Nabil Sabri Enattah

Independent Introduction of Two Lactase-Persistence Alleles into Human Populations Reflects Different History of Adaptation to Milk Culture

The T(-13910) variant located in the enhancer element of the lactase (LCT) gene correlates perfectly with lactase persistence (LP) in Eurasian populations whereas the variant is almost nonexistent among Sub-Saharan African populations, showing high prevalence of LP. Here, we report identification of two new mutations among Saudis, also known for the high prevalence of LP. We confirmed the absence of the European T(-13910) and established two new mutations found as a compound allele: T/G(-13915) within the -13910 enhancer region and a synonymous SNP in the exon 17 of the MCM6 gene T/C(-3712), -3712 bp from the LCT gene. The compound allele is driven to a high prevalence among Middle East population(s). Our functional analyses in vitro showed that both SNPs of the compound allele, located 10 kb apart, are required for the enhancer effect, most probably mediated through the binding of the hepatic nuclear factor 1 alpha (HNF1 alpha). High selection coefficient (s) approximately 0.04 for LP phenotype was found for both T(-13910) and the compound allele. The European T(-13910) and the earlier identified East African G(-13907) LP allele share the same ancestral background and most likely the same history, probably related to the same cattle domestication event. In contrast, the compound Arab allele shows a different, highly divergent ancestral haplotype, suggesting that these two major global LP alleles have arisen independently, the latter perhaps in response to camel milk consumption. These results support the convergent evolution of the LP in diverse populations, most probably reflecting different histories of adaptation to milk culture.

Transcriptional regulation of the lactase-phlorizin hydrolase gene by polymorphisms associated with adult-type hypolactasia

Background and aims: The mechanism of the developmental downregulation of the lactase-phlorizin hydrolase (LPH) gene underlying adult-type hypolactasia is unknown. We have determined the functional significance of the recently identified two single nucleotide polymorphisms (SNPs), C/T−13910 and G/A−22018, associated with adult-type hypolactasia by studying LPH mRNA levels in intestinal biopsy samples with different genotypes. Methods: Intestinal biopsy samples were taken from 52 patients with abdominal complaints. Hypolactasia was diagnosed by determining lactase and sucrase activities and calculating their ratio (L/S ratio). The functional effect of the C/T−13910 and G/A−22018 genotype on expression of LPH mRNA was demonstrated in patients heterozygous for the C/T−13910 and G/A−22018 polymorphism and an informative expressed SNP located in the coding region of the LPH mRNA. Reverse transcription-polymerase chain reaction followed by solid phase minisequencing was used for accessing the relative expression levels of the LPH alleles using informative SNPs located in exons 1, 2, 6, 10, 13, or 17 as markers. Results: Statistically significant differences between the three different genotypes CC−13910 GG−22018, CT−13910 GA−22018, and TT−13910 AA-22018 and their respective L/S ratios were observed. Relative quantitation of the expressed LPH alleles showed that the persistent allele represented 92 (6)% (mean (SEM), range 78–99%; n=14) of the expressed LPH mRNA. The patient with the homozygous persistent TT−13910 AA−22018, as well as hypolactasic patients with CC−13910 GG−22018, showed equal expression of both alleles (47 (1)%; n=7). Conclusions: Expression of LPH mRNA in the intestinal mucosa in individuals with T−13910 A−22018 alleles is several times higher than that found in individuals with C−13910, G−22018 alleles. These findings suggest that the two SNPs, C/T−13910 and G/A−22018, associated with adult-type hypolactasia, are associated with the transcriptional regulation of the LPH gene. The presence of the T−13910 A−22018 allele also shows significant elevation of the L/S ratio.

A genetic test which can be used to diagnose adult-type hypolactasia in children

Background/Aims: Adult-type hypolactasia (primary lactose malabsorption) affects most of world’s human population and limits the use of fresh milk due to lactose intolerance. The diagnosis of adult-type hypolactasia has been difficult to establish because of unsatisfactory diagnostic methods. C/T-13910 single nucleotide polymorphism residing 13910 base pairs from the 5′ end of the lactase gene has been shown to be associated with lactase persistence. The aim of the study was to assess the applicability of the C/T-13910 variant as a diagnostic test for adult-type hypolactasia during childhood. Methods: Intestinal biopsies were obtained from 329 children and adolescents of African, Finnish, and other White origins aged 0.1–20 years undergoing upper gastrointestinal endoscopy because of abdominal complaints. The biopsies were assayed for lactase, sucrase, and maltase activity and genotyped for the C/T-13910 variant using polymerase chain reaction minisequencing. Results: The frequency of the C/C-13910 genotype defining lactase non-persistence was well in agreement in this study with published figures for the prevalences of adult-type hypolactasia in Africans and Whites. The C/C-13910 genotype was associated with very low lactase activity (<10 U/g protein) in the majority of children tested at 8 years of age and in every child older than 12 years of age giving a specificity of 100% and sensitivity of 93% for the genetic test. The decline of lactase activity was somewhat earlier in African compared with Finnish children with C/C-13910 genotype (p<0.03). Conclusions: Genetic test of C/T-13910 polymorphism can be used as a first stage screening test for adult-type hypolactasia.

Evidence of still-ongoing convergence evolution of the lactase persistence T-13910 alleles in humans

A single-nucleotide variant, C/T(-13910), located 14 kb upstream of the lactase gene (LCT), has been shown to be completely correlated with lactase persistence (LP) in northern Europeans. Here, we analyzed the background of the alleles carrying the critical variant in 1,611 DNA samples from 37 populations. Our data show that the T(-13910) variant is found on two different, highly divergent haplotype backgrounds in the global populations. The first is the most common LP haplotype (LP H98) present in all populations analyzed, whereas the others (LP H8-H12), which originate from the same ancestral allelic haplotype, are found in geographically restricted populations living west of the Urals and north of the Caucasus. The global distribution pattern of LP T(-13910) H98 supports the Caucasian origin of this allele. Age estimates based on different mathematical models show that the common LP T(-13910) H98 allele (approximately 5,000-12,000 years old) is relatively older than the other geographically restricted LP alleles (approximately 1,400-3,000 years old). Our data about global allelic haplotypes of the lactose-tolerance variant imply that the T(-13910) allele has been independently introduced more than once and that there is a still-ongoing process of convergent evolution of the LP alleles in humans.

Assignment of the Locus for Congenital Lactase Deficiency to 2q21, in the Vicinity of but Separate from the Lactase-Phlorizin Hydrolase Gene

Congenital lactase deficiency (CLD) is an autosomal recessive, gastrointestinal disorder characterized by watery diarrhea starting during the first 1-10 d of life, in infants fed lactose-containing milks. Since 1966, 42 patients have been diagnosed in Finland. CLD is the most severe form of lactase deficiency, with an almost total lack of lactase-phlorizin hydrolase (LPH) activity on jejunal biopsy. In adult-type hypolactasia, the most common genetic enzyme deficiency in humans, this enzyme activity is reduced to 5%-10%. Although the activity of intestinal LPH has been found to be greatly reduced in both forms, the molecular pathogenesis of lactase deficiencies is unknown. On the basis of the initial candidate-gene approach, we assigned the CLD locus to an 8-cM interval on chromosome 2q21 in 19 Finnish families. At the closest marker locus, a specific allele 2 was present in 92% of disease alleles. On the basis of a genealogical study, the CLD mutation was found to be enriched in sparsely populated eastern and northern Finland, because of a founder effect. The results of both the genealogical study and the haplotype analysis indicate that one major mutation in a novel gene causes CLD in the Finnish population. Consequently, the critical region could be restricted further, to an approximately 350-kb interval, by ancient-haplotype and linkage-disequilibrium analyses. Surprisingly, the LPH gene was shown to lie outside the critical CLD region, excluding it as a causative gene for CLD. The LPH locus was found to reside >2 Mb from the critical CLD region.

Genetically defined adult-type hypolactasia and self-reported lactose intolerance as risk factors of osteoporosis in Finnish postmenopausal women.

Objective:To study the relationships of molecularly defined lactose malabsorption (LM) and self-reported lactose intolerance (LI) to bone mineral density (BMD) and fractures among Finnish postmenopausal women.Design:A cross-sectional study of two cohorts.Setting:Helsinki University Central Hospital.Subjects:One cohort was population-based and comprised 453 women, aged 62–78 (mean 69) y. Another comprised 52 women, aged 69–85 (mean 75) y, with osteoporotic fractures and 59 control women, aged 69–83 (mean 74) y, without osteoporosis.Methods:A single nucleotide polymorphism of the lactase (LCT) gene at chromosome 2q21–22 was studied. It shows complete association with intestinal disaccharidase activity, with the genotype CC−13 910 meaning adult-type hypolactasia (primary LM) and the genotypes CT−13 910 and TT−13 910 lactose absorption. BMD of the heel was measured by dual-energy X-ray absorptiometry (DXA).Results:In the population-based cohort, 16.0% of women had self-reported LI but only 15.3% of them had the CC−13 910 genotype. Calcium intake from dairy products (P=0.10) and BMD, adjusted for age, weight, height, exercise, smoking, and estrogen use (P=0.71) were similar for the genotypes. Women with self-reported LI had reduced calcium intake from dairy products (P<0.0001) but they were more frequent users of calcium supplements than lactose-tolerants (P<0.0001). Adjusted BMD was similar for lactose intolerant and tolerant women (P=0.60). Of 104 women with previous fracture in the population-based cohort, 13.5% had the CC−13 910 genotype, which did not differ from the prevalence of 19.3% among 347 women without fractures (P=0.29). The frequency of the CC−13 910 genotype (23.1%) for 52 women with established osteoporosis was similar as for 59 control women (15.3%) (P=0.19).Conclusion:Molecularly defined LM and self-reported LI are not risk factors for osteoporosis, if calcium intake from diet and/or supplements remains sufficient. Our study confirms the poor correlation between self-reported LI and LM established by different techniques.Sponsorhip:Supported by the Research Funding from Helsinki University Central Hospital (Erityisvaltionosuus) and by the Miina Sillanpää Foundation, Helsinki, Finland.

Genetic variant of lactase-persistent C/T-13910 is associated with bone fractures in very old age.

Objectives: To determine the relation between the C/T‐13910 single‐nucleotide polymorphism residing 13,910 base pairs from the 5′ end of the lactase gene associated with lactase persistence and the occurrence of bone fractures in elderly people.

Molecularly Defined Lactose Malabsorption, Peak Bone Mass and Bone Turnover Rate in Young Finnish Men

Lactose malabsorption (LM; adult-type hypolactasia), an autosomal recessive condition, results from the down-regulation of the activity of lactase enzyme in the intestinal wall. In previous studies the effect of LM on bone mass, bone turnover rate, development of osteoporosis and osteoporotic fractures has remained controversial. We have recently identified a single nucleotide polymorphism (SNP), a C to T change residing 13910 base pairs upstream of the lactase (LCT) gene at chromosome 2q21-22, which shows complete association with lactase persistence, with the C/C–13910 genotype defining LM and the genotypes C/T–13910 and T/T–13910 lactase persistence. The present study was undertaken to examine the relationship of the C/T–13910 polymorphism to peak bone mass, bone turnover rate, and stress fractures among young Finnish men. The study population comprised 234 young men, aged 18.3 to 20.6 years, 184 men were recruits of the Finnish Army, and 50 were men of similar age who had postponed their military service for reasons not related to health. Bone mineral content (BMC), density (BMD), and scan area were measured in the lumbar spine and upper femur by dual-energy X-ray absorptiometry (DXA). Blood was sampled for genotyping of the C/T–13910 polymorphism and determination of serum 25-hydroxyvitamin D (25OHD), intact parathyroid hormone (iPTH), type I procollagen aminoterminal propeptide (PINP), and tartrate-resistant acid phosphatase 5b (TRACP5b). Second-void urine samples were collected for the determination of type I collagen aminoterminal telopeptide (NTX). The prevalence of the C/C–13910-genotype of these young adults did not differ significantly from the corresponding population prevalence of C/C–13910 (17.1% vs 18.1%) among Finnish blood donors. Fifteen recruits of the army experienced a stress fracture; 3 of them (20%) had the C/C–13910-genotype. Calcium intake was similar for the three genotypes as were the unadjusted BMCs, scan areas, and BMDs at different measurement sites. The adjustments for age, height, weight, smoking, alcohol consumption, and physical exercise in the multiple regression analysis did not reveal any significant relationships between the lactase genotypes and BMDs at lumbar (P = 0.16), femoral neck (P = 0.99) or total hip (P = 0.96) sites. Serum 25OHD, iPTH, and bone marker levels were similar for the C/C–13910 C/T–13910 and T/T–13910 genotypes. In summary, in young Finnish men, molecularly defined lactose malabsorption does not alter bone turnover rate and impair the acquisition of peak bone mass. Moreover, the C/C–13910 genotype does not seem to be a risk factor for stress fractures in army recruits.

Transcriptional downregulation of the lactase (LCT) gene during childhood

Adult-type hypolactasia, characterised by bloating, gas formation, and diarrhoea after ingestion of lactose containing food, affects half of the world’s population.1 The molecular background of lactase non-persistence/persistence trait has been shown to associate with a single nucleotide polymorphism (SNP) C/T−13910 residing 13910 base pairs upstream from the 5′ end of the lactase (LCT) gene in an intron of the minichromosome maintenance 6 (MCM6) gene.2–4 We have demonstrated a trimodal distribution of lactase activity in the intestinal mucosa in adults, with low lactase activity (4–9 U/g protein) in those with the C/C−13910 genotype.3 The C−13910 and T−13910 allele show differential regulation of lactase promoter activity and binding capacity for the nuclear proteins in electromobility shift assay.5,6 Our recent analysis in a paediatric population demonstrated that the main time period for lactase downregulation in Finns and in Somalians is from five to 10 years of age.4 To further assess the role …

The genetic variant of lactase persistence C (-13910) T as a risk factor for type I and II diabetes in the Finnish population

Objective: Lactase persistence (LP), the ability to maintain a high lactase activity throughout life, has been suggested to be a possible risk factor for diabetes. Recently, a single nucleotide polymorphism C (−13910) T, residing 14 kb from the 5′ end of the lactase (LCT) gene was shown to be associated with LP. Here we have studied the relationship between C (13910) T polymorphism and diabetes in the Finnish population.Patients and design: In all, 1455 patients with type I and 615 with type II diabetes and 446 nondiabetic controls in the Finnish population were genotyped for the C (-13910) T polymorphism by PCR minisequencing.Results: No differences were detected in the LP genotype frequencies (CT&TT) between diabetic and nondiabetic subjects.Conclusions: We conclude that the C (−13910) T polymorphism associated with lifelong LP is not a risk factor for type I or type II diabetes in the Finnish population.