Segmental duplications in genome-wide significant loci and housekeeping genes; warning for GAPDH and ACTB

Normalizing quantitative polymerase chain reaction (qPCR) data to a housekeeping gene is a critical step in qPCR analyses. Our bioinformatics analysis of 1978 housekeeping genes revealed that 348 of them, including GAPDH and ACTB, are not reliable normalizers for qPCR validation of genomic copy number variants because they overlap highly homologous segmental duplications. For RNA-based qPCR, it is also critical to ensure that the cDNA is not contaminated with genomic DNA if GAPDH or ACTB is used as an endogenous control. Furthermore, we observed that 138 significant single nucleotide polymorphisms (SNPs) reported in 134 published genome-wide association studies (GWAS) (out of 1093 GWAS) are mapped to regions affected by segmental duplications. This observation is important, because these SNPs could potentially tag copy number variations that might explain the GWAS signal. However, it is essential to ensure that the association between disease and such a SNP is not a false positive finding (due to incorrect genotype calls) or the result of an association with another homologous genomic region.