SLC10A1 rs2296651 variant (S267F mutation) predicts biochemical traits, hepatitis B virus infection susceptibility and the risk of gallstone disease.

Sodium taurocholate co-transporting polypeptide (NTCP), a bile acid transporter, plays a crucial role in regulating bile acid levels and influencing the risk of HBV infection. Genetic variations in the SLC10A1 gene, which encodes NTCP, affect these functions. However, the impact of SLC10A1 gene variants on the metabolic and biochemical traits remained unclear. We aimed to investigate the association of SLC10A1 gene variants with the clinical and biochemical parameters, and the risk of different HBV infection statuses and gallstone disease in the Taiwanese population. Genotyping data from 117,679 Taiwan Biobank participants were analyzed using the Axiom genome-wide CHB arrays. Regional-plot association analysis demonstrated genome-wide significant association between the SLC10A1 rs2296651 genotypes and lipid profile, gamma glutamyl transferase (γGT) level and anti-HBc-positivity. Genotype-phenotype association analyses revealed significantly lower total cholesterol, low-density lipoprotein (LDL) cholesterol and uric acid levels, a higher γGT level and a higher gallstone incidence in rare rs2296651-A allele carrier. Participants with the rs2296651 AA-genotype exhibited significantly lower rates of anti-HBc-positivity and HBsAg-positivity. Compared to those with the GG-genotype, individuals with non-GG-genotypes had reduced risks for various HBV infection statuses: the AA-genotype showed substantially lower risks, while the GA-genotype demonstrated modestly lower risks. Predictive tools also suggested that the rs2296651 variant potentially induced protein damage and pathogenic effects. In conclusion, our data revealed pleiotropic effects of the SLC10A1 rs2296651 genotypes on the levels of biochemical traits and the risk of HBV infection and gallstone disease. This confirms SLC10A1's versatility and implicates its genotypes in predicting both biochemical traits and disease susceptibility.

Development and internal validation of a multifactorial risk prediction model for gallbladder cancer in a high-incidence country.

Since 2006, Chile has been implementing a gallbladder cancer (GBC) prevention program based on prophylactic cholecystectomy for gallstone patients aged 35 to 49 years. The effectiveness of this prevention program has not yet been comprehensively evaluated. We conducted a retrospective study of 473 Chilean GBC patients and 2137 population-based controls to develop and internally validate three GBC risk prediction models. The Baseline Model accounted for gallstones while adjusting for sex and birth year. Enhanced Model I also included the non-genetic risk factors: body mass index, educational level, Mapuche surnames, number of children and family history of GBC. Enhanced Model II further included Mapuche ancestry and the genotype for rs17209837. Multiple Cox regression was applied to assess the predictive performance, quantified by the area under the precision-recall curve (AUC-PRC) and the number of cholecystectomies needed (NCN) to prevent one case of GBC at age 70 years. The AUC-PRC for the Baseline Model (0.44%, 95%CI 0.42-0.46) increased by 0.22 (95%CI 0.15-0.29) when non-genetic factors were included, and by 0.25 (95%CI 0.20-0.30) when incorporating non-genetic and genetic factors. The overall NCN for Chileans with gallstones (115, 95%CI 104-131) decreased to 92 (95%CI 60-128) for Chileans with a higher risk than the median according to Enhanced Model I, and to 80 (95%CI 59-110) according to Enhanced Model II. In conclusion, age, sex and gallstones are strong risk factors for GBC, but consideration of other non-genetic factors and individual genotype data improves risk prediction and may optimize allocation of financial resources and surgical capacity.

Differential Effects of ABCG5/G8 Gene Region Variants on Lipid Profile, Blood Pressure Status, and Gallstone Disease History in Taiwan.

ABCG5 and ABCG8 are two key adenosine triphosphate-binding cassette (ABC) proteins that regulate whole-body sterol trafficking. This study aimed to elucidate the association between ABCG5/G8 gene region variants and lipid profile, cardiometabolic traits, and gallstone disease history in Taiwan. A total of 1494 Taiwan Biobank participants with whole-genome sequencing data and 117,679 participants with Axiom Genome-Wide CHB Array data were enrolled for analysis. Using genotype-phenotype and stepwise linear regression analyses, we found independent associations of four Asian-specific ABCG5 variants, rs119480069, rs199984328, rs560839317, and rs748096191, with total, low-density lipoprotein (LDL), and non-high-density lipoprotein (HDL) cholesterol levels (all p ≤ 0.0002). Four other variants, which were in nearly complete linkage disequilibrium, exhibited genome-wide significant associations with gallstone disease history, and the ABCG8 rs11887534 variant showed a trend of superiority for gallstone disease history in a nested logistic regression model (p = 0.074). Through regional association analysis of various other cardiometabolic traits, two variants of the PLEKHH2, approximately 50 kb from the ABCG5/G8 region, exhibited significant associations with blood pressure status (p < 10-6). In conclusion, differential effects of ABCG5/G8 region variants were noted for lipid profile, blood pressure status, and gallstone disease history in Taiwan. These results indicate the crucial role of individualized assessment of ABCG5/G8 variants for different cardiometabolic phenotypes.

Adeno-associated virus-based caveolin-1 delivery via different routes for the prevention of cholesterol gallstone formation.

BACKGROUND: Hepatic caveolin-1 (CAV1) is reduced in cholesterol gallstone disease (CGD). Mice with CAV1 deficiency were prone to develop CGD. However, it remains unknown whether restored hepatic CAV1 expression prevents the development of CGD. METHODS: C57BL/6 mice were injected with adeno-associated virus 2/8 (AAV2/8) vectors carrying the CAV1 gene (AAV2/8CAV1) via intravenous (i.v.) or intraperitoneal (i.p.) route and then subjected to a lithogenic diet (LD) for 8 weeks. Uninjected mice were used as controls. The functional consequences of rescuing CAV1 expression by either i.v. or i.p. AAV2/8CAV1 treatment for CGD prevention and its subsequent molecular mechanisms were examined. RESULTS: CAV1 expression was reduced in the liver and gallbladder of LD-fed CGD mice. We discovered that AAV2/8CAV1 i.p. delivery results in higher transduction efficiency in the gallbladder than tail vein administration. Although either i.v. or i.p. injection of AAV2/8CAV1 improved liver lipid metabolic abnormalities in CGD mice but did not affect LD feeding-induced bile cholesterol supersaturation. In comparison with i.v. administration route, i.p. administration of AAV2/8CAV1 obviously increased CAV1 protein levels in the gallbladder of LD-fed mice, and i.p. delivery of AAV2/8CAV1 partially improved gallbladder cholecystokinin receptor (CCKAR) responsiveness and impeded bile cholesterol nucleation via the activation of adenosine monophosphate-activated protein kinase (AMPK) signaling, which induced a reduction in gallbladder mucin-1 (MUC1) and MUC5ac expression and gallbladder cholesterol accumulation. CONCLUSION: CGD prevention by i.p. AAV2/8CAV1 injection in LD-fed mice was associated with the improvement of gallbladder stasis, which again supported the notion that supersaturated bile is required but not sufficient for the formation of cholesterol gallstones. Additionally, AAV treatment via the local i.p. injection offers particular advantages over the systemic i.v. route for much more effective gallbladder gene delivery, which will be an excellent tool for conducting preclinical functional studies on the maintenance of normal gallbladder function to prevent CGD.

Patients with Primary and Secondary Bile Duct Stones Harbor Distinct Biliary Microbial Composition and Metabolic Potential.

Introduction: Cholelithiasis has a high incidence worldwide and limited treatment options due to its poorly understood pathogenesis. Furthermore, the role of biliary microbiota in cholelithiasis remains understudied. To address these questions, we performed microbial sequencing from biliary samples from primary bile duct stone (PBDS) and secondary bile duct stone (SBDS) patients. Results: We analyzed in total 45 biliary samples, including those from cholelithiasis patients with PBDS or SBDS and people with other digestive diseases. 16S rRNA sequencing showed the bacteria family Alcaligenaceae increased in relative abundance in the lithiasis group compared with the non-lithiasis group. In addition, the PBDS group showed significantly lower bacterial diversity than SBDS, with Propionibacteriaceae, Sphingomonadaceae, and Lactobacillaceae as the most significant bacteria families decreased in relative abundance. We further performed whole metagenomic shotgun sequencing (wMGS) and found increased ability of biofilm synthesis and the ability to sense external stimuli in PBDS based on functional annotation of mapped reads. From genome-resolved analysis of the samples, we identified 36 high-quality draft bacterial genome sequences with completion ≥70% and contamination ≤10%. Most of these genomes were classified into Proteobacteria, Firmicutes, or Actinobacteria. Conclusions: Our findings indicated that there is a subtle impact on biliary microbiome from cholelithiasis while the difference is more pronounced between the PBDS and SBDS. It was revealed that the diversity of biliary microbiota in PBDS is lower, while some metabolic pathways are up-regulated, including those linked to higher incidence of different types of cancer, providing new insights for the understanding of cholelithiasis with different origin.

Biliary microbiota and mucin 4 impact the calcification of cholesterol gallstones.

BACKGROUND: Cholesterol gallstones account for over 80% of gallstones, and the pathogenesis of gallstone formation involves genetic and environmental factors. However, data on the evolution of cholesterol gallstones with various densities are limited. This study aimed to determine the roles of microbiota and mucins on the formation of calcified cholesterol gallstones in patients with cholelithiasis. METHODS: Paired gallbladder tissues and bile specimens were obtained from cholelithiasis patients who were categorized into the isodense group and calcified group according to the density of gallstones. The relative abundance of microbiota in gallbladder tissues was detected. Immunohistochemistry and enzyme-linked immunosorbent assay were performed to detect the expression levels of MUC1, MUC2, MUC3a, MUC3b, MUC4, MUC5ac and MUC5b in gallbladder tissues and bile. The correlation of microbiota abundance with MUC4 expression was evaluated by linear regression. RESULTS: A total of 23 patients with gallbladder stones were included. The density of gallstones in the isodense group was significantly lower than that of the calcified group (34.20 ± 1.50 vs. 109.40 ± 3.84 HU, P < 0.0001). Compared to the isodense group, the calcified group showed a higher abundance of gram-positive bacteria at the fundus, in the body and neck of gallbladder tissues. The concentrations of MUC1, MUC2, MUC3a, MUC3b, MUC5ac and MUC5b in the epithelial cells of gallbladder tissues showed no difference between the two groups, while the concentrations of MUC4 were significantly higher in the calcified group than that in the isodense group at the fundus (15.49 ± 0.69 vs. 10.23 ± 0.54 ng/mL, P < 0.05), in the body (14.54 ± 0.94 vs. 11.87 ± 0.85 ng/mL, P < 0.05) as well as in the neck (14.77 ± 1.04 vs. 10.85 ± 0.72 ng/mL, P < 0.05) of gallbladder tissues. Moreover, the abundance of bacteria was positively correlated with the expression of MUC4 (r = 0.569, P < 0.05) in the calcified group. CONCLUSIONS: This study showed the potential clinical relevance among biliary microbiota, mucins and calcified gallstones in patients with gallstones. Gram-positive microbiota and MUC4 may be positively associated with the calcification of cholesterol gallstones.

The Role of Gallstones in Gallbladder Cancer in India: A Mendelian Randomization Study.

BACKGROUND: Past history of gallstones is associated with increased risk of gallbladder cancer in observational studies. We conducted complementary observational and Mendelian randomization (MR) analyses to determine whether history of gallstones is causally related to development of gallbladder cancer in an Indian population. METHODS: To investigate associations between history of gallstones and gallbladder cancer, we used questionnaire and imaging data from a gallbladder cancer case-control study conducted at Tata Memorial Hospital, Mumbai, Maharashtra, India (cases = 1,170; controls = 2,525). We then used 26 genetic variants identified in a genome-wide association study of 27,174 gallstone cases and 736,838 controls of European ancestry in an MR approach to assess causality. The association of these genetic variants with both gallstones and gallbladder cancer was examined in the gallbladder cancer case-control study. Various complementary MR approaches were used to evaluate the robustness of our results in the presence of pleiotropy and heterogeneity, and to consider the suitability of the selected SNPs as genetic instruments for gallstones in an Indian population. RESULTS: We found a strong observational association between gallstones and gallbladder cancer using self-reported history of gallstones [OR = 4.5; 95% confidence interval (CI) = 3.5-5.8] and with objective measures of gallstone presence using imaging techniques (OR = 2.0; 95% CI = 1.5-2.7). We found consistent causal estimates across all MR techniques, with ORs for gallbladder cancer in the range of 1.3-1.6. CONCLUSIONS: Our findings indicate a causal relationship between history of gallstones and increased risk of gallbladder cancer, albeit of a smaller magnitude than those found in observational analysis. IMPACT: Our findings emphasize the importance of gallstone treatment for preventing gallbladder cancer in high-risk individuals.

Epigenome-Wide Analysis of Methylation Changes in the Sequence of Gallstone Disease, Dysplasia, and Gallbladder Cancer.

BACKGROUND AND AIMS: Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-income and middle-income countries, and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide. APPROACH AND RESULTS: To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of formalin-fixed, paraffin-embedded gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Preprocessed, quality-controlled data from 82 samples (gallstones n = 32, low-grade dysplasia n = 13, high-grade dysplasia n = 9, GBC n = 28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs). The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of cytosine-guanine dinucleotide islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected cyclin-dependent kinase inhibitor 2A, MDM2 proto-oncogene, tumor protein P53, and cyclin D1 genes. Gains in the targetable Erb-B2 receptor tyrosine kinase 2 gene were detected in 14% of GBC samples. CONCLUSIONS: Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (high-grade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.

Depletion of hepatic forkhead box O1 does not affect cholelithiasis in male and female mice.

Cholelithiasis is one of the most prevalent gastroenterological diseases and is characterized by the formation of gallstones in the gallbladder. Both clinical and preclinical data indicate that obesity, along with comorbidity insulin resistance, is a predisposing factor for cholelithiasis. Forkhead box O1 (FoxO1) is a key transcription factor that integrates insulin signaling with hepatic metabolism and becomes deregulated in the insulin-resistant liver, contributing to dyslipidemia in obesity. To gain mechanistic insights into how insulin resistance is linked to cholelithiasis, here we determined FoxO1's role in bile acid homeostasis and its contribution to cholelithiasis. We hypothesized that hepatic FoxO1 deregulation links insulin resistance to impaired bile acid metabolism and cholelithiasis. To address this hypothesis, we used the FoxO1LoxP/LoxP-Albumin-Cre system to generate liver-specific FoxO1-knockout mice. FoxO1-knockout mice and age- and sex-matched WT littermates were fed a lithogenic diet, and bile acid metabolism and gallstone formation were assessed in these animals. We showed that FoxO1 affected bile acid homeostasis by regulating hepatic expression of key enzymes in bile acid synthesis and in biliary cholesterol and phospholipid secretion. Furthermore, FoxO1 inhibited hepatic expression of the bile acid receptor farnesoid X receptor and thereby counteracted hepatic farnesoid X receptor signaling. Nonetheless, hepatic FoxO1 depletion neither affected the onset of gallstone disease nor impacted the disease progression, as FoxO1-knockout and control mice of both sexes had similar gallstone weights and incidence rates. These results argue against the notion that FoxO1 is a link between insulin resistance and cholelithiasis.

Cholesterol depletion sensitizes gallbladder cancer to cisplatin by impairing DNA damage response.

Gallbladder cancer (GBC) is the common malignancy of the bile tract system with extremely poor clinical outcomes, owing to its metastatic property and intrinsic resistance to the first-line drugs. Although it is well-established that cholesterol abnormity contributes to gallstone formation, a leading risk factor for GBC, the link of cholesterol homeostasis with GBC has not been investigated. The present study systematically examined the genes implicated in cholesterol homeostasis, and revealed altered gene expressions of de novo cholesterol biosynthesis and sterol sulfonation (SULT2B1), reduced bile acid synthesis (CYP7B1 and CYP39A1) and impaired sterol efflux (ABCA1, ABCG5, LCAT, and CETP) in GBC tissues. Suppression of cholesterol biosynthesis by lovastatin inhibited GBC cell proliferation possibly through attenuating the DNA repair process. Further investigation revealed lovastatin sensitized GBC cells to cisplatin-induced apoptosis and suppressed the activation of CHK1, CHK2, and H2AX during DNA damage response. By using chemically distinct statins, HMGCR depletion or supplementing mevalonate, the product of HMGCR, we showed the inhibitory effects on DNA repair process of lovastatin were due to the blockage of the mevalonate pathway. Subcutaneous xenograft mice model suggested lovastatin promoted the therapeutic efficacy of cisplatin, and significantly prolonged the survival times of tumor-bearing mice. Moreover, HMGCR ablation repressed tumor growth in vivo, which can be rescued partially by restored expression of HMGCR, suggesting the on-target effects of lovastatin. Therefore, our study provides the clinical relevance of cholesterol homeostasis with GBC progression, and highlights a novel intervention of combined use of lovastatin and cisplatin for GBC.

Variants in ABCG8 and TRAF3 genes confer risk for gallstone disease in admixed Latinos with Mapuche Native American ancestry.

Latin Americans and Chilean Amerindians have the highest prevalence of gallstone disease (GSD) and gallbladder cancer (GBC) in the world. A handful of loci have been associated with GSD in populations of predominantly European ancestry, however, they only explain a small portion of the genetic component of the disease. Here, we performed a genome-wide association study (GWAS) for GSD in 1,095 admixed Chilean Latinos with Mapuche Native American ancestry. Disease status was assessed by cholecystectomy or abdominal ultrasonography. Top-10 candidate variants surpassing the suggestive cutoff of P < 1 × 10-5 in the discovery cohort were genotyped in an independent replication sample composed of 1,643 individuals. Variants with positive replication were further examined in two European GSD populations and a Chilean GBC cohort. We consistently replicated the association of ABCG8 gene with GSD (rs11887534, P = 3.24 × 10-8, OR = 1.74) and identified TRAF3 (rs12882491, P = 1.11 × 10-7, OR = 1.40) as a novel candidate gene for the disease in admixed Chilean Latinos. ABCG8 and TRAF3 variants also conferred risk to GBC. Gene expression analyses indicated that TRAF3 was significantly decreased in gallbladder (P = 0.015) and duodenal mucosa (P = 0.001) of GSD individuals compared to healthy controls, where according to GTEx data in the small intestine, the presence of the risk allele contributes to the observed effect. We conclude that ABCG8 and TRAF3 genes are associated with GSD and GBC in admixed Latinos and that decreased TRAF3 levels could enhance gallbladder inflammation as is observed in GSD and GSD-associated GBC.

Genetics of gallstone disease revisited: updated inventory of human lithogenic genes.

PURPOSE OF REVIEW: Gallstone disease is one of the most frequent diseases in gastroenterology and treatment by endoscopy and surgery causes high costs in our health-care systems. Family and twin studies have demonstrated that gallstones are, in part, genetically determined. Here we review all recent genome-wide and phenome-wide studies of gallstones in humans and provide an updated 'inventory of human lithogenic genes.' RECENT FINDINGS: The largest population attributable risk is conferred by the common variant (p.D19H) of the hepatic and intestinal cholesterol transporter ABCG5/G8. A second ABC transporter, the hepatic phosphatidylcholine translocase ABCB4, increases the risk for gallstone disease, gallbladder cancer and chronic liver diseases in general, whereas the common PNPLA3 risk variant p.I148M decreases gallstone risk. SUMMARY: Better understanding of the pathomechanisms of gallstone disease might help to overcome the current invasive treatment of this exceptionally prevalent and economically significant digestive disease by personalized prevention in at-risk patients.

Gallstone Disease and Type 2 Diabetes Risk: A Mendelian Randomization Study.

The presence of gallstone disease (GSD) was reported to be positively associated with diabetes risk. Whether the association is causal remains unclear. We aim to examine the potential causal association between GSD and type 2 diabetes risk using a Mendelian randomization analysis. Observational study was conducted among 16,299 participants who were free of cancer, heart disease, stroke, and diabetes at baseline in the Dongfeng-Tongji cohort study. GSD was diagnosed by experienced physicians by abdominal B-type ultrasound inspection and type 2 diabetes was defined according to the criteria of the American Diabetes Association. Cox proportional hazard regression model was used to examine the association of GSD with type 2 diabetes risk. A genetic risk score (GRS) for GSD was constructed with eight single nucleotide polymorphisms that were derived from the previous genome-wide association studies. The causal associations of the score for GSD with type 2 diabetes were tested among 7,000 participants in Mendelian randomization analysis. We documented 1,110 incident type 2 diabetes cases during 73,895 person-years of follow-up from 2008 to 2013 (median 4.6 years). Compared with participants without GSD, the multivariate-adjusted hazard ratio of type 2 diabetes risk in those with GSD was 1.22 (95% confidence interval [CI], 1.03-1.45, P = 0.02). Each 1 SD (0.23) increment in the weighted GRS was associated with a 17% increment of type 2 diabetes risk (odds ratio = 1.17, 95% CI, 0.90-1.52) without statistical significance (P = 0.25). Conclusion: The present study supported a positive but not a causal association of GSD with type 2 diabetes risk. More studies are needed to verify our findings.

Identification and Replication of Six Loci Associated With Gallstone Disease.

Gallstone disease is a common complex disease that confers a substantial economic burden on society. The genetic underpinnings of gallstone disease remain incompletely understood. We aimed to identify genetic associations with gallstone disease using publicly available data from the UK Biobank and two large Danish cohorts. We extracted genetic associations with gallstone disease from the Global Biobank Engine (GBE), an online browser of genome-wide associations in UK Biobank participants (14,940 cases and 322,268 controls). Significant associations (P < 5 × 10-8 ) were retested in two Copenhagen cohorts (comprising 1,522 cases and 18,266 controls). In the Copenhagen cohorts, we also tested whether a genetic risk score was associated with gallstone disease and whether individual gallstone loci were associated with plasma levels of lipids, lipoproteins, and liver enzymes. We identified 19 loci to be associated with gallstone disease in the GBE. Of these, 12 were replicated in the Copenhagen cohorts, including six previously unknown loci (in hepatocyte nuclear factor 4 alpha [HNF4A], fucosyltransferase 2, serpin family A member 1 [SERPINA1], jumonji domain containing 1C, AC074212.3, and solute carrier family 10A member 2 [SLC10A2]) and six known loci (in adenosine triphosphate binding cassette subfamily G member 8 [ABCG8], sulfotransferase family 2A member 1, cytochrome P450 7A1, transmembrane 4 L six family member 4, ABCB4, and tetratricopeptide repeat domain 39B). Five of the gallstone associations are protein-altering variants, and three (HNF4A p.Thr139Ile, SERPINA1 p.Glu366Lys, and SLC10A2 p.Pro290Ser) conferred per-allele odds ratios for gallstone disease of 1.30-1.36. Individuals with a genetic risk score >2.5 (prevalence 1%) had a 5-fold increased risk of gallstones compared to those with a score <1.0 (11%). Of the 19 lithogenic loci, 11 and ten exhibited distinct patterns of association with plasma levels of lipids and liver enzymes, respectively. Conclusion: We identified six susceptibility loci for gallstone disease.

[Relationship Between LEP G2548A Polymorphism and Cholesterol Gallstone].

OBJECTIVES: To determine the associations of single nucleotide polymorphism (SNP) in leptin (LEP) genes and environmental factors with cholesterol gallstone in southeast Han populations. METHODS: A 1:2 matched case-control study was conducted involving 200 patients with cholesterol gallstone. Genotyping of the SNP was examined on the LightCycler480 PCR platform using in-house high resolution melting (HRM) approaches. Detection correctness was validated through direct sequencing. Multifactor dimensionality reduction (MDR) analysis was applied to examine the effects of potential gene-environment interactions. RESULTS: Three genotypes of LEP G2548A were obtained by HRM genotyping, including 52 cases of GG wild type, 192 cases of GA mutant heterozygosity and 356 cases of AA mutation homozygous type. The genotype distribution of the SNP locus in the control group was in line with the Hardy-Weinberg genetic balance (P>0.05). The AA genotype carriers of LEP G2548A had significantly higher serum leptin than the GA/GG genotype carriers (H=6.83, P<0.05). The conditional logistic regression revealed that high serum leptin [odds ratio (OR)=5.012, 95% confidence interval (CI): 3.248-7.734], AA genotype of LEP G2548A site (OR=2.292, 95%CI: 1.012-5.193), family history of gallstones (OR=2.984, 95%CI: 1.329-6.700), high SBP (OR=1.927, 95%CI: 1.140-3.255) and smoking (OR=1.717, 95%CI: 1.006-2.928) were predictors of cholesterol gallstone. However, regular drinking of strong tea (OR=0.552, 95%CI: 0.336-0.907) and exercise (OR=0.591, 95%CI: 0.395-0.882) were protecting factors for cholesterol gallstone. The results of MDR analysis indicated that tea drinking, genotype of LEP G2548A site and serum leptin formed the optimal gene-environment interaction model. CONCLUSIONS: Individuals who drink less tea, carry AA genotype and have high serum leptin are more susceptible to cholesterol gallstone.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.

Primary Extragastrointestinal Stromal Tumours in the Hepatobiliary Tree and Telocytes.

The first decade of the twenty-first century witnessed the presence and light microscopic, immunophenotypic, and ultrastructural characterization of interstitial Cajal-like cells (coined as 'telocytes') in virtually every extragastrointestinal site of the human body by Laurentiu M. Popescu and his co-workers. Not surprisingly, stromal tumours, immunophenotypically similar to that of telocytes [CD117 (c-KIT) +/CD34 +], have also been sporadically reported outside the tubular gut (so-called extragastrointestinal stromal tumours, EGISTs), including the gall bladder, liver, and pancreas. A meticulous literature search from January 2000 to November 2015 have found 9 such case reports of EGISTs in the gall bladder, 16 in the liver, and 31 occurring in the pancreas. The site wise mean age at presentation for these tumours were reported to be 62.2 ± 16.6, 50.9 ± 20.1, and 55.3 ± 14.3 years, respectively. Six of nine EGISTs in the gall bladder were associated with gallstones. On pathological evaluation, these tumours exhibited prominent spindled cell morphology and consistent expression of CD117/c-KIT and CD34 on immunohistochemistry and variable expression of vimentin and α-smooth muscle actin. The biological behaviour of hepatic and pancreatic lesions was favourable compared to that in the gall bladder, following definitive surgery with or without imatinib therapy. While the exact pathophysiologic role played by telocytes in various organs is yet to be fully elucidated, there seems to be a direct link between these enigmatic stromal cells and pathogenesis of gallstones and origin of EGISTs, and a hope for targeted therapies. Furthermore, the possible role of telocytes in hepatic regeneration and liver fibrosis opens a new dimension for futuristic research.

Global methylation profiling to identify epigenetic signature of gallbladder cancer and gallstone disease.

Promoter methylation in various tumor suppressor genes is reported to influence gallbladder carcinogenesis. Here, we aimed to identify methylation status in gallbladder cancer (GBC) by performing a comprehensive genome-wide DNA methylation profiling. The methylation status of 485,577 CpG sites were investigated using Illumina's Infinium Human Methylation 450 BeadChip array in 24 tissues (eight each of tumor, adjacent non-tumor, and gallstone). About 33,443 differentially methylated sites (DMRs) were obtained in the whole human genome, of which 24,188 (72 %) were hypermethylated and 9255 (28 %) were hypomethylated. The data also revealed that majority of the DMRs are localized on the proximal promoter region [Transcription start sites (TSS200, TSS1500) and 5' untranslated region (5'UTR)] and first exon. Exclusion of first exon detected a total of 10,123 (79 %) hypermethylated and 2703 (21 %) hypomethylated sites. Comparative analysis of the later with our differential proteomics data resulted in identification of 7 hypermethylated or down-regulated (e.g., FBN1, LPP, and SOD3) and 61 hypomethylated or up-regulated markers (e.g., HBE1, SNRPF, TPD52) for GBC. These genes could be further validated on the basis of their methylation/expression status in order to identify their utility to be used as biomarker/s for early diagnosis and management of GBC.

Effects of sphincter of Oddi motility on the formation of cholesterol gallstones.

AIM: To investigate the mechanisms and effects of sphincter of Oddi (SO) motility on cholesterol gallbladder stone formation in guinea pigs. METHODS: Thirty-four adult male Hartley guinea pigs were divided randomly into two groups, the control group (n = 10) and the cholesterol gallstone group (n = 24), which was sequentially divided into four subgroups with six guinea pigs each according to time of sacrifice. The guinea pigs in the cholesterol gallstone group were fed a cholesterol lithogenic diet and sacrificed after 3, 6, 9, and 12 wk. SO manometry and recording of myoelectric activity were obtained by a multifunctional physiograph at each stage. Cholecystokinin-A receptor (CCKAR) expression levels in SO smooth muscle were detected by quantitative real-time PCR (qRT-PCR) and serum vasoactive intestinal peptide (VIP), gastrin, and cholecystokinin octapeptide (CCK-8) were detected by enzyme-linked immunosorbent assay at each stage in the process of cholesterol gallstone formation. RESULTS: The gallstone formation rate was 0%, 0%, 16.7%, and 83.3% in the 3, 6, 9, and 12 wk groups, respectively. The frequency of myoelectric activity in the 9 wk group, the amplitude of myoelectric activity in the 9 and 12 wk groups, and the amplitude and the frequency of SO in the 9 wk group were all significantly decreased compared to the control group. The SO basal pressure and common bile duct pressure increased markedly in the 12 wk group, and the CCKAR expression levels increased in the 6 and 12 wk groups compared to the control group. Serum VIP was elevated significantly in the 9 and 12 wk groups and gastrin decreased significantly in the 3 and 9 wk groups. There was no difference in serum CCK-8 between the groups. CONCLUSION: A cholesterol gallstone-causing diet can induce SO dysfunction. The increasing tension of the SO along with its decreasing activity may play an important role in cholesterol gallstone formation. Expression changes of CCKAR in SO smooth muscle and serum VIP and CCK-8 may be important causes of SO dysfunction.

Clinical and ABCB11 profiles in Korean infants with progressive familial intrahepatic cholestasis.

AIM: To investigate clinical profiles and mutations of ABCB11 in Koreans with progressive familial intrahepatic cholestasis 2 and review the differences between Koreans and others. METHODS: Of 47 patients with neonatal cholestasis, five infants had chronic intrahepatic cholestasis with normal γ-glutamyl transpeptidase. Direct sequencing analyses of ABCB11, including exons and introns, were performed from peripheral blood. RESULTS: Living donor-liver transplantation was performed in four patients because of rapidly progressive hepatic failure and hepatocellular carcinoma. Three missense mutations were found in two patients: compound heterozygous 677C>T (S226L)/3007G>A (G1003R) and heterozygous 2296G>A (G766R). The mutations were located near and in the transmembranous space. CONCLUSION: Alterations in the transmembrane of the bile salt export pump in the Korean infants were different from those previously reported in Chinese, Japanease, Taiwanese, and European patients.