Computational protocol: Characterization of the DYX2 locus on chromosome 6p22 with reading disability, language impairment, and IQ

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Protocol publication

[…] We developed a SNP marker panel in an attempt to capture the common variation in the DYX2 locus. TagSNPs in the locus were selected using the association study design server of Han et al. (). The final DYX2 panel contained 195 SNPs with an estimated average power of 83 and 68 % to capture known common and rare variants, respectively, in the DYX2 locus spanning approximately 1.4 Mb. Markers were genotyped on the Sequenom MassARRAY platform (San Diego, CA) following manufacturer’s guidelines at the Yale Center for Genome Analysis (West Haven, CT). Briefly, markers were genotyped in nine multiplex reactions of 30–36 markers each, totaling 300 markers (Supplemental Table 1). A subset of markers was not in the DYX2 locus and was not included in the subsequent characterization of the DYX2 locus. In addition to quality control via call rate and Hardy–Weinberg, the histogram plot for each marker was manually evaluated, and a total of 15 markers showing aberrant patterns were excluded. To control for errors in labeling and manipulation of plates, the samples were also genotyped for four sex-determining SNPs in the genes AMELXY and ZNFXY in the pseudoautosomal regions of the X and Y chromosomes. These SNPs correctly determined sex for 99.5 % of samples; the remaining samples were excluded.Markers that deviated substantially from Hardy–Weinberg equilibrium (p < 0.0001), or that had an overall call rate <85 %, were not used for genetic analyses. In the discovery ALSPAC cohort, single marker SNP analyses of case–control status and quantitative traits were completed using SNP and variation suite (SVS) v7.6.4 (Bozeman, MT). Linkage disequilibrium was assessed and haplotype blocks were constructed using the four-gamete rule option in HaploView v4.2. Haplotype-based association tests were performed with haplotypes that had frequencies ≥1 % using PLINK v1.07 (Barrett et al. ; Purcell et al. ). To correct for multiple testing, we used a Bonferroni threshold of 0.000256 (0.05 divided by 195 markers) for discovery association tests in the ALSPAC cohort. However, associations with p < 0.001 are also reported for the ALSPAC discovery cohort to indicate suggestive results.We tested SNPs that had single marker or within-haplotype associations with p < 0.001 in the ALSPAC for replication in the Iowa LI, Italian RD, and Colorado RD cohorts. Iowa LI was analyzed using SVS v7.6.4 (Bozeman, MT), while the family-based Italian RD and Colorado RD cohorts were examined using PLINK v1.07 (Purcell et al. ). We moved suggestive ALSPAC results forward to our replication analyses to emphasize replication of associations over statistical corrections for multiple testing. Replications with p < 0.05 in the Iowa LI, Italian RD, and Colorado RD cohorts are reported. […]

Pipeline specifications

Software tools SVS, Haploview, PLINK
Application GWAS
Organisms Homo sapiens