Analysis of nsSNP rs1468384 of NPC1L1 and its association with plasma cholesterol levels in general population

摘要

Niemann-Pick C1–like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte. It effects intestinal cholesterol absorption and intracellular transport and as such is an integral part of complex process of cholesterol homeostasis. The study of population data for the distribution of these SNPs of NPC1L1 has lead to the identification of six nsSNPs (non-synonymous single nucleotide polymorphism). The in vitro analysis using the software MuPro and StructureSNP shows that nsSNP M510I (rs1468384), which involves A→G base pair change leads to decrease in the stability of the protein. A reproducible and a cost-effective PCR-RFLP based assay was developed to screen for the SNP distribution amongst the North Western Indian population as a test case. This SNP has been studied in Caucasian, Asian and African American populations. Till date, no data is available on Indian population. The allele distribution in Indian Population differs significantly from that of other populations. Moreover on investigating the effect of this nsSNP on the plasma lipid levels in the general population we found a profound association of this nsSNP with higher ranges of plasma lipid levels. Introduction Niemann-Pick C1–like 1 (NPC1L1) protein, a newly identified sterol influx transporter, located at the apical membrane of the enterocyte, which may actively facilitate the uptake of cholesterol by promoting the passage of sterols across the brush border membrane of the enterocyte (1,2). The protein has been characterized by the presence of a signal peptide, 13 putative transmembrane regions, a conserved NPC1 domain and a sterol sensing domain (SSD). Although expressed within several tissues in the body, the expression is predominant in liver and small intestine (3, 4, and 5). In rodents, NPC1L1 is highly expressed on the surface of jejunal absorptive cells whereas in humans the expression is more in the hepatoma cells in the liver (6). The protein plays a key role in cholesterol uptake and intracellular cholesterol trafficking from the plasma membrane to the endoplasmic reticulum (7). A fact further strengthened by the observation of Gracio-Calvo et al., 2005 (8) that the protein was the direct molecular target of ezetimibe, a drug that inhibits cholesterol absorption. Recently Temel et al., 2007 (9) have suggested the presence of NPC1L1 on the canalicular membrane of hepatocytes which may modulate biliary cholesterol excretion. Thus this protein is actively involved in the cholesterol homeostasis pathway (10, 11).Single nucleotide polymorphisms (SNPs), together with copy number variation, are the primary source of variability in the human genome. As amino acid substitutions currently account for approximately half of the known gene lesions responsible for human inherited disease, study of nsSNPs are important in delineating the etiology of many such disorders (12, 13). These SNPs may lead to changes in protein confirmation and may be associated with altered response to drug treatment, susceptibility to disease, and other phenotypic variations (14). The study of population data for the distribution of NPC1L1 has lead to the identification of six nsSNPs (non-synonymous single nucleotide polymorphism). Among the 6 identified nsSNPs, M510I (rs1438384) shows decrease in the stability of the protein as analysed in silico by MuPro (15) and StructureSNP (16) softwares. The M510I polymorphism is the result of a nucleotide change G to A at position 2993 of the cDNA sequence in exon 2, and it results in the substitution of isoleucine for methionine at amino acid 510 of the NPC1L1 protein. The SNP has already been studied in Caucasian, Asian and African American populations by sequencing as given in NCBI database. Till date, no data is available on Indian population. Thus,