Research On Variants Of ABCA1 And Its Relationship With Cerebral Infarct

Background and purposeIn recent decades, with the human standard of living improve, antherosclerosis become the major cause of death. Adenosine triphosphate binding cassette transporter A1 (ABCA1) play an important role in the pathogenesis of atherosclerosis. ABCA1 gene is located at 9q31, the length of 149kb, contains 50 exons and 49 introns, encoding 2261 amino acid composition of the integrity of the transmembrane protein, weight of 250kD. ABCA1 to ATP for energy, the promotion of intracellular free cholesterol and phospholipids of the outflow, combined with the cell surface of apoA1, the formation of nascent high density lipoprotein (HDLC), and activate reverse cholesterol transport process. ABCA1 mutations cause Tangier disease (TD) and familial hyperlipoproteinemiaα(FHA), clinical manifestations of HDL and apoA1 defect, early-onset atherosclerosis, associated with hepatosplenomegaly and peripheral neuropathy, etc. At present, human ABCA1 gene has 81 mutations, including 54 missense mutations, three splicing mutations, 20 insertion or deletion mutation, four mutations in the regulatory mutations(http://www.hgmd.org),These mutations can cause outflow lipid barriers, and promote the development of atherosclerosis. ABCA1 gene coding region and non-coding region contains many SNP, the study shows that these SNP are associated with plasma lipid levels as well as atherosclerosis. The study about ABCA1 gene mutation present in Chinese Han population are rare, mostly single nucleotide polymorphisms are susceptibility SNP research, has not found the mutations in Chinese population study. The study of SNPs most focused on cardiovascular disease, and less susceptibility to cerebral infarction. The purpose of this study was to explore the distribution of variation of ABCA1 gene in Chinese Han population and susceptibility to cerebral infarction; simultaneously for the functional studies of mutant.Methods1 using PCR-RFLP, denaturing polyacrylamide gel electrophoresis and DNA sequencing techniques, 72 cases of HDL <0.8 mmol / L and apoA1 <0.8 mmol / L of atherosclerosis in patients with cerebral infarction of the ABCA1 gene mutation cluster part of exon (exon 7,14,17,19,49) and its intron junction area variation detection; for the newly discovered mutation site at 200 cases of normal controls for authentication, and using Panther software analysis its function changes.2 using PCR-RFLP techniques to case - control study of 324 cases of the way patients with cerebral infarction (atherosclerotic cerebral infarction group and 193 cases lacunar infarction Group 131 cases) and 152 cases of normal control of ABCA1 gene 1051G→A (Arg219Lys) and 2826G→A (Val825Ile) polymorphism detection, comparison of different genotypes with lipid levels and its relationship with individual susceptibility to cerebral relevance; compare different synergistic interaction between genotype.3 Construction of ABCA1-EGFP eukaryotic expression plasmid of wild-type and mutants, the transfected 293T cells to confocal immunofluorescence microscopy of wild-type and mutant cells in the expression of ABCA1 protein (morphological changes); another aspects of transfected 293T cells 24 hours after add with dehydroergosterol (DHE) cell culture medium, using high-performance liquid chromatography (HPLC) detection of wild-type and mutant cells to DHE transhipment capacity.ResultThe SNPs in Chinese Han population has been reported on the 7th exon R219K and V825I in exon 17. In atherosclerotic cerebral infarction (ACI) Group R219K genotype frequencies and allele frequencies with the control group there was a significant difference ((χ2 = 9.89, P <0.01;χ2 = 9.16, P <0.005); at the age of more than 60-year-old subgroup, ACI group compared with the control group, KK genotype and K allele frequency frequency difference was significant (χ2 = 13.91, P <0.005;χ2 = 11.46, P <0.005); in the male subgroup, ACI group compared with the control group, KK genotype frequency and allele frequencies of K there was a significant difference (χ2 = 13.24, P <0.005;χ2 = 8.60, P <0.005); carry KK genotype RR carriers than individuals suffering from atherosclerotic cerebral infarction significantly reduced the risk (OR: 0.30, 95% CI: 0.14 ~ 0.66, P = 0.002); in hypertensive patients, compared with the KK genotype, RK and RR genotype ACI increased risk of cancer (OR: 3.09, 95% CI: 1.54-6.19, P = 0.002; OR: 4.81, 95% CI: 1.94-11.94, P = 0.001); Similar results can also be found in VI and VV compared with the II genotype; compared with the KK and II carriers, RR and VI of the sub-carriers suffering from ACI significantly increased risk (OR: 2.69, 95% CI: 1.39-5.22, P = 0.003). V825I locus and atherosclerosis is not clearly related to cerebral infarction. Compared with the RR type, RK, KK genotype ApoA1 serum levels were gradually increasing trend (P = 0.04; P = 0.03); in female patients who are relatively RR type, to carry K allele of serum ApoA1 and HDL-C level high (P = 0.01). V825I is no obvious relationship with lipid levels. This study did not find reported in the literature at home and abroad R230C, M233V (7 exons) and N831D (17 exon); at 49 exon and intron were found at the junction of 6645 +55 G→C mutation loci, genotype frequencies were 44.07%, 40.68% and 15.25%, allele frequency were 35.59%; simultaneously at exon 49 was found on a case of 2206 points, the TAC→GAC heterozygotes (Y2206D), the plasma HDL level of 0.66 mmol / L, apoA1 level of 0.61 mmol / L, while 200 cases of normal control group did not find the mutation site, and its frequency of occurrence is less than 1% more likely to support the mutation; Panther software analysis of Y2206D SubPSEC: -8.746, Pdeleterlous: 0.9968, suggesting that the mutation site of the devastating effects of a strong (http://www.Pantherd.org/tools); cell morphology studies showed that 2206 mutant at positioning happened change, ABCA1 protein is located in many intracellular endoplasmic reticulum, not like the wild-type as on location in the cell membrane; HPLC detection of 2206 mutation on the DHE transit barriers, cellular DHE content is more than the wild-type, the difference has statistical significance (P <0.05).ConclusionThere are three SNPs(R219K, V825I and 6645 +55 G→C) existing in Han Chinese atherosclerosis in patients with cerebral infarction; no mutations are detected on exon 14 and exon 19. ABCA1 gene R219K possible with the Chinese Han population in atherosclerotic cerebral infarction related to genetic susceptibility, particularly in the older than 60 years of age and male subgroups, as well as patients suffering from hypertension 219K protective effect of a more prominent; Y2206D loci in Chinese Han population are probably a new mutation with abnormal lipid metabolism.