Association of APOE-C1 gene cluster polymorphisms with gallstone disease

Digestive and Liver Disease 38 (2006) 397–403

Liver, Pancreas and Biliary Tract

Association of APOE-C1 gene cluster polymorphisms with gallstone disease M. Dixit a , G. Choudhuri b , B. Mittal a,∗ a b

Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India Received 27 November 2005; accepted 14 February 2006 Available online 19 April 2006

Abstract Background. Genetic polymorphisms in apolipoprotein genes may be associated with alteration in lipid profile and susceptibility to gallstone disease. Aim. To find out the association of APOE HhaI and APOC1 HpaI polymorphisms with gallstone disease. Subjects. HhaI polymorphism of APOE and HpaI polymorphism of APOC1 were analysed in DNA samples of 214 gallstone patients and 322 age- and sex-matched healthy controls. Methods. For genotyping DNA samples of all study subjects were amplified using polymerase chain reaction, followed by restriction digestion. All statistical analyses were done using SPSS v11.5 and ARLEQUIN v2.0 softwares. Result. APOC1 HpaI polymorphism was found to be significantly associated with gallstone disease. Frequency of H2H2 was significantly higher (P = 0.017) in patients than in controls and it was imposing very high risk (OR 9.416, 95% CI 1.125–78.786) for gallstone disease. When data were stratified in male and female, H2H2 was associated (P = 0.011) with disease in females only. Analysis at allele level revealed no association. APOE HhaI polymorphism and APOE-C1 haplotypes showed no association with gallstone disease. Conclusion. APOC1 HpaI polymorphism is associated with gallstone disease and shows gender-specific differences. APOE HhaI polymorphism may not be associated with gallstone disease. © 2006 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. Keywords: APOC1 HpaI polymorphism; APOE-C1 haplotype; APOE HhaI polymorphism; Gallstone disease; Gene polymorphisms

1. Introduction Gallstones result from complex interactions between genetic and environmental factors. The disease shows very high incidence in certain ethnic groups. High concordance of cholelithiasis in monozygotic twins [1,2] and clustering of cases in families in which gallstone disease is diagnosed in childhood [3] have provided evidence for a genetic basis of the disease. A recent study on 43,141 twin pairs found ∗ Corresponding author. Tel.: +91 522 2668805-8x2323 (O)/x2329 (Lab); fax: +91 522 2668973/8078/8017. E-mail addresses: [email protected] (M. Dixit), [email protected] (G. Choudhuri), [email protected], bml [email protected] (B. Mittal).

that concordances and correlations were higher in monozygotic twins compared with dizygotic twins, both for males and females [4]. A prerequisite for the development of gallstone is lithogenic bile, which is often the result of enhanced cholesterol synthesis or a reduced bile acid pool size, or both [5]. A number of epidemiological surveys have shown an association between altered plasma lipid levels and gallstone disease [6–8]. Apo E and apo C-I play a central role in lipid metabolism and redistribution and thus are involved in both physiological and pathophysiological processes. APOE and APOC1 have been mapped to chromosome 19q13 in human. APOC1 is located 5 kb downstream of the APOE on chromosome 19 in the same transcriptional orientation [9,10]. Genetic variants of APOE and APOC1

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may affect lipid levels and ultimately the risk for gallstone disease. APOE is polymorphic and 30 genetic variants have been identified in various populations [11]. Among them, HhaI polymorphism is the most widely studied. Based on this polymorphism, apo E is found in three isoforms (E2, E3 and E4), which are genetically controlled by three different alleles ␧2, ␧3 and ␧4. These isoforms show different affinity for low-density lipoprotein (LDL) receptor [12] that affect the intestinal absorption of cholesterol in the form of chylomicrons [13], and the clearance of the major cholesterol carrying lipoprotein (LDL) from the plasma [14]. In most human populations it is found that individuals with apo E2 display high levels of apo E and low levels of plasma cholesterol, LDL-cholesterol and apo B, whereas those with apo E4 show the opposite [11,15,16]. Association of APOE HhaI polymorphism with gallstone disease has been studied by various groups, but the results are conflicting [17–24]. Most studied HpaI polymorphism of APOC1 is localised to a site 317 bp 5 to the apo C-I transcription initiation site [25] and is produced due to a CGTT insertion at position −317. The role of APOC1 HpaI polymorphism in type III hyperlipidemia [26] and in determination of plasma levels of remnant particles [27] has been suggested. The increased levels of plasma apo C-I and very low-density lipoprotein (VLDL) in hypertriglyceridaemic subjects have been associated with increased production of apo C-I [28]. However, there is no study of APOC1 polymorphism in gallstone disease in any population. In India, gallstone disease imposes significant economic burden and with increasing rate of urbanisation and privileged circumstances, rate of gallstone disease may further increase in future. Establishment of genetic factors associated with gallstone disease may help in better management of the disease. Since there is no study about the genetic risk factors in Indian population, present study was undertaken to find out association of APOE HhaI and APOC1 HpaI polymorphisms with gallstone disease.

2. Materials and methods 2.1. Subjects Study comprised of 214 gallstone patients (mean age 44.71 ± 13.20 year) and 322 controls (mean age 43.98 ± 11.46 year). All subjects were from North India. The gallstone patients were recruited from inpatients undergoing cholecystectomy and outpatients attending the clinics of Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences. Initially 350 controls were recruited from the healthy staff members of the institute and general population of the region. Finally 322 age- and sex-matched control subjects who were found to be negative for gallstone (by ultrasound), diabetes mellitus,

obesity, and other chronic debilitating disease were included in the study. Female subjects taking hormones in any form were excluded from the study. Patient and control females were also matched for the number of pregnancies. Study was approved by ethical committee of the institute. After an informed consent, blood was taken in EDTA for analysis of DNA. The genomic DNA was extracted from peripheral blood leucocytes pellet using the standard salting out method [29]. 2.2. Genotyping Fragment encompassing APOE HhaI variations were amplified and genotyped as described by Hixson and Vernier [30]. Genomic DNA was amplified in a DNA thermal cycler (DNA Engine PTC-100, MJ Research Inc.) using specific primer sequences and digested with 5 U HhaI (Fermantas Inc., USA) at 37 ◦ C for 6 h. PCR product of 244 bp also contains four nonpolymorphic sites for HhaI which cuts it into various fragments. Depending upon the presence or absence of polymorphic restriction site, allelic pattern in study subjects was determined [30]. For APOC1 HpaI polymorphism, flanking 450 bp region was amplified using primers described previously [31]. Amplified PCR product was digested with 5 U HpaI (Fermantas Inc., USA) for 6 h at 37 ◦ C. The digested samples were genotyped on 1.5% agarose gel. Presence of H1 allele gave 450 bp band and H2 allele gave two bands of 334 bp and 116 bp. 2.3. Cholesterol content in gallstones Stone sample was available in 67 gallstone patients. Soon after cholecystectomy stone sample was collected, washed with distilled water, air-dried and stored at room temperature. At the time of analysis whole stone was taken, weighed, and finely powdered using mortar and pestle. Twenty-milligram gallstone powder was dissolved in 500 ␮l isopropyl alcohol and centrifuged at 5000 rpm for 5 min to palate down debris. In 10 ␮l supernatant cholesterol content was estimated (in triplicate) using commercially available kits (Accurex Biomedical Pvt. Ltd., Mumbai, India). Cholesterol content was expressed as percent of dry weight. Gallstones were classified as cholesterol stones if the cholesterol content was more than 50% and as pigment stones if the cholesterol content was less than 20% of the dry weight of stone [32]. When cholesterol content was between 20% and 50%, stones were classified as mixed type stones. 2.4. Statistical evaluation To examine whether the genotype frequencies were in Hardy–Weinberg equilibrium, goodness-of-fit χ2 test was used. Haplotype frequencies were determined by maximum likelihood method, using expectation maximisation algorithm. Pair wise linkage disequilibrium between each pair of APOE-C1 loci was analysed using a likelihood-ratio test,

M. Dixit et al. / Digestive and Liver Disease 38 (2006) 397–403 Table 1 Demographic profile of gallstone patients and controls

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3.1. APOE HhaI polymorphism

Demographic profile

Gallstone patients

Controls

P-value

Age (years) Sex (M/F) BMI (kg/m2 )

44.71 ± 13.20 69/145 22.94 ± 3.90

43.98 ± 11.46 116/206 23.13 ± 3.85

0.500 0.368 0.732

For this polymorphism, study population was in Hardy–Weinberg equilibrium for controls (P = 0.077) and patients (P = 0.842). APOE HhaI polymorphism showed similar frequency of genotypes in patient group and control group (Table 2). Separate analysis in male and female showed that in male population frequency of ␧3␧3 was slightly lower (77.27% versus 80.17%) in patients. On the contrary, frequency of ␧2␧3 was higher (12.12% versus 5.17%) in patients than in controls. Genotype ␧2␧4 was present (5.17%) only in control population. These differences were not statistically significant. In female population, striking differences in frequency of genotypes were not observed. Frequency of ␧2, ␧3 and ␧4 alleles were similar in patient and control groups (Table 3). When we analysed male and female population separately, same pattern of frequency distribution was observed.

whose empirical distribution was obtained by a permutation procedure. Above calculations were performed using ARLEQUIN v2.0 software. All other analyses were done using SPSS v11.5 in whole study population and, in male and female population separately. Genotype and allele frequencies were determined by direct counting and compared by χ2 test or Fisher’s exact test. Haplotype frequency was also compared using χ2 test, between patients and controls.

3. Results

3.2. APOC1 HpaI polymorphism Table 1 shows age, male/female ratio and body mass index (BMI) in gallstone patients and controls. Differences in age, male/female ratio and BMI were insignificant. Stone samples were available in 67 gallstones patients. Cholesterol type of stone was present in 83.58% samples and mixed type was present in 14.93% samples. Pigment type stone was present only in one patient, which was excluded from the study.

For this promoter region polymorphism of APOC1, study population was found to be in Hardy–Weinberg equilibrium for controls (P = 0.322) and patients (P = 0.528). This polymorphism was found to be associated with gallstone disease (Table 4). Frequency of H2H2 was significantly higher (P = 0.017) in patients than in controls and it was

Table 2 APOE HhaI polymorphism genotype frequency in total subjects and stratified in male and female subjects Genotype

Total

Male

Patienta

␧2␧2 ␧3␧3 ␧4␧4 ␧2␧3 ␧2␧4 ␧3␧4 a

Female

Control (No. = 322) N (%)

P-value

Patient (No. = 66) N (%)

Control (No. = 116) N (%)

P-value

Patient (No. = 141) N (%)

Control (No. = 206) N (%)

P-value

(No. = 207) N (%) 0 (0) 158 (76.33) 2 (0.97) 16 (7.73) 3 (1.45) 28 (13.53)

1 (0.31) 247 (76.71) 1 (0.31) 21 (6.52) 7 (2.17) 45 (13.98)

0.422 0.920 0.327 0.595 0.550 0.884

0 (0) 51 (77.27) 0 (0) 8 (12.12) 0 (0) 7 (10.61)

1 (0.86) 93 (80.17) 0 (0) 6 (5.17) 6 (5.17) 10 (8.62)

1.000 0.644 – 0.091 0.088 0.658

0 (0) 107 (75.89) 2 (1.42) 8 (5.67) 3 (2.13) 21 (14.89)

0 (0) 154 (74.76) 1 (0.49) 15 (7.28) 1 (0.49) 35 (16.99)

– 0.811 0.569 0.554 0.308 0.602

Out of 214 gallstone patient DNA samples genotyping could be done in 207 samples.

Table 3 APOE HhaI and APOC1 HpaI polymorphism allele frequency in total subjects and stratified in male and female subjects Polymorphism allele

Total

Male

Female

Patient (No.a = 414) N (%)

Control (No.a = 644) N (%)

P-value

Patient (No.a = 132) N (%)

Control (No.a = 232) N (%)

P-value

Patient (No.a = 282) N (%)

Control (No.a = 412) N (%)

P-value

APOE HhaI ␧2 ␧3 ␧4

19 (4.59) 360 (86.96) 35 (8.45)

30 (4.66) 560 (86.96) 54 (8.39)

0.958 1.00 0.969

8 (6.06) 117 (88.64) 7 (5.30)

14 (6.03) 202 (87.07) 16 (6.90)

0.992 0.662 0.548

11(3.90) 243 (86.17) 28 (9.93)

16 (3.88) 358 (86.89) 38 (9.22)

0.991 0.784 0.756

APOC1 HpaI H1 H2

358 (86.06) 58(13.94)

569 (89.47) 67 (10.53)

0.095 0.095

122 (89.71) 14 (10.29)

214 (93.04) 16 (6.96)

0.261 0.261

236 (84.29) 44 (15.71)

355 (87.44) 51 (12.56)

0.240 0.240

a

Total number of chromosomes.

0.572 0.860 0.011 152 (74.88) 51 (25.12) 0 (0) 101(72.14) 34(24.29) 5 (3.57) 0.635 (0.282–1.430) 1.546 (0.669–3.571) 1.701 (0.105–27.652) 55(80.88) 12(17.65) 1 (1.47) H1H1 H1H2 H2H2

156(75.00) 46(22.12) 6 (2.88)

252 (79.25) 65 (20.44) 1 (0.31)

0.254 0.645 0.017

0.786 (0.519–1.189) 1.105 (0.722–1.692) 9.416 (1.125–78.786)

100 (86.96) 14 (12.17) 1 (0.87)

0.270 0.305 1.000

P-value Control (No. = 203) N (%) Patient (No. = 140) N (%) OR (95% CI) P-value Patient (No. = 68) N (%) Patient (No. = 208) N (%)

Control (No. = 318) N (%)

P-value

OR (95% CI)

Control (No. = 115) N (%)

Female Male Total Genotype

Table 4 APOC1 HpaI polymorphism genotype frequency in total subjects and stratified in male and female subjects

0.869 (0.534–1.414) 0.956 (0.580–1.576) –

M. Dixit et al. / Digestive and Liver Disease 38 (2006) 397–403

OR (95% CI)

400

Table 5 APOE HhaI–APOC1 HpaI haplotype frequency in gallstone patients and controls Haplotype

Patients (frequency) (No.a = 402)

Controls (frequency) (No.a = 636)

P-value

␧2-H1 ␧2-H2 ␧3-H1 ␧3-H2 ␧4-H1 ␧4-H2

0.016 0.029 0.827 0.049 0.023 0.057

0.022 0.025 0.839 0.032 0.033 0.049

0.495 0.719 0.591 0.158 0.342 0.569

a

Total number of chromosomes.

imposing very high risk (OR 9.416, 95% CI 1.125–78.786) for gallstone disease. Genotypes H1H1 and H1H2 were not distributed differently between patients and controls. When data was stratified in male and female, H2H2 was associated (P = 0.011) with disease in females only. Differences in the frequencies of other genotypes were not statistically significant. Analysis at allele level showed that frequency of H2 allele was higher in patients than in controls but statistically no association of H1 or H2 allele with gallstone disease was seen (Table 3). Separate analysis of male and female population also did not reveal any association. 3.3. APOE-C1 haplotype Haplotypes were constructed using expectation maximisation (EM) algorithm in 201 gallstone patients and 318 controls. APOE-C1 haplotypes for total population are shown in Table 5. No haplotype was found to be associated with gallstone disease. Haplotypes were also constructed for male and female population separately (data not shown). Even after the stratification of data gender wise, no significant difference in the APOE-C1 haplotype frequency was observed.

4. Discussion During last decade, mouse model studies have provided important information about different genetic loci and candidate genes influencing gallstone formation [33]. Since most of the mouse genes have human orthologues, their role in gallstone disease in human can be delineated. Genetic association studies in human have statistical power to reveal contribution by risk alleles in a polygenic disease like gallstone formation. Several association studies have been carried out in the past in gallstone disease. Various genetic variants of APOE, APOB and CETP were implicated in the susceptibility for gallstone disease but lot of variations in published results has been observed [18,22,24,34,35]. Part of the problem may be sample size, which was usually small. Moreover, the association studies are sensitive to population/ethnicity and geographical variation. Till date there was no study in Indian popula-

M. Dixit et al. / Digestive and Liver Disease 38 (2006) 397–403

tion. Therefore, we have carried out case–control analysis of APOE-C1 polymorphisms. In APOE HhaI polymorphism, the changes in the amino acids at 112 and 158 are believed to affect the 3-D structure involved in stabilisation of receptor-binding domain [36]. The receptor-binding domain of apo E is present in the Nterminal from amino acid 136–150. These three isoforms of apo E show different affinity for LDL receptor [12] that affects the intestinal absorption of cholesterol in the form of chylomicrons [13], and the clearance of the major cholesterol carrying lipoproteins [14]. Because the efficiency of lipoprotein uptake by the liver increases in the sequence ␧2 < ␧3 < ␧4, the polymorphism of APOE influences hepatic cholesterol content and possibly cholesterol gallstone formation [17]. To test the hypothesis, whether APOE, ␧4 confers increased risk for gallstone disease, various groups have carried out case–control studies, but the results are contradictory. Bertomeu et al. [17] showed that the E4/3 phenotype was enriched in female patients with gallstones and those who underwent cholecystectomy, with significantly (P < 0.006) higher ␧4 allele frequencies than controls [OR 2.67 (95% CI 1.23–5.93) and 3.62 (95% CI 1.49–8.91), respectively]. However, the differences in males were not significant. Niemi et al. [19] observed that in women with ␧2 (phenotypes E2/2, 2/3, and 2/4) as compared with those without ␧2 (E3/3, 4/3, and 4/4), the odds ratio for gallstone disease was 0.28 (95% CI 0.08–0.92), suggesting the protective role of ␧2. However, no protection was observed in men. The ␧4 allele has also been shown to confer higher risk of stone recurrences after clearance of gallstones by extracorporeal shock-wave lithotripsy [18,22]. It may be pointed out that most of the association studies of APOE with gallstone disease found association in a particular sub-group. There are several other reports where ␧4 allele was not found to confer increased risk of gallstone disease. Since pregnancy is a risk factor for the development of gallstone disease, APOE polymorphism was studied in the pregnant women but no overall association of ␧4 was observed with the development of new gallbladder sludge or stones in pregnancy [20]. According to recent study, bile lipid composition and cholesterol saturation index were similar in the patients as well as control subjects harbouring the ␧4 allele and those without ␧4. Furthermore, the prevalence of the ␧4 allele was similar in patients with cholesterol gallstone disease and in controls [23]. A recent study also did not find any association of gallstone disease with APOE polymorphism [24]. Our study does not support association of ␧4 allele with gallstone disease. Inconsistent results observed in various studies might be due to population-to-population variation. As we have already discussed, most of the association studies of APOE with gallstone disease either did not find any association or found association in a particular sub-group. In previous studies sample size was comparatively small [18,19,22] which may give false association. APOE may not be directly involved in gallstone disease. It is now believed that association of ␧4 allele observed by some researchers might be due to linkage disequilibrium with another polymorphism present

401

on the same gene or nearby gene. One of the possible genes is APOC1. We for the first time report association of APOC1 HpaI polymorphism and found significantly higher frequency of APOC1 H2H2 in female gallstone patients (P = 0.017) (Table 4). Gender-specific difference can be attributed to interaction between gender-specific hormones and genetic variants. Till date, there is no study showing the molecular mechanism of gene–hormone interaction in gallstone disease. In total population, H2H2 genotype showed very high risk (OR 9.416) but the range of confidence interval was wide (1.125–78.786), showing low power of association. Large sample size may be needed to get more authentic results. We observed this association only in homozygous form, while in heterozygous form not even trends were present. Here we suggest that H2 allele is recessive and its effect is evident only in homozygous form. Due to dominant nature of H1 allele we could not observe any association in heterozygous form. Recently, Shachter et al. [37] suggested that the mechanism of the previously described association of APOC1 HpaI polymorphism with plasma triglyceride is related to the correlation of the polymorphism with the level of expression of apo C-I. APOC1 HpaI polymorphism arises due to insertion of 4 bp (CGTT) at position −317 in the promoter [10,26]. This polymorphism might be affecting transcription rate and it has been reported to have 1.5-fold increase in APOC1 gene expression with 4 bp insertion (H2 allele). APOC1 H2 allele sequence exhibited decreased binding of a nuclear protein that is specifically bound by H1 allele. H2 allele may disrupt the binding of a negatively acting transcriptional factor to produce a positive effect on transcription [31]. The H2H2 carriers may have increased levels of apo C-I which influences triglyceride levels. The role of APOC1 HpaI polymorphism in type III hyperlipidemia [26] and in determination of plasma levels of remnant particles [27] has been suggested. Xu et al. [31] reported that this polymorphism increases gene transcription and exhibits an ethnically distinct pattern of linkage disequilibrium with the alleles of APOE. In present study at APOE-C1 locus, APOE polymorphism was not associated with the disease but APOC1 H2H2 was associated with gallstone disease. As APOE and APOC1 are present as cluster, haplotype analysis of APOE-C1 locus was carried out. However, the analysis did not show any association, suggesting no overall contribution of both polymorphisms as haplotype, which might be due to no association of APOE polymorphism with gallstone disease. Other polymorphisms present on APOE, APOC1, and other genes need to be considered to calculate overall contribution of genetic factors. Recent twin study has detected 25% variance for heritability [4]. The genes involved in gallbladder motility (CCKAR, CCK), cholesterol synthesis (HMG-CoA), bile acid synthesis (cholesterol 7-␣ hydroxylase) and mucins are important candidates for genetic studies in gallstone disease. Identification of risk alleles will provide definite contribution of risk due to genetic factors. In conclusion, APOC1 HpaI polymorphism is associated with gallstone disease and show gender-specific differences.

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