression that is not downregulated, as the original path is. Interestingly, in vivo studies further revealed that, even though the LP phenotype is considered a binary trait encoded dominantly, lactase activity is instead a codominant quantitative trait, with a clear trimodal distribution [ 76]. This difference in expression between homozygous and heterozygous likely has a minor effect on the ability to efficiently break down lactose, but as suggested by Swallow, it could become important under certain conditions, such as stress or disease. Although Enattah et al. (2002) [ 69] also reported another mutation associated with the LP phenotype, the −22.018:G>A (rs182549), it has been demonstrated that it is in complete LD with −13.910:C>T, and not able to drive LP by itself. Actually, −13.910 and −22.018 might interact epistatically; in vitro studies have shown that the −22.018 region is a weak silencer of the enhancer activity driven by −13.910 [ 16, 39, 66]. As can be observed in our map, −13.910:C>T is not only a European mutation but also underlies the LP phenotype all over Asia, including in various populations from Russia, Pakistan, and Iran; Central Asia (prevalence of 30% in herders); and Nepal (prevalence of up to 34% in herders from the north) [ 77, 78]. It is also the primary LP-associated mutation in Algerians (17-33%), Mozabites (22%), and other Berber populations (14-23%) from North Africa, as well as in Fulani (23-48%) from both Central Africa (Cameroon) and East Africa (South Sudan). Interestingly, the −13.910:C>T has also been found at a moderate frequency in South America (22% in communities from the Coquimbo region in Chile) [ 25, 79], Central America (20% in rural areas from Mexico), and in North America (21-82%).

Although it was thought that in all of Eurasia the LP phenotype was monogenic, a second variant, the −13495:C>T, was described and found to be widely spread worldwide (87% prevalence in Norway, 96% in Ireland, 78% in UK, 50% in Spain, Italy, and Portugal, as well as a frequency higher than 15% in other Eurasians regions such as Azerbaijan, Georgia, Uzbekistan, Russia, Mongolia, Pakistan, and India). In all the populations where the −13495:C>T has been found, the variant exhibits very similar frequencies to the −13.910:C>T, with a practically complete LD described for both markers (D' = 0.99 according to the 1000 Genomes Project Phase 3). Located just outside the enhancer MCM6 region, the −13495:C>T has been shown to occur as a derived allele on an ancestral haplotype and to be older than the −13.910:C>T [ 9].

The situation is different in the Arabian peninsula and East Africa, where four different mutations have been found to be associated with LP: −13.907:C>G (rs41525747), −13.915:T>G (rs41380347), −14.009:T>G (ss820486563), and −14.010:G>C (rs145946881), all of which cluster in the intron 13 of the MCM6, within a 100-base-pair interval of each other that includes the −13.910:C>T [ 12, 14, 16, 65, 66, 74]. All of these mutations result in an increase of lactase activity in vitro [ 10, 12, 13, 16, 39, 65, 66, 70, 71, 72, 73, 74], and two of them, −13.907:C>G and −13915:T>G, seem to affect binding of Oct-1 [ 10, 73]. While −14.010:G>C is most prevalent as one goes to East Africa (32-46%) [ 12] and South Africa (13-20%) [ 80, 81], −13.907:C>G and −14.009:T>G are most prevalent among the Beja people of Sudan and in Ethiopia. On the other hand, −13915:T>G is the most common variant in camel herders from the Middle East (72-88%) [ 10, 82], and is also extended among almost all Central-East African populations (Kenya, Sudan, Cameroon, Nigeria, Chad).

Although increasing data are becoming available for American populations, these have been exclusively focused on the −13.910:C>T variant. Given the great diversity of Afro-descendants living in the American continent, more studies focused on the new variants identified by Tishkoff et al. (2007) [ 12] (−13.907:C>G (rs41525747), −13.915:T>G (rs41380347), −14.009:T>G (ss820486563), and −14.010:G>C (rs145946881)) would be of much interest, and would complete the picture for worldwide LP alleles distributions.

3.6. Evolutionary Genetics of Lactase Persistence

All LP alleles show strong signals of positive selection [ 83] and have spread through migration [ 84]. Their evolutionary advantage has been argued in the ability they confer to humans to consume animal milk during adulthood without risk of symptoms of lactose intolerance [ 12, 84, 85, 86, 87, 88, 89]. Besides a positive selection mechanism, present-day frequencies of these alleles have also been clearly influenced by other processes, including population expansion, allele surfing, and cultural/environmental processes [ 84]. According to ancient DNA studies, the earliest occurrences of an LP allele (the −13.910:C>T) have been reported in Spain, dated to about 5000 years BP according to PCR data [ 90], or in Germany and Sweden about 4000 BP according to NGS data [ 85, 88, 91, 92]. This is in tune with performed extended haplotype homozygosity (EHH) analyses of the sequences surrounding LP SNPs, which have shown that the haplotypes carrying the derived LP-associated alleles are much longer than their ancestral counterparts, supporting a recent origin of these variants [ 93]. Accordingly, by means of other population genetic and modeling approaches, some authors have estimated the strength and timing of selection from a sample of European Americans, and found that the −13.910:C>T allele arose 2188-20,650 years BP, and that this selection was favored with a selection coefficient of 0.014-0.15 [ 10, 86]. Such selection coefficients place the LP as one of the strongest examples of positive selection in the human genome, even ahead of the resistance to malaria (0.04-0.09) [ 94], and skin pigmentation in Europeans (0.03) [ 95].

Four main theories of evolutionary advantage have been proposed to explain the observed strong LP selection coefficients and the high LP frequencies found in the majority of dairying populations worldwide [reviewed in [ 7, 96, 97]:

Lifelong access to nutrient-rich milk;

Lifelong access to carbohydrate and fluid source, critical to pastoralists living in hot, arid environments;

Lactose enhances calcium absorption, which may be compromised by low vitamin D synthesis in high latitude (northern) environments;

Human consumption of bovine milk may accelerate reproductive maturation or physical growth, or contribute to larger adult size, possibly due to milk's stimulatory effects on insulin-like growth factor-I.

For non-dairying groups, however, the above hypotheses suggest that LP mutations did not spread, as they did not confer any advantage.

Although these four theories are widely accepted and serve as the best explanation for the majority of scenarios, there remain some questions regarding the evolutionary origin of LP. This is particularly the case with some herders from the Middle East, where low LP frequencies have been found even though they have relied heavily on pastoralism for millennia (12% and 24% in long-term Mongol and Kazakh herders, respectively) ( http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software) [ 97]. Central Asian herders are not the only exception to the expected pattern. Indeed, the Sami reindeer herders in Scandinavia have a lower LP frequency than the rest of the Swedish population (33% versus 91%) despite a higher dependence on pastoralism (60% versus 30%). Similarly, some African pastoral ethnic groups who consume milk (50-90% pastoralism) have low LP frequencies, as in the case of the Dinka (LP frequency of 0-25%) and Nuer (22%) in Sudan, the Somali in Ethiopia (24%), and the Herero in South Africa (3%). Furthermore, in the areas where animals were first domesticated (notably in Turkey) and more generally around the Mediterranean, populations that have used milk for millennia have moderate LP frequencies.

Two main hypotheses have been postulated to explain such discrepancies: (1) The existence of patterns of admixture between pastoral and non-pastoral populations, which could have limited the efficacy of natural selection, and (2) the ability to transform milk into derived dairy products, which would has been further affected by a combination of cultural, nutritional, and environmental issues (such as the preference or need to ferment milk, the stability and availability of other sources of energy, seasonality and mobility limitations or the type of livestock). The admixture between pastoral and non-pastoral populations could explain the situation found in Central Asia, which is a migratory crossroads, and is located in the middle of non-pastoral groups. Otherwise, the ability to ferment milk might account for the intermediate LP frequencies found in populations around the Mediterranean and north of the Middle East (39%), where dairy animals were first domesticated, given that these populations consume moderate amounts of fresh milk (102 L per person per year) and transform a large proportion of milk into cheese and fermented milk (38% on average) [ 98]. On this matter, it has been demonstrated that variability in fermenting practices correlates better with LP frequency than the levels of pastoralism [ 97].

3.7. New Insights into the Genetic Basis of Lactase Phenotypes: Epigenetics Alterations

Despite the identification of LP-associated alleles, some additional molecular basis of the trait remains to be uncovered. Indeed, two populations with substantial LP have been reported to completely lack known LP-associated mutations: the Hadza from Tanzania, who have an LP frequency of 47% [ 12, 99], and the Wolof from Senegal, who have an LP frequency of 51% [ 74, 100]. The case of the Hadza is particularly intriguing because no associated mutation has been found despite a large sequencing effort, including intron 9 of MCM6 (1.3 kb), intron 13 of MCM6 (3.2 kb), and the LCT promoter (2 kb) [ 12, 99]. As complementary underlying mechanisms, some authors have postulated that lactase phenotypes could also be directed by epigenetic modifications [ 31, 63, 101, 102, 103]. The first evidence supporting this hypothesis was published by Labrie et al. (2016) [ 63], who demonstrated that the C allele from the −13910:C>T was associated with the appearance and accumulation of age-related methylations at both the MCM6 and the LCT regions [ 63]. Indeed, they demonstrated that these DNA variation-dependent methylations further elicit a silencing on LCT expression, thereby being a plausible contributor to LNP in mammals. In line with Labrie's findings, another work demonstrated how certain epigenetic signatures, at enhancer and promoter LCT elements, align with transcriptional variation of LCT in mouse enterocytes [ 103], further reporting how age and phenotype-specific environmental cues (lactose exposure after weaning) induce changes to these epigenetics modifications and alter the intestinal LCT mRNA gradient [ 103]. Finally, a recent study identified putative LCT meQTLs (methylation quantitative trait loci), which are differentially methylated between LP and LNP individuals [ 31]. Interestingly, these authors also found that DNA methylations in the enhancer and promoter sites of the LCT, rather than the expression of intestinal transcription factors such as CDX2, POU2F1, GATA4/6 or HNF1-α, were predictive of LNP/LP () [ 31]. Indeed, DNA methylation variation presented a higher power to predict lactase phenotypes than the classic LP alleles alone.

Altogether, results from these studies suggest a plausible role of epigenetics in the appearance and regulation of lactase phenotypes, and reinforce the utility of DNA methylation as a complementary biomarker for LP diagnosis. More studies would be needed in this area in order to completely elucidate the epigenetic etiology of the trait. If demonstrated, a worldwide distribution map for such epigenetic signatures would also be of much value for the scientific community.

3.8. Lactose Intolerance and Intestinal Microbiota

Another aspect of special relevance for the understanding of lactase phenotypes is the human microbiota, which has been described to elicit a direct influence on the appearance, or amelioration, of lactose intolerance clinical symptoms [ 104, 105]. This relationship results from the ability of human colonic microbiota (the sum of all microorganisms present in the colon) and their genes (intestinal microbiome) to ferment undigested lactose. This fermentation ability varies widely among individuals, and depends mostly on the composition of each colonic microbiota. This is important to the point that we may observe an amelioration or a worsening of lactose intolerance symptoms according to the abundance of certain strains of bacteria in the colon of individuals. For example, if colonic bacteria are efficient at fermenting lactose, then the lactose osmotic shock (which leads to diarrhea) will be reduced, but the quantity of gases might increase. In parallel, if methanogenic archaebacteria are prevalent, then carbon dioxide will be transformed mostly into methane, leading to constipation rather than diarrhea, as observed in 30% of intolerant individuals [ 106]. In addition, consumption of dairy products by LNP individuals can influence the colonic microbiota composition and lead to a reduction of intestinal symptoms [ 107, 108]. Based on the fact that intestinal microbiota can be modulated by supplementation, many studies have tried to induce a "healthy" microbiota in LNP subjects, in order to alleviate the signs and symptoms of lactose intolerance. As a result, a positive relationship has been found between probiotics and lactose intolerance, in relation to specific strains and concentrations [ 105]. Taking all this into consideration, diet-induced particularities in human microbiota could be an additional explanation for those discrepancies encountered in LP/LNP phenotypes and discussed in the present review.

4. Conclusions

Lactase phenotypes (LP and LNP) in humans present a complex genetic basis that has been widely investigated during the last decades. Although great advances have been achieved, the exact molecular mechanisms underlying the appearance of LNP after the weaning phase remain not fully understood. Probably, the combined action of transcription factors and epigenetics alterations on the LCT gene is the most plausible explanation. In the present review, we have summarized all proposed explanatory theories and gathered them in a figure (). The mechanisms responsible for LP, on the other hand, are known in better detail, and respond to the existence of cis-element mutations mapping a gene other than LCT; the regulatory enhancer region MCM6. Particularly, there have been a total of twenty-three SNPs within the MCM6 associated with LP so far in human populations. These variants seem to have arisen during the same period but independently in different human populations, the reason why LP has become a textbook example of convergent regulatory evolution and gene-culture co-evolution.

The distribution of lactase phenotypes, and their associated SNPs across human populations, has also been widely studied though not recently reviewed. All available information has always been presented in the form of static world maps or large-dimension tables, so that it would benefit from the newly available visualization tools, such as interactive world maps. In the present review, besides gathering all information available to date for LP phenotype and genetic frequencies, we have also created two online interactive resources ( http://bionit.ugr.es/pages/investigacion/software/bioinformatics-methods-software). As fully expandable and interactive graphics, our tools further constitute an upgrade over previously published static world maps, allowing users a personalized data exploration at the same time as accessing complete reports by population or ethnicity. Despite the great availability of data, we have detected that more studies are needed, especially for certain Central and South America regions, for which LP phenotype/genetics data lack entirely. Likewise, there is also a need for more genetics studies on the frequency of genetic variants other than the −14010:G>C (especially in non-Caucasian populations). Finally, and due to the existence of certain studies pointing to epigenetics as a plausible contributor to lactase phenotypes, it would also be interesting to carry out more population studies focused on the distribution of LCT epigenetics signatures associated with LP/LNP.

Concerning evolutionary genetics, LP is one of the strongest examples of positive selection found in the human genome. According to the latest studies, the evolutionary advantage conferred by LP alleles mainly lies in the lifetime access to nutrient-rich milk in populations that have traditionally practiced cattle domestication. However, there are still some discrepancies unexplained, as is the case of several ancient pastoral populations with a very low or null frequency of LP. As possible explanations, variability in milk fermentation practices and population admixture events have been proposed. On this matter, more nutritional anthropological studies investigating the amount, type, and seasonality of milk and dairy products consumed, and the perception of these foods in traditional populations would be helpful in understanding why some populations took up drinking fresh milk, whereas others mostly transformed it.

Human gut microbiome is, in a way, an extension of the human genome; as thousands of metabolic processes performed by members of the microbial community directly influence host physiology, including their host's ability to utilize lactose and other carbohydrates. Hence, further studies of utilization of lactose by the human microbiome are needed to explain discrepancies found in LP/NLP phenotypes.

Research works like the present review lay the foundations for the study of the genetic bases of lactase phenotypes in humans, and represent a new paradigm in the way of visualizing genetic and phenotype data at the population level.

# data[].attributes.attachment_link.title
Genetics of Lactose Intolerance: An Updated Review and Online Interactive World Maps of Phenotype and Genotype Frequencies

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Lactose is the main carbohydrate present in milk and one of the main sources of energy during the nursing period in mammals. To take advantage of milk lactose, mammals have to first hydrolyze it into glucose and galactose, monosaccharides that can be easily absorbed by the intestinal tract [ 1].

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Convergent adaptation of human lactase persistence in Africa and Europe

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Convergent adaptation of human lactase persistence in Africa and Europe

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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2672153/

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Nat Genet. Author manuscript; available in PMC 2009 Apr 23.

Published in final edited form as:

Sarah A Tishkoff, 1, ⁹ Floyd A Reed, 1, ⁹ Alessia Ranciaro, 1, ² Benjamin F Voight, ³ Courtney C Babbitt, ⁴ Jesse S Silverman, ⁴ Kweli Powell, ¹ Holly M Mortensen, ¹ Jibril B Hirbo, ¹ Maha Osman, ⁵ Muntaser Ibrahim, ⁵ Sabah A Omar, ⁶ Godfrey Lema, ⁷ Thomas B Nyambo, ⁷ Jilur Ghori, ⁸ Suzannah Bumpstead, ⁸ Jonathan K Pritchard, ³ Gregory A Wray, ⁴ and Panos Deloukas ⁸

Abstract

A SNP in the gene encoding lactase ( LCT) (C/T-13910) is associated with the ability to digest milk as adults (lactase persistence) in Europeans, but the genetic basis of lactase persistence in Africans was previously unknown. We conducted a genotype-phenotype association study in 470 Tanzanians, Kenyans and Sudanese and identified three SNPs (G/C-14010, T/G-13915 and C/G-13907) that are associated with lactase persistence and that have derived alleles that significantly enhance transcription from the LCT promoter in vitro. These SNPs originated on different haplotype backgrounds from the European C/T-13910 SNP and from each other. Genotyping across a 3-Mb region demonstrated haplotype homozygosity extending >2.0 Mb on chromosomes carrying C-14010, consistent with a selective sweep over the past ~7,000 years. These data provide a marked example of convergent evolution due to strong selective pressure resulting from shared cultural traits-animal domestication and adult milk consumption.

In most humans, the ability to digest lactose, the main carbohydrate present in milk, declines rapidly after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase (LPH). LPH is predominantly expressed in the small intestine, where it hydrolyzes lactose into glucose and galactose, sugars that are easily absorbed into the bloodstream1. However, some individuals, particularly descendants from populations that have traditionally practiced cattle domestication, maintain the ability to digest milk and other dairy products into adulthood. These individuals have the 'lactase persistence' trait. The frequency of lactase persistence is high in northern European populations (>90% in Swedes and Danes), decreases in frequency across southern Europe and the Middle East (~50% in Spanish, French and pastoralist Arab populations) and is low in non-pastoralist Asian and African populations (~1% in Chinese, ~5%-20% in West African agriculturalists)1⁻3. Notably, lactase persistence is common in pastoralist populations from Africa (~90% in Tutsi, ~50% in Fulani)1,3.

Lactase persistence is inherited as a dominant mendelian trait in Europeans1,2,4. Adult expression of the gene encoding LPH ( LCT), located on 2q21, is thought to be regulated by cis-acting elements5 ( Fig. 1 ). A linkage disequilibrium (LD) and haplotype analysis of Finnish pedigrees identified two single SNPs associated with the lactase persistence trait: C/T-13910 and G/A-22018, located ~14 kb and ~22 kb upstream of LCT, respectively, within introns 9 and 13 of the adjacent minichromosome maintenance 6 ( MCM6) gene4 ( Fig. 1 ). The T-13910 and A-22018 alleles were 100% and 97% associated with lactase persistence, respectively, in the Finnish study4, and the T-13910 allele is ~86%-98% associated with lactase persistence in other European populations6⁻8. Although these alleles could simply be in LD with an unknown regulatory mutation6, several additional lines of evidence, including mRNA transcription studies in intestinal biopsy samples9 and reporter gene assays driven by the LCT promoter in vitro 10⁻12, suggest that the C/T-13910 SNP regulates LCT transcription in Europeans.

It is hypothesized that natural selection has had a major role in determining the frequencies of lactase persistence in different human populations since the development of cattle domestication in the Middle East and North Africa ~7,500-9,000 years ago2,3,6,13⁻18. A region of extensive LD spanning >1 Mb has been observed on European chromosomes with the T-13910 allele, consistent with recent positive selection6,14,16⁻18. Based on the breakdown of LD on chromosomes with the T-13910 allele, it is estimated14 that this allele arose within the past ~2,000-20,000 years within Europeans, probably in response to strong selection for the ability to digest milk as adults.

Although the T-13910 variant is likely to be the causal variant for the lactase persistence trait in Europeans, analyses of this SNP in culturally and geographically diverse African populations indicated that it is present (and at low frequency (<14%)) in only a few West African pastoralist populations, such as the Fulani (or Fulbe) and Hausa from Cameroon15,19,20. It is absent in all other African populations tested, including East African pastoralist populations with a high prevalence of the lactase persistence trait19. Thus, the lactase persistence trait has evolved independently in most African populations owing to distinct genetic events15,19,20.

Here, we examine genotype-phenotype associations in 470 East Africans, and we identify three previously undescribed variants associated with the lactase persistence trait, each of which arose independently from the European T-13910 allele and resulted in enhanced transcriptional activity in LCT promoter-driven reporter gene assays. We demonstrate that the most common variant in Kenyans and Tanzanians spread rapidly to high frequency in East Africa over the past ~7,000 years owing to the strong selective force of adult milk consumption, and we show that chromosomes with these variants have one of the strongest genetic signatures of natural selection yet reported in humans.

RESULTS

Frequency of lactase persistence in East African populations

We classified individuals as having lactase persistence, lactase intermediate persistence (LIP) or lactase non-persistence (LNP) by examining the maximum rise in blood glucose levels after administration of 50 g of lactose using a lactose tolerance test (LTT)21 in 470 individuals from 43 ethnic groups originating from Tanzania, Kenya and Sudan. These populations speak languages belonging to the four major language families present in Africa (Afro-Asiatic, Nilo-Saharan, Niger-Kordofanian and Khoisan) and practice a wide range of subsistence patterns ( Fig. 2 and Supplementary Table 1 online). Because genetic substructure can result in false genotype-phenotype associations22, we analyzed data from samples separated by geographic region and language family, with the exception of the Sandawe and Hadza (both click-speaking Khoisan), whom we analyzed independently ( Fig. 2 ). We made these groupings to minimize population structure, based on a global analysis of ~1,200 unlinked nuclear markers (S.A.T. and F.A.R., unpublished data). The frequency of lactase persistence was highest in the Afro-Asiatic-speaking Beja pastoralist population from Sudan (88%) and lowest in the Khoisian-speaking Sandawe hunter-gatherer population from Tanzania (26%) ( Fig. 2a and Supplementary Table 1).

SNPs associated with lactase persistence in Africans

To identify SNPs associated with regulation of the lactase persistence trait, we sequenced 3,314 bp of intron 13 and 1,761 bp of intron 9 of MCM6 ( Fig. 1c,d ) in 40 LNP individuals and 69 lactase-persistent individuals at the extremes of the phenotype distribution ( Supplementary Fig. 1 online). A newly discovered SNP, G/C-14010, showed a significant association with the lactase persistence trait in Kenyans ( n = 53; χ ² = 14.4, d.f. = 2 and P = 0.0007) and Tanzanians ( n = 31; χ ² = 10.9, d.f. = 2 and P = 0.0043) ( Fig. 1d ). A second newly and discovered SNP, T/G-13915, was significantly associated with lactase persistence in Kenyans ( n = 53, d.f. = 1, χ ² = 4.70, P = 0.0302) and a third newly discovered SNP, C/G-13907, was marginally significantly associated with lactase persistence in the Beja population from northern Sudan ( n = 11, d.f. = 1, χ ² = 2.93, P = 0.0869) ( Fig. 1d ). Sequencing of these regions in a panel of great apes indicated that the C-14010, G-13915 and G-13907 alleles are derived.

In order to determine regional haplotype structure and further characterize the frequency and degree of association of these alleles, we genotyped 123 SNPs (including G/C-14010, T/G-13915 and C/G-13907) across a 3-Mb region flanking the MCM6 and LCT genes in the full set of 470 individuals with reliable phenotype data and in 24 additional individuals ( Fig. 1a and Supplementary Table 2 online). We determined the genotype-phenotype distribution and χ ² tests of association for our three candidate SNPs ( Fig. 3a-d ) with data partitioned according to classification of lactase persistence, LIP or LNP in major geographic regions. Additionally, we used a linear-regression approach23,24 (which accounts for the continuous phenotype distribution) to test for an association between all 123 SNPs and a rise in blood glucose after digestion of lactose. G/C-14010 is the most significantly associated SNP in the Kenyan Nilo-Saharan and Tanzanian Afro-Asiatic populations ( r ² = 0.19 and 0.16, and P = 2.67 × 10 −7 and 2.79 × 10 −4, respectively; Fig. 3e ) as well as in overall populations combined in the meta-analysis ( P = 2.9 × 10 −7; Fig. 3f ). Although C/G-13907 and T/G-13915 are associated with the phenotype, this association was not statistically significant after Bonferroni correction in either the individual populations or in the meta-analysis ( Fig. 3e,f ). The C-14010, G-13915 and G-13907 alleles in Africans exist on haplotype backgrounds that are divergent from each other and from the European T-13910 haplotype background ( Fig. 4 ).

Based on analysis of variance (ANOVA) of the phenotypes for each of the six classes of observed compound G/C-14010, T/G-13915 and C/G-13907 genotypes, ~20% of the total phenotypic variation is accounted for by the genotypes in the pooled sample, suggesting that there are environmental and/or measurement factors, and possibly unidentified genetic factors, influencing the LTT phenotype in this data set.

Frequency of G/C-14010, T/G-13915 and C/G-13907 in Africans

Genotype frequencies for G/C-14010, T/G-13915 and C/G-13907 are shown in Figure 2b , whereas Supplementary Table 1 gives allele frequencies for these SNPs as well as the European lactase persistence-associated SNPs C/T-13910 and G/A-22018. The T-13910 allele is absent in all of the African populations tested, and we observed the A-22018 allele in a single heterozygous Akie individual from Tanzania. The C-14010 allele is common in Nilo-Saharan populations from Tanzania (39%) and Kenya (32%) and in Afro-Asiatic populations from Tanzania (46%) but is at lower frequency in the Sandawe (13%) and Afro-Asiatic Kenyan (18%) populations and is absent in the Nilo-Saharan Sudanese and Hadza populations ( Fig. 2b and Supplementary Table 1). The C-13907 and G-13915 alleles are at ≥5% frequency only in the Afro-Asiatic Beja populations (21% and 12%, respectively) and in the Afro-Asiatic Kenyan populations (5% and 9%, respectively).

C-14010, G-13915 and G-13907 affect expression in vitro

In order to test whether the C-14010, G-13915 and G-13907 variants affect expression from the LCT core promoter, we transfected the human intestinal cell line Caco-2 with luciferase expression vectors driven by the basal 3-kb promoter alone or by the promoter fused to one of five haplotypes of the 2-kb MCM6 intron 13 region: one haplotype with ancestral alleles at the three candidate SNPs (G-14010, T-13915, C-13907), two haplotypes that differed only at the derived C-14010 or G-13915 alleles, one haplotype that differed at the derived G-13907 allele as well as at a linked T-13495 allele and one haplotype that has the ancestral lactase persistence-associated alleles, with a T at position -13945 (to control for the effect of this variant). Differences in luciferase expression between the basal 3-kb LCT core promoter and the promoter plus any of the five MCM6 intron sequence constructs were highly significant (paired t test, P < 0.001), resulting in more than a twenty-fold increase in expression over the core promoter alone ( Fig. 5 ).

Notably, we also observed differences in expression between the five MCM6 intron 13 haplotypes that were functionally tested using the dual-luciferase reporter assay ( Fig. 5 ). The C-14010-, G-13915- and G-13907-derived haplotypes consistently drove higher expression (from ~18%-30%) than the haplotypes with the ancestral alleles. There was no statistically significant difference in expression between the constructs with the C-14010, G-13907/T-13495 and G-13915 alleles.

Evidence for positive selection of the C-14010 allele

If a mutation provides a large enough benefit to its carriers (in this case, the ability to digest milk as adults), resulting in more viable offspring, it is expected to rise rapidly to high frequency in the population, together with linked variants (that is, genetic hitchhiking)25. Under neutrality, one expects common mutations to be older and to have lower levels of LD with flanking markers. In contrast, one of the genetic signatures of an incomplete selective sweep is a region of extensive LD (termed extended haplotype homozygosity or EHH) and low variation on high-frequency chromosomes carrying the derived beneficial mutation relative to chromosomes with the ancestral allele17,26. Over time, this pattern will degrade owing to recombination and newly occurring mutations. Thus, by measuring the frequency of the haplotype and extent of LD in the region, it is possible to estimate the age and strength of a beneficial mutation.

In order to visually assess the evidence for selection on chromosomes with the C-14010 allele, we constructed plots depicting EHH for ancestral (G) and derived (C) alleles using both unphased data ( Fig. 6 ), as well as phase-inferred data ( Fig. 7 ). For the unphased data, we plotted continuous homozygosity at each of the 123 genotyped SNPs for individuals homozygous for the ancestral (G/G-14010) and derived (C/C-14010) alleles ( Fig. 6a ). For comparison, we plotted EHH for the 101 SNPs genotyped in Eurasians14 for individuals homozygous for the ancestral (C/C-13910) and derived (T/T-13910) lactase persistence-associated alleles ( Fig. 6b ). The average homozygous tract length in C/C-14010 homozygotes ( n = 51) was 1.8 Mb (with a maximum of 3.15 Mb), compared with 1,800 bp in G/G-14010 homozygotes ( n = 228). In Eurasians, the average homozygous tract length in T/T-13910 homozygotes ( n = 61) was 1.4 Mb (with a maximum of 2.1 Mb), compared with 1,900 bp in C/C-13910 homozygotes ( n = 38). We observed a similar result in the individual African populations using phase-inferred data, with EHH extending as far as 2.18-2.90 Mb (1.6-2.2 cM) ( Table 1 and Fig. 7 ). Chromosomes with the G-13907 and G-13915 alleles show EHH spanning ~1.4 Mb (0.56 cM) and 1.1 Mb (0.37 cM), respectively ( Supplementary Fig. 2 online).

Table 1

Dominant model

Additive model

Population

Sample size (n)

Frequency (C-14010)

iHS

P (simulated)

P (empirical)

Span (cM)

Span (Mb)

s (95% c.i.)

Age (years) (95% c.i.)

s (95% c.i.)

Age (years) (95% c.i.)

Tanzania-Niger-
Kordofanian

61

0.230

−2.61

<0.003

0.00032

2.22

2.90

0.077
(0.026-0.142)

2,778
(1,219-6,049)

0.097
(0.036-0.148)

4,075
(2,304-9,533)

The high frequency of the C-14010 allele and the very long stretch of homozygosity (>2 Mb) for haplotypes containing the C-14010 allele are consistent with the action of positive selection elevating this allele and the surrounding linked variation to high frequency. To test the neutrality of this SNP, we used a modification of the EHH test26, the integrated haplotype score (iHS)17 (sample sizes for G-13915 and G-13907 alleles were too small for sufficient power with the iHS test). For most populations, the iHS score was statistically more extreme relative to iHS scores for data simulated both under a neutral model with constant population size ( P < 0.002) and under an assortment of demographic population expansion and contraction models ( Supplementary Table 3 online). All populations had statistically more extreme scores relative to the empirical distribution of iHS scores observed in the Yoruban HapMap data, for alleles at matching frequency ( P < 0.05) ( Table 1 ). Furthermore, as predicted, the direction of the score was consistent with the action of positive selection on the lactase persistence-associated haplotype.

Age of variants and estimates of selection coefficients

We estimated the age of the C-14010 allele using coalescent simulations under a model incorporating selection and recombination27. The simulations assumed either an additive ( h = 0.5) or dominant ( h = 1) model for fitness (Supplementary Methods online) and were designed to match several aspects of the data, including SNP ascertainment and density, allele frequency, sample size, recombination profile and phase uncertainty17. We estimated selection intensity and ages by matching simulated data to the observed cM span and the observed frequency of the derived allele in each population. We estimated these values ( Table 1 ) and found extremely recent (within the last ~3,000-7,000 years; confidence interval (c.i.) 1,200-23,200 years ago) and strong ( s = 0.04-0.097, c.i. 0.01-0.15) positive selection in many African populations.

DISCUSSION

Role of G/C-14010, T/G-13915 and C/G-13907 in LCT expression

Although we cannot rule out the possibility that G/C-14010 is in LD with another causative SNP, our data suggest that G/C-14010 regulates LCT gene expression. First, this SNP shows significant statistical association with the LTT phenotype in Kenyan and Tanzanian populations ( Fig. 3 ). Although most individuals with a C-14010 allele have moderate to high increases in blood glucose (mean of 2.04 and 2.45 mM in heterozygotes and homozygotes, respectively; Fig. 2b ), many individuals who are homozygous for the ancestral G-14010 allele are also LIP or lactase persistent ( Fig. 3 ), probably because of genetic heterogeneity of this trait, as discussed further below. Additionally, there is likely to be phenotype measurement error due to working in field conditions and to the relative insensitivity of the LTT test (see Methods). Also, individuals with the C-14010 allele may be classified as LNP if they have had damage to intestinal cells caused by infectious disease21.

Second, we observe extensive LD on chromosomes with the C-14010 allele, with haplotype homozygosity extending >2 Mb (Figs. ​(Figs.6 6 and ​and7). 7 ). Of the 123 SNPs genotyped, high LD ( D' > 0.9, LOD score ≥ 2) extends farthest for SNP G/C-14010 ( Supplementary Fig. 3 online) and is inconsistent with demographic models that incorporate even extreme bottlenecks. In fact, this region of haplotype identity, spanning 2.18-2.9 Mb (1.6-2.2 cM), is more extensive than any span of identity derived from HapMap data from global populations16,17. These results suggest that chromosomes with the C-14010 allele have rapidly risen to high frequency in East African populations owing to strong positive selection, consistent with a functional role of this variant. Last, analyses of transcriptional regulation of the LCT promoter in vitro indicate that otherwise identical constructs with a C-14010 allele consistently produce ~18% more luciferase than constructs with the G-14010 allele ( Fig. 5 ), an increase in transcription similar to that observed for the T-13910 allele in Europeans10,11 (Supplementary Discussion online).

We have also identified two additional variants, G-13907 and G-13915, at ≥5% frequency only in the Afro-Asiatic-speaking Beja from Sudan and in Afro-Asiatic-speaking Kenyans, that are on haplotype backgrounds that increase gene expression by ~18%-30% compared with the ancestral haplotypes ( Fig. 4 and Supplementary Discussion). Although SNPs T/G-13915 and C/G-13907 are associated with a mean rise in blood glucose of 3.18 and 3.99 mM in heterozygotes, respectively ( Fig. 2b ), these associations are not significant in the subpopulations or in the meta-analysis ( Fig. 3 ), possibly because of small sample size and loss of power for these SNPs. Additionally, chromosomes with the G-13907 and G-13915 alleles show EHH spanning ~1.4 Mb and ~1.1 Mb, respectively ( Supplementary Fig. 2). Although these results suggest that G-13915 and G-13907 are probable candidate LCT regulatory mutations, larger sample sizes from populations containing these alleles are required to test for an association with the lactase persistence trait and to rule out the possibility that they are simply in LD with a different causal SNP. Identification of transcription factors that bind to the sites of the C-14010, T-13915 and G-13907 variants would also be informative for clarifying the possible role of these variants in regulating LCT expression.

Adaptive significance and the origins of pastoralism

Archeological evidence suggests that cattle domestication originated in southern Egypt as early as ~9,000 years ago but no later than ~7,700 years ago and in the Middle East ~7,000-8,000 years ago28, consistent with the age estimate of ~8,000-9,000 years (95% c.i. ~2,200-19,200 years) for the T-13910 allele in Europeans. The more recent age estimate of the C-14010 allele in African populations, ~2,700-6,800 years (95% c.i. ~1,200-23,000 years), is consistent with archeological data indicating that pastoralism did not spread south of the Sahara and into northern Kenya until ~4,500 years ago and into southern Kenya and northern Tanzania ~3,300 years ago28,29. The ability to digest milk as adults is likely to be adaptive owing to the increased nutritional benefits from milk (carbohydrates as well as fat, protein and calcium) and also because milk is an important source of water in arid regions2,28,30,31. Considering the symptoms of lactose intolerance, which includes water loss from diarrhea, individuals who had the lactase persistence-associated alleles and could tolerate milk could have had a very strong selective advantage2. This is supported by our high estimates for the selection coefficient ( s = 0.035-0.097). Because the selective force, adult milk consumption, is associated with the cultural development of cattle domestication, the recent and rapid spread of the lactase persistence-associated alleles, together with the practice of pastoralism in East Africa, is an excellent example of ongoing adaptation in humans32 and coevolution of genes and culture3.

We observe the oldest age estimates of the C-14010 allele, ~6,000-7,000 years (95% c.i. ~2,000-16,000 years), in the Kenyan Nilo-Saharan and Tanzanian Afro-Asiatic populations ( Table 1 ). We also observe an old age estimate in the Tanzanian Sandawe, but its low frequency suggests it was introduced via recent gene flow (Supplementary Discussion). However, we cannot distinguish with certainty whether this allele first arose in the Cushitic-speaking Afro-Asiatic populations, who are thought to have migrated into Kenya and Tanzania from Ethiopia ~5,000 years ago33 and practice a mixture of agriculture and pastoralism, or in the Nilotic-speaking Nilo-Saharan populations, who are thought to have migrated into Kenya and Tanzania from southern Sudan within the past ~3,000 years33 and are strict pastoralists28. These results are consistent with both linguistic34 and genetic data (F.A.R. and S.A.T., unpublished data) indicating cultural exchange and genetic admixture between these groups. The absence of C-14010 in the southern Sudanese Nilo-Saharan-speaking populations suggests that this allele either originated in or was introduced to the Kenyan Nilo-Saharan populations after their migration from southern Sudan. Regardless of the population origins of the C-14010 allele, it spread rapidly throughout the region along with the cultural practice of pastoralism, consistent with a demic diffusion model of genetic and cultural expansion35.

Implications for identifying disease-associated variants

It has been hypothesized that genetic variants associated with both mendelian diseases (such as sickle cell anemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency) and common complex diseases (such as hypertension, diabetes, obesity and asthma) may be at high frequency in modern populations because they were adaptive in ancient environments16,17,36⁻38. Thus, identification of loci that are targets of natural selection could be informative for identifying disease-risk alleles. The rapid increase in frequency of geographically restricted lactase persistence-associated alleles is an example of local adaptation that would have been missed by studying other African populations, such as the Yoruba, which do not show a signature of selection at LCT in the HapMap data set16,17. Because of the possibility that disease-associated alleles may also be geographically restricted owing to recent, local adaptation, these results suggest the importance of resequencing analyses in multiple populations, even from within a single geographic region such as Africa.

Our study also indicates how challenging it may be to identify alleles that are targets of selection. Networks of the 98-kb region encompassing the LCT and MCM6 genes ( Fig. 4 ) indicate several haplotypes that are at high frequency in global populations and that have ancestral alleles at the lactase persistence-associated SNPs (that is, haplotypes D and E) ( Fig. 4 ). Based on a single-factor ANOVA test, neither of these haplotypes is significantly associated with the lactase persistence phenotype ( P = 0.20 and P = 0.058, respectively). The only difference between lactase persistence-associated haplotype F and the ancestral haplotype E is the single G→C substitution at position 14010. The presence of these globally common haplotypes that are identical over at least 98 kb raises the possibility that there have been additional selective sweeps in the LCT- MCM6 gene region, possibly unrelated to LCT gene expression and confounding the haplotype-based inference of selection at LCT (Supplementary Discussion).

Convergent evolution of LP-associated variants

These data suggest that at least two, and probably four or more, distinct causal variants associated with lactase persistence (T-13910 in Europeans and C-14010, G-13907 and G-13915 in Africans) have evolved independently in European and African populations owing to convergent evolution in response to a strong selective force, adult milk consumption. These variants arose on highly divergent haplotype backgrounds that are geographically restricted ( Fig. 4b and Supplementary Discussion), but they do not account for all of the phenotypic variation, particularly in the Nilo-Saharan Sudanese and Hadza ( Fig. 2 ). Therefore, it is likely that there are additional lactase persistence-associated variants in Africans.

Notably, the Hadza population of Tanzania, who speak a click language and subsist by hunting and gathering, have the lactase persistence phenotype at ~50% frequency ( Fig. 2a ), suggesting that either the Hadza descend from a pastoralist population or that the lactase persistence trait may be adaptive for something other than milk digestion (Supplementary Discussion). These results, which should be confirmed in a larger sample, add to the mystery of the origins of the Hadza and their relationship to other click-speaking populations in Africa.

In conclusion, multiple independent variants have allowed various human populations to quickly modify LCT expression and have been strongly adaptive in adult milk-consuming populations, emphasizing the importance of regulatory mutations in recent human evolution39. Further resequencing and genotype-phenotype analyses in Africa, particularly in populations that lack the C-14010 allele, will be necessary for identifying additional lactase persistence-associated variants. Once these variants are identified, genotype analyses in a broader set of African populations will be informative for reconstructing an even more complete history of adaptation to pastoralism in Africa.

METHODS

DNA samples

Tanzanian DNA samples were collected from individuals residing in the Arusha and Dodoma provinces of Tanzania. Kenyan samples were collected in the Rift Valley, Nyanza and Eastern provinces of Kenya. Sudanese samples were collected in the Khartoum and Kasala provinces of the Sudan. Institutional Review Board approval for this project was obtained from the University of Maryland at College Park. Written informed consent was obtained from all participants, and research permits from the Tanzanian Commission for Science and Technology, Tanzanian National Institute for Medical Research, the Kenya Medical Research Institute and the University of Khartoum were obtained prior to sample collection. Samples were grouped according to self-identified ethnolinguistic ancestry from unrelated individuals. Ethnic groups, number of individuals sampled, language classification and subsistence classification are given in Supplementary Table 1. White cells were isolated in the field from whole blood using a modified salting-out procedure40, and DNA was extracted in the laboratory using a Purgene DNA extraction kit (Gentra).

Phenotype test

The LTT measures elevation in blood glucose levels after consumption of 50 g of lactose (equivalent to ~1-2 l of cow's milk)21. Blood was obtained via a finger prick and baseline glucose levels were measured by an Accucheck Advantage glucose monitor and Accucheck Comfort Strips (Roche). Blood glucose levels were obtained 20, 40 and 60 min after consumption of 50 g of lactose (Quintron) dissolved in 250 ml water. Based on manufacturer recommendation, glucose values were adjusted based on previously determined error associated with use of the Comfort Strip Curves according to the following regression equation: y = 0.985 x - 7.5, where x is the measured glucose value. We determined the maximum rise in glucose level compared with baseline values. We used the following definitions to classify individuals: an individual with a rise of >1.7 mM was classified as 'lactase persistent'; one with a rise of <1.1 mM was classified as 'lactase non-persistent' and one with a rise of 1.1-1.7 mM was considered ambiguous and classified as 'lactase intermediate persistent'21. There is likely to be some error in phenotype classification owing to administration of the test under field conditions. The LTT test is less reliable than determining lactase enzyme activity directly by intestinal biopsy2,21, with a false negative rate (that is, lactase-persistent individuals being misclassified as LNP) as high as 23%-30% (ref. 21). Although more accurate indirect tests exist (such as determination of urinary galactose after inclusion of ethanol with the lactose load or a hydrogen breath test21), these were not feasible in remote locations in Africa. In addition, we were not able to ensure that participants had fasted for at least 8 h prior to administration of the test, as recommended in clinical settings2, although most participants indicated that they had not eaten for at least several hours prior to testing (Supplementary Methods).

Sequence analysis

A 3,314-bp region encompassing intron 13 of MCM6 and a 1,761-bp region encompassing intron 9 were amplified by PCR ( Fig. 1c,d ) in 110 individuals (69 lactase-persistent and 40 LNP): 16 lactase-persistent and 10 LNP from Sudan, 36 lactase-persistent and 17 LNP from Kenya and 17 lactase-persistent and 14 LNP from Tanzania (primers and PCR conditions are given in Supplementary Methods). PCR products were prepared for sequencing with shrimp alkaline phosphatase and exonuclease I (US Biochemicals). All nucleotide sequence data were obtained using the ABI Big Dye v3.1 terminator kit and 3730 xl automated sequencer (Applied Biosystems). Sequence files were aligned and SNPs identified using Sequencher software (v. 4.0.5; GeneCodes).

SNP genotyping

We selected 146 SNPs for genotyping from ref. 14, dbSNP and the resequencing of introns 9 and 13 of MCM6 in the individuals listed above. All SNPs were genotyped in 494 samples. Following ref. 14, the SNPs were chosen to represent a large area on chromosome 2 but with increased density in the LCT and MCM6 gene regions ( Fig. 1a ). SNPs were also included that had previously been shown to be associated with lactase persistence in Europeans (C/T-13910 and G/A-22018) or that seemed to be associated with lactase persistence based on the initial resequencing screen described above. SNP assays were designed with SpectroDESIGNER software (Sequenom). SNP typing was performed with the Homogeneous Mass Extend assay (Sequenom) as described elsewhere41. Genotyping was carried out at a multiplex level of up to ten SNPs per well, and data quality was assessed by duplicate DNAs ( n = 7 in triplicate). SNPs with more than one discrepant call or those showing self-priming in the negative control (water) were removed. Finally, we removed SNPs with call rates below 70% and flagged markers that departed from Hardy-Weinberg equilibrium ( P < 0.001). A total of 123 SNPs (of which seven were monomorphic) passed quality control and were included in the final analysis; these included 79 SNPs from ref. 14, 34 SNPs from dbSNP and ten SNPs from resequencing (five from intron 9 and five from intron 13) ( Supplementary Table 2).

Genotype-phenotype association tests

We determined genotype-phenotype association for data binned into lactase-persistent, LNP and LIP classifications using a χ ² test. The degrees of freedom for the χ ² test are calculated as (number of phenotypes − 1) × (number of genotypes − 1). In cases where there were low expected cell counts (<5), cells were pooled to satisfy Cochran's guidelines42. Because the phenotype (rise in blood glucose) is a continuous trait, we also used a least-squares linear regression approach to test for significant genotype-phenotype associations24. This method avoids the loss of information that may arise from binning the phenotype into discrete categories. For each SNP, different homozygotes were assigned to values of 0 or 1, and heterozygotes were assigned an intermediate genotype value of 0.5 (assuming an additive model). Next, a linear regression was fit to the x-axis genotype values and y-axis phenotypes (rise in glucose). The resulting r ² and P values were recorded as measures of the degree of association. Because of the large amount of multiple testing (123 SNPs), a significant association was determined after applying a conservative Bonferroni P-value correction.

Combined population meta-analysis

In order to both gain statistical power and avoid the issues of population stratification, we conducted a meta-analysis on the results of the association tests in the individual geographic-linguistic populations. This was done by combining the P values for each SNP over k populations in an unweighted Z transform test according to the following equation43:

where Zi is the Z score of the standard normal curve corresponding to the P value from an individual population phenotype-genotype regression, and Z meta is the Z score for the combined meta-analysis. This method tests for a skew in the overall distribution of P values (from tests in individual populations) regardless of the significance of any individual test and allows us to regain some of the power that was lost by dividing the data into smaller groups.

ANOVA

A single-factor ANOVA was used to test for a significant difference in phenotypes between the two common haplotypes (D and E) in the LCT- MCM6 region ( Fig. 4a ) and all other haplotypes, after individuals carrying a C-14010 and/or a G-13907 and/or a G-13915 allele (or unknown genotypes at any of these three markers) had been removed. An ANOVA was also used to quantify the overall variation in phenotype measures explained by G/C-14010, T/G-13915 and C/G-13907; each of the ten compound genotypes found in the data set was treated as a category.

Homozygosity plots

To visualize the extent of homozygosity on chromosomes with the lactase persistence-associated alleles, individuals that were homozygous for the ancestral and derived alleles at G/C-14010 and C/T-13910 SNPs were selected and the extent of continuous homozygosity at each assayed SNP, in each direction, was plotted. Note that this is the actual measured homozygosity and thus is independent of haplotype phase estimation but sensitive to inbreeding.

Haplotype phase estimation

fastPHASE44 was used, with population label information, in order to estimate phased haplotype backgrounds.

Calculation of iHS scores

We calculated iHS scores as in ref. 17 for each subpopulation for all SNPs in the region. In calculating the scores, we used an interpolated recombination map estimated from the HapMap project Yoruba data set16. iHS scores were standardized using estimates of the mean and s.d. obtained via coalescent simulation under a variety of demographic models. These simulations were tailored to match the frequency spectrum, SNP density and recombination profile of the observed data. Alternative demographic models included either exponential growth or a bottleneck (which varied in onset, severity, duration and population size recovery after the bottleneck). We simulated 1,000 repetitions of each demographic model and calculated the distribution of iHS scores for sites matching the frequency (within 2.5%) as well as position of C-14010. Supplementary Table 3 contains a description of the demographic models (and results) and also gives empirical P-values that count the number of simulated iHS scores for each model that exceeded (that is, were more negative than) the observed iHS statistic. In addition, iHS scores were standardized empirically by comparison with the Yoruba HapMap data for alleles at the same frequency as C-14010.

Estimating selection intensity and sweep ages

We applied a rejectionsampling approach using the cM span surrounding the selected site to estimate selection intensity and ages of the candidate lactase persistence-associated mutations for each population45 (Supplementary Methods). Point estimates for the selection intensity and ages are presented, assuming an additive or fully dominant fitness effect. Although our model assumes constant population size, previous studies have demonstrated that for an allele that rapidly increases in frequency, population demographic history has only a modest effect on allele age estimates38,46.

Because of the way that SNPs were ascertained, the allele frequency spectrum departs from the expectation for DNA sequence data. To model the effect of ascertainment bias of SNPs selected for genotyping, we followed the approach in ref. 17 (Supplementary Methods). In addition, the observed data vary in SNP density, showing a dense central core region flanked by regions with lower SNP density (on average). To match this feature of the data, a secondary rejection step was applied such that the average SNP density for central and flanking regions (both left and right) matched the observed density. With respect to recombination, for each simulation we chose to exactly match the recombination map estimated from the data using the Li and Stephens algorithm47. For all populations, we calculated cM spans assuming the estimated population genetic map for the Yoruba HapMap data set16 and calculated those distances assuming the rates estimated from the deCODE genetic map48 across 40 Mb flanking this region on chromosome 2.

Network analyses

Haplotype networks were generated using the median-joining algorithm of Network 4.1.1.1 (ref. 49) for SNPs within the LCT and MCM6 gene regions from rs1042712 to rs309125, spanning 98 kb. The root was inferred assuming the chimpanzee allelic state at each SNP is ancestral.

Vector construction, transfection and expression assay

The LCT 'core' promoter, starting 3,083 bp upstream of LCT at position −3 of the transcription start site, was amplified by PCR using high-fidelity Phusion polymerase (Finnzyme). PCR products were then cloned and ligated into a pGL3-basic luciferase reporter (Promega). Constructs including intron 13 of MCM6 were assembled by cloning 2,035 bp, beginning at position −14354 relative to LCT, 5′ of the 'core' promoter. Caco-2 cells were then transfected with these constructs. We lysed cells 48 h after transfection and measured luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) and a Veritas Microplate Luminometer (Turner BioSystems). Transfections of cells were performed six times for control and 'core' promoters and 12 times for vectors with the intron from MCM6. The expression data were analyzed using paired t tests (Supplementary Methods).

ACKNOWLEDGMENTS

We thank K. Panchapakesan, E. King, S. Morrow and T. Severson for technical assistance. We thank E. Sibley and L.C. Olds for sharing advice and materials and T. Bersaglieri and J. Hirschhorn for sharing data. We thank S.J. Deo, P. Lufungulo, W. Ntandu, A. Mabulla, J.L. Mountain, J. Hanby, D. Bygott, A. Tibwitta, D. Kariuki, L. Alando, E. Aluvala, F. Mohammed, A. Teia and A.A. Mohamed for their assistance with sample collection. We thank A. Clark for critical review of the manuscript and for helpful suggestions and we thank L. Peltonen, N. Enattah and C. Ehret for discussion. We thank the African participants who generously donated DNA and phenotype information so that we might learn more about their population history and the genetic basis of lactase persistence in Africa. This study was funded by L.S.B. Leakey and Wenner Gren Foundation grants, US National Science Foundation (NSF) grants BSC-0196183 and BSC-0552486, US National Institutes of Health (NIH) grant R01GM076637 and David and Lucile Packard and Burroughs Wellcome Foundation Career Awards to S.A.T. K.P. and H.M.M. were funded by NSF grant IGERT-9987590 to S.A.T. F.A.R. was supported by US National Institutes of Health (NIH) grant F32HG03801. B.F.V. and J.K.P. were supported by NIH grant HG002772-1. The Institute for Genome Sciences and Policy of Duke University supported the work of C.C.B., J.S.S. and G.A.W. The Wellcome Trust supported the work of J.G., S.B. and P.D.

Footnotes

AUTHOR CONTRIBUTIONS

S.A.T. conceived and supervised the study. S.A.T., K.P., H.M.M., A.R., J.B.H., M.O., M.I., S.A.O., G.L. and T.B.N. were involved in DNA collection and phenotype testing. A.R. performed the resequencing and initial identification of association of candidate SNPs with the phenotype. S.A.T. and F.A.R. selected the SNPs to be genotyped and samples to test for gene expression. P.D., J.G. and S.B. performed the SNP design and genotyping. F.A.R. processed and phased the raw data and performed the genotype-phenotype association analyses, plots of haplotype homozygosity from unphased data, dominance estimates and pairwise plot of LD. B.F.V. performed, and J.K.P. co-supervised, the iHS test to detect positive selection and plots of haplotype homozygosity from phased data as well as rejection-sampling analyses to estimate age of alleles and selection parameters. H.M.M. constructed the haplotype networks. C.C.B., J.S.S. and G.A.W. built the expression constructs, carried out transcription assays and analyzed the results of expression assays. The paper was written primarily by S.A.T., with contributions from F.A.R., B.F.V., J.K.P., C.C.B., G.A.W. and P.D. The supplementary information was written by S.A.T. and F.A.R. with contributions from B.F.V., J.K.P., C.C.B., G.A.W. and P.D.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturegenetics/

Reprints and permissions information is available online at Published online at http://npg.nature.com/reprintsandpermissions/

Note: Supplementary information is available on the Nature Genetics website.

References

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19. Mulcare CA, et al. The T allele of a single-nucleotide polymorphism 13.9 kb upstream of the lactase gene ([[ LCT) (C-13.9kbT) does not predict or cause the lactase-persistence phenotype in Africans. Am. J. Hum. Genet. 2004;74:1102-1110. PMC free article] [ PubMed] Google Scholar]

23. Reed FA, Reeves RG, Aquadro CF. Evidence of susceptibility and resistance to cryptic X-linked meiotic drive in natural populations of [ Drosophila melanogaster. Evolution Int. J. Org. Evolution. 2005;59:1280-1291. [ PubMed] Google Scholar]

29. Ambrose S. Chronology of the Later Stone Age and food production in East Africa. J. Arch. Sci. 1998;25:377-391. [ Google Scholar]

31. Cook GC. Did persistence of intestinal lactase into adult life originate in the Arabian peninsula? Man. 1978;13:418-427. [ Google Scholar]

38. Tishkoff SA, et al. Haplotype diversity and linkage disequilibrium at human [ G6PD: recent origin of alleles that confer malarial resistance. Science. 2001;293:455-462. [ PubMed] Google Scholar]

42. Cochran WG. Some methods for strengthening the common chi-square test. Biometrics. 1954;10:417-451. [ Google Scholar]

# data[].attributes.attachment_link.title
Convergent adaptation of human lactase persistence in Africa and Europe

# data[].attributes.attachment_link.description
A SNP in the gene encoding lactase (LCT) (C/T-13910) is associated with the ability to digest milk as adults (lactase persistence) in Europeans, but the genetic basis of lactase persistence in Africans was previously unknown. We conducted a genotype-phenotype ...

# data[].attributes.headline
Development of a novel SNP assay to detect lactase persistence associated genetic variants

# data[].attributes.subject
Development of a novel SNP assay to detect lactase persistence associated genetic variants

# data[].attributes.body
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8572811/

# data[].attributes.attachment_link.readable_text
Molecular Biology Reports

Pasquale De Luca, ¹ Daniela Iaconis, ¹ Elio Biffali, ¹ Coluccia Enza, ² Laura de Magistris, ² Gabriele Riegler, ² Diego Pappalardo, ² Maria Rosaria Amato, ² Patrizia Iardino, ³ Concetta Montanino, ⁴ and Bruna De Felice ⁴

Abstract

Background

In adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs). The present research was aimed to develop a novel genetic method to test lactase non persistence more powerfully.

Methods and results

In our study, we selected eight different SNPs that are associated with lactase persistence from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations to set up an approach to detect all the eight different SNPs at the same time in the same sample. This technique is centred on the identification of SNPs with a single nucleotide primer extension method using Sanger sequencing and capillary electrophoresis.

Conclusions

Our method allowed us to check the genotype asset of eight SNPs related to lactase persistence simultaneously and in a very efficient manner. It could be applied to a higher number of SNPs in a single reaction.

Keywords: SNP, Lactase, Fingerprint, Genomic

Introduction

The most common worldwide food adverse reaction is Lactose intolerance. It is the consequence of the lack or decreased activity of the enzyme lactase, expressed as a member of the brush border on top of microvilli in the small intestine. After the intake of dairy food, individuals bearing such a condition show clinical symptoms such as cramping, abdominal pain, flatulence, diarrhoea, nausea and vomiting. Symptoms are the consequence of bacterial fermentation in large intestine, where free lactose becomes a substrate metabolised into lactic acid, methane, hydrogen and other products [ 1, 2].

Primary lactose intolerance (adult acquired hypolactasia) is a natural process, involving a gradual disappearance of the activity of Lactase-phlorizin hydrolase (LPH) in the small intestinal brush border, starting between the age of 1-5 years, progressively declining after weaning [ 3], and depending on the ethnic group. Such phenomenon is very frequent among Asians, in the Mediterranean, and among black individuals, in 60% to 100% of the adult population.

Persistence (LP) or non-persistence (LNP) of lactase expression into adult life is a polymorphic trait that has been initially attributed to the single nucleotide polymorphism C/T-13910 (rs4988235) in an enhancer element 13.9 kb upstream of the lactase gene on chromosome 2 [ 4], specifically in a sequence known to be target for the transcription factor Oct-1. The − 13910*T allele occurs at very high frequency in northern Europeans and increases the affinity of the surrounding sequence for Oct-1 [ 5], providing a possible mechanism for up-regulation of LCT expression in lactase persistence. It is located in intron 13 of an adjacent gene, MCM6 (minichromosome maintenance 6). Later, other SNPs have been identified within the same enhancer region. Two of them, the abovementioned C/T -13910 (rs4988235) and G/A -22018 (rs182549), showed complete co-segregation with LNP/LP trait in Europeans [ 4]. The relation between the C/T -13910 and G/A -22018 polymorphisms and the incidence of hypolactasia is well known.

A decrease in lactase activity, progressing with age (LNP), is generated by genotypes -13910CC and -22018GG, while lifelong high lactase activity (LP) is produced by the presence of -13910CT and -13910TT as well as -22018GA and -22018AA genotypes. Recently, the role of DNA methylation in LNP/LP has also been reported [ 6].

Age gradual decrease in lactase activity can be associated to increasing methylation. Research suggests that LNP haplotypes containing the -13910*C allele accumulates modified cytosines that silence the regulatory elements in LCT rather than the haplotype containing the -13910*T allele. Age specific down-regulation and inter-individual variation of lactase activity in different populations may explain the epigenetic aging. Therefore, the epigenetic clock to regulate lactase expression is established by individual genetic background. Other genetic polymorphisms are also significant in other ethnic groups [ 4]. C/T -13914 (rs773131166) has been identified in some East Europeans [ 7], G/C -13907 (rs41525747) and A/C -13915 (rs41380347) in Middle East populations [ 8, 9], A/G -13913 (rs41456145) and C/G -14010 (rs145946881) are present in African populations and G/A -13908 (rs4988236) in Far East Asians [ 10]. These results suggest that LP alleles have emerged independently in several geographic/ethnic groups, a phenomenon most likely driven by recent positive selection due to the amount of dairy milk products used as food supply, as well as demographic factors (for example, migration) [ 11].

In countries in which the population is a mix of different ethnic groups, a method capable of testing all the possible SNP variations in one single reaction could be standardized and used, so that there is no need to tailor the investigation on patients' origin.

To date, several diagnostic procedures are available to test lactose intolerance. The most common is Lactose breath test (LBT) that is a specific application of breath testing based on gaseous production from intestinal microflora [ 12]. LBT is highly specific and it has been considered the golden standard for the diagnosis of lactose intolerance; it however requires the patients to spend at least 4 h in the hospital; moreover, patients have to prepare themselves along the previous days. Genetic testing is a standard technique for the diagnosis of primary adult lactase persistence; it is a quick test, based on DNA extraction, usually from an EDTA blood sample or buccal swab [ 13], followed by real-time PCR protocol for the detection of a single SNP per reaction and Sanger sequencing, that is the most used method [ 14- 17]. Other approaches have recently been developed, based on different techniques, like pyrosequencing [ 18], melting curve analysis [ 19], or loop-mediated isothermal amplification and melting curve analysis [ 20]. All these methods allow the simultaneous detection of several variants in the same reaction, although with different efficiencies. To recognize a secondary lactase deficiency can be crucial the contemporary administration of LBT and genetic testing [ 2, 21].

Secondary lactase deficiency is the consequence of many conditions leading to either a reduction of absorptive capacity or down-regulation of lactase expression in the small intestine, such as severe malnutrition, mucosal damage due to celiac disease and Inflammatory bowel diseases (IBD), Small bowel bacterial overgrowth (SIBO), etc. [ 22]. A secondary lactase deficiency is often reversible, when the cause is removed.

The present research was aimed to develop a novel genetic method to test lactase persistence more efficiently than the usual above-mentioned real-time PCR approach. This technique is based on the identification of SNPs with a single nucleotide primer extension method based on the principles of Sanger sequencing and capillary electrophoresis.

In this study, we selected eight different SNPs that are associated with lactase persistence [ 7- 10] from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations (see Table ​Table1) 1 ) and are often very close to each other (some of them are adjacent in the genomic sequence). We set up an approach to detect all the eight different SNPs at the same time on the same sample.

Table 1

SNP

Variant

Population

SNP primer

TM

Name

Materials and methods

The study was approved by the Ethics committee of University of Campania Luigi Vanvitelli. All enrolled subjects provided informed consent to participate.

Patients undergoing a Lactose breath test (LBT) for suspected lactose intolerance, over a twelve months period, were examined from Gastroenterology Unit of Azienda Policlinico Università della Campania L. Vanvitelli. 125 compliant subjects were enrolled, 75 females and 50 males mean age ± SD: 40.3 ± 14.8.

4 mL of peripheral whole blood was collected to perform genetic testing to the enrolled patients. Exclusion criteria were: age < 18 and > 75 years, major pathological and/or metabolic diseases and pregnancy.

DNA extraction

Genomic DNA was extracted from EDTA anticoagulated blood with the QIAmp Mini Spin Column (QIAGEN, Hilden, Germany), following manufacturer's protocol.

Briefly, 20 µl of protease-k and 200 µl of lysis buffer were added to 200 µl of sample (whole blood). The tube was vortexed for 15 s and incubated at 56 °C for 10 min, followed by centrifugation at 5000 rpm for 1 min. 200 µl of Ethanol were added and mixed for 15 s. After short centrifugation, the content of each tube was pipetted into the corresponding microfilter, and then centrifuged at 8000 rpm for 1 min. The flow-through was discarded and the micro filter washed with 500 µl of washing solution A, then with 500 µl of solution B. Genomic DNA was eluted in 100 μl of milliQ H ₂ O.

Quantity and quality of the samples were measured with the Nanodrop LITE spectrophotometer (Thermo Scientific).

DNA amplification and SNP analysis

Two primer pairs were designed to amplify the regions of interest in the lactase enhancer:

MDM12F- ACTACTCCCCTTTTACCTCGTT, MDM12R- TCTGTTTATCTCTGCTCTCATCAT amplify a 400 bp region centered on the − 13910 position (rs4988235), and MDM22F- AGCTGGGACCACAAGCAC, MDM21R-CATTATCAGCCAACATCAAAGC amplify a 250 bp region surrounding the − 22018 position (rs182549).

The PCR reaction was conducted in a final volume of 25 μl with 0.5 μM of each primer and 1 unit of Taq Expand High Fidelity PCR System (Roche); the PCR protocol was 95 °C 3ʹ, then 35 cycles of 95 °C 30″, AT 30″, 72 °C 45″, followed by a final elongation step at 72 °C for 5ʹ. Annealing temperature was 56 °C for the MDM12F/12R primer pair and 58 °C for MDM22F/MDM21R primer pair; 0.5% DMSO were added in the MDM22F/MDM21R reactions.

For each sample, 5 μl of each PCR product were mixed and purified with ExoSap (Thermo Fisher), following manufacturer's protocol. The DNA concentration for each PCR product was close to 10 ng/μl.

The following primers were designed for SNP detection (Table ​(Table1 1 ):

A GACT repeat was added to each primer to obtain the desired length. SNPs reaction was conducted with the SNaPshot Multiplex Kit (Thermo Fisher) following manufacturer's protocol, with a final concentration of 0.2 μM for each primer (0.4 μM for the primers SNPMDM4 and SNPMDM5). Reactions were treated with Calf Intestinal Alkaline Phosphatase (CIAP, Thermo Fisher). 2 μl of each reaction was mixed with 0.2 μl GeneScan™ 120 LIZ™ dye Size Standard (Applied Biosystems), then loaded onto the 3730 DNA Analyzer (Thermo Fisher) following manufacturer's instructions. Results were analysed with the GeneMapper 5 software (Thermo Fisher).

DNA sequence analysis

Sequencing of the amplicons was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), with the same primers described previously, in a 3730 DNA Analyzer (Thermo Fisher). The sequences were matched using BLAST ( https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results

Lactose intolerance was investigated in 125 individuals with a SNP analysis with the goal of developing a more reliable genetic test to evaluate lactase persistence. All the selected individuals were originally from Southern Italy and, due to the domination history of this region, we could not exclude a priori the presence of SNPs of different ethnic origin. The locus region of the LCT gene was analysed and, with the goal of obtaining a widely relevant genetic test, eight SNP previously reported to be associated with lactase persistence in different populations were identified (Fig. 1 ).

A primer for each of them was designed, with a pairing region of 18-29 nucleotides preceded by a neutral GACT repetition, so that each final primer differed in length from the others of at least five nucleotides (Table ​(Table1), 1 ), ranging from 36 to 71 bases in length. We tested all the primers in individual reactions with 2 μl of each purified PCR product to assess their performance before the setup of the multiplex assay with the SNaPshot multiplex kit (ThermoFisher) on the 3730 DNA Analyzer (ThermoFisher). We had to slightly increase the DNA amount for the multiplex reaction (5 μl of each PCR product) and, after testing different primer concentrations, we decided to double the amount of SNPMDM4 and SNPMDM5. We then performed a SNP analysis on all the 125 samples. With this approach we observed no variation for the positions − 13907, − 13908, − 13913, − 13914, − 13915 and − 14010, as expected in a caucasic population. Six subjects showed a C/T − 13910 heterozygous variation, seven were heterozygous C/T in position − 22018; 19 individuals were heterozygous in both positions (see Fig. 2 for representative results). We didn't observe any homozygous variation in − 13910 nor in − 22018 SNPs (Table ​(Table2 2 ).

Table 2

− 22018

− 13910

GG

GA

AA

CC

93

6

0

(0%)

(4.8%)

(74.4%)

CT

7

19

0

(0%)

(15.2%)

(5.6%)

TT

0

0

0

(0%)

(0%)

(0%)

In order to demonstrate the strength of our approach we decided to Sanger sequencing all the samples, to confirm the SNaPshot results in each case (Fig. 3 ). All the genotypes detected were confirmed by sequencing.

Discussion

Lactose intolerance is widely recognized as one of the most common food adverse reactions. Although the hydrogen breath test is considered a golden standard to diagnose lactose intolerance, it poses some disadvantages, the most important being that it is not possible to distinguish between a primary or a secondary intolerance with this approach. The genetic basis of Lactase persistence (LP) or Non-persistance (LNP) is now well known, so genetic testing is becoming more and more used to obtain a better diagnosis of lactose intolerance and its clinical value increases with age [ 23], being maximal in adulthood. A bias in this approach is the presence of different SNPs in the enhancer region of the lactase gene, strictly associated with the ethnicity of individuals [ 24]. The usual genetic test, the detection by a real time PCR approach, commonly investigates one SNP per reaction, implies the knowledge of the ethnicity of each individual. To overcome this limitation, we developed a SNP assay to investigate different genetic variants associated with lactase persistence at the same time in the same reaction. We tested 125 individuals and confirmed all the obtained results by Sanger sequencing, finding 100% congruence.

In the modern, globalized world, more and more countries have their population composed of people from different geographical areas. Studies of the genetic predisposition to lactase persistence showed a strong association between the ethnicity and a given polymorphism. Our work demonstrated that the SNP approach, while as informative as the Real Time PCR technique, has the advantage of a single standardized reaction applicable to all known lactase polymorphisms independently of the ethnicity of the patients.

Lactase persistent phenotype is incredibly variable globally. In Europe, a North-South distribution can be detected; in fact, 90% of adults can digest lactose in Scandinavian countries, however, lactose tolerant adults are less than 50% in Mediterranean. In Italy, frequencies of 62, 3% non-persistent genotypes while in southern Italy 67, 1% have been recorded [ 25, 26].

The observed percentage of non-persistent genotypes in this study is higher (i.e.: 74, 4%) than the above reported values, possibly because patients were already suspected to have lactose intolerance and enrolled during a lactose intolerance screening.

LCT genotyping could replace or complement hydrogen breath testing, which is laborious for both patients and physicians, and can be affected by age, and other minor causes as celiac disease [ 27]. Moreover, beside venous blood used here, LCT genotyping could be performed using buccal swab procedure that is suitable for large population-based genetic studies [ 28] and saliva, which has been shown to be an excellent source of human genomic DNA [ 29]. Checking saliva we can obtain a less invasive method compared to blood or cotton swab samples.

The traditional Real Time PCR approach allows the detection of a single SNP per reaction, but it is increasingly interesting the development of techniques that permit the simultaneous detection of different SNPs in one reaction, especially in countries target of human migratory flows. Other methods have already been developed, all having advantages and disadvantages. Pyrosequencing [ 18] is efficient, but it is useful only for SNPs that are within a short distance from each other, it would not be possible to analyse, for example, positions − 13910 and − 22018 in the same reaction. Melting curve analysis [ 19] and loop-mediated isothermal amplification and melting curve analysis [ 20] are easier than sequencing or SNPs detection, but they often do not resolve some different genotypes, and they both request the use of standard sequences to identify the correct SNPs in genomic DNAs [ 19, 20].

We think that the SNP Assay that has been validated in this study represents a valuable tool for routine diagnosis, especially if applied to saliva genomic DNA, and even if it requires DNA extraction and an automated sequencer.

In fact the combination of eight different SNP allows the accurate molecular typing of multiple known LCT variants and overcomes diagnostic pitfalls reported for other methodologies. It is worth to underline that the potential of this approach could be wider because more than eight SNPs could still be analysed in each sample setting up a single reaction tube per patient.

Acknowledgements

We are grateful to Mrs Elvira Mauriello and Mr Raimondo Pannone for their technical help in Sanger sequencing and SNPs runs.

Author contributions

PDL: Conceptualization, Investigation, Data curation, Writing - review and editing. BDF: Conceptualization, Writing - original draft. CM: Conceptualization, Investigation. DI: Investigation. CE, LdM, GR, DP, MRA, PI: Conceptualization, Investigation. EB: Conceptualization, Writing - review and editing.

Funding

Open access funding provided by Università degli Studi della Campania Luigi Vanvitelli within the CRUI-CARE Agreement.

Declarations

Conflict of interest

The authors declare that they have no competing interests.

Footnotes

Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Pasquale De Luca, Email: ti.nzs@aculed.elauqsap.

Bruna De Felice, Email: ti.ainapmacinu@ecilefed.anurb.

References

12. Gasbarrini A, Corazza GR, Gasbarrini G (promoters of the Consensus), et al. (Componenets of the expert study groups) (2009) Methodology and indications of H ₂-breath testing in gastrointestinal diseases: the Rome Consensus Conference. Aliment Pharmacol Ther 29 (Suppl. 1): 1-14 [ PubMed]

13. Adriaanse MPM, Vreugdenhil ACE, Vastmans V, Goeneveld L, Molenbroeck S, Shott DA, Voorter CEM, Tilanus MGJ. Human leukocyte antigen typing using buccal swabs as accurate and non-invasive substitute for venipuncture in children at risk for celiac disease. JGH. 2016;31:1711-1716. [[ PubMed] Google Scholar]

17. Enko D, Pollheimer V, Németh S, Pühringer H, Stolba R, Halwachs-Baumann G, Kriegshäuser G. Lactase non-persistence genotyping: comparison of two real-time PCR assays and assessment of concomitant fructose/sorbitol malabsorption rates. Clin Lab. 2016;62(4):727-730. [[ PubMed] Google Scholar]

18. Nilsson TK, Olsson LA. Simultaneous genotyping of the three lactose tolerance-linked polymorphisms LCT -13907C>G, LCT -13910C>T and LCT -13915T>G with Pyrosequencing[ TM technology. Clin Chem Lab Med. 2008;46(1):80-84. doi: 10.1515/CCLM.2008.015. [ PubMed] [ CrossRef] Google Scholar]

26. Zadro C, Dipresa S, Zorzetti G, Pediroda A, Menegoni F. Lactase non-persitent genotype distribution in Italy. Minerva Gastroenterol Dietol. 2017;63:264-269. [[ PubMed] Google Scholar]

# data[].attributes.attachment_link.title
Development of a novel SNP assay to detect lactase persistence associated genetic variants

# data[].attributes.attachment_link.description
Molecular Biology Reports Pasquale De Luca, 1 Daniela Iaconis, 1 Elio Biffali, 1 Coluccia Enza, 2 Laura de Magistris, 2 Gabriele Riegler, 2 Diego Pappalardo, 2 Maria Rosaria Amato, 2 Patrizia Iardino, 3 Concetta Montanino, 4 and Bruna De Felice 4 In adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs).

# data[].attributes.headline
Country, regional, and global estimates for lactose malabsorption in adults: a systematic review and meta-analysis

# data[].attributes.subject
Country, regional, and global estimates for lactose malabsorption in adults: a systematic review and meta-analysis

# data[].attributes.body
https://pubmed.ncbi.nlm.nih.gov/28690131/

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Affiliations

¹ Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway.

² Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway; Department of Clinical Dentistry, University of Bergen, Norway; Department of Addiction Medicine, Haukeland University Hospital, Bergen, Norway. Electronic address: lars.fadnes@uib.no.

Affiliations

¹ Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway.

² Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway; Department of Clinical Dentistry, University of Bergen, Norway; Department of Addiction Medicine, Haukeland University Hospital, Bergen, Norway. Electronic address: lars.fadnes@uib.no.

Abstract

Background: Studies have shown wide variation in the prevalence of lactose malabsorption across the world, but no systematic reviews or meta-analyses have recently assessed the prevalence of lactose malabsorption in different geographical areas. We aimed to present an updated systematic review and meta-analysis on the prevalence of lactose malabsorption in adults, by countries and regions, and to assess the variation between different testing methods.

Methods: Studies reporting on prevalence of lactose malabsorption and lactase persistence were identified by searching MEDLINE and Embase from database inception to Nov 2, 2016. We evaluated studies presenting lactose malabsorption or lactase persistence prevalence data in adults and children aged 10 years or older, including cross-sectional and prospective studies, using genotyping, hydrogen breath tests, lactose tolerance tests, and other testing methods. We excluded studies in children younger than 10 years, studies using self-reported data, and studies including inpatients and outpatients at gastroenterological wards. Studies were screened by two authors (CLS and SKF) and data values were extracted by two authors (CLS and SKF) independently. The primary outcome was the prevalence of lactose malabsorption. This study is registered with PROSPERO, number CRD42017064802.

Findings: We screened 2665 records, and 306 study populations from 116 full-text articles were included (primary sources); data for 144 additional study populations from 59 articles were obtained from review articles, because full-text primary articles could not be obtained (secondary sources). Of the 450 study populations included, 231 were assessed by genotyping, 83 by hydrogen breath tests, 101 by lactose tolerance tests, and 35 by other methods or methods that were not described sufficiently. The studies included 62 910 participants from 89 countries (covering 84% of the world's population). When standardising for country size, the global prevalence estimate of lactose malabsorption was 68% (95% CI 64-72), ranging from 28% (19-37) in western, southern, and northern Europe to 70% (57-83) in the Middle East. When assessing the global prevalence using genotyping data only, the estimate was 74% (69-80), whereas prevalence was 55% (46-65) using lactose tolerance test data, and 57% (46-67) using hydrogen breath test data. Risk of bias was assessed based on ten indicators; 12 of the articles had a score of ten, indicating low risk of bias, 76 had a score of nine, 26 a score of eight, and two articles a score of seven (indicating higher risk of bias). There was substantial heterogeneity between studies within most of the assessed countries.

Interpretation: Lactose malabsorption is widespread in most of the world, with wide variation between different regions and an overall frequency of around two-thirds of the world's population. Acknowledging regional patterns of lactose malabsorption is important to guide management of gastrointestinal symptoms.

Funding: None.

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# data[].attributes.attachment_link.title
Country, regional, and global estimates for lactose malabsorption in adults: a systematic review and meta-analysis - PubMed

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1 Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway. 2 Centre for International Health, Department of Global Public Health and Primary Care, University of Bergen, Norway; Department of Clinical Dentistry, University of Bergen, Norway; Department of Addiction Medicine, Haukeland University Hospital, Bergen, Norway.

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