| Objective:Dyslipidemia represents an important risk factor for cardiovascular disease,and cardiovascular disease is the primary cause of death worldwide.The prevalance of dyslipidemia is as high as 40.40%and increasing in China.Serum cholesterol,such as total cholesterol?TC?,low density lipoprotein cholesterol?LDL-C?,and high density lipoprotein cholesterol?HDL-C?,is the common indicator of diagnosing dyslipidemia.The serum cholesterol level is mediated by both genetic and environmental factors.Here,we aimed to further elucidate the the magnitude of genetic impact on serum TC,HDL-C,LDL-C variation and further searched for genetic susceptibility loci for them using genome-wide association study?GWAS?.Methods:The subjects were selected from 2012-2013 years Qingdao Twin Registration system following specific inclusion criteria and exclusion criteria.All included twins were investigated by questionnaires,physical examination and laboratory examination.Fasting serum cholesterol levels were measured using an automatic biochemical analyzer according to the standard procedure.Sex,ABO blood type and 16multiple short tandem sequence repeat DNA markers combined to identify the zygosity.Genome-wide SNPs were genotyped using the Illumina's Infinium Omni2.5Exome-8v1.2 Bead-Chip platform.We performed stringent genotype quality control procedures for typed SNPs.We used the IMPUTE2 software to impute un-typed SNPs based on the LD information from1000 Genomes Project Phase 3 reference panel.Meanwhile,a strict quality control standards were used to screen the imputation SNPs.We used the structural equation models?SEM?to evaluate the genetic variance components with Mx software.Heritability?h2?was calculated in the best-fitting model based on the ratio of additive genetic variance to total phenotypic variance,with adjusting for the effect of age,sex and education level.We used the genome-wide efficient mixed-model association?GEMMA?to test the association between serum cholesterol level and SNP genotypes after adjusting for the following covariates:sex,age,and education level.We used Quantile-quantile?Q-Q?plots and Manhattan plots to illustrate significant SNPs.We analyzed the enrichment of cell-type enhancers using online HaploReg v4.1 software.The gene-based test was implemented in Versatile Gene-based Association Study-2?VEGAS2?.Pathway scoring algorithm?PASCAL?software was used to compute pathway-scored.Results:The final sample consisted of 382 twin pairs for heritability analysis and139 DZ twin pairs for GWAS analysis with a mean age±SD of 51.6±7.7 years.The mean values±SD of serum TC,HDL-C,and LDL-C levels for all subjects were 4.9±1.2umol/L,1.5±0.5 umol/L,and 2.8±0.9 umol/L,respectively.Since MZ twin correlations for TC,HDL-C,LDL-C levels?TC:rMZ=0.61,HDL-C:rMZ=0.74,LDL-C:rMZ=0.65?were all less than 2 times of DZ twin correlation?TC:rDZ=0.35,HDL-C:rDZ=0.61,LDL-C:rDZ=0.35?,the complete ACE model was determined.Then the nested model was selected by using likelihood Chi-square test and Akaike's information criterion.Finally,the ACE model was the best fit model with additive genetic parameter?A?accounting for 26.6%,common or shared environmental parameter?C?accounting for 47.8%,unique or non-shared environmental parameter?E?accounting for 25.6%for the variance in serum HDL-C level.The AE model was the best fit model for serum TC level?A:61.4%,E:38.6%?and serum LDL-C level?A:65.5%,E:34.5%?.The power of our heritability analysis calculated by Mx software was above 90%.A sample of 139 DZ twin pairs including 1,365,181 qualified SNPs was included for the present GWAS.While no SNPs reached the genome-wide significance level (P<5×10-8),8,14,9 SNPs exceeded the suggestive significance level(P<1×10-5)for serum TC,HDL-C,LDL-C levels,respectively.The promising genetic regions for serum TC,HDL-C,LDL-C were on chromosome 11 around rs7107698,chromosome 5 around rs12518218,chromosome 2 around rs10490120,respectively.Cell-type specific enhancers of primary neutrophils from peripheral blood and ovary were identified for serum TC level.HUES6 cells,HUES64 cells and iPS-18 cells may be related to serum LDL-C level.Although post-imputation Manhattan plots showed that no SNPs reached the genome-wide significance level(P<5×10-8),imputation still increased the number of SNPs at the suggestive significance level(P<1×10-5).There were 39,55,and 59 SNPs exceeding the threshold for suggestive significance level for TC,HDL-C,and LDL-C,respectively.The strongest associations were on chromosome 2 around rs77348447,chromosome 5 around rs79775842,chromosome 1 around rs56047090 for serum TC,HDL-C,LDL-C,respectively.We also compared our post-imputation SNPs results with34,421 East Asians lipids GWAS meta-analysis results.32,50,39 SNPs were repeated for TC,HDL-C,LDL-C level,respectively.Gene-based analysis found 1038,1033 and 1090 genes nominally associated with serum TC,HDL-C,LDL-C level?P<0.05?,especially FAF1,KLKB1 etc.for TC level,KLKB1 etc.for HDL-C level,and NTRK1,FAF1,SNTB2 etc.for LDL-C level,respectively.The number of common related genes among TC,HDL-C and LDL-C was71,including FAF1,KLKB1,etc.PASCAL pathway enrichment analysis discovered well-known related pathways,zinc transporters,metal ion SLC transporters for serum TC level,cell adhesion molecules CAMs and IL-6 signaling for serum HDL-C level,FC epsilon RI signaling pathway and NFAT pathway for serum LDL-C level,respectively.Conclusions:The serum TC?h2=61.4%?and LDL-C?h2=65.5%?levels were moderately heritable and the HDL-C?h2=26.6%?level is lowly heritable in Qingdao Twin population.The relevant SNPs loci including rs7107698,rs12518218,rs10490120 etc.,functional genes including FAF1,KLKB1,NTRK1,SNTB2 etc.,and biological pathways including zinc transporters,metal ion SLC transporters,cell adhesion molecules CAMs,IL-6 signaling,FC epsilon RI signaling pathway,NFAT pathway etc.were significantly associated with serum cholesterol level.Our study can provide a useful reference for the GWAS study of serum TC,HDL-C,LDL-C levels in East Asian population,and provide a basis for future molecular biology research. |
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