Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome

Abstract

Objective: IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase-isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase-isomaltase (SI) gene variants for their potential relevance in IBS.

Design: We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population.

Results: CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case-control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05).

Conclusions: SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Keywords: DIARRHOEA; GENETICS; IRRITABLE BOWEL SYNDROME; POLYMORPHIC VARIATION.

Figure 1

Properties of sucrase–isomaltase (SI) mutants and common coding polymorphisms. Left: schematic representation of SI protein structure and functional domains; the position of congenital sucrase–isomaltase deficiency (CSID) mutations and common coding variants is reported and colour-coded according to their functional effects (red=damaging, green=benign). Right: the variants, corresponding dbSNP IDs (http://www.ncbi.nlm.nih.gov/SNP), SI protein domain location (TM, transmembrane; I, isomaltase; S, sucrase), allele frequency, PHRED-like score (range 1–99, ranking a variant relative to all possible substitutions in the human genome) and predicted functional consequences are reported. ^#Exome Aggregation Consortium browser (http://exac.broadinstitute.org); ^Combined Annotation-Dependent Depletion database (http://cadd.gs.washington.edu/info). SNP, single nucleotide polymorphism.

Figure 2

Functional characterisation of the p.Val15Phe coding polymorphism. COS-1 cells were transiently transfected with either 15Val or 15Phe cDNAs and studied 48 hours after transfection. Individual values for 15Val and 15Phe cells from the same experiment are indicated with identical symbols. Net differences are reported with red bars as per cent average relative to 15Val arbitrarily set as 100% reference. (A) Cell surface localisation via immunofluorescence. Non-permeabilised cells were immunostained with a mixture of anti-sucrase–isomaltase (SI) antibodies and Alexa 488 secondary antibody, and analysed by confocal laser scanning microscopy on the xy (scale bar 25 µm) and xz planes (scale bar 10 µm). (B) Quantification of cell surface expression. SI surface proteins were labelled with biotin and immunoprecipitated using anti-Si antibodies after cell lysis. Immunoprecipitates were divided into two equal aliquots and analysed by immunoblotting with either anti-SI or anti-streptavidin antibodies. Relative quantification of surface-bound SI versus total cell SI was performed, and results are expressed in relation to values obtained for 15Val, which is set to 100%. (C) Quantification of association with sphingolipid/cholesterol-rich microdomains (lipid rafts) via detergent-resistant membrane (DRM) analysis. Following non-ionic detergent cell lysis, SI proteins were immunoprecipitated, fractionated by ultracentrifugation into insoluble (pellet, raft) and soluble (supernatant, non-raft) fractions, and DRM association (raft) quantified by immunoblotting with anti-SI antibodies. (D) Quantification of enzymatic activity. Sucrase activity was determined on immunoprecipitated SI proteins by measuring glucose release with the GOD–PAP method, upon normalisation for total protein amount by immunoblotting. *p<0.05.

Figure 3

Correlation between p.Val15Phe genotype and stool frequency. Mean (±SD) number of bowel movements per day (stool frequency, y-axis) is reported for Population-based Colonoscopy Study individuals (with available diary data) stratified according to the genotype at the p.Val15Phe single nucleotide polymorphism site (x-axis). The Spearman's p value for the correlation test is also reported.