Four novel mutations in the lactase gene (LCT) underlying congenital lactase deficiency (CLD).
ABSTRACT: BACKGROUND: Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder of newborns. The diagnosis is challenging and based on clinical symptoms and low lactase activity in intestinal biopsy specimens. The disease is enriched in Finland but is also present in other parts of the world. Mutations encoding the lactase (LCT) gene have recently been shown to underlie CLD. The purpose of this study was to identify new mutations underlying CLD in patients with different ethnic origins, and to increase awareness of this disease so that the patients could be sought out and treated correctly. METHODS: Disaccharidase activities in intestinal biopsy specimens were assayed and the coding region of LCT was sequenced from five patients from Europe with clinical features compatible with CLD. In the analysis and prediction of mutations the following programs: ClustalW, Blosum62, PolyPhen, SIFT and Panther PSEC were used. RESULTS: Four novel mutations in the LCT gene were identified. A single nucleotide substitution leading to an amino acid change S688P in exon 7 and E1612X in exon 12 were present in a patient of Italian origin. Five base deletion V565fsX567 leading to a stop codon in exon 6 was found in one and a substitution R1587H in exon 12 from another Finnish patient. Both Finnish patients were heterozygous for the Finnish founder mutation Y1390X. The previously reported mutation G1363S was found in a homozygous state in two siblings of Turkish origin. CONCLUSION: This is the first report of CLD mutations in patients living outside Finland. It seems that disease is more common than previously thought. All mutations in the LCT gene lead to a similar phenotype despite the location and/or type of mutation.
Project description:Congenital lactase deficiency (CLD) is a severe gastrointestinal disorder characterized by watery diarrhea in infants fed with breast milk or other lactose-containing formulas. We initially assigned the CLD locus by linkage and linkage disequilibrium on 2q21 in 19 Finnish families. Here we report the molecular background of CLD via characterization of five distinct mutations in the coding region of the lactase (LCT) gene. Twenty-seven patients out of 32 (84%) were homozygous for a nonsense mutation, c.4170T-->A (Y1390X), designated "Fin(major)." Four rare mutations--two that result in a predicted frameshift and early truncation at S1666fsX1722 and S218fsX224 and two point mutations that result in substitutions Q268H and G1363S of the 1,927-aa polypeptide--confirmed the lactase mutations as causative for CLD. These findings facilitate genetic testing in clinical practice and enable genetic counseling for this severe disease. Further, our data demonstrate that, in contrast to common adult-type hypolactasia (lactose intolerance) caused by a variant of the regulatory element, the severe infancy form represents the outcome of mutations affecting the structure of the protein inactivating the enzyme.
Project description: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.
Project description:Congenital lactase deficiency (CLD) is a rare severe autosomal recessive disorder, with symptoms like watery diarrhea, meteorism and malnutrition, which start a few days after birth by the onset of nursing. The most common rationales identified for this disorder are missense mutations or premature stop codons in the coding region of the lactase-phlorizin hydrolase (LPH) gene. Recently, two heterozygous mutations, c.4419C > G (p.Y1473X) in exon 10 and c.5387delA (p.D1796fs) in exon 16, have been identified within the coding region of LPH in a Japanese infant with CLD.Here, we investigate the influence of these mutations on the structure, biosynthesis and function of LPH. Therefore the mutant genes were transiently expressed in COS-1 cells.We show that both mutant proteins are mannose-rich glycosylated proteins that are not capable of exiting the endoplasmic reticulum. These mutant proteins are misfolded and turnover studies show that they are ultimately degraded. The enzymatic activities of these mutant forms are not detectable, despite the presence of lactase and phlorizin active sites in the polypeptide backbone of LPH-D1796fs and LPH-Y1473X respectively. Interestingly, wild type LPH retains its complete enzymatic activity and intracellular transport competence in the presence of the pathogenic mutants suggesting that heterozygote carriers presumably do not show symptoms related to CLD.Our study strongly suggests that the onset of severe forms of CLD is elicited by mutations in the LPH gene that occur in either a compound heterozygous or homozygous pattern of inheritance.
Project description:The ability to digest the milk sugar lactose as an adult (lactase persistence) is a variable genetic trait in human populations. The lactase-persistence phenotype is found at low frequencies in the majority of populations in sub-Saharan Africa that have been tested, but, in some populations, particularly pastoral groups, it is significantly more frequent. Recently, a CT polymorphism located 13.9 kb upstream of exon 1 of the lactase gene (LCT) was shown in a Finnish population to be closely associated with the lactase-persistence phenotype (Enattah et al. 2002). We typed this polymorphism in 1,671 individuals from 20 distinct cultural groups in seven African countries. It was possible to match seven of the groups tested with groups from the literature for whom phenotypic information is available. In five of these groups, the published frequencies of lactase persistence are >/=25%. We found the T allele to be so rare that it cannot explain the frequency of the lactase-persistence phenotype throughout Africa. By use of a statistical procedure to take phenotyping and sampling errors into account, the T-allele frequency was shown to be significantly different from that predicted in five of the African groups. Only the Fulbe and Hausa from Cameroon possessed the T allele at a level consistent with phenotypic observations (as well as an Irish sample used for comparison). We conclude that the C-13.9kbT polymorphism is not a predictor of lactase persistence in sub-Saharan Africans. We also present Y-chromosome data that are consistent with previously reported evidence for a back-migration event into Cameroon, and we comment on the implications for the introgression of the -13.9kb*T allele.
Project description:Adult-type hypolactasia is a common phenotype caused by the lactase enzyme deficiency. The -13910 C>T polymorphism, located 14 Kb upstream of the lactase gene (LCT) in the MCM6 gene was associated with lactase persistence (LP) in Europeans. This polymorphism is rare in Africa but several other variants associated with lactase persistence were observed in Africans. The aims of this study were to identify polymorphisms in the MCM6 region associated with the lactase persistence phenotype and to determine the distribution of LCT gene haplotypes in 981 individuals from North, Northeast and South Brazil. These polymorphisms were genotyped by PCR based methods and sequencing. The -13779*C,-13910*T, -13937*A, -14010*C, -14011*T LP alleles previously described in the MCM6 gene region that acts as an enhancer for the LCT gene were identified in Brazilians. The most common LP allele was -13910*T. Its frequency was highly correlated with European ancestry in the Brazilian populations investigated. The -13910*T was higher (0.295) in southern Brazilians of European ancestry and lower (0.175) in the Northern admixed population. LCT haplotypes were derived from the 10 LCT SNPs genotyped. Overall twenty six haplotypes previously described were identified in the four Brazilian populations studied. The Multidimensional Scaling analysis showed that Belém, in the north, was closer to Amerindians. Northeastern and southern Afro-descendants were more related with Bantu-speaking South Africans whereas the Southern population with European ancestry grouped with Southern and Northern Europeans. This study shows a high variability considering the number of LCT haplotypes observed. Due to the highly admixed nature of the Brazilian populations, the diagnosis of hypolactasia in Brazil, based only in the investigation of the -13910*T allele is an oversimplification.
Project description:In humans the expression of lactase changes during post-natal development, leading to phenotypes known as lactase persistence and non-persistence. Polymorphisms within the lactase gene (LCT) enhancer, in particular the -13910C > T, but also others, are linked to these phenotypes. We were interested in identifying dynamic mediators of LCT regulation, beyond the genotype at -13910C > T. To this end, we investigated two levels of lactase regulation in human intestinal samples obtained from New England children and adolescents of mixed European ancestry: differential expression of transcriptional regulators of LCT, and variations in DNA methylation, and their relation to phenotype. Variations in expression of CDX2, POU2F1, GATA4, GATA6, and HNF1α did not correlate with phenotype. However, an epigenome-wide approach using the Illumina Infinium HM450 bead chip identified a differentially methylated position in the LCT promoter where methylation levels are associated with the genotype at -13910C > T, the persistence/non-persistence phenotype and lactase enzymatic activity. DNA methylation levels at this promoter site and CpGs in the LCT enhancer are associated with genotype. Indeed, taken together they have a higher power to predict lactase phenotypes than the genotype alone.
Project description:BACKGROUND:Inflammatory Bowel Disease (IBD), which includes both Crohn's disease (CD) and ulcerative colitis (UC), is caused by a complex interplay involving genetic predisposition, environmental factors and an infectious agent. Mycobacterium avium subsp. paratuberculosis (MAP) is a promising pathogen candidate since it produces a chronic intestinal inflammatory disease in ruminants that resembles CD in humans. MAP is a ubiquitous microorganism, although its presence in the food chain, especially in milk from infected animals, is what made us think that there could be an association between lactase persistence (LP) and IBD. The LCT mutation has brought adaptation to dairy farming which in turn would have increased exposure of the population to infection by MAP. NOD2 gene mutations are highly associated to CD. METHODS:In our study, CD and UC patients and controls from the North of Spain were genotyped for the lactase gene (LCT) and for three NOD-2 variants, R702W, G908R and Cins1007fs. MAP PCR was carried out in order to assess MAP infection status and these results were correlated with LCT and NOD2 genotypes. RESULTS:As for LP, no association was found with IBD, although UC patients were less likely to present the T/T-13910 variant compared to controls, showing a higher C-allele frequency and a tendency to lactase non-persistence (LNP). NOD2 mutations were associated to CD being the per-allele risk higher for the Cins1007fs variant. MAP infection was more extended among the healthy controls (45.2%) compared to CD patients (21.38%) and UC patients (19.04%) and this was attributed to therapy. The Asturian CD cohort presented higher levels of MAP prevalence (38.6%) compared to the Basque CD cohort (15.5%), differences also attributed to therapy. No interaction was found between MAP infection and LCT or NOD2 status. CONCLUSIONS:We conclude that LP is not significantly associated with IBD, but that MAP infection and NOD2 do show not mutually interacting associations with IBD.
Project description:The lactase persistent (LP) or lactase non-persistent (LNP) state in European adults is genetically determined by a single nucleotide polymorphism (SNP) located 13.9 kb upstream of the lactase (LCT) gene, known as LCT C>T(-13910) (rs4988235). The LNP condition leads to an inability to digest the milk sugar lactose leading to gastrointestinal symptoms and can affect nutrient and calcium intake in certain populations.The authors studied a group of 51 Chilean patients to assess whether this SNP influences the LP/LNP state in this population, and determined the prevalence of LCT C>T(-13910) genotypes in a representative sample of 216 Hispanics and 43 Amerindians with correlation to digestive symptoms.Case-control study done in Chilean patients with clinical suspicion of LNP that were assessed using clinical survey, hydrogen breath test (HBT) and SNP genotyping. The population sample of Hispanics and Amerindians was assessed by clinical survey and SNP genotyping.Of the 51 patients with clinical suspicion of LNP, 29 were HBT-positive. The CC genotype (LNP) was present in 89.7% of the patients with positive HBT and in only 4.7% of those with negative HBT. The prevalence of the CC genotype was 56.9% in the Hispanic population and 88.3% in Amerindians, and was associated with a higher self-reported clinical intolerance to ingestion of dairy products.The LP/LNP state is determined by the LCT C>T(-13910) variant in Chileans. This variant predicts digestive symptoms associated with the ingestion of lactose and is a good tool for the diagnosis of primary adult hypolactasia. The LCT T(-13910) allele is rare in the Amerindian population and is suggestive of European ancestry in this contemporary population.
Project description:Recent data from mainly homogeneous European and African populations implicate a 140-bp region 5' to the transcriptional start site of LCT (the lactase gene) as a regulatory site for lactase persistence and nonpersistence. Because there are no studies of US nonhomogeneous populations, we performed genotype/phenotype analysis of the -13910 and -22018 LCT single nucleotide polymorphisms (SNPs) in New England children, mostly of European ancestry.Duodenal biopsies were processed for disaccharidase activities, RNA quantification by reverse transcription polymerase chain reaction (RT-PCR), allelic expression ratios by PCR, and genotyping and SNP analysis. Results were compared with clinical information.Lactase activity and mRNA levels, and sucrase-to-lactase ratios of enzyme activity and mRNA, showed robust correlations with genotype. None of the other LCT SNPs showed as strong a correlation with enzyme or mRNA levels as did -13910. Data were consistent, with the -13910 being the causal sequence variant instead of -22018. Four individuals heterozygous for -13910T/C had allelic expression patterns similar to individuals with -13910C/C genotypes; of these, 2 showed equal LCT expression from the 2 alleles and a novel variant (-13909C>A) associated with lactase persistence.The identification of -13910C/C genotype is likely to predict lactase nonpersistence, consistent with prior published studies. A -13910T/T genotype will frequently, but not perfectly, predict lactase persistence in this mixed European-ancestry population; a -13910T/C genotype will not predict the phenotype. A long, rare haplotype in 2 individuals with -13910T/C genotype but equal allele-specific expression contains a novel lactase persistence allele present at -13909.
Project description:Congenital lactase deficiency is an extremely rare gastrointestinal disorder characterized by neonatal-onset watery diarrhoea and failure to thrive. We present the first genetically confirmed case of congenital lactase deficiency in Central Europe.After an uneventful pregnancy and birth, a male newborn of consanguineous parents of Turkish origin presented with watery diarrhoea. On day 17, he was admitted to hospital with weight loss, hypertonic dehydration, and metabolic acidosis. Additionally, the patient showed an elevated calcium concentration in blood and urine as well as nephrocalcinosis. Diarrhoea stopped during intravenous rehydration and when feeding a glucose-, galactose-, and lactose-free formula. Therefore, glucose-galactose-malabsorption was assumed. However, genetic testing of the SGLT1 (SLC5A1) gene was negative and, indeed, feeding maltodextrine did not result in recurrence of diarrhoea. In contrast, lactose feeding immediately caused watery diarrhoea, suggesting congenital lactase deficiency. Genetic testing of the LCT gene revealed homozygosity for a 1-bp deletion in exon 8 (c.3448delT). Because of the nature of the mutation, causing a frame shift and a premature termination of translation, congenital lactase deficiency was confirmed and intestinal biopsies were unnecessary. The patient's general condition improved substantially on a lactose-free diet, including hypercalcaemia, hypercalciuria, and nephrocalcinosis which, however, only disappeared after months.This case demonstrates (a) that congenital lactase deficiency should be considered in cases of severe neonatal diarrhoea, (b) that intestinal biopsies can be avoided in typical cases that are confirmed by genetic testing, and