ABCMdb

PMID: 20594224

Siddapuram SP, Mahurkar S, Duvvuru NR, Mitnala S, Guduru VR, Rebala P, Mansard MJ
Hepatic cholesterol transporter ABCG8 polymorphisms in gallstone disease in an Indian population.
J Gastroenterol Hepatol. 2010 Jun;25(6):1093-8., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Studies have identified single nucleotide polymorphisms (SNP) D19H and T400K in the cholesterol transporter gene ATP-binding cassette, subfamily G, member 8 (ABCG8) in patients with cholesterol gallstones. Login to comment 4 ABCG8 p.Asp19His ABCG8 p.Thr400Lys The aim of this study was to analyze the relationship between D19H and T400K polymorphisms in the ABCG8 gene and GSD in an Indian population, and the effects of these polymorphisms on cholesterol levels in sera and bile. Login to comment 7 ABCG8 p.Asp19His ABCG8 p.Thr400Lys The genotype of SNP D19H and T400K of ABCG8 was analyzed in 226 patients and 222 control samples. Login to comment 8 ABCG8 p.Asp19His ABCG8 p.Thr400Lys SNP D19H was analyzed by direct sequencing, and SNP T400K genotyping was assayed by the amplification refractory mutation system-polymerase chain reaction. Login to comment 9 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Results: There was no significant difference in the allelic distribution of SNP T400K between the GSD and gallstone-free groups (P > 0.05), but the distribution of the SNP variant, D19H, was significantly higher (P = 0.017, odds ratio = 2.274) in patients compared to controls. Login to comment 11 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Conclusion: SNP D19H, but not SNP T400K, in the ABCG8 gene is significantly associated with GSD in an Indian population. Login to comment 18 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Principally, gallstones are composed of cholesterol monohydrate crystals.14 Previous studies have found that the single nucleotide polymorphism (SNP) D19H of ABCG8 was stronger in patients with cholesterol gallstones (odds ratio =3.3), suggesting that H19 might be associated with more efficient transport of cholesterol into the bile; SNP T400K is found in Chinese males.15,16 The present study was undertaken to investigate, for the first time, the relationship between these two polymorphisms and GSD within an Indian population and to analyze their association with cholesterol in sera and bile. Login to comment 25 ABCG8 p.Asp19His ABCG8 p.Thr400Lys DNA was isolated from lymphocytes using standard methods.17 Two SNP, rs 4148217 = c.1199C>A (p.T400K) and rs 11887534 = c.55G>C (p.D19H) in the ABCG8 gene were analyzed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR; tetra-primer based) and direct sequencing methods, respectively. Login to comment 26 ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys ABCG8 T400K analysis SNP T400K genotyping was assayed by tetra-primer ARMS-PCR that adopts principles of both the tetra-primer PCR method and the ARMS. Login to comment 38 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 D19H analysis The SNP D19H of ABCG8 was analyzed by direct sequencing, as the tetra-primerARMS-PCR assay did not give satisfactory results for this region. Login to comment 40 ABCG8 p.Asp19His The primers (Table 1) amplified the 306-bp DNA fragment encompassing D19H of ABCG8. Login to comment 42 ABCG8 p.Asp19His ABCG8 p.Thr400Lys The PCR reaction mixture and PCR conditions described for T400K were similar to SNP D19H, with the exception of the annealing temperature, which was 68°C. Login to comment 45 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys Low-density lipoprotein (LDL) cholesterol was Table 1 Polymerase chain reaction primer details of single nucleotide polymorphisms D19H and T400K of the ABCG8 gene Polymorphisms Primer sequence Annealing temperature Amplicon size D19H Forward primer: 68°C 306 bp 5'-ACTCGCAGCCCTGTTTCTAG 3' Reverse primer: 5'-GATGGGCTTTCACCTTGTTG 3' T400K Forward inner primer (A allele): 5'-ATGCCTGGGGCGGTGCAGCAGTTCAA3' Reverse inner primer (C allele): 5'-AAATGACAGATAATTACCGGATCAGCGCCG3' Forward outer primer (5'-3'): 5'-ACATCCCCTGCTTGCAGTGGACCTGACC3' Reverse outer primer (5'-3'): 5'-AGATGGGCTTTCACCTTGTTGCCCAGGC3' 65°C 287 bp (A allele) 365 bp (C allele) 596 bp (from two outer primers) Genetic basis of gallstone disease in India SP Siddapuram et al. 1094 Journal of Gastroenterology and Hepatology 25 (2010) 1093-1098 calculated using the Friedewald formula: T/5 = very low density lipoprotein (VLDL) TC-(HDL + VLDL). Login to comment 54 ABCG8 p.Thr400Lys 2 3 4 5 6 7 8 9 10 11 12 13 14 M 596 bp 365 bp 287 bp Figure 1 Electrophoresis of single nucleotide polymorphism T400K amplification refractory mutation system-polymerase chain reaction products. Login to comment 55 ABCG8 p.Thr400Lys Tetra-primer-based genotyping of the ABCG8 T400K polymorphism: 596-bp product is common to both mutant and wild-type alleles; 365-bp product is specific for wild-type alleles; and 287-bp product is specific for mutant alleles. Login to comment 59 ABCG8 p.Asp19His Figure 2 Chromatogram showing the sequence of single nucleotide polymorphism D19H (c.55G>C) of ABCG8. Login to comment 69 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys Distribution of SNP T400K and D19H between the GSD and GSF groups In the present study, 226 patients and 222 controls were analyzed for two SNP: T400K and D19H of the ABCG8 gene. Login to comment 71 ABCG8 p.Asp19His ABCG8 p.Thr400Lys No significant difference was observed in the distribution of genotypic frequency of SNP T400K when compared to the GSD and GSF groups (P = 0.520), whereas the distribution of SNP D19H showed a significant difference between the patient and control groups (P = 0.046). Login to comment 74 ABCG8 p.Asp19His ABCG8 p.Thr400Lys No significant difference was noted in any allele distribution of SNP T400K between the GSD and GSF groups, but the distribution of the heterozygous variant allele of SNP D19H was significantly higher (P = 0.048, OR = 2.219, CI: 1.054-4.672) in the GSD patients compared to the GSF individuals (Table 4). Login to comment 75 ABCG8 p.Asp19His Further, the mutant allelic (heterozygous + mutant homozygous) distribution of D19H was also assessed between the two groups, where the mutant alleles were statistically significant (P = 0.017, OR = 2.274, CI: 1.171-4.416) in the GSD cohort compared to the GSF group. Login to comment 76 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Thus, in the present study, allelic distribution infers that the T400K genotype might not be associated with gallstone formation in our cohort, whereas SNP D19H was shown to be associated with the disease. Login to comment 78 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Thr400Lys A strong association between elevated bile cholesterol and SNP D19H (P < 0.001) was observed in the analysis, where the mean bile cholesterol for SNP T400K (heterozygous + homozygous variants) was 155.13 mg/dL, and that of SNP D19H (heterozygous + homozygous variants) was 385.37 mg/dL Table 2 Demography and plasma lipid profile of gallstone disease and gallstone-free individuals Parameter Patient group Control group P-value Mean SD Mean SD Age (years) 45.88 14.840 39.92 11.864 0.063 BMI (kg/m2 ) 26.430 10.830 24.727 5.003 0.018 Plasma CH CH (mg/dL) 182.623 55.996 224.979 0.352 0.0001 HDL-C (mg/dL) 32.168 9.130 39.081 9.771 0.0001 LDL-C (mg/dL) 120.305 43.311 138.896 39.712 0.0001 TG (mg/dL) 149.672 83.945 147.207 89.148 0.324 BMI, body mass index; CH, cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SD, standard deviation; TG, triglycerides. Login to comment 79 ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Asp19His ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys Table 3 Frequency of genotype distribution of single nucleotide polymorphisms T400K and D19H between gallstone disease and gallstone-free individuals Single nucleotide polymorphisms of ABCG8 Wild type Heterozygous Homozygous P-value T400K (patients/controls) 69.47%/67.56% 29.20%/31.98% 1.32%/0.45% 0.520 D19H (patients/controls) 86.72%/93.69% 10.17%/4.95% 3.09%/1.35% 0.046 Table 4 Statistical analysis of single nucleotide polymorphisms T400K and D19H of ABCG8 between the gallstone disease and gallstone-free groups for possible alleles Allele P-value c2 -test Odds ratio 95% confidence interval T400K TT vs TK 0.608 0.33 0.888 0.594-1.329 TT vs. Login to comment 80 ABCG8 p.Asp19His TK + KK 0.685 0.18 0.916 0.614-1.364 D19H DD vs DH† 0.048 4.5 2.219 1.054-4.672 DD vs DH + HH‡ 0.017 6.1 2.274 1.171-4.416 Statistically-significant genotypes between the †gallstone disease and ‡gallstone-free groups. Login to comment 84 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Discussion Genotyping The aim of present study was to determine whether SNP T400K and D19H of the ABCG8 gene were associated with GSD. Login to comment 85 ABCG8 p.Asp19His Our results indicated that, of these common polymorphisms in the ABCG8 gene, D19H could be associated with GSD in an Indian population. Login to comment 87 ABCG8 p.Asp19His ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys A previous study mapped a susceptibility locus for gallstone formation to the murine ortholog of the ABCG5/ABCG8 genes, using a quantitative trait locus analysis in experimental crosses of inbred mouse strains.20 A recent linkage study of 715 individuals from 39 Mexican-American families with gallstone disease provided suggestive evidence of linkage to gallbladder disease on chromosome 2p21, the site of the ABCG5/ABCG8 genes.21 A genetic association study demonstrated that the ABCG8 D19H variant was significantly associated with the presence of gallstones in cases; the 19H (c.55C) allele was significantly more common in cases than in controls (OR = 3.018, P = 0.017).15 Carriers of the 19H allele, whether homozygous or heterozygous, were significantly overrepresented in the cases compared with the controls (OR = 3.118, P = 0.019), and in a case-control study, heterozygous 19H carriers (OR = 2.2) indicated a robust association of ABCG8 with gallstones in different populations.22 Previous studies have indicated the role of T400K polymorphisms in lipid parameters and the formation of gallstones.12 The frequencies of the K400 allele (12.4% vs 6.2%, P = 0.018) of ABCG8, and the TK/KK genotype (24.8% vs 12.4%, P = 0.025) were significantly higher in GSD groups than in GSF groups.23 In contrast, the present study frequencies of SNP genotypes T400K were not significantly different (Table 4) between patients and controls. Login to comment 88 ABCG8 p.Asp19His ABCG8 p.Thr400Lys ABCG8 p.Thr400Lys Demography and lipid profile Several studies have reported that gallstones are more common in women than men and at all age groups, as the present study has indicated.24 In the present study, of the 226 patients, 56.7% (128) were females and 43.3% (98) were males. The demographic analysis in the present study showed a significantly higher BMI in GSD patients, which supports an earlier study.16 Hypersecretion of cholesterol from the liver and supersaturation of bile with cholesterol represents the common defects in patients with cholesterol gallstones.25,26 Another study suggested that the ABCG8 T400K polymorphism affects the reduction in total plasma cholesterol and LDL cholesterol levels.23 In earlier studies, researchers found a lower concentration of serum campesterol in 19H carriers, and hypothesized that the H allele leads to increased ABCG8transporter activity.10 Further, this hypothesis was supported by the observation that 19H carriership is associated with lower total serum cholesterol levels.27 Perhaps the basic function of both the T400K and D19H polymorphisms is the same to regulate cholesterol transport. Login to comment 89 ABCG8 p.Asp19His Similarly, our lipid profile data showed an association between the D19H polymorphism and reduced total plasma cholesterol and LDL cholesterol levels in patients. Login to comment 90 ABCG8 p.Asp19His Previous studies reported that low serum HDL cholesterol and high serum triglyceride concentrations are associated risk factors of GSD.28 In contrast, in a family study of a normal population, the D19H substitution of the ABCG8 gene resulted a different situation: no association with serum lipid levels was found, nor any compensatory upregulation of cholesterol synthesis.10 However, the present study shows similarity with earlier studies, where low serum HDL cholesterol and high serum triglyceride concentrations are associated with GSD. Login to comment 91 ABCG8 p.Asp19His Although the GSD risk is clearly attributable to the D19H variant in the ABCG8 gene in the present study, the limitation of the study could be the possibility of other ABCG5/ ABCG8 SNP and other Lith genes that contribute to gallstone susceptibility, and the elucidation of different gallstones types. Login to comment 92 ABCG8 p.Asp19His ABCG8 p.Thr400Lys In conclusion, our study shows for the first time in India that the D19H SNP of ABCG8 was shown to have a strong association with GSD, whereas the T400K SNP of ABCG8 was not found to be significant with the disease in the study cohort. Login to comment 96 ABCG8 p.Asp19His ABCG8 p.Thr400Lys Our sincere thanks to Dr Jamuna Cherri (Asian Healthcare Foundation) for her constant help in the plasma lipid profile analysis. We are extremely thankful to Ms Mary Thomas and Ms Vasantha Rani (Asian Healthcare Foundation) for 3040N = Group D19hT400k Bilecholesterol 700 600 500 400 300 200 100 0 Figure 3 Distribution of bile cholesterol in gallstone patients between mutant alleles of single nucleotide polymorphisms T400K and D19H of ABCG8. Login to comment