Computational protocol: Replication of association between schizophrenia and ZNF804A in the Irish Case Control Study of Schizophrenia (ICCSS) sample

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Protocol publication

[…] Following discussion with the Cardiff group, we genotyped 3 of the most promising SNPs from the report of genomewide association in the Cardiff sample, rs1344706 in ZNF804A, rs6490121 in NOS1 and rs9922369 in RGRIP1L. Based on the results in these markers, we then completed genotyping of 11 additional LD-tagging SNPs (tSNPs) in ZNF804A. We first manually included rs1344706 (associated in the original study) in the test alleles set and then selected additional tSNPs using the TAGGER algorithm with default criteria of r2≥0.8 and minor allele frequency (MAF)≥0.2 as implemented in Haploview 4.0 using HapMap data release 22/Phase II, Apr 07. Including rs1344706, we genotyped a total of 12 SNPs in ZNF804A. Markers are shown in ; orientation and alleles are reported on the genomic (+) strand (rs1344706 is reported here as A/C, not T/G as in the original report).All markers were genotyped with Taqman Assays-on-Demand (Applied Biosystems, Foster City, CA). To ensure uniformity and accuracy, all reaction steps were performed using the Eppendorf 5075 automated liquid handling platform. Genotypes were called using an automated allele scoring platform. For quality control, we exclude any individual with >50% missing genotypes. We analyzed duplicate genotype data for 35 duplicate pairs (11 cases and 24 controls) and use discordant genotypes in these duplicates to estimate genotyping error rates. [...] We assessed sequence around SNP rs1344706 in the UCSC Genome Browser (http://genome.ucsc.edu). Based on evidence of conservation, we sought to identify transcription-factor bindingsites altered by the alternative rs1344706 alleles. A 53 bp sequencesurrounding rs1344706 (chr2:185,486,646-185,486,698) was analyzed using MatInspector v7.7.3.1 and SNPinspector v2.2 (http://www.genomatix.de/). In both cases the vertebrate transcription factor matrices and optimized matrix threshold were used to reduce the incidence of false-positives.Based on the results of these predictions, the SMRI CON samples were genotyped for rs1344706 for analysis of expression differences due to variation at this position. Genotypes are collected exactly as described above using the same instrumentation and reagents.For the allelic test, expression values were binned by rs1344706 allele and the means of the bins were then compared. Each individual’s expression value is used twice: for homozygotes, twice in the same bin, for heterozygotes, once in each. As a result, the variation in the homozygotes may be less than that in heterozygotes, and we therefore compare the two group means using the unpaired t-test with Welch’s correction to account for possible heteroscedastic variances. […]

Pipeline specifications

Software tools Tagger, Haploview, MatInspector, SNPInspector
Databases UCSC Genome Browser
Applications GWAS, Genome data visualization
Organisms Homo sapiens