A Klothoβ Variant Mediates Protein Stability and Associates With Colon Transit in Irritable Bowel Syndrome With Diarrhea

doi:10.1053/j.gastro.2011.02.063

Gastroenterology

Volume 140, Issue 7, June 2011, Pages 1934-1942

https://doi.org/10.1053/j.gastro.2011.02.063 Get rights and content

Background & Aims

Bile acid (BA) malabsorption of moderate severity is reported in 32% of patients with chronic unexplained diarrhea, including diarrhea-predominant irritable bowel syndrome (IBS-D). We hypothesized that variants of genes regulating hepatic BA synthesis play a role in IBS-D.

Methods

In 435 IBS and 279 healthy subjects, we tested individual associations of 15 common single nucleotide polymorphisms (SNPs) from 7 genes critical to BA homeostasis with symptom-based subgroups using dominant genetic models. In a subset of 238 participants, we tested association with colonic transit. SNP-SNP interactions were investigated based on known protein interactions in BA homeostasis. The function of SNP rs17618244 in Klothoβ (KLB) was evaluated using a protein stability assay in HEK293 cells.

Results

SNP rs17618244 (Arg728Gln in KLB) is associated with colonic transit at 24 hours. G allele (Arg728) compared with A allele (Gln728) is associated with accelerated colonic transit (P = .0007) in the overall cohort; this association was restricted to IBS-D (P = .0018). Interaction tests of KLB rs17618244 with 3 nonsynonymous SNPs of fibroblast growth factor receptor 4 (FGFR4) revealed that rs1966265 (Val10Ile) and rs351855 (Gly388Arg) modulate rs1768244's association with colonic transit in IBS-D (P = .0025 and P = .0023, respectively). KLB Arg728 significantly reduced protein stability compared with KLB Gln728, demonstrating KLB rs17618244's functional significance. No significant associations with symptom-based subgroups of IBS were detected.

Conclusions

A functional KLB gene variant mediating protein stability associates with colonic transit in IBS-D. This association is modulated by 2 genetic variants in FGFR4. The FGF19-FGFR4-KLB pathway links regulation of BA synthesis to colonic transit in IBS-D.

Section snippets

Subjects

Our study assessed a total of 717 subjects: 435 patients with a functional gastrointestinal disorder (Rome II criteria positive consisting of 177 patients with IBS-C, 174 with IBS-D, 84 with alternating-type IBS) and 282 healthy volunteers. Of these, 239 participants had undergone scintigraphic colonic transit measurement. Baseline characteristics of the full cohort of 714 participants and the subset of 238 participants with colonic transit and genotyping information are shown in Table 1. The

Hardy–Weinberg Equilibrium for Candidate SNPs

Table 2 lists the 15 SNPs analyzed, P values for Hardy–Weinberg equilibrium, and predicted MAFs and observed MAFs separately for the healthy participants (n = 279) within the entire cohort of 714 subjects (with genotype data) and the healthy participants (n = 55) within the subset of 238 subjects with colonic transit measurements.

In the healthy participants, FGFR4 rs376618 was the only SNP of the 15 candidate SNPs to differ significantly from Hardy–Weinberg equilibrium, both in the full cohort

Discussion

In this study, we show that a functional gene variant mediating stability of KLB, a protein regulating BA synthesis, associates with colonic transit in IBS-D. This association is modulated by 2 independent genetic variants in FGFR4, whose protein product directly interacts with KLB.³⁴ This study links common polymorphisms in human genes regulating BA synthesis to colonic transit at 24 hours, a well-characterized intermediate phenotype, in IBS-D. These data support the hypothesis that genetic

References (41)

D.A. Drossman
The functional gastrointestinal disorders and the Rome III process
Gastroenterology
(2006)
Y.A. Saito et al.
Genetic approaches to functional gastrointestinal disorders
Gastroenterology
(2010)
F.J. Zieve et al.
Results of the glucose-lowering effect of WelChol study (GLOWS): a randomized, double-blind, placebo-controlled pilot study evaluating the effect of colesevelam hydrochloride on glycemic control in subjects with type 2 diabetes
Clin Ther
(2007)
J.Y. Chiang
Bile acids: regulation of synthesis
J Lipid Res
(2009)
J.R. Walters et al.
A new mechanism for bile acid diarrhea: defective feedback inhibition of bile acid biosynthesis
Clin Gastroenterol Hepatol
(2009)
S. Ito et al.
Molecular cloning and expression analyses of mouse betaklotho, which encodes a novel Klotho family protein
Mech Dev
(2000)
T.A. Kerr et al.
Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis
Dev Cell
(2002)
L. Wang et al.
Redundant pathways for negative feedback regulation of bile acid production
Dev Cell
(2002)
V. Andresen et al.
Is there an association between GNbeta3-C825T genotype and lower functional gastrointestinal disorders?
Gastroenterology
(2006)
H. Kurosu et al.
Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21
J Biol Chem
(2007)

B.C. Lin et al.

Liver-specific activities of FGF19 require Klotho beta

J Biol Chem

(2007)

M. Camilleri

Genetics and irritable bowel syndrome: from genomics to intermediate phenotype and pharmacogenetics

Dig Dis Sci

(2009)

S. Eusufzai

Bile acid malabsorption in patients with chronic diarrhoea

Scand J Gastroenterol

(1993)

F. Fernandez-Banares et al.

Systematic evaluation of the causes of chronic watery diarrhea with functional characteristics

Am J Gastroenterol

(2007)

G. Sciarretta et al.

Absence of histopathological changes of ileum and colon in functional chronic diarrhea associated with bile acid malabsorption, assessed by SeHCAT test: a prospective study

Am J Gastroenterol

(1994)

L. Wedlake et al.

Systematic review: the prevalence of idiopathic bile acid malabsorption as diagnosed by SeHCAT scanning in patients with diarrhoea-predominant irritable bowel syndrome

Aliment Pharmacol Ther

(2009)

S. Wildt et al.

Bile acid malabsorption in patients with chronic diarrhoea: clinical value of SeHCAT test

Scand J Gastroenterol

(2003)

S.T. Odunsi-Shiyanbade et al.

Effects of chenodeoxycholate and a bile acid sequestrant, colesevelam, on intestinal transit and bowel function

Clin Gastroenterol Hepatol

(2009)

P. Oelkers et al.

Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2)

J Clin Invest

(1997)

M. Montagnani et al.

Absence of dysfunctional ileal sodium-bile acid cotransporter gene mutations in patients with adult-onset idiopathic bile acid malabsorption

Scand J Gastroenterol

(2001)

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Intestinal epithelial β Klotho is a critical protective factor in alcohol-induced intestinal barrier dysfunction and liver injury
2022, eBioMedicine
Citation Excerpt :
Brain KLB knockout mice exhibit an increase in alcohol preference.17 In the gut, the decreased KLB expression caused by gene variation is associated with increased intestinal permeability in patients with irritable bowel syndrome with diarrhea,12 suggesting that the downregulated KLB may play a role in alcohol-induced intestinal epithelial dysfunction and liver injury. From apical-to-basal, the paracellular space between intestinal epithelial cells is sealed with tight junction (TJ, zonula occludens), adherens junction (zonula adherens), and desmosomes.18
Intestinal barrier dysfunction is crucial in alcohol-associated liver disease (ALD). The decreased beta-Klotho (KLB) expression caused by gene variation is associated with hyperpermeability in patients with irritable bowel syndrome. Here we investigated the roles of intestinal KLB in maintaining the intestinal epithelial barrier in ALD and the underlying mechanisms.
We constructed the intestine-specific overexpression KLB mice to investigate the role of KLB on alcohol-induced intestinal barrier dysfunction and liver injury in an ALD mouse model. To investigate the molecular mechanism in vitro, Caco2 cells were cultured and infected with the KLB overexpression lentivirus, or transfected with KLB/TRPV6 siRNA, or TRPV6/FXR1 overexpression plasmid, and treated with or without ethanol.
The upregulation of KLB in enterocytes effectively protected mice from alcohol-induced intestinal barrier hyperpermeability, thereby ameliorating hepatic steatosis and inflammation. KLB competitively suppressed FXR1 binding to the TRPV6 mRNA, increasing TRPV6 mRNA stability and protein abundance in intestinal epithelial cells. Furthermore, KLB formed a complex with TRPV6 and tight junction (TJ) proteins, protecting against alcohol-induced TJ proteins endocytosis and degradation as well as intestinal barrier impairment.
This work suggested that KLB attenuated alcohol-induced intestinal epithelial barrier dysfunction and liver injury through FXR1/TRPV6/TJ proteins pathway.
National Natural Science Foundation of China, Chongqing Natural Science Foundation, Talent Project of Chongqing and the Science and Technology Research Program of Chongqing Municipal Education Commission.
GWAS of stool frequency provides insights into gastrointestinal motility and irritable bowel syndrome
2021, Cell Genomics
Gut dysmotility is associated with constipation, diarrhea, and functional gastrointestinal disorders like irritable bowel syndrome (IBS), although its molecular underpinnings are poorly characterized. We studied stool frequency (defined by the number of bowel movements per day, based on questionnaire data) as a proxy for gut motility in a GWAS meta-analysis including 167,875 individuals from UK Biobank and four smaller population-based cohorts. We identify 14 loci associated with stool frequency (p ≤ 5.0 × 10⁻⁸). Gene set and pathway analyses detected enrichment for genes involved in neurotransmitter/neuropeptide signaling and preferentially expressed in enteric motor neurons controlling peristalsis. PheWAS identified pleiotropic associations with dysmotility syndromes and the response to their pharmacological treatment. The genetic architecture of stool frequency correlates with that of IBS, and UK Biobank participants from the top 1% of stool frequency polygenic score distribution were associated with 5× higher risk of IBS with diarrhea. These findings pave the way for the identification of actionable pathological mechanisms in IBS and the dysmotility syndromes.
β-Klotho gene variation is associated with liver damage in children with NAFLD
2020, Journal of Hepatology
Citation Excerpt :
These previous data suggested that the decrease of hepatic Klotho protein in pediatric NAFLD could be ascribable to the beta isoform (KLB), which is the main form in the liver.25–28 Recently, two functional genetic variants modulating the activity of the KLB/FGFR4 pathway, namely rs17618244 G>A KLB and rs1966265 G>A FGFR4, encoding for the R728Q and V10I aminoacidic substitutions have been associated with accelerated transit in irritable bowel syndrome,29 supporting a possible functional impact on the regulation of the gut-liver axis via modulation of FGF19 signaling.30 Therefore, the aim of this study was to examine the impact of the rs17618244 G>A KLB and rs1966265 G>A FGFR4 variants on liver damage severity in pediatric patients with NAFLD.
Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in adults and children. Along with obesity, diabetes and insulin resistance, genetic factors strongly impact on NAFLD development and progression. Dysregulated bile acid metabolism and the fibroblast growth factor 19 (FGF19) pathway play a pivotal role in NAFLD pathogenesis. However, the mechanism through which the FGF19 receptor system is associated with liver damage in NAFLD remains to be defined.
We evaluated the impact of the rs17618244 G>A β-Klotho (KLB) variant on liver damage in 249 pediatric patients with biopsy-proven NAFLD and the association of this variant with the expression of hepatic and soluble KLB. In vitro models were established to investigate the role of the KLB mutant.
The KLB rs17618244 variant was associated with an increased risk of ballooning and lobular inflammation. KLB plasma levels were lower in carriers of the rs17618244 minor A allele and were associated with lobular inflammation, ballooning and fibrosis. In HepG2 and Huh7 hepatoma cell lines, exposure to free fatty acids caused a severe reduction of intracellular and secreted KLB. Finally, KLB downregulation obtained by the expression of a KLB mutant in HepG2 and Huh7 cells induced intracellular lipid accumulation and upregulation of p62, ACOX1, ACSL1, IL-1β and TNF-α gene expression.
In conclusion, we showed an association between the rs17618244 KLB variant, which leads to reduced KLB expression, and the severity of NAFLD in pediatric patients. We can speculate that the KLB protein may exert a protective role against lipotoxicity and inflammation in hepatocytes.
Genetic and environmental factors strongly impact on the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD). The FGF19/FGFR4/KLB pathway plays a pivotal role in the pathogenesis of NAFLD. The aim of the study was to investigate the impact of a genetic variant in the KLB gene on the severity of liver disease. Our data suggest that the KLB protein plays a protective role against lipotoxicity and inflammation in hepatocytes.
Irritable bowel syndrome
2019, Clinical and Basic Neurogastroenterology and Motility
Irritable bowel syndrome (IBS) remains a highly prevalent condition that constitutes a large disease burden, and negatively impacts quality of life for a significant segment of the population. As such, IBS represents a common diagnosis in both primary care and specialty care settings. Reflecting the varied pathophysiologic mechanisms that contribute to IBS, patients similarly may present with an array of symptoms, including abdominal pain, diarrhea and constipation. Treatment choices also reflect the broad range of underlying mechanisms which may contribute to symptoms among patients with IBS. Therapy should be tailored to specific symptoms of IBS and may be further targeted to likely underlying mechanisms of IBS in an individual patient. In this chapter, we review and update the diagnostic evaluation, pathophysiologic mechanisms, and management of IBS.
Bile acids and FXR in functional gastrointestinal disorders
2018, Digestive and Liver Disease
Citation Excerpt :
It has been shown that hydrophobic BAs augmented small intestinal permeability either through nicotinic cholinergic mechanisms or changes within the small intestinal brush border membrane (changes in fragility and fluidity of the membrane) and caused higher influx of fluid and electrolytes to the intestinal lumen that contributed to the loose stool consistency. Genetic variations in the regulation of endogenous BA synthesis play a role in the etiology of IBS-D. Two genes, klotho B and FGFR4, were identified to modulate BA pool and contribute to worsening of symptoms in patients with accelerated colonic transit and IBS-D patients [80]. More specifically, differences in protein stability were detected between two alleles within klotho B (rs17618244) which provide a biologically plausible mechanism by which the single nucleotide polymorphisms may affect intestinal motility [80].
Functional gastrointestinal disorders (FGIDs), such as irritable bowel syndrome (IBS) and chronic constipation (CC), are commonly diagnosed conditions in clinical practice which create a substantial global burden. Since the farnesoid X receptor (FXR) and bile acids (BAs) are responsible for maintaining homeostasis in the GI tract, any disturbances in the expression of FXR or the composition of BAs may contribute to the development of the GI symptoms. Alterations in the mechanism of action of FXR directly affect the BAs pool and account for increased intestinal permeability and changes in abundance and diversity of gut microbiota leading to intestinal dysmotility.
Current review focuses on the correlation between the FXR, BAs and the composition of gut microbiota and its influence on the occurrence of GI symptoms in FGIDs.
Association of the rs1966265 and rs351855 FGFR4 Variants with Colorectal Cancer in a Mexican Population and Their Analysis In Silico
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: Conflicts of interest The authors disclose no conflicts.

: Funding Dr Camilleri's research in irritable bowel syndrome is supported by National Institutes of Health grants R01-DK079866 and 1RC1-DK086182.

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Clinical—Alimentary TractA Klothoβ Variant Mediates Protein Stability and Associates With Colon Transit in Irritable Bowel Syndrome With Diarrhea

Background & Aims

Methods

Results

Conclusions

Section snippets

Subjects

Hardy–Weinberg Equilibrium for Candidate SNPs

Discussion

Gastroenterology

Gastroenterology

Clin Ther

J Lipid Res

Clin Gastroenterol Hepatol

Mech Dev

Dev Cell

Dev Cell

Gastroenterology

J Biol Chem

J Biol Chem

Genetics and irritable bowel syndrome: from genomics to intermediate phenotype and pharmacogenetics

Dig Dis Sci

Bile acid malabsorption in patients with chronic diarrhoea

Scand J Gastroenterol

Systematic evaluation of the causes of chronic watery diarrhea with functional characteristics

Am J Gastroenterol

Absence of histopathological changes of ileum and colon in functional chronic diarrhea associated with bile acid malabsorption, assessed by SeHCAT test: a prospective study

Am J Gastroenterol

Systematic review: the prevalence of idiopathic bile acid malabsorption as diagnosed by SeHCAT scanning in patients with diarrhoea-predominant irritable bowel syndrome

Aliment Pharmacol Ther

Bile acid malabsorption in patients with chronic diarrhoea: clinical value of SeHCAT test

Scand J Gastroenterol

Effects of chenodeoxycholate and a bile acid sequestrant, colesevelam, on intestinal transit and bowel function

Clin Gastroenterol Hepatol

Primary bile acid malabsorption caused by mutations in the ileal sodium-dependent bile acid transporter gene (SLC10A2)

J Clin Invest

Absence of dysfunctional ileal sodium-bile acid cotransporter gene mutations in patients with adult-onset idiopathic bile acid malabsorption

Scand J Gastroenterol

Clinical—Alimentary Tract
A Klothoβ Variant Mediates Protein Stability and Associates With Colon Transit in Irritable Bowel Syndrome With Diarrhea