Evaluation of genetic association of 40 SNPs in candidate genes with cholesterol gallstone disease in north Indian population

Meta Gene 21 (2019) 100579

Contents lists available at ScienceDirect

Meta Gene journal homepage: www.elsevier.com/locate/mgene

Evaluation of genetic association of 40 SNPs in candidate genes with cholesterol gallstone disease in north Indian population Tripty Chauhana, R.D. Mittalb, B. Mittala, a b

T

⁎

Department of Biotechnology, Baba Saheb Bhimrao Ambedkar University, Lucknow, UP, India Department of Urology, SGPGIMS, Lucknow, UP, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Genetic susceptibility Gallstone disease Genetic association SNP Polymorphism GENEMANIA

Background: In north Indian population, Gallbladder Stone Disease (GSD) affects 7% of population, with majority of stones containing > 50% cholesterol. In general, GSD susceptibility has been suggested to include the combination of predisposing alleles of multiple lithogenic (LITH) genes and environmental factors. Materials and methods: Study included 610 GSD patients (USG positive) and 315 healthy controls. A panel of 48 SNPs was prepared for candidate genes based on pathobiology of GSD, MAF in Hapmap and 1000 genome database, and Tagger SNPs in Linkage Disequilibrium. Genotyping was carried out by Sequenom Mass ARRAY platform and statistical analyses were performed using SPSS version 16.0, Bonferroni adjustment was applied for multiple testing corrections. Potential functional consequences of the exonic polymorphisms were determined using Polyphen2, SIFT score and interaction networks of associated genes by GENEMANIA. Results: Genotypes of 40 out of 48 SNPs followed Hardy Weinberg Equilibrium in controls and subjected to present analysis. We observed significant associations of genotypes/alleles of FGFR4, CYP7A1, APOE, TM4SF4, NR1H2, IL10, PLCE1, CYP1A1, SLC10A2, MMP2, CYP8B1, Gab1 with GSD whereas remaining SNPs revealed no association. Analysis after gender stratification and Bonferroni correction revealed that in males SNPs in TM4SF4, PLCE1 and Gab1 were significantly associated with GSD while in females APOE and CYP8B1 polymorphisms were significantly associated with GSD. All but three significantly associated SNPs were intronic. The potential functional consequences of associated exonic SNPs by Polyphen2 predicted probable damaging effects of FGFR4 and APOE variants, while PLCE1 SNP showed benign effect. Further SIFT score predicted probable tolerated effects of FGFR4 and PLCE1, while APOE variant showed deleterious effect. Conclusions: Our study suggests that FGFR4, CYP7A1, APOE, TM4SF4, NR1H2, IL10, PLCE1, CYP1A1, SLC10A2, MMP2, CYP8B1 genetic variants confer significant risk for gallstone disease in North Indian population. The association of many genes with disease risk was found to be sex-specific.

1. Introduction

the lowest incidence (< 5%) (Kratzer and Mason, 1999). These geographic and ethnic differences in gallstone prevalence imply that genetic factors influence risk of gallstone formation. In a large study of Swedish twins, the contribution of genetic factors to the development of symptomatic GSD was estimated (Katsika et al., 2005). The report found that the concordance rates in monozygotic twins of both the genders (12%) were significantly higher than in dizygotic twins (6%) in 43,141 Swedish twins, provides strong evidence for a genetic component in GSD. The inbred strains of mice have shown great variations in susceptibility to gallstone formation when fed diet with high cholesterol and cholic acid (Khanuja et al., 1995). Quantitative trait loci (QTL) genetic

Gallstones are quite prevalent in humans and the disease occurs in 5–10% of adult population (Lowenfels and Velema, 1992). The disease prevalence has been found to be around at least 6% in adult population of North India (Khuroo et al., 1989). Long-standing gallstones are major risk for carcinoma of the gallbladder. Epidemiologically, the highest prevalence (48%) has been found in Pima Indians, some North and South American Indians, Chileans (Everhart et al., 2002; Sampliner et al., 1970; Weiss et al., 1984). American (white), European (20%), and Asian populations (5–20%) show intermediate rates (Everhart et al., 1999; Lammert and Sauerbruch, 2005), whereas Africans register ⁎

Corresponding author. E-mail address: [email protected] (B. Mittal).

https://doi.org/10.1016/j.mgene.2019.100579 Received 30 October 2018; Received in revised form 5 March 2019; Accepted 23 April 2019 Available online 25 April 2019 2214-5400/ © 2019 Published by Elsevier B.V.

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

analysis yielded several chromosomal regions (Lith loci) that contribute towards gallstone formation and many candidate genes have been identified (Lammert et al., 2001; Lammert et al., 2002; Wang et al., 2010). All these findings suggest that genetic factors play significant role in the development of gallstone disease. One of the important approaches to derivee genes in human polygenic diseases is case control association studies which can narrowly estimate association of particular genetic variant with disease in the population. In Gallstone disease, numerous association studies have been performed in the past decade. Several genetic variants of APOE, APOB and CETP were associated with the susceptibility for gallstone disease but the published results have shown inconsistency in the outcome of concluding results (Jiang et al., 2004; Juvonen et al., 1995; Portincasa et al., 1996; Tan et al., 2003). Perhaps problem partly may be due to the sample size, which was usually small. Furthermore, the association studies are elusive to population/ethnicity and geographical variation. Till date there was no satisfactory study in Indian population that could bring light to the complex pathophysiology of GSD. Therefore, we have carried out case–control analysis. On the basis of pathophysiology and epidemiology of GSD, cholesterol metabolism, cholesterol transport, sex steroids and their receptors, bile acid transport mucins, gallbladder motility and adrenergic receptor pathways, we hypothesized that the genetic variants in genes belonging to these pathways may provide some more insights into pathophysiology of gallstone formation. Consequently, a panel of 48 SNPs was prepared for candidate gene association study, using SNP database from Hapmap, 1000 genome and Tagger SNPs in Linkage Disequilibrium. Based on literature, all 48 SNPs from 47 candidate genes have been proposed to play a substantial role in the genetic predisposition of gallbladder stone disease particularly in European populations.

Table 1 Selected markers for the study.

2. Materials and methods 2.1. Subjects The present case control study is both a retrospective and prospective study. DNA samples from previous study were also used after Institutional Ethical Committee approval. At the same time, new patients and controls were also enrolled. The study comprised a total of 610 consequently diagnosed cholesterol gallstone patients (USG positive) recruited among patients attending the clinics of Department of Gastroenterology and Gastro-surgery of Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS) Lucknow, UP India. Patients with a history of Inflammatory Bowel Disease (Crohn's disease and Ulcerative Colitis), diabetes mellitus, renal failure any other chronic and debilitating disease were excluded from the gallstone group. A total of 315 healthy controls were recruited from unrelated individuals from north India. The recruited controls were from the hospital that serves as a tertiary referral center so the lack of enough USG based gallstone-free of healthy control participants restricted our control subjects merely up to 315. The inclusion criteria for controls were absence of Inflammatory Bowel Disease (Crohn's disease and Ulcerative Colitis), diabetes mellitus, renal failure any other chronic, debilitating disease. After obtaining the informed consent, all the individuals were personally interviewed for information on ethnicity, food habits, occupation and tobacco usage. Both patients and controls had similar ethnicity. The study was approved by local ethics committee of the institute. Blood samples from all the subjects were collected in ethylene diamine tetra acetic acid (EDTA) and stored at −70 °C until further use.

S. No.

Gene symbol

SNP Id

Chromosomal location

MAF*

1 2 3 4

LDLR CCK SLCO1B1 CXCL8

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

TGFB1 EGFR RXRB ESR1 FABP1 MMP2 LPL NR1H2 FGFR4 MTTP MUC1 EGF COMT IL18 CYP7A1 PTGS2 HNF1A APOE SLC10A2 TM4SF4 FTO DYNC2LI1 NR1H4 MMP7 ALCAM ABCG8 CLDN2 GCKR ESR2 SSTR5 CETP CCKAR IL10 PLCE1 CR1 MLN CYP17A1 CYP1A1 CYP8B1 Gab1 ABCB11 TLR4 TIMP2

rs1003723 rs10865918 rs11045819 rs12506479 rs2227306 rs1800469 rs2017000 rs2076310 rs2234693 rs2241883 rs2285053 rs263 rs2695121 rs351855 rs3816873 rs4072037 rs4444903 rs4633 rs5744247 rs6471717 rs689466 rs7310409 rs7412 rs9514089 rs9843304 rs9939609 rs1025447 rs11110385 rs11568818 rs1157 rs11887534 rs12014762 rs1260326 rs1271572 rs169068 rs1800775 rs1800855 rs1800871 rs2274223 rs2274567 rs2281820 rs2486758 rs2606345 rs3732860 rs3805246 rs497692 rs4986791 rs8179090

19p13.2 3p22.1 12p12.1 4q13.3 4q13.3 19q13.1 7p11.2 6p21.32 6q25.1-q25.2 2p11.2 16q12.2 8p21.3 19q13.3 5q35.2 4q23 1q22 4q25 22q11.21 11q23.1 8q12.1 1q31.1 12q24.31 19q13.32 13q33.1 3q25.1 16q12.2 2p21 12q23.1 11q22.2 3q13.11 2p21 Xq22.3 2p23.3 14q23.2-q23.3 16p13.3 16q13 4p15.2 1q32.1 10q23.33 1q32.2 6p21.31 10q24.32 15q24.1 3p22.1 4q31.21 2q31.1 9q33.1 17q25.3

0.36 0.22 0.06 0.33 0.31 0.38 0.45 0.37 0.38 0.11 0.11 0.18 0.23 0.38 0.35 0.49 0.49 0.41 0.09 0.29 0.14 0.48 0.05 0.37 0.39 0.24 0.17 0.17 0.36 0.23 0.02 0.24 0.19 0.46 0.42 0.38 0.36 0.40 0.26 0.48 0.30 0.26 0.36 0.35 0.48 0.31 0.10 0.13

Gujarati Indians in Houston (GIH) Minor Allele Frequency (MAF*) in. Hapmap and 1000 genome database.

pathobiology of GSD, MAF in Hapmap and 1000 genome database, and Tagger SNPs in Linkage Disequilibrium (Tables 1 and 2). Genotyping of the SNPs was carried out using the Sequenom Mass ARRAY platform (Sequenom, San Diego, CA). All SNPs genotyped by Sequenom Mass ARRAY platform passed the genotyping call-rate threshold (> 95%). To explore the gender-specific effect of these polymorphisms, analysis was done after stratification of subjects according to gender. 2.3. Statistical analysis Genotype and allele distribution was compared between gallstone and healthy subjects using a χ2 test. The independent segregation of alleles was tested for HWE, comparing the observed genotype frequencies with those expected (χ2test). For genotype-disease association studies, differences in genotype distributions were calculated, applying a log additive logistic regression model adjusted for sex and age. All statistical analyses were performed using SPSS software version 16.0 (SPSS, Chicago, IL).

2.2. Genotyping The genomic DNA was extracted from peripheral blood leucocytes pellet using the standard salting-out method (Miller et al., 1988). A panel of 48 SNPs was prepared for candidate genes based on the basis of 2

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Table 2 The various candidate genes catagorized in the 9 selected pathways. S. No.

Selected pathways

Candidate genes

1.

Cholesterol metabolism

2. 3. 4.

Gallbladder motility Hormone related Regulation of extracelluar matrix (ECM) components Inflammatory Signaling transduction pathway Lipid metabolism Insulin sensitivity Obesity related

ABCG8, CYP17A1, ApoE, CETP, LDLR, CYP7A1, FGFR4, CYP8B1, GCKR, SLC10A2, SLCO1B1, NR1H2, ABCB11, HNF1A, DYNC2LI1, CLDN2 CCKAR, CCK, SSTR5, MLN ESR2, COMT, CYP1A1 MMP2, MMP7, TIMP2, ALCAM

5. 6. 7. 8. 9.

IL8, TGFB1, IL10, IL18, CR1, TLR4 EGF, EGFR, Gab1, TM4SF4 FABP1, MTTP, APOE, LDLR, LPL, NR1H2, PTGS2, PLCE1, TIMP2 RXRB FTO

95% CI =1.93(1.14–3.27)] and [G] allele [p = .043; OR and 95% CI = 1.2(1.01–1.53)] seemed to impose a high risk with GSD. The genotypes of APOE rs7412 [CT] [p = .002; OR and 95% CI =0.48(0.30–0.77)] and its [T] allele [p = .003; OR and 95% CI = 0.49(0.314–0.79)] also marked low risk with gallstone disease. In TM4SF4 rs9843304 gene patients with the [TC] genotype [p = .048; OR and 95% CI =0.73(0.54–0.99)] also imposed low risk with gallstone disease. While no association was observed with its allele frequency. In IL10 rs1800871 gene Patients with the [GA] [p = .021; OR and 95% CI =0.69(0.50–0.94)] had low risk with gallstone disease but no association was observed with its allele frequency. PLCE1 rs2274223 gene genotype [GG] [p = .013; OR and 95% CI =0.49(0.27–0.86)] and the [G] allele [p = .054; OR and 95% CI = 0.81(0.65–1.00)] showed significant low risk with GSD. In gene CYP1A1 rs2606345 genotype [AA] [p = .043; OR and 95% CI =1.63(1.01–2.61)] seemed to impose a high risk with GSD while the allele frequency marked no association with the disease. In Gab1 rs3805246 gene genotypes [GA] and [GG] [p = .014; OR and 95% CI =1.48(1.08–2.03)], [p = .028; OR and 95% CI =1.54(1.04–2.26)] respectively and [G] allele [p = .016; OR and 95% CI = 1.3(1.05–1.54)] revealed significant high risk associated with cholesterol gallstone disease. The remaining SNPs revealed no association with cholesterol gallstone disease (Supplementary Table 2). To explore the gender-specific effect of these polymorphisms, analysis was done after stratification of subjects according to gender. Bonferroni correction was applied for multiple testing of the data where P < 0·025 was considered statistically significant and the present study indicated that in males the genotypes of TM4SF4 rs9843304 gene with genotype [TC] [p = .021; OR and 95% CI =0.61(0.39–0.93)] also had low risk with gallstone disease. The PLCE1 rs2274223 gene, genotype [GG] [p = .007; OR and 95% CI =0.29(0.12–0.71)] revealed low risk with gallstone disease. In Gab1 rs3805246 gene, genotype [GG] [p = .007; OR and 95% CI =2.1(1.21–3.48)] was significantly associated with a high risk of cholesterol gallstone disease. In females, the APOE rs7412 gene, genotype [CT] [p = .003; OR and 95% CI =0.35(0.18–0.69)] showed low risk with gallstone disease. CYP8B1 rs3732860 genotypes [CT] [p = .006; OR and 95% CI =0.47(0.28–0.81)] was significantly associated with a low risk of cholesterol gallstone disease. The results of our present study were compared with previous studies and tabulated in Table 5 and Supplementary Table 3.

Power calculations were carried out to establish the strength of the study. Power of the study was > 80% except for three SNPs (SLCO1B1 rs11045819, IL18 rs5744247 and ABCG8 rs11887534) whose MAF was low. 2.4. In silico analysis An interaction networks of all associated genes were determined by GENEMANIA (http://www.genemania.org/). The potential functional consequences of the exonic polymorphisms were assessed by use of the predictive programs Polyphen2 and SIFT score. 3. Results Patient characteristics and genotype frequencies demographic characteristics of gallstone patients and controls are summarized in Table 3. The mean age of GSD patients and healthy controls was 48.42 ± 12.057 and 33.55 ± 11.95 respectively. In the total recruited patients, 398(65.2%) of the subjects were females while in case of controls 96(30.5%) subjects were females. The controls followed Hardy-Weinberg equilibrium (HWE) in 40 out of 48 selected SNPs and observed frequencies were similar to Gujarati Indians in Houston mentioned in Hap Map and 1000 Genomes Browser. The deviation from HWE was observed in 8 selected SNPs and were removed from further analyses (Supplementary Table 1). 3.1. Single locus logistic regression analysis of gallstone disease For genotype association study, differences in genotype distributions were calculated applying a log additive logistic regression model adjusted for gender and age. Table 4 show the single locus logistic regression analysis of the 40 investigated genetic variants with gallstone disease. Logistic regression analysis revealed significant associations (P < .05) of following SNPs with cholesterol gallstone disease risk whereas the remaining SNPs revealed no association (P > .05). The study found that in FGFR4 rs351855 patients with genotypes [GA] and [AA] [p = .035; OR and 95% CI =0.72(0.53–0.97)], [p = .025; OR and 95% CI =0.62(0.40–0.94)] respectively and the [A] allele [p = .012; OR and 95% CI = 0.78(0.64–0.95)] showed significant low risk with gallstone disease. CYP7A1 rs6471717 genotype [GG] [p = .014; OR and

3.2. In silico analysis Table 3 Characteristic profile of healthy controls and gallstone patients. Demographic profile

Healthy controls Mean ± SD [n = 315]

Gallstone patients Mean ± SD [n = 610]

P value

Age (years) Gender Males Females

33.55 ± 11.95

48.42 ± 12.06

0.178

n = 219(69.5%) n = 96(30.5%)

n = 212(34.8%) n = 398(65.2%)

– –

An interaction network of all of the associated genes was generated by GENEMANIA. This network revealed direct and indirect interactions among them via co-expression, gene interactions, physical interactions, shared protein domains, and pathways, as shown in Fig. 1. Through GENEMANIA we observed that APOE, CETP, CYP8B1, CYP1A1, CYP7A1, SLC10A2, FGFR4, TM4SF4, IL10, PLCE1, MMP2 and Gab1 were in interactions among them via co-expression. CYP1A1, CYP7A1 and CYP8B1 interacted among them via Shared protein 3

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Table 4 Significant association of the investigated genetic variants and allele with Gallstone patients and controls: Gene

SNP Id

Controls N (%)

Patients N (%)

P-value

OR (95%CI)

FGFR4 Total

rs351855 GG GA AA

102(32.5%) 161(51.3%) 51(16.2%)

247(40.8%) 282(46.6%) 76(12.6%)

0.035 0.025

ref 0.72(0.53–0.97) 0.62(0.40–0.940)

G A

365(58.1%) 263(41.9%)

776(64.1%) 434(35.9%)

0.012

ref 0.78(0.64–0.95)

GG GA AA

74(33.8%) 112(51.1%) 33(15.1%)

78(37.0%) 111(52.6%) 22(10.4%)

0.770 0.152

ref 0.94(0.62–1.42) 0.63(0.34–1.18)

GG GA AA rs7412

28(29.5%) 49(51.6%) 18(18.9%)

169(42.9%) 171(43.4%) 54(13.7%)

0.035a 0.040a

ref 0.58(0.35–0.96) 0.49(0.26–0.97)

CC CT TT

274(87.8%) 38(12.2%) 0

569(93.7%) 38(6.3%) 0

0.002

ref 0.48(0.30–0.772)

C T

586(93.9%) 38(6.1%)

1176(96.9%) 38(3.1%)

0.003

ref 0.49(0.314–0.79)

CC CT TT

193(89.4%) 23(10.6%) 00

196(93.3%) 14(6.7%) 00

0.148

ref 0.59(0.30–1.19)

CC CT TT rs9843304

81(84.4%) 15(15.6%) 00

373(94.0%) 24(6.0%) 00

0.003b

ref 0.35(0.18–0.69)

TT TC CC

98(31.5%) 163(52.4%) 50(16.1%)

223(36.8%) 272(44.9%) 111(18.3%)

0.048 0.906

ref 0.73(0.54–0.99) 0.98(0.64–1.47)

T C

359(57.7%) 263(42.3%)

718(59.2%) 494(40.8%)

0.530

ref 0.94(0.77–1.14)

TT TC CC

68(31.5%) 116(53.7%) 32(14.8%)

83(39.2%) 86(40.6%) 43(20.3%)

0.021b 0.736

ref 0.61(0.39–0.93) 1.1(0.63–1.93)

TT TC CC rs2274223

30(31.6%) 47(49.5%) 18(18.9%)

140(35.5%) 186(47.2%) 68(17.3%)

0.525 0.525

ref 0.85(0.51–1.41) 0.81(0.0.42–1.55)

AA GA GG

153(49.2%) 132(42.4%) 26(8.4%)

327(53.7%) 255(41.9%) 27(4.4%)

0.487 0.013

ref 0.90(0.68–1.20) 0.49(0.27–0.86)

A G

438(70.4%) 184(29.6%)

909(74.6%) 309(25.4%)

0.054

ref 0.81(0.65–1.00)

AA GA GG

94(43.5%) 102(47.2%) 20(9.3%)

115(54.5%) 89(42.2%) 7(3.3%)

0.093 0.007b

ref 0.71(0.48–1.05) 0.29(0.12–0.71)

AA GA GG rs3805246

59(62.1%) 30(31.6%) 6(6.3%)

212(53.3%) 166(41.7%) 20(5.0%)

0.081 0.878

ref 1.5(0.95–2.49) 0.93(0.36–2.42)

AA GA GG

109(35.2%) 139(44.8%) 62(20.0%)

161(26.5%) 305(50.2%) 141(23.2%)

0.014 0.028

ref 1.48(1.08–2.03) 1.54(1.04–2.26)

A G

357(57.6%) 263(42.4%)

627(51.6%) 587(48.4%)

0.016

ref 1.3(1.05–1.54)

Allele

Men

Women

APOE Total

Allele

Men

Women

TM4SF4 Total

Allele

Men

Women

PLCE1 Total

Allele

Men

Women

Gab1 Total

Allele

(continued on next page)

4

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Table 4 (continued) Gene

SNP Id

Controls N (%)

Patients N (%)

P-value

OR (95%CI)

AA GA GG

72(33.5%) 96(44.7%) 47(21.9%)

47(22.2%) 102(48.1%) 63(29.7%)

0.038a 0.007b

ref 1.6(1.03–2.58) 2.1(1.21–3.48)

AA GA GG rs6471717

37(38.9%) 43(45.3%) 15(15.8%)

114(28.9%) 203(51.4%) 78(19.7%)

0.092 0.123

ref 1.5(0.93–2.52) 1.7(0.87–3.28)

AA AG GG

153(48.9%) 139(44.4%) 21(6.7%)

271(44.8%) 262(43.3%) 72(11.9%)

0.670 0.014

ref 1.1(0.80–1.416) 1.93(1.14–3.27)

A G

445(71.1%) 181(28.9%)

804(66.4%) 406(33.6%)

0.043

ref 1.2(1.01–1.53)

AA AG GG

106(48.8%) 96(44.2%) 15(6.9%)

102(48.1%) 88(41.5%) 22(10.4%)

0.811 0.245

ref 0.95(0.64–1.42) 1.5(0.75–3.10)

AA AG GG rs1800871

47(49.0%) 43(44.8%) 6(6.2%)

169(43.0%) 174(44.3%) 50(12.7%)

0.618 0.069

ref 1.1(0.71–1.79) 2.3(0.94–5.74)

GG GA AA

90(29.1%) 162(52.4%) 57(18.4%)

221(36.5%) 275(45.4%) 110(18.2%)

0.021 0.241

ref 0.69(0.50–0.94) 0.79(0.52–1.17)

G A

342(55.3%) 276(44.7%)

717(59.2%0 495(40.8%)

0.118

ref 0.86(0.70–1.04)

GG GA AA

62(28.8%) 114(53.0%) 39(18.1%)

71(33.5%) 98(46.2%) 43(20.3%)

0.196 0.893

ref 0.75(0.49–1.16) 0.96(0.56–1.67)

GG GA AA rs2606345

28(29.8%) 48(51.1%) 18(19.1%)

150(38.1%) 177(44.9%) 67(17.0%)

0.155 0.278

ref 0.69(0.41–1.15) 0.69(0.36–1.34)

CC CA AA

131(41.9%) 153(48.9%) 29(9.3%)

236(38.9%) 285(47.0%) 85(14.0%)

0.821 0.043

ref 1.03(0.77–1.38) 1.63(1.01–2.61)

C A

415(66.3%) 211(33.7%)

757(62.5%) 455(37.5%)

0.105

ref 1.2(0.96–1.45)

CC CA AA

91(41.6%) 107(48.9%) 21(9.6%)

79(37.3%) 104(49.1%) 29(13.7%)

0.584 0.153

ref 1.1(0.75–1.68) 1.6(0.84–3.01)

CC CA AA rs2285053

40(42.6%) 46(48.9%) 8(8.5%)

157(39.8%) 181(45.9%) 56(14.2%)

0.992 0.166

ref 1.0(0.62–1.61) 1.8(0.79–4.04)

CC CT TT

257(82.6%) 51(16.4%) 3(1.0%)

480(79.3%) 113(18.7%) 12(2.0%)

0.357 0.241

ref 1.2(0.825–1.707) 2.1(0.599–7.658)

C T

565(90.8%) 57(9.2%)

1073(88.7%) 137(11.3%)

0.156

ref 1.3(0.91–1.75)

CC CT TT

169(78.6%) 43(20.0%) 3(1.4%)

160(75.8%) 47(22.3%) 4(1.9%)

0.546 0.657

ref 1.2(0.72–1.84) 1.4(0.31–6.4)

CC CT TT rs9514089

88(91.7%) 8(8.3%) 0(0.0%)

320(81.2%) 66(16.8%) 8(2.0%)

0.037a 0.999

ref 2.3(1.1–4.90) 4.4(0.00)

Men

Women

CYP7A1 Total

Allele

Men

Women

IL10 Total

Allele

Men

Women

CYP1A1 Total

Allele

Men

Women

MMP2 Total

Allele

Men

Women

SLC10A2

(continued on next page)

5

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Table 4 (continued) Gene

SNP Id

Controls N (%)

Patients N (%)

P-value

OR (95%CI)

TT TC CC

134(43.2%) 136(43.9%) 40(12.9%)

235(39.0%) 295(48.9%) 73(12.1%)

0.156 0.859

ref 1.23(0.92–1.65) 1.04(0.67–1.61)

T C

404(65.2%) 216(34.8%)

765(63.4%) 441(36.6%)

0.466

ref 1.1(0.88–1.32)

TT TC CC

85(39.7%) 98(45.8%) 31(14.5%)

83(39.3%) 97(46.0%) 31(14.7%)

0.949 0.936

ref 1.01(0.67–1.53) 1.0(0.57–1.83)

TT TC CC rs3732860

49(51.0%) 38(39.6%) 9(9.4%)

152(38.8%) 198(50.5%) 42(10.7%)

0.032a 0.310

ref 1.7(1.05–2.69) 1.5(0.68–3.31)

CC CT TT

107(34.4%) 158(50.8%) 46(14.8%)

239(39.4%) 289(47.7%) 78(12.9%)

0.191 0.209

ref 0.82(0.60–1.1) 0.76(0.49–1.16)

C T

372(59.8%) 250(40.2%)

767(63.3%) 445(36.7%)

0.146

ref 0.86(0.71–1.1)

CC CT TT

85(39.2%) 102(47.0%) 30(13.8%)

84(39.6%) 102(48.1%) 26(12.3%)

0.955 0.671

ref 1.0(0.67–1.52) 0.88(0.48–1.60)

CC CT TT rs2695121

22(23.4%) 56(59.6%) 16(17.0%)

155(39.3%) 187(47.5%) 52(13.2%)

0.006b 0.034a

ref 0.47(0.28–0.81) 0.46(0.23–0.94)

CC TC TT

144(46.3%) 136(43.7%) 31(10.0%)

295(49.0%) 251(41.7%) 56(9.3%)

0.478 0.609

ref 0.90(0.67–0.20) 0.88(0.54–1.428)

C T

424(68.2%) 198(31.8%)

841(69.9%) 363(30.1%)

0.460

ref 0.92(0.75–1.1)

CC TC TT

96(44.7%) 100(46.5%) 19(8.8%)

110(52.9%) 73(35.1%) 25(12.0%)

0.030a 0.680

ref 0.64(0.42–0.96) 1.1(0.59–2.21)

CC TC TT rs1800775

48(50.0%) 36(37.5%) 12(12.5%)

185(47.0%) 178(45.2%) 31(7.9%)

0.308 0.288

ref 1.3(0.79–2.07) 0.67(0.32–1.40)

AA CA CC

112(35.8%) 156(49.8%) 45(14.4%)

229(37.6%) 287(47.1%) 93(15.3%)

0.488 0.960

ref 0.90(0.66–1.21) 1.01(0.66–1.54)

A C

380(60.7%) 246(39.3%)

745(61.2%) 473(38.8%)

0.847

ref 0.98(0.81–1.20

AA CA CC

67(30.7%) 118(54.1%) 33(15.1%)

84(39.6%) 97(45.8%) 31(14.6%)

0.048a 0.334

ref 0.66(0.43–0.99) 0.75(0.42–1.34)

AA CA CC

45(47.4%) 38(40.0%) 12(12.6%)

145(36.5%) 190(47.9%) 62(15.6%)

0.075 0.188

ref 1.6(0.96–2.51) 1.6(0.79–3.24)

Total

Allele

Men

Women

CYP8B1 Total

Allele

Men

Women

NR1H2 Total

Allele

Men

Women

CETP Total

Allele

Men

Women

Abbreviations: 95% CI, 95% confidence interval; P-value. ‘a’ non-significant after Bonferroni correction and ‘b’ Significant after Bonferroni correction. Rest of the 27 SNPs in CCK, SLCO1B1, IL8, TGFB1, EGFR, RXRB, ESR1, FABP1, LPL, MTTP, COMT, IL18, HNF1A, FTO, MMP7, ALCAM, ABCG8, GCKR, SSTR5, CCKAR, CR1, MLN, CYP17A1, ABCB11, TLR4 and TIMP2 were not associated with GSD. The values in bold show significant association with Gallstone disease.

were predicted to have probable damaging effects. While one of the markers showed benign effect (Table 6). On the basis of SIFT score assessment 2 of the exonic SNPs showed tolerated effect, while one of the variants showed deleterious effect (Table 7).

domains. On the other hand, Genetic interactions were observed among IL10, SLC10A2, TM4SF4, CYP7A1, CETP, NR1H2, MMP2 and Gab1 genes. The potential functional consequences of the exonic SNPs were assessed by use of the predictive programs Polyphen2. Two of the 13 SNPs 6

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Table 5 Comparison of significant association in present study to previous published association studies. Genes/SNP

Present study

Previous studies

Genotype/allele

95% CI

MMP2 rs2285053

Women

[CT]a increased risk

(1.1–4.90)

NR1H2 rs2695121 FGFR4 rs351855

Men Total

[TC]a decreased risk [GA] decreased risk [AA] decreased risk [A] decreased risk [GA]a decreased risk [AA]a decreased risk [GG] increased risk [G] increased risk [CT] decreased risk

(0.42–0.96) (0.53–0.97) (0.40–0.940) (0.64–0.95) (0.35–0.96) (0.26–0.97) (1.14–3.27) (1.01–1.53) (0.30–0.772)

[T] decreased risk [CT]b decreased risk [TC]a increased risk [TC] decreased risk [TC]b decreased risk [CA]a decreased risk [GA] decreased risk [GG] decreased risk [G] decreased risk [GG]b decreased risk [AA] increased risk [CT]b decreased risk [TT]a decreased risk [GA] increased risk [GG] increased risk [G] increased risk [GA]a increased risk [GG]b increased risk

(0.314–0.79) (0.18–0.69) (1.05–2.69) (0.54–0.99) (0.39–0.93) (0.43–0.99) (0.50–0.94) (0.27–0.86) (0.65–1.00) (0.12–0.71) (1.01–2.61) (0.28–0.81) (0.23–0.94) (1.08–2.03) (1.04–2.26) (1.05–1.54) (1.03–2.58) (1.21–3.48)

Allele Women CYP7A1 rs6471717 APOE rs7412

Total Allele Total

CYP1A1 rs2606345 CYP8B1 rs3732860

Allele Women Women Total Men Men Total Total Allele Men Total Women

Gab1 rs3805246

Total

SLC10A2 rs9514089 TM4SF 4 rs9843304 CETP rs1800775 IL10 rs1800871 PLCE1 rs2274223

Allele Men

Sharma et al. (2012) - Positive association

Sharma et al. (2013a) - Positive association Chen et al. (2012) - No association.

Joshi et al. (2016) - Positive association Risk with APOE4 Sánchez-Cuén et al. (2010) - no association Martinez-Lopez et al. (2015) - positive association Pinheiro-Junior et al. (2012) -no association Renner et al. (2009) - Positive association, Tönjes et al. (2011) - No association Joshi et al. (2016) - Positive association Xu et al. (2011) - Negative association Vishnoi et al. (2007) - Positive association Sharma et al. (2013b) - Positive association

Park et al. (2009)- Positive association Qin et al. (2011)- Positive association; Qin et al. (2013) - Positive association Meng et al. (2014) - Positive association

‘a’ non-significant after Bonferroni correction and ‘b’ significant after Bonferroni correction.

4. Discussion

associated positively with gallstone disease risk which is in concordance with the result of present study. SLC10A2 gene encodes a sodium/bile acid cotransporter. Bile acids are the catabolic product of cholesterol metabolism, so this protein is also critical for cholesterol homeostasis. The study by Tönjes A et al. reported no significant association of rs9514089 with gallstone risk (Tönjes et al., 2011). The study by Renner et al. (2009) reported a significant association of the polymorphism rs9514089 with gallstone disease. The present study also showed a positive association with the risk of gallstone disease. Fibroblast growth factor receptor 4 (FGFR4) plays an important role in maintaining bile acid homeostasis by regulating the expression of cholesterol 7α-hydroxylase (CYP7A1). The Gly388Arg (G-388R) polymorphism of FGFR4 affects stabilization and activation of FGFR4. The study conducted by Chen et al. (2012) reported that this polymorphism is a genetic risk factor contributing to aggravation of gallstone disease but this result is not in concordance with the result of our study. Lipid metabolism is another important alleyway to GSD risk. The study reported that the APOE, NR1H2 and PLCE1 are associated with GSD through this pathway. In the polymorphism APOE HhaI, the variations in the amino acids at 112 and 158 are supposed to affect the 3-D structure required for stabilization of receptor-binding domain (Wilson et al., 1991). The receptor-binding domain of APO E exists in the N terminal from amino acid 136–150. Because the efficiency of lipoprotein uptake by the liver increases in the sequence e2 < e3 < e4, of APOE polymorphism influences hepatic cholesterol content and possibly cholesterol gallstone formation (Bertomeu et al., 1996). In the present study we have selected only one of the SNP rs7412 of APOE showing low risk of GSD. Juvonen et al. (1993) studied for the first time the relationship between APOE polymorphism and gallstones. Patients with e4 allele had higher cholesterol content in stones, rapid cholesterol crystallization and shorter median nucleation time in the Finnish population(Juvonen et al., 1993). But other studies (Bertomeu et al.,

In our exploration study involving 40 SNPs from various genes, we found that SNPs belonging to genes from various pathways modify risk of gallstone disease. Since cholesterol metabolism is supposed to be the vital pathway prominent to gallstone disease, SNPs in genes such as CETP, CYP8B1, CYP7A1, SLC10A2 and FGFR4 revealed significant association with gallstone disease. CETP, also called plasma lipid transfer protein, is a plasma protein that facilitates transport of cholesteryl esters and triglycerides between lipoproteins. The study by Xu et al. (2011) did not find an association between CETP variants and biliary tract stones or cancer. In previous studies, CETP variants were associated with gallstones in Finnish and Chinese populations, but not in an Indian population (Juvonen et al., 1995; Dixit et al., 2006; Zhang et al., 1999). The CYP8B1 gene encoded protein, CYP8B1, is a P450 cytochrome expressed exclusively in the liver, which acts as a key enzyme meditating cholic acid (CA) synthesis and maintenance of the CA to chenodeoxycholic acid (CDCA) ratio (Kosters et al., 2003). In a study by Qin et al. (2016), SNP rs3732860 in the 3′UTR of the gene reflected significant association to GSD, with carriers of the G allele reported the risk of GSD. In another study by Qin J et al. SNP rs3732860 of CYP8B1 gene was found to be associated with gallstone disease in Chinese population where A allele may play a protective role in the pathogenesis of gallstone (Qin et al., 2011) and this result is in concordance with result of the present study. CYP7A1 gene encodes a member of the cytochrome P450 superfamily of enzymes. Cytochrome P450 proteins are the monooxygenases that catalyze several reactions concerned with metabolism of drug and synthesis of steroids, cholesterol, and other lipids. Polymorphisms in the promoter of this gene are associated with defects in bile acid synthesis. A study by Joshi et al. (2016) reported that rs6471717 was 7

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Fig. 1. Interaction network of all associated genes generated by GENEMANIA (Associated genes in bold). Table 6 Potential functional consequences of the markers. Gene

SNP identifier

Amino acid change

Polyphen 2 (score)

FGFR4 APOE PLCE1

rs351855 rs7412 rs2274223

Gly388Arg R176C His1927Arg

Probably damaging (0.998) Probably damaging (1.000) Benign (0.000)

(sensitivity: 0.27; specificity: 0.99) (sensitivity: 0.00; specificity: 1.00) (sensitivity: 1.00; specificity: 0.00)

harboring gallstones. Phospholipase C epsilon 1 (PLCE1) plays critical roles in carcinogenesis and progression of esophageal and gastric cancers. The study by Sharma et al. (2013b) reported that PLCE1 rs2274223 genotype was significantly associated with an increased risk of GBC in females GBC patients with stones but the analysis in present study reported a protective association with gallstone disease. Present study observed two of the GSD associated markers TM4SF4 and Gab1 related to signal transduction pathway. The protein encoded by TM4SF4 gene is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. The study by Joshi et al. (2016) reported that rs9843304 was associated positively with gallstone disease

1996; Ko et al., 2000; Van Erpecum et al., 1998) had contradictory results. There were reports that median nucleation time (Bertomeu et al., 1996) was similar in patients between the genotypes and that the cholesterol saturation index (Van Erpecum et al., 1998) was lower in patients with e4 allele. Nuclear receptor subfamily 1 (NR1H2) is a ligand activated transcription factor belonging to the family of nuclear receptors. It plays role in control of lipid homeostasis, glucose metabolism, inflammation, and proliferation. NR1H2 are expressed in gallbladder cholangiocytes. The study by Sharma et al. (2013a) found a new genetic association between the NR1H2 (rs2695121) with gallbladder cancer patients 8

Meta Gene 21 (2019) 100579

TOLERATED 40 3.11 1 CDS ENSP00000360438 ENST00000371385 ENSG00000138193 PLCE1 H1619R G A 96066341 rs2274223

rs2274223

rs2274223

rs2274223

rs7412

rs7412

rs7412

rs351855

rs351855

risk but a protective association was observed in our study. GRB2-associated-binding protein 1 (Gab1) is activated through the binding of Grb2 on two SH3 binding sites to form a Gab1-Grb2 complex and thereafter combine with activated receptor molecule to form a Gab1Grb2-receptor complex which in turn is directed by the SH2 domain of Grb2 (Ortiz-Padilla et al., 2013). The study carried out by Meng (2014) reported that A/A genotype and A allele of rs3805246 in Gab1 were significantly higher in BTC group than in GSD group or controls and the present study also reported a positive association with gallstone disease. From hormone related pathway CYP1A1 was found to be associated with GSD. The CYP1A1 gene encodes the CYP1A1 enzyme, which catalyzes estradiol to 2-hydroxy-estradiol (2-OH-E2) (Schwarz et al., 2000). Upregulation of estrogen have been associated with biliary tract cancers due to decreased gallbladder motility, thereby increasing the formation of gallstones and the risk of inflammation in the biliary tract (Everson et al., 1991; Kritz-Silverstein et al., 1990). In study by Park et al. (2009), CYP1A1 gene marker conferred risk for biliary tract cancers but no association in the case of GSD but in present study a positive association was observed with the risk of gallstone disease. Inflammatory pathway registered one marker i.e. IL10 to be associated with GSD risk. Anti-inflammatory cytokine interleukin10 (IL10) gene polymorphisms have been associated with susceptibility to various inflammatory diseases and cancers. The study by Vishnoi et al. (2007) reported that the frequency of IL10 − 819C/T genotypes were comparable in GBC, gallstone patients, and healthy subjects. Nevertheless, interplay of sex hormones and IL10 − 819C/T polymorphism may lead to the susceptibility of gallstone-mediated GBC and the results of present study displayed a protective association of the marker with gallstone disease. Apart from these one of the markers namely MMP2 from ECM pathway was found to be associated with the Gallstone disease. Matrix metalloproteinase belongs to family of pericellular collagenases that degrade extracellular matrix (ECM), and is involved in the modulation and susceptibility of various carcinomas. The study by Sharma et al. (2012) reported that the genotypes of MMP2 were significantly associated with increased risk of GBC probably through gallstone-mediated inflammatory pathway and the present study also reported a significant association of the variant with the risk of gallstone disease. Women between 20 and 60 years of age are twice more likely to develop gallstones than men (Everhart et al., 1999). The reasons why women are at higher risk of gallstone disease are presumably hormonal. Estrogen increases biliary cholesterol saturation, and progesterone may inhibit gallbladder contraction (Sharma et al., 2012). It has been proposed that fertility period, pregnancies, and exogenous female sex hormones account for all sexual differences observed in gallbladder disease (Jørgensen, 1988). Gender based analysis of the present study after Bonferroni correction revealed that the gene Gab1 showed positive association in males. On the other hand gene TM4SF4 and PLCE1 gene showed low risk in males while gene APOE and CYP8B1 gene showed low risk in females for gallstone disease but none of the genes showed association in both the sexes for gallstone disease, Based on findings from present study, it appears that action of differential susceptibility genes may be differently modulated by hormonal milieu. A large GWAS in European population has strongly established the crucial role of ABCG8 in gallstone disease (Buch et al., 2007). The [3H]cholesterol export assays of allelic constructs harboring lithogenic genetic variants ABCG8-19H demonstrated increased transport activity (von Kampen et al., 2013). Previous studies of our group had also suggested weak but significant associations of ABCG8 variants with cholesterol gallstone disease (Srivastava et al., 2010) in our population. However, the association of ABCG8 D19 H (rs11887534) did not reach statistical significance in the present cohort. In-silico analysis of the interaction network of all of the associated genes with gallstone risk generated by GENEMANIA revealed direct and indirect connections among them via co-expression, gene interactions,

10

TOLERATED 35 3.15 1 CDS ENSP00000360431 ENST00000371380 ENSG00000138193 PLCE1 H1927R G A 96066341 10

TOLERATED 40 3.11 1 CDS ENSP00000360426 ENST00000371375 ENSG00000138193 PLCE1 H1619R G A 96066341 10

TOLERATED 35 3.15 1 CDS ENSP00000260766 ENST00000260766 ENSG00000138193 PLCE1 H1927R G A 96066341 10

DELETERIOUS 25 3.04 0.002 CDS ENSP00000413653 ENST00000434152 ENSG00000130203 APOE R202C T C 45412079 19

DELETERIOUS 25 2.82 0.002 CDS ENSP00000252486 ENST00000252486 ENSG00000130203 APOE R176C T C 45412079 19

DELETERIOUS 24 3.02 0.001 CDS ENSP00000413135 ENST00000446996 ENSG00000130203 APOE R176C T C 45412079 19

TOLERATED 106 2.36 0.358 CDS ENSP00000424960 ENST00000502906 ENSG00000160867 FGFR4 G388R A 176520243 5

G

TOLERATED 106 2.36 0.358 CDS ENSP00000292408 ENST00000292408 ENSG00000160867 FGFR4 G388R A G 176520243 5

TOLERATED 79 2.86 0.356 CDS ENSP00000424670 ENST00000511076 ENSG00000160867 FGFR4 G23R A G 176520243

Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 Homo_sapiens/ GRCh37.74 rs351855

5

Coordinate CHR organism/build SNP

Table 7 Potential functional consequences of the markers.

Ref allele

Alt allele

Amino acid change

Gene name

Gene ID

Transcript ID

Protein ID

Region

Sift score

Sift median

No of seqs At position

Sift prediction

T. Chauhan, et al.

9

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

physical interactions and shared protein domains. This network may have an important role in GSD pathogenesis. The potential functional consequences of exonic SNPs using predictive programs Polyphen2 revealed FGFR4 rs351855 and APOE rs7412 to have probable damaging effects but PLCE1 rs2274223 SNP showed benign effect. On the basis of SIFT score assessment 2 of the exonic SNPs FGFR4 rs351855 and PLCE1 rs2274223 SNP showed tolerated effect, while one of the variants APOE rs7412 showed deleterious effect. To conclude, the present study reports that genetic variation in several selected genes play an important role in gallbladder disease in northern India. CYP7A1 rs6471717, CYP1A1 rs2606345, Gab1 rs3805246 gene variants conferred high risk whereas FGFR4 rs351855, APOE rs7412, TM4SF4 rs9843304, IL10 rs1800871, PLCE1 rs2274223 showed lower risk of cholesterol gallstones. Sex-specific association with disease risk was also observed with several genetic variants. Since gallstones are major risk factor for gallbladder cancer which has very high incidence in North India, the genes associated with gallstone disease may play role in gallbladder cancer pathogenesis through gallstone mediated pathway. Limitations of this study include the relatively average sample size particularly of controls which limits the statistical power to detect some significant associations, especially in the stratified gender based analysis results. It is worth stating that the association studies are population-specific and inconsistency in the outcome requires future epidemiologic studies, preferably GWAS, in larger sample sizes. Further, correlation of genetic studies are required with physiological and biochemical findings in humans. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.mgene.2019.100579.

Four susceptibility loci for gallstone disease identified in a meta-analysis of genomewide association studies. Gastroenterology 151 (2), 351–363 (e28). Juvonen, T., Kervinen, K., Kairaluoma, M.I., Lajunen, L.H.J., Kesäniemi, Y.A., 1993. Gallstone cholesterol content is related to apolipoprotein E polymorphism. Gastroenterology 104 (6), 1806–1813. Juvonen, T., Savolainen, M.J., Kairaluoma, M.I., Lajunen, L.H., Humphries, S.E., YA, Kesà ¤niemi, 1995. Polymorphisms at the apoB, apoA-I, and cholesteryl ester transfer protein gene loci in patients with gallbladder disease. J. Lipid Res. 36 (4), 804–812. Katsika, D., Grjibovski, A., Einarsson, C., Lammert, F., Lichtenstein, P., Marschall, H.U., 2005. Genetic and environmental influences on symptomatic gallstone disease: a Swedish study of 43,141 twin pairs. Hepatology 41 (5), 1138–1143. Khanuja, B., Cheah, Y.-C., Hunt, M., Nishina, P.M., Wang, D.Q., Chen, H.W., et al., 1995. Lith1, a major gene affecting cholesterol gallstone formation among inbred strains of mice. Proc. Natl. Acad. Sci. 92 (17), 7729–7733. Khuroo, M.S., Mahajan, R., Zargar, S.A., Javid, G., Sapru, S., 1989. Prevalence of biliary tract disease in India: a sonographic study in adult population in Kashmir. Gut 30 (2), 201–205. Ko, C.W., Beresford, S.A.A., Alderman, B., Jarvik, G.P., Schulte, S.J., Calhoun, B., et al., 2000. Apolipoprotein E genotype and the risk of gallbladder disease in pregnancy. Hepatology 31 (1), 18–23. Kosters, A., Jirsa, M., Groen, A.K., 2003. Genetic background of cholesterol gallstone disease. Biochimica et Biophys. Acta (BBA)-Mol. Basis Dis. 1637 (1), 1–19. Kratzer, W., Mason, R.A., 1999. Kächele V. prevalence of gallstones in sonographic surveys worldwide. J. Clin. Ultrasound 27 (1), 1–7. Kritz-Silverstein, D., Barrett-Connor, E., Wingard, D.L., 1990. The relationship between reproductive history and cholecystectomy in older women. J. Clin. Epidemiol. 43 (7), 687–692. Lammert, F., Sauerbruch, T., 2005. Mechanisms of disease: the genetic epidemiology of gallbladder stones. Nat. Rev. Gastroenterol. Hepatol. 2 (9), 423. Lammert, F., Carey, M.C., Paigen, B., 2001. Chromosomal organization of candidate genes involved in cholesterol gallstone formation: a murine gallstone map. Gastroenterology 120 (1), 221–238. Lammert, F., Wang, D.Q., Wittenburg, H., Bouchard, G., Hillebrandt, S., Taenzler, B., et al., 2002. Lith genes control mucin accumulation, cholesterol crystallization, and gallstone formation in a/J and AKR/J inbred mice. Hepatology 36 (5), 1145–1154. Lowenfels, A.B., Velema, J.P., 1992. Estimating gallstone incidence from prevalence data. Scand. J. Gastroenterol. 27 (11), 984–986. Martinez-Lopez, E., Curiel-Lopez, F., Hernandez-Nazara, A., Moreno-Luna, L.E., RamosMarquez, M.E., Roman, S., et al., 2015. Influence of ApoE and FABP2 polymorphisms and environmental factors in the susceptibility to gallstone disease. Ann. Hepatol. 14 (4), 515–523. Meng, L.Q., 2014. Essential role of polymorphism of Gab1, EGFR, and EGF for the susceptibility of biliary tract cancer. Tumor Biol. 35 (12), 12497–12508. Miller, S.A., Dykes, D.D., Polesky, H., 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16 (3), 1215. Ortiz-Padilla, C., Gallego-Ortega, D., Browne, B.C., Hochgrafe, F., Caldon, C.E., Lyons, R.J., et al., 2013. Functional characterization of cancer-associated Gab1 mutations. Oncogene 32 (21), 2696. Park, S.K., Andreotti, G., Sakoda, L.C., Gao, Y.-T., Rashid, A., Chen, J., et al., 2009. Variants in hormone-related genes and the risk of biliary tract cancers and stones: a population-based study in China. Carcinogenesis 30 (4), 606–614. Pinheiro-Junior, S., Pinhel, M.A.S., Nakazone, M.A., Pinheiro, A., Amorim, G.F.S., Florim, G.M.S., et al., 2012. Effect of genetic variants related to lipid metabolism as risk factors for cholelithiasis after bariatric surgery in Brazilian population. Obes. Surg. 22 (4), 623–633. Portincasa, P., van Erpecum, K.J., van De Meeberg, P.C., Dallinga-Thie, G.M., de Bruin, T.W., van Berge-Henegouwen, G.P., 1996. Apolipoprotein E4 genotype and gallbladder motility influence speed of gallstone clearance and risk of recurrence after extracorporeal shock-wave lithotripsy. Hepatology 24 (3), 580–587. Qin, J., Jiang, Z.Y., Niu, Z.M., Zhang, K.Y., Hua, Q., Jiang, Z.H., et al., 2011. Association of single nucleotide polymorphism in human CYP8B1 gene with gallstone disease. Zhonghua Yi Xue Za Zhi 91 (30), 2092–2095. Qin, J., Han, T.Q., Yuan, W.T., Zhang, J., Fei, J., Jiang, Z.Y., et al., 2016. A single-nucleotide polymorphism in the 3'-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple-negative breast cancer. J. Gastroenterol. Hepatol. 28 (4), 717–722. Renner, O., Harsch, S., Schaeffeler, E., Winter, S., Schwab, M., Krawczyk, M., et al., 2009. A variant of the SLC10A2 gene encoding the apical sodium-dependent bile acid transporter is a risk factor for gallstone disease. PLoS One 4 (10), e7321. Sampliner, R.E., Bennett, P.H., Comess, L.J., Rose, F.A., Burch, T.A., 1970. Gallbladder disease in Pima Indians: demonstration of high prevalence and early onset by cholecystography. N. Engl. J. Med. 283 (25), 1358–1364. Sánchez-Cuén, J., Aguilar-Medina, M., Arámbula-Meraz, E., Romero-Navarro, J., Granados, J., Sicairos-Medina, L., Ramos-Payán, R., 2010. ApoB-100, ApoE and CYP7A1 gene polymorphisms in Mexican patients with cholesterol gallstone disease. World J Gastroenterol: WJG 16 (37), 4685. Schwarz, D., Kisselev, P., Schunck, W.-H., Chernogolov, A., Boidol, W., Cascorbi, I., et al., 2000. Allelic variants of human cytochrome P450 1A1 (CYP1A1): effect of T461N and I462V substitutions on steroid hydroxylase specificity. Pharmacogenet. Genomics 10 (6), 519–530. Sharma, K.L., Misra, S., Kumar, A., Mittal, B., 2012. Higher risk of matrix metalloproteinase (MMP-2, 7, 9) and tissue inhibitor of metalloproteinase (TIMP-2) genetic variants to gallbladder cancer. Liver Int. 32 (8), 1278–1286. Sharma, K.L., Misra, S., Kumar, A., Mittal, B., 2013a. Association of liver X receptors (LXRs) genetic variants to gallbladder cancer susceptibility. Tumor Biol. 34 (6), 3959–3966.

Acknowledgments This study was supported by research grant to BM from Department of Science and Technology (DST), Govt. of India. DST NO: SERB/F/ 3512/2014-15. Dr. Ashok Kumar (Department of Surgical Gastroenterology S.G.P.G.I.M.S. Lucknow, UP.) is gratefully acknowledged for his help in manuscript editing. Declaration of competing interests The author(s) declare that they have no competing interests. References Bertomeu, A., Ros, E., Zambon, D., Vela, M.A., Perez-Ayuso, R.M., Targarona, E., et al., 1996. Apolipoprotein E polymorphism and gallstones. Gastroenterology 111 (6), 1603–1610. Buch, S., Schafmayer, C., Valzke, H., Becker, C., Franke, A., von Eller-Eberstein, H., et al., 2007. A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease. Nat. Genet. 39 (8), 995. Chen, Q., Li, W.J., Wan, Y.Y., Yu, C.D., Li, W.G., 2012. Fibroblast growth factor receptor 4 Gly388Arg polymorphism associated with severity of gallstone disease in a Chinese population. Genet. Mol. Res. 11 (1), 548–555. Dixit, M., Choudhuri, G., Mittal, B., 2006. Association of lipoprotein receptor, receptorassociated protein, and metabolizing enzyme gene polymorphisms with gallstone disease: a case control study. Hepatol. Res. 36 (1), 61–69. Everhart, J.E., Khare, M., Hill, M., Maurer, K.R., 1999. Prevalence and ethnic differences in gallbladder disease in the United States. Gastroenterology 117 (3), 632–639. Everhart, J.E., Yeh, F., Lee, E.T., Hill, M.C., Fabsitz, R., Howard, B.V., et al., 2002. Prevalence of gallbladder disease in American Indian populations: findings from the strong heart study. Hepatology 35 (6), 1507–1512. Everson, G.T., McKinley, C., Kern, F., 1991. Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism. J. Clin. Invest. 87 (1), 237–246. Jiang, Z.-Y., Han, T.-Q., Suo, G.-J., Feng, D.-X., Chen, S., Cai, X.-X., et al., 2004. Polymorphisms at cholesterol 7Î ± -hydroxylase, apolipoproteins B and E and low density lipoprotein receptor genes in patients with gallbladder stone disease. World J. Gastroenterol. 10 (10), 1508. Jørgensen, T., 1988. Gall stones in a Danish population: fertility period, pregnancies, and exogenous female sex hormones. Gut 29 (4), 433–439. Joshi, A.D., Andersson, C., Buch, S., Stender, S., Noordam, R., Weng, L.-C., et al., 2016.

10

Meta Gene 21 (2019) 100579

T. Chauhan, et al.

Cancer 38 (1), 46–51. von Kampen, O., Buch, S., Nothnagel, M., Azocar, L., Molina, H., Brosch, M., et al., 2013. Genetic and functional identification of the likely causative variant for cholesterol gallstone disease at the ABCG5/8 lithogenic locus. Hepatology 57 (6), 2407–2417. Wang, H.H., Portincasa, P., Afdhal, N.H., Wang, D.Q.H., 2010. Lith genes and genetic analysis of cholesterol gallstone formation. Gastroenterol. Clin. 39 (2), 185–207. Weiss, K.M., Ferrell, R.E., Hanis, C.L., Styne, P.N., 1984. Genetics and epidemiology of gallbladder disease in New World native peoples. Am. J. Hum. Genet. 36 (6), 1259. Wilson, C., Wardell, M.R., Weisgraber, K.H., Mahley, R.W., Agard, D.A., 1991. Threedimensional structure of the LDL receptor-binding domain of human apolipoprotein E. Science 252 (5014), 1817–1822. Xu, H.-L., Cheng, J.-R., Andreotti, G., Gao, Y.-T., Rashid, A., Wang, B.-S., et al., 2011. Cholesterol metabolism gene polymorphisms and the risk of biliary tract cancers and stones: a population-based case-control study in Shanghai, China. Carcinogenesis 32 (1), 58–62. Zhang, M., Xiao, L., Lin, Q., 1999. Polymorphism at cholesteryl ester transfer protein gene loci in patients with gallstone. Hua xi yi ke da xue xue bao= J. West China Univ. Med. Sci.= Huaxi yike daxue xuebao 30 (1), 68–71.

Sharma, K.L., Umar, M., Pandey, M., Misra, S., Kumar, A., Kumar, V., et al., 2013b. Association of potentially functional genetic variants of PLCE1 with gallbladder cancer susceptibility in north Indian population. J. Gastrointest. Cancer 44 (4), 436–443. Srivastava, A., Srivastava, A., Srivastava, K., Choudhuri, G., Mittal, B., 2010. Role of ABCG8 D19H (rs11887534) variant in gallstone susceptibility in northern India. J. Gastroenterol. Hepatol. 25 (11), 1758–1762. Tan, Y.F., Yang, S., Yu, R.B., Shen, C., Ding, W.L., Zhou, W.M., et al., 2003. Relationship among the XhaI and EcoRI locus polymorphisms of apolipoprotein B gene, serum lipid metabolism and gallstone disease. Zhonghua Yi Xue Za Zhi 83 (10), 844–847. Tönjes, A., Wittenburg, H., Halbritter, J., Renner, O., Harsch, S., Stange, E.F., et al., 2011. Effects of SLC10A2 variant rs9514089 on gallstone risk and serum cholesterol levelsmeta-analysis of three independent cohorts. BMC Med. Genet. 12 (1), 149. Van Erpecum, K.J., Van Berge-henegouwen, G.P., Eckhardt, E.R., Portincasa, P., Van De Heijning, B.J., Dallinga-Thie, G.M., Groen, A.K., 1998. Cholesterol crystallization in human gallbladder bile: relation to gallstone number, bile composition, and apolipoprotein E4 isoform. Hepatology 27 (6), 1508–1516. Vishnoi, M., Choudhuri, G., Mittal, B., 2007. Is IL-10-819C/T gene polymorphism modulating the risk of gallbladder disease in north Indian population? J. Gastrointest.

11