Computational protocol: Evaluation of WISP1 as a candidate gene for bone mineral density in the Old Order Amish

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Protocol publication

[…] Study participants were genotyped using either the Affymetrix 500k or Affymetrix v6.0 SNP chips by the Genomics Core Laboratory at the University of Maryland. SNPs with a minor allele frequency (MAF) ≥1%, a call rate exceeding 95% and conforming to the expectations of Hardy-Weinberg equilibrium (p > 10−6) were used for imputation with IMPUTE-2 using 1000 G CEU reference sample phase2. SNPs with Imputation quality score (INFO) ≥30% were considered. Selection of the tagSNPs was performed based on the OOA genotyping data. Using the aggressive tagger mode of Haploview version 4.2 (http://www.broadinstitute.org/haploview/), we selected 58 tagSNPs which cover all common genetic variation within 5 kb upstream and downstream to WISP1 gene (Chr8: 134198282–134248933, GRCh37.p13) (Table ). Association analysis including these 58 tagSNPs was performed. [...] Summary statistics of baseline clinical characteristics were expressed as unadjusted means ± standard deviations (SD) using the SPSS statistics version 23 (IBM Corporation, N.Y., NY, USA). The association analyses were carried out using in-house software called MMAP (https://mmap.github.io/). The polygenic component was modeled using the relationship matrix derived from the complete 14-generation Amish pedigree structure. We included family structure, study, age, sex, age*sex, as covariates in the association models. BMI was associated with BMD on univariate analysis and was therefore included as a covariate in model 2. Subgroup analyses to determine whether there were differences in gender were performed. Estimation of the additive genetic heritability follows basic quantitative genetic theory, which models the phenotypic covariance (conditional upon covariate effects) between two individuals in a pedigree as a function of their degree of biologic relatedness. Maximum likelihood methods were used to estimate the values of the parameters, such as heritability, that resulted in highest likelihood obtained across all of the pedigrees. Covariates for BMD heritability analysis were study, sex and age. P-values less than 0.05 were considered as significant. Correction for multiple testing was performed using the Bonferroni method for the number of SNPs and traits tested (P = 0.05/(58 × 5) = 1.72 × 10−4). [...] Analysis of linkage disequilibrium (LD) statistics (r2) surrounding variants of interest was performed using Haploview version 4.2 (http://www.broadinstitute.org/haploview/). Prediction of histone marks and DNAse hypersensitivity sites was performed using HaploReg v4.1, and the five SNPs were annotated in regulatory elements cataloged in Encyclopedia of DNA Elements (ENCODE) project according to UCSC Genome Bioinformatics website (http://genome.ucsc.edu/). The eQTL analyses were performed in GTEx (https://www.gtexportal.org/home/), ExSNP (http://www.exsnp.org/), and primary osteoblasts (obtained from bone biopsies). […]

Pipeline specifications

Software tools IMPUTE, Tagger, Haploview, SPSS, HaploReg
Databases GTEx
Applications Miscellaneous, GWAS
Organisms Homo sapiens