Association of lipoprotein receptor, receptor-associated protein, and metabolizing enzyme gene polymorphisms with gallstone disease: A case–control study

Hepatology Research 36 (2006) 61–69

Association of lipoprotein receptor, receptor-associated protein, and metabolizing enzyme gene polymorphisms with gallstone disease: A case–control study Manjusha Dixit a , Gourdas Choudhuri b , Balraj Mittal c,∗ a

Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India b Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India c Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India Received 13 March 2006; received in revised form 22 May 2006; accepted 31 May 2006 Available online 11 July 2006

Abstract Introduction: To identify high risk alleles for gallstone disease, we analyzed association of LDLR AvaII, LRPAP1 insertion/deletion, CETP TaqI B, and LPL HindIII polymorphisms with gallstone disease. Methods: In DNA samples of 214 gallstone patients and 322 age and sex matched controls, specific region containing polymorphisms was PCR amplified and digested with restriction enzymes except for LRPAP1 insertion/deletion polymorphism. Results: LRPAP1 gene insertion/deletion polymorphism was found to be significantly associated with gallstone disease. Genotype II was conferring significant risk for gallstone disease in females only (P = 0.019; OR 2.577, 95% CI 1.144–5.806). LDLR AvaII, CETP TaqI B, and LPL HindIII polymorphisms were not found to be associated with gallstone disease either at genotype or allele level. Conclusions: LRPAP1, II genotype carrier females may have increased risk for gallstone disease. On the other hand, LDLR AvaII, CETP TaqI B, and LPL HindIII polymorphisms may not be associated with gallstone disease. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Gallstone disease; Genetic polymorphism; LDLR AvaII polymorphism; LRPAP1 insertion/deletion polymorphism; CETP TaqI B polymorphism; LPL HindIII polymorphism

1. Introduction Gallstone disease is the most common gastrointestinal disease that afflicts 10–15% of the western population. In North India also its frequency is high and with westernization of culture, frequency is likely to increase in future. It is multifactorial disease and several lines of evidence indicate that genetic factors play an important role in pathogenesis of disease [1–3]. In North Indian population, 70–80% stones were found to be cholesterol type [4]. Super saturation of bile with ∗ Corresponding author. Tel.: +91 522 2668005 8x2322 (O)/2329 (Lab); fax: +91 522 2668973/2668078/2668017. E-mail addresses: [email protected] (M. Dixit), [email protected] (G. Choudhuri), [email protected], bml [email protected] (B. Mittal).

1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.05.005

cholesterol is prerequisite for stone formation, which results from imbalance in cholesterol homeostasis. Genetic variants present in lipid metabolizing genes may be important determinant of cholesterol homeostasis in liver and may also determine genetic susceptibility for gallstone disease. There are some studies about determination of genetic factors in certain populations but no such study has been reported from India. We undertook this study to find out the association of LDLR (low-density lipoprotein receptor) AvaII, LRPAP1 (LDL receptor related protein associated protein 1) insertion/deletion, CETP (cholesterol ester transfer protein) TaqI B, and LPL (lipoprotein lipase) HindIII polymorphism with gallstone disease in North Indian population. Plasma cholesterol levels in human are largely controlled by the interactions between the LDL and its receptor LDLR [5]. AvaII RFLP is widely studied polymorphism of LDLR

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M. Dixit et al. / Hepatology Research 36 (2006) 61–69

gene, which is present in exon 13 and C to T change at 1959 bp creates the site for AvaII [6]. LDLR AvaII polymorphism has been found to be associated with familial hypercholesterolemia [7,8]. Studies in mice have reported that receptor-associated protein (RAP) is molecular chaperone present within the endoplasmic reticulum to assist the folding of certain LDL receptor family members [9]. LRPAP1 is human orthologue of RAP. Large (37 bp) insertion/deletion polymorphism in intron 5 of LRPAP1 has been linked to regulation of cholesterol metabolism in coronary artery disease [10]. Our preliminary study in gallstone disease found this polymorphism to be significantly associated with gallstone disease; we carried out further study to confirm the results in larger sample size [11]. CETP plays an important role in HDL (high density lipoprotein) and apo A-I catabolism and in the determination of HDL size and subclass distribution. The CETP has been localized to chromosome 16q21 in humans ([12,13]). TaqI B polymorphism is among the most widely studied CETP variants, a silent base change affecting the 277th nucleotide in the first intron of the CETP [12]. Absence of the TaqI B restriction site has been associated with decreased CETP activity and, in turn, increased HDL-cholesterol levels [14–17]. The association studies of TaqI B polymorphism in gallstone disease have reported contradictory results [18,19]. LPL is crucial in removing triglyceride from both chylomicron and VLDL [20,21]. The human LPL gene is located on chromosome 8p22 [22]. LPL HindIII polymorphism is most widely studied polymorphism [23]. This polymorphism arises due to a replacement of a thymine (T) with a guanine (G) base at position 1495 in intron 8 and abolishes a HindIII restriction enzyme recognition site [24]. It has been associated with elevated triglyceride levels [25,26], low HDL-cholesterol levels [25,27–31], increased apolipoprotein C-III [32] and increased apolipoprotein B levels [33]. The main goal of this study was to gain insights into the genetic predisposition in gallstone disease and identification of high risk or protective alleles using case–control approach. We carried out an association of genetic variants present due to LDLR AvaII, LRPAP1 insertion/deletion, CETP TaqI B and, LPL HindIII polymorphisms in gallstone disease.

2. Methods 2.1. Subjects Study comprised of 214 gallstone patients (mean age 44.71 ± 13.20 yr) and 322 controls (mean age 43.98 ± 11.46 yr). All subjects were from North India. The gallstone patients were recruited among 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 institute and general population of the region and 322 age and sex

matched subjects who were found to be negative for gallstone (by ultrasound), diabetes mellitus, obesity, and other chronic debilitating disease, were included in the study. Study was approved from local 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 [34]. 2.2. Genotyping Gene fragment containing intron 5 insertion/deletion polymorphism of LRPAP1, AvaII polymorphism of LDLR, intron 1 TaqI B polymorphism of the CETP, and HindIII polymorphism of LPL were amplified using primers reported previously [35–37,10]. DNA was amplified by polymerase chain reaction in a DNA thermal cycler (DNA Engine PTC100, MJ Research, Inc.). Each amplification was performed using 200–800 ng of genomic DNA in a volume of 25 ␮l using 12.5 pmol of each primer, 200 ␮M each dNTPs, 15 mM MgCl2 , 100 mM Tris and 2 U of Taq polymerase (Bangalore Genei, India). LDLR AvaII polymorphism PCR products were digested with 5 U AvaII (Fermantas Inc., USA) restriction enzyme for 6 h at 37 ◦ C in a water bath. Depending upon the restriction pattern on 2% agarose gel it was designated A1 allele when site was absent and a single band of 255 bp was obtained. In the presence of alternative allele A2, two fragments of 185 and 70 bp were obtained. LRPAP1 insertion allele is 37 bp larger than deletion allele, this difference in size was detected by gel electrophoresis PCR products. Alleles (185 bp for D, 222 bp for I) were determined by running PCR products on 2% agarose gel. For CETP TaqI B polymorphism analysis PCR products were digested with 10 U of TaqI B restriction enzyme (MBI Fermentas, USA) at 65 ◦ C for 3 h. TaqI B genotype was determined by electrophoresis of restricted PCR product on 1.5% agarose gel followed by ethidium bromide staining. Presence of TaqI site (B1 allele) gave two bands of 174 and 361 bp and absence (B2 allele) showed one band of 535 bp. For LPL HindIII polymorphism genotyping PCR products were digested with 5 U HindIII (Fermantas Inc., USA) restriction enzyme for 6 h at 37 ◦ C in a water bath. Genotype was determined by electrophoresis of restricted PCR product on 2% agarose gel followed by ethidium bromide staining. Depending upon the restriction pattern it was designated L1 allele when site was absent and a single band of 365 bp was obtained. In the presence of alternative allele L2 two fragments of 205 and 170 bp were obtained. 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

M. Dixit et al. / Hepatology Research 36 (2006) 61–69

63

0.627 0.922 0.625 35.47 46.31 18.23

% N

72 94 37 38.03 45.77 16.20

% N

54 65 23 0.889 0.936 0.797

%

28.45 50.86 20.69 33 59 24

N % %

32.92 47.96 19.12

0.581 0.938 0.565

p-value

OR (95% CI)

1.109 (0.768–1.601) 0.986 (0.696–1.398) 0.875 (0.555–1.379)

N

29.41 51.47 19.12

N N

20 35 13

Patient (no. = 68) Control (no. = 319) Patient (no. = 210)

Control (no. = 116) Male

LDLR AvaII polymorphism was in Hardy–Weinberg equilibrium (P = 0.925) in control and gallstone patients (P = 0.975) population. Table 2 shows the frequency of LDLR AvaII genotypes. This polymorphism was not found to be associated with gallstone disease. In stratified male and female population also same trends were observed in frequency but no differences were statistically significant.

Total

3.1. LDLR AvaII polymorphism

Genotype

Table 1 shows age, male/female ratio and 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.

Table 2 LDLR AvaII polymorphism genotype frequency in total subjects and stratified in male and female subjects

3. Results

p-value

2.4. Statistical evaluation Sample size was calculated using software QUANTO Version 1.0 (http://www.hydra.usc.edu/gxe) for each genetic marker [39]. To examine whether the genotype frequencies were in Hardy–Weinberg equilibrium Goodness of fit χ2 -test was used. 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. Logistic regression analysis was used to find out contribution of genetic polymorphisms to the risk of disease. Models were constructed to obtain odds ratios for each allele after adjustment for the effect of age, sex, and BMI (body mass index).

1.048 (0.542–2.026) 1.025 (0.563–1.865) 0.906 (0.427–1.924)

OR (95% CI)

Female

Control (no. = 203)

finely powdered using mortar and pestle. Twenty milligrams of 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 % 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 [38]. When cholesterol content was between 20 and 50%, stones were classified as mixed type stones

105 153 61

0.500 0.368 0.732

%

43.98 ± 11.46 116/206 23.13 ± 3.85

35.24 47.62 17.14

44.71 ± 13.20 69/145 22.94 ± 3.90

74 100 36

Age (yr) Sex (M/F) Body mass index (kg/m2 )

A1A1 A1A2 A2A2

P-value

OR (95% CI)

Controls

p-value

Gallstone patients

Patient (no. = 142)

Demographic profile

1.116 (0.716–1.741) 0.979 (0.637–1.505) 0.867 (0.490–1.535)

Table 1 Demographic profile of gallstone patients and controls

M. Dixit et al. / Hepatology Research 36 (2006) 61–69

0.940 0.212 0.019 %

58.33 36.76 4.90 119 75 10

N %

57.93 30.34 11.72 84 44 17

N

0.615 (0.337–1.123) 1.406 (0.769–2.570) 2.148 (0.557–8.291) N

57.02 39.47 3.51 65 45 4 44.93 47.83 7.25 57.86 37.74 4.40 53.74 184 35.98 120 10.28 14

%

%

0.347 0.681 0.008

P-value

OR (95% CI)

0.846 (0.597–1.199) 0.927 (0.647–1.329) 2.488 (1.243–4.980)

N

% N N

31 33 5

%

0.112 0.268 0.301

P-value Control (no. = 114) Patient (no. = 69)

LDLR is receptor for LDL, and is present on the surface of the plasma membrane of the cell. LDLR AvaII polymorphism is present in exon 13 but it does not lead to change in amino acid. We first time studied this polymorphism in gallstone disease patients and found no association (Table 2). Another

Control (no. = 318)

4. Discussion

Patient (no. = 214)

To determine the contribution of different alleles to the risk of gallstone disease logistic regression analysis was used (Table 7). Different models were constructed to find out contribution of risk alleles alone and in association with environmental factors for all four polymorphisms. None of the polymorphisms showed significant risk for gallstone disease. Very less (0.3–0.8%) variance was being explained after adjustment for environmental factors.

Male

3.5. Risk assessment

Total

Studied control and patient population was in Hardy– Weinberg equilibrium (P = 0.089 and 0.429, respectively) for this polymorphism. This polymorphism was not associated with gallstone disease (Table 6). At allele level also no differences in frequency of L1 and L2 alleles were observed (data not shown).

Genotype

3.4. LPL HindIII polymorphism

Table 3 LRPAP1 insertion/deletion polymorphism genotype frequency in total subjects and stratified in male and female subjects

For CETP TaqI B polymorphism studied population was in Hardy–Weinberg equilibrium for controls (P = 0.377) and patients (P = 0.842) both. CETP TaqI B polymorphism was not associated with gallstone disease (Table 5). Alleles B1 and B2 were distributed similarly between both groups. Even after the gender based stratification no difference was observed (data not shown).

OR (95% CI)

3.3. CETP TaqI B polymorphism

115 77 22

Patient (no. = 145)

Female

Control (no. = 204)

P-value

OR (95% CI)

LRPAP1 insertion/deletion polymorphism was found to be in Hardy–Weinberg equilibrium in controls (P = 0.560) and in patients (P = 0.256). LRPAP1 gene insertion/deletion polymorphism was found to be associated with gallstone disease. Genotype II was significantly associated with the gallstone disease (P = 0.008; OR 2.488, 95% CI 1.243–4.980), but DD and ID genotypes were not distributed differently in patients and controls. After analyzing male and female population separately, II was associated with disease in females only (P = 0.019; OR 2.577, 95% CI 1.144–5.806). In males difference in II frequency was not statistically significant (Table 3). Frequency of I allele was higher in gallstone patients than in controls in total study population as well as, after stratification into male and female but the difference was not statistically significant (Table 4).

0.984 (0.639–1.514) 0.749 (0.476–1.180) 2.577 (1.144–5.806)

3.2. LRPAP1 insertion/deletion polymorphism

DD ID II

64

Table 4 LRPAP1 insertion/deletion polymorphism allele frequency in total subjects and stratified in male and female subjects Allele

D I

Male

Patient (no.a = 428)

Control (no.a = 636)

N

%

N

%

307 121

71.73 28.27

488 148

76.73 23.27

P-value

OR (95% CI)

0.066 0.066

0.769 (0.582–1.018) 1.300 (0.983–1.719)

Female

Patient (no.a = 138)

Control (no.a = 228)

N

%

N

%

95 43

68.84 31.16

175 53

76.75 23.25

P-value

OR (95% CI)

0.095 0.095

0.669 (0.417–1.074) 1.495 (0.931–2.40)

Patient (no.a = 290)

Control (no.a = 408)

N

%

N

%

212 78

73.10 26.90

313 95

76.72 23.28

P-value

OR (95% CI)

0.276 0.276

0.825 (0.583–1.167) 1.212 (0.857–1.714)

P-value

OR (95% CI)

0.922 0.730 0.602

1.025 (0.626–1.679) 1.079 (0.700–1.664) 0.869 (0.512–1.475)

Total chromosome number.

M. Dixit et al. / Hepatology Research 36 (2006) 61–69

a

Total

Table 5 CETP TaqI B polymorphism genotype frequency in total subjects and stratified in male and female subjects Genotype

B1B1 B1B2 B2B2

Total

Male

Patient (no. = 206)

Control (no. = 310)

N

%

N

54 107 45

26.21 78 51.94 167 21.84 65

P-value

OR (95% CI)

0.788 0.667 0.812

1.057 (0.706–1.581) 0.925 (0.650–1.317) 1.054 (0.686–1.618)

% 25.16 53.87 20.97

Female

Patient (no. = 68)

Control (no. = 107)

N

%

N

%

18 33 17

26.47 48.53 25.00

26 62 19

24.30 57.94 17.76

P-value

OR (95% CI)

0.747 0.223 0.248

1.122 (0.559–2.251) 0.684 (0.371–1.261) 1.544 (0.737–3.235)

Patient (no. = 138)

Control (no. = 203)

N

%

N

36 74 28

26.09 53.62 20.29

52 105 46

% 25.62 51.72 22.66

65

M. Dixit et al. / Hepatology Research 36 (2006) 61–69 0.945 (0.397–2.247) 0.978 (0.635–1.506) 1.037 (0.673–1.597) 0.897 0.919 0.868 6.83 47.32 45.85

CETP TaqI B

Polymorphism

%

6.47 46.76 46.76 9 65 65

N

1.363 (0.353–5.259) 0.813 (0.442–1.497) 1.152 (0.631–2.104) 0.729 0.507 0.644 %

4.39 44.74 50.88 5 51 58

N % N

5.88 39.71 54.41 5.96 46.39 47.65 6.28 19 44.44 148 49.28 152

%

%

0.879 0.661 0.715

P-value

OR (95% CI)

1.058 (0.511–2.192) 0.924 (0.650–1.314) 1.067 (0.752–1.515)

4 27 37

N N

13 92 102 L1L1 L1L2 L2L2

Patient (no. = 139) Patient (no. = 68) Control (no. = 319) Patient (no. = 207)

Control (no. = 114)

P-value

OR (95% CI)

Female Male Total Genotype

A1a

P-value

OR

R2

95% CI Lower

Upper

0.580 0.584

1.073 1.073

0.835 0.835

1.379 1.378

0.005 0.005

Ia Ib

0.057 0.060

1.312 1.309

0.992 0.989

1.736 1.733

0.008 0.008

B1a B1b

1.000 0.979

1.000 0.997

0.779 0.776

1.284 1.280

0.003 0.003

LPL HindIII

L2a L2b

0.855 0.856

1.026 1.026

0.780 0.780

1.350 1.349

0.004 0.004

a

Table 6 LPL HindIII polymorphism genotype frequency in total subjects and stratified in male and female subjects

Model

A1b

LDLR AvaII

14 97 94

N

%

LRPAP1 I/D

Control (no. = 205)

OR (95% CI)

Table 7 Adjusted odds ratios for gallstone disease according to alleles

P-value

66

b

Adjusted for age and sex. Adjusted for age, sex, and BMI.

polymorphism of LDLR, i.e. 3 dinucleotide repeat, have also shown contradictory results in Chinese population [40,41]. As we have discussed earlier, this polymorphism does not change amino acid so it might not be directly involved in lipid metabolism. However, studies in Alzheimer’s disease have shown significant interaction of LDLR gene polymorphism with other genes [42,43] Therefore, we suggest that positive associations in previous studies might be due to linkage disequilibrium of this polymorphism with other polymorphisms present on same or other genes. LRPAP1 codes for a molecular chaperon, which assists in the folding of LDL receptor family members and may also influence cholesterol homeostasis. In vitro studies show that RAP, which is a gene product of mouse orthologue of LRPAP1, interacts with LDL receptor related protein and prevents premature ligand interaction by maintaining receptor in functionally inactive state during its trafficking [44]. RAP regulates the amount of functional LDL receptor on the cell surface which is the main receptor for apo E [45]. We studied large (37 bp) insertion/deletion polymorphism in intron 5 of LRPAP1. We first time report the association of LRPAP1 insertion/deletion polymorphism with gallstone disease. Genotype II was imposing high risk (OR 2.577) for gallstone disease in females (Table 3). Our previous study in small sample size also showed association of LRPAP1, I allele with gallstone disease [11]. This polymorphism has been linked to coronary artery disease and Alzheimer’s disease [10,46,47]. A recent study revealed a strong association of a silent polymorphism (C/T) in exon 5 of LRPAP1 gene with metabolic syndrome in women coronary artery disease patients [48]. Here it was noticeable that the syndrome was also characterized by decreased levels of HDL-cholesterol and supports the role of LRPAP1 in cholesterol homeostasis. Carriers of I allele of LRPAP1 might have imbalance of cholesterol homeostasis which lead to gallstone formation. Study in coronary artery disease also found that D allele of LRPAP1 increases plasma HDL-cholesterol and apo A-I in dominant manner. In other words, I allele was associated with decreased plasma HDL-cholesterol [10]. RAP is inhibiter of LRP, and

M. Dixit et al. / Hepatology Research 36 (2006) 61–69

tetranucleotide polymorphism of LRP has been associated with plasma HDL2 -cholesterol levels [49]. RAP also inhibits the endocytosis of HDL by cubilin (receptor for intrinsic factor Vitamin B12 complex) [50]. These observations suggest the link between RAP and HDL. I allele carriers might have imbalanced HDL-cholesterol homeostasis which leads to gallstone formation. Molecular basis of this LRPAP1, intron 5 insertion/deletion polymorphism is not known. The polymorphism is present in intronic region, which is not implicated directly in protein structure. It may have role in regulation of gene expression or may be in linkage disequilibrium with other functional polymorphism either in the same gene or in other nearby genes. Here we hypothesize that I allele carriers might have decreased levels of HDL-cholesterol levels, which is a well known risk factor for gallstone disease. We observed association of LRPAP1 insertion/deletion polymorphism only in females not in males. The gallstone disease is more common in females than males. The difference can partly 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 gallstone disease both genetic and environmental factors contribute to the risk significantly (62%) and disease may be collective effect of both factors [3]. Among environmental factors, gender specific hormones are important contributory factors. Stratification of population based on gender neutralizes major environmental factors and we can dissect out association of genetic factor. Various polymorphisms associated with CETP have been studied; among them TaqI B is most widely studied. CETP TaqI B, B2 allele has been shown to be associated with higher HDL-cholesterol levels [14,15,17,51,52]. We did not find this polymorphism significantly associated with gallstone disease (Table 5). Other studies on gallstone disease and CETP TaqI B polymorphism have reported inconsistent results. The distribution of CETP TaqI B polymorphism in the cholesterol gallstones patients from Finland differed significantly from that in the controls, with the B1B1 subjects (P = 0.036) higher in controls [18]. A study from China revealed just opposite result with the B1 allele and B1B1 genotypes more frequent in gallstone patients than controls (0.523 versus 0.346 and 0.288 versus 0.1176, respectively) [19]. These contradictory results might be due to some other polymorphism linked to TaqI B polymorphism. From above studies it is apparent that TaqI B polymorphism might not be implemented directly as determinant of plasma HDL-cholesterol levels. This polymorphism is present in intron region, so on the basis of present knowledge it is difficult to term it as determinant of protein structure or expression level. Some other polymorphism present on the same or nearby gene may be in linkage disequilibrium, which has direct role in protein functionality [53] also found that the TaqI B polymorphism is not instrumental in determining CETP or HDL-cholesterol levels, but is a marker for the −629 promoter variant and the −2708 and −971 polymorphisms are likely to play a role in determining CETP concentration.

67

Lipid processing proteins, receptors and associated proteins might be important determinants of cholesterol levels in bile. Lipoprotein lipase is one of the key enzymes in the metabolism of the TG-rich lipoproteins. LPL is crucial in removing triglyceride from both chylomicron and VLDL. The primary function of LPL is the hydrolysis of the core triglycerides of circulating chylomicrons and VLDL thus initiating the lipolytic cascade required to convert VLDL into LDL. A number of common sequence variants have been reported, among them HindIII polymorphism is most widely studied. It has been hypothesized that the more common H1 allele (presence of cutting site) is associated with a lower LPL activity compared with the rare H2 allele (absence of the restriction site). In studies of healthy individuals, H2 allele has been associated with higher values of HDL-C and lower values of plasma triglyceride but other reports failed to note such effects [25,29–31,54,55]. It has also been associated with increased apolipoprotein C-III [32], apo B [33], and premature coronary heart disease [26,28,32,33]. We did not find any association of LPL HindIII polymorphism with disease or lipid variation (Table 6). There is no other study in gallstone disease. In present study, analysis of four polymorphisms present in lipoprotein metabolizing protein genes showed little variance for gallstone disease when adjusted for environmental factors (Table 7). Recent twin study in gallstone disease has detected 25% variance for heritability [3]. 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 all risk alleles will provide definite contribution of risk due to genetic factors. Since this is the first study of its kind more studies in different populations on large sample size are needed to confirm it.

5. Conclusion LRPAP1 insertion/deletion polymorphism is associated with gallstone disease and shows gender specific differences. LRPAP1, II genotype carrier females may have increased risk for gallstone disease. On the other hand, LDLR AvaII, CETP TaqI B, and LPL HindIII polymorphisms may not be associated with gallstone disease.

Acknowledgement MD is thankful to university grant commission for providing senior research fellowship.

References [1] Harvald B, Hauge M. A catamnestic investigation of Danish twins: a preliminary report. Dan Med Bull 1956;3:151–8.

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