Computational protocol: Lack of association between SREBF-1c gene polymorphisms and risk of non-alcoholic fatty liver disease in a Chinese Han population

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Protocol publication

[…] The genomic region harbouring SREBF-1c was examined to select SNPs based on the LD patterns of Chinese Han population in Beijing (CHB) from the International HapMap Project SNP database [HapMap Data Rel. 28 Phase II + III, August 10, on National Center for Biotechnology Information (NCBI) B36 assembly, dbSNP b126, accessed April 2013]. We then used the pairwise tagging method of the Haploview v4.2 software (Broad institute, Cambridge, MA, USA) to capture SNPs with a minimum minor allele frequency (MAF) of > 0.02 and a minimum r2 of > 0.8. Additionally, the SNPs were prioritized according to the following criteria i): known SNPs associated with metabolic traits from the literature; ii) SNPs within coding regions (exon); iii) SNPs within the promoter region (2,500 bp before the start codon); iv) SNPs within 3′ untranslated region (UTR) (500 bp after the stop codon); and v) SNPs within 100 bp before an exon-intron splicing boundaries. As result, four SNPs (rs11868035, rs2297508, rs13306741 and rs62064119) were selected from unrelated Han Chinese individuals in Beijing. DNA was isolated from EDTA-treated blood samples following standard procedures. These polymorphisms were genotyped by iPlex technology based on a MassARRAY platform in all subjects. Primers for the amplification and extension reactions were designed using the Mass Array Assay Design Version 3.1 software (Sequenom, San Diego, CA, USA), and SNP genotypes were determined according to the iPLEX protocol provided by the manufacturer. Genotyping assays were performed by laboratory personnel who were blinded to the NAFLD status of each subject. The genotyping quality was examined using a detailed QC procedure consisting of a > 95% successful call rate, duplicate calling of genotypes, internal positive control samples and HWE testing. [...] Unless otherwise indicated, phenotypic quantitative data were expressed as mean ± standard deviation. To evaluate differences in clinical characteristics between cases and controls, Student’s t-test, Pearson’s Chi-squared test or the nonparametric Mann–Whitney U tests were used as appropriate. HWE was checked both in cases and controls by using the chi-squared test. In the case–control studies, each SNP was tested for association with NAFLD in a logistic regression analysis, adjusted for age, sex, marriage, education or income, smoking, tea drinking and clinical characteristics. When the genotype frequency for homozygotes of the minor allele was less than 5%, carriers (heterozygotes and homozygotes individuals) of the minor allele were grouped. P-values were adjusted for multiple tests.LD between SNPs was estimated in the case–control studies using Haploview4.2 ( The Haplo.stats software package was developed using the R language and was used to estimate adjusted ORs and 95% CIs for each haplotype. To assess statistical significance, we performed permutation procedures to correct the P-value of single-locus association results for multiple testing. Simulations were run 1,000 times for empirical P-values. All statistical analyses were performed using R statistical software. […]

Pipeline specifications

Software tools Haploview, haplo.stats
Databases dbSNP International HapMap Project
Application GWAS
Organisms Homo sapiens
Diseases Non-alcoholic Fatty Liver Disease
Chemicals Cholesterol