MPP440807.indd E-Mail karger@karger.com Original Paper Med Princ Pract 2016;25:18–20 DOI: 10.1159/000440807 The Frequency of the LCT * –13910C>T Polymorphism Associated with Lactase Persistence Diverges among Euro-Descendant Groups from Brazil Stefanie Epp Boschmann   a Angelica Beate Boldt   a, b Ilíada Rainha de Souza   c Maria Luiza Petzl-Erler   b Iara Jose Messias-Reason   a   a   Laboratório de Imunopatologia Molecular, Hospital de Clínicas, Alto da Glória, and b   Laboratório de Genética Molecular Humana, Setor de Ciências Biológicas, Universidade Federal do Paraná, Curitiba , c   Laboratório de Polimorfismos Genéticos, Universidade Federal de Santa Catarina, Florianópolis, Brazil tiba displayed differences when compared to all other Brazil- ian groups (p < 0.0001), even to Euro-Brazilians from a differ- ent geographic region (p = 0.0003), but were similar to those from Porto Alegre (p = 0.2). Conclusion: Differences in the –13910 * T- associated lactase persistence distribution among Euro-Brazilian groups reflect the ancestry and admixture of each particular group and should be considered for adult- type hypolactasia screening. © 2015 S. Karger AG, Basel Introduction The lactase enzyme hydrolyzes lactose into galactose and glucose at the brush-border membrane of the small intestine. This enzyme is encoded by the lactase gene LCT located at chromosome 2q21, with 17 exons encoding an mRNA of 6,279 nucleotides [1, 2] . Although lactase ex- pression and activity are high in newborns, they become downregulated after weaning in approximately 65% of in- dividuals, causing adult-type hypolactasia or lactase non- persistence [3] . A lower capacity to hydrolyze lactose can result in gastrointestinal symptoms such as abdominal pain and distention, flatulence, diarrhea and vomiting, a condition known as lactose intolerance disorder [4] . A Key Words LCT gene · Lactase persistence · Hypolactasia · Euro-Brazilians · Mennonites Abstract Objective: The aim of this study was to investigate the fre- quency of the LCT * –13910C>T polymorphism associated with a high expression of lactase in the small intestine dur- ing adulthood, and to infer the lactase persistence and adult-type hypolactasia phenotypes among Euro-Brazilians and Mennonites from South Brazil. Materials and Meth- ods: A sequence-specific PCR method to genotype the LCT * –13910C>T polymorphism in 292 Euro-Brazilians and 151 Mennonites (a group with European ancestry and a long history of endogamy) was developed. Using an exact test of population differentiation, the genotype and allele frequen- cy between these and other Brazilian populations were com- pared. Results: The frequency of –13910 * T was significantly higher among the Mennonites when compared to the Euro- Brazilian cohort (0.63 vs. 0.33, p < 0.000001). Accordingly, Mennonites had a higher prevalence of the lactase persis- tence genotype (88.1 vs. 55.5%, p < 0.000001). The distribu- tion of –13910 * T differed between Mennonites and all other Brazilian groups (p < 0.0001). The Euro-Brazilians from Curi- Received: November 4, 2014 Accepted: September 2, 2015 Published online: November 14, 2015 Prof. Dr. Iara Jose Messias-Reason Laboratório de Imunopatologia Molecular, Hospital de Clínicas Universidade Federal do Paraná, Rua General Carneiro 181 Alto da Glória, Curitiba, PR 80035-050 (Brazil) E-Mail iarareason   @   hc.ufpr.br © 2015 S. Karger AG, Basel 1011–7571/15/0251–0018$39.50/0 www.karger.com/mpp Th is is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Un- ported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribu- tion permitted for non-commercial purposes only. http://dx.doi.org/10.1159%2F000440807 The Frequency of LCT * –13910C>T Med Princ Pract 2016;25:18–20 DOI: 10.1159/000440807 19 high prevalence of lactase persistence (LP) occurs in pop- ulations with a long history of pastoralism and milking, mainly in northern Europe and among nomads in Africa and the Middle East [3] . This phenotype is characterized by high levels of lactase during adulthood and a higher capacity to digest lactose. Enattah et al. [4] identified the major polymorphism responsible for this trait in Europe- ans, a C>T substitution in an enhancer region 13.9 kb upstream of the LCT gene (–13910C>T) , in intron 13 of the MCM6 gene. Other causative single-nucleotide poly- morphisms for LP occur in Africans and Middle-Eastern populations, all of them located in the same enhancer re- gion [3] . The –13910*T allele is associated with enhanced mRNA expression of the LCT gene, causing LP in adult- hood, even among heterozygotes, thus being inherited as a dominant trait [5] . Brazilian admixture has occurred since the Portuguese met different Amerindian tribes and Africans from An- gola and Mozambique, brought in as slaves between the 16th and 19th century. After the abolishment of slavery in 1850, the Brazilian government encouraged and sup- ported the immigration of Europeans. Although Euro- pean ancestry is predominant in Brazil, the range of ad- mixture diverges across the geographic regions of the country [6] . The Mennonites are a pacifist group of Ger- man and Dutch ancestry, well-suited for genetic and epi- demiological studies due to successive bottleneck effects caused by persecution in the 16th century, endogamy and successive migratory waves to the Soviet Union at the end of the 18th century and from there to North America, Paraguay and Brazil in the early 20th century [7] . Although LCT * –13910C>T has been indicated as a good genetic marker for adult-type hypolactasia in Eu- rope and in non-European countries [3, 8, 9] , its frequen- cy is unknown for Mennonites and is poorly estimated for Euro-Brazilians [8, 9] . Hence, the goal of this work was to estimate the prevalence of –13910C -associated adult-type hypolactasia among Mennonites and Euro-Brazilians from Curitiba (a major city of Paraná State) and to com- pare our results to those for other Brazilian groups. Port Alegre – Euro1 Curitiba – Euro3 Mennonites3 SP – Euro2 SP – Admixed2 Recife – Admixed1 Belém – Admixed1 68% SP – African* 2 Port Alegre – African** 1 48.6% 51.4% 55.5% 32% 88.1% 44.5% 11.9% 56.6% 56.9% 43.4% 43.1% 80% 20% 36.6% 63.4% 31% 69% RR AM AC RO MT PA AP MA PI CE RN PB PE AL SE ES RJ BA TO DF GO MS PR RS SC SP MG Fig. 1. Distribution of the –13910C>T- associated LP (dark gray) and hypolactasia (light gray) among different Brazilian groups. *  Admixed people of African ancestry were not included; * *  admixed people of African ances- try may have been included. 1   [8] ; 2   [9] ; 3  our study. http://dx.doi.org/10.1159%2F000440807 Boschmann/Boldt/de Souza/Petzl-Erler/ Messias-Reason Med Princ Pract 2016;25:18–20 DOI: 10.1159/000440807 20 Materials and Methods A total of 151 Mennonites (78 females and 73 males aged 18–89 years) and 292 Euro-Brazilians (150 females and 142 males aged 18–64 years) from South Brazil were included. The Euro-Brazil- ians were mainly blood donors from two blood banks of Curitiba. They were classified as ‘European-derived’ based on physical char- acteristics and self-reported ancestry. The project was approved by the Ethics Committee of the Hospital de Clínicas, Universidade Federal do Paraná. All participants were informed about the aims of the study and their written informed consent was obtained. Peripheral blood samples were collected with EDTA and nu- cleic acid (DNA) extracted and amplified as previously published [10] using a touchdown strategy (10 cycles at 64   °   C, 10 at 62   °   C and 10 at 60   °   C). A fragment of 431 bp of the human growth hormone gene was coamplified in each reaction [10] . The LCT * –13910C>T was identified using PCR sequence-specific (SSP) amplification of a 140-bp fragment with the forward primer LCTf (5 ′ -GTT- TATAGAGTGCATAAAGACG-3 ′ ) and the specific reverse prim- ers LCT–13910Cr (5 ′ -GTTCCTTTGAGGCCAGGGG-3 ′ ) or LCT–13910Tr (5 ′ -GTTCCTTTGAGGCCAGGGA-3 ′ ). Genotype and allele frequencies were obtained by direct counting and ana- lyzed using the Arlequin 3.5 software and Fisher’s exact test. Results Both groups were in Hardy-Weinberg equilibrium. The allele frequency of –13910 * T was almost twice as high in Mennonites (0.65) as in Euro-Brazilians (0.33). The genotypes for LCT * –13910T -associated LP, –13910C/T and T/T , were observed in 88% of Mennonites and in 55.5% of Euro-Brazilians (p < 0.000001). Conversely, the genotype for LCT * –13910C -associated adult-type hypo- lactasia ( –13910C/C ) was observed in 12% of Mennonites and in 44.5% of Euro-Brazilians (p < 0.000001). In other words, Mennonites had an almost 6 times higher chance of presenting the –13910T -associated LP than Euro-Bra- zilians [OR 5.93 (95% CI 3.44–10.21)]. The distribution of –13910 * T differed significantly between Mennonites, Euro-Brazilians and other Brazilian groups (p < 0.0001). The Euro-Brazilians were similar to those from Porto Alegre (p = 0.2), a city also located in South Brazil, but differed from all other Brazilian groups (p < 0.0001), even from Euro-Brazilians from a different geographic region (p = 0.0003; fig. 1). Discussion According to our results, across all Brazilian groups investigated, Mennonites had the highest frequency of the LCT * –13910C>T polymorphism and, accordingly, of the LP-associated genotypes, followed by Euro-Brazilians from South Brazil. The Mennonites differed from Euro- Brazilians who live in the same geographic area, which could be due to the founder effects suggested to have oc- curred among them [7] . The –13910 * T frequency was higher in Euro-Brazilians than in admixed and Afro-Bra- zilian groups. Thus, the allelic distribution of –13910 * T may reflect the ancestry and different admixture rates across various Brazilian geographical regions. Conclusion Differences in the –13910 -associated LP distribution across the various Brazilian groups are remarkable. They should thus be considered for adult-type hypolactasia screening as well as for dietary genetic counseling regard- ing milk consumption and digestive problems. Since there are other causative variants for LP, further studies are needed to investigate whether the distribution of the –13910 * T polymorphism is consistent with the distribu- tion of the LP phenotype in Brazil. 1 Boll W, Wagner P, Mantei N: Structure of the chromosomal gene and cDNAs coding for lac- tase-phlorizin hydrolase in humans with adult- type hypolactasia or persistence of lactase. 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