Single Nucleotide Polymorphism Of NPC1L1 Gene In Association With Cholesterol Gallstone Disease

Background:Cholesterol gallstone disease is a common disease.Multi-genetic susceptibility leading to abnormalities in entero-hepatic cholesterol and bile acid metaboslisms that contributes to bililary cholesterol saturation,impaired gallbladder motility and the disbalance of pro-and anti-nucleation cholesterol in bile is the recognized pathway of gallstone formation so far.Niemann Pick C1 like 1（NPC1L1）protein is a critical protein regulating cholesterol level.It is expressed in proximal jejunum and liver in human.In the proximal jejunum,it is responsible for cholesterol absorption into enterocytes and in liver,NPC1L1 localizes at hepatic canalicular membranes,re-uptaking biliary cholesterol back into hepatocytes.This study aims to investigate the difference of distribution of single nucleotide polymorphism（SNP）of NPC1L1 gene between patient with and without gallstone disease,and to study the difference of transcription by NPC1L1 promoter carrying different SNP.At last,the effect on apoptosis and autophagy in cells after influecing intracellular cholesterol level were also evaluated.Material and manners:Collected venous blood from 25 gallstone patients（GS）,extracted DNA from white blood cell,by means of PCR amplified NPC1L1 exons and sequenced,acquired distribution of NPC1L1 gene single nucleotide polymorphism（SNP）.Collected venous blood from299 gallstone patients and 249 gallstone-free patients（GSF）,extracted DNA from white blood cell,used Taqman probe based on quantitative PCR to analyzed the distribution of genotype and haplotype of common SNP in NPC1L1.Utilized dual-luciferase report gene to detect different expression level of NPC1L1 associated with SNP located in promoter region of NPC1L1 gene.Use ezetimibe to inhibit cholesterol absorption and simvastatin to reduce cholesterol biosynthesis in cells induce apoptosis and autophagy changes.Results:Foure SNPs of NPC1L1 were found using genomic DNA from 25 patients with gallstone disease:rs2073548（g.-762T>C）,rs17655652（g.-133T>C）,rs41279633（g.-18G>T）,rs2072183（g.1679C>G）.The polymorphic loci and frequency of genotypes were listed as followed:rs2073548（alleleic frequency T=0.64,C=0.36,genotype frequency CT=0.48,TT=0.40,CC=0.12）,rs17655652（alleleic frequency T=0.98,C=0.02,genotype frequency TT=0.96,CT=0.04）,rs41279633（alleleic frequency G=0.98,T=0.02,genotype frequency GG=0.96,GT=0.04）,rs2072183（allelic frequency C=0.62,G=0.38,genotype frequency CG=0.44,CC=0.40,GG=0.16）.Comparing with GSF group,the frequency of C allele of rs2073548 loci was significantly higher in GS group（36.3%vs 30.5%,P<0.05）and the frequency of G allele of rs2072183 was higher in GS group as well（37.8%vs 31.5%,P<0.05）.Combined with data from our previous study,the frequency of C allele of rs2073548 loci did not differ between GS group and GSF group（35.9%vs32.4%,P>0.05）.The frequency of G allele of rs2072183 loci was still significantly higher in GS group than in GSF group（38.8%vs 32.8%,P<0.01）.Furthermore,the frequency of T^-762/G¹⁶⁷⁹haplotype of GS group was significant higher than GSF group（P=0.02）.Dual-luciferase report assay showed no difference for the activity of promoter carrying either T or C allele of rs2073548（g.-762T>C）.Neither was there any difference between the two promoter in the response to either simvastatin or ezetimibe（P>0.05）.Reducing intracellular cholesterol by either simvastatin or ezetimibe level could promote apoptosis in cells.Ezetimibe,by inhibiting NPC1L1,enhanced the expression of autophagy specific protein LC3.Conclusion:1)The polymorphism of rs2072183（g.1679C>G）is associated with cholesterol gallstone disease.Individuals carryintthe G allele of rs2072183 and T^-762/G¹679679 haplotype are at risk to gallstone disease.The polymorphism of rs2073548（g.-762T>C）does not affect the transcriptional activity of NPC1L1 promoter.2)Inhibiting NPC1L1 can promote autophagy.