Identification of truncating mutations in HPCX1 families with NMD technology

In this study, nonsense-mediated mRNA decay (NMD) inhibition was used for the discovery of truncating mutations. Six prostate cancer (PRCA) patients and their healthy brothers were selected from a group of HPCX1-linked families. mRNA was isolated from their lymphoblastic cells after pharmacological treatment. Expression analyses were done using Agilent 44K oligoarrays, and selected genes were screened for mutations by sequencing. In order to identify genes containing inactivating mutations in the Xq27-q28 region, an NMD microarray analysis with Agilent 44K Whole Human Genome oligonucleotide microarrays was performed in the families showing the strongest linkage to HPCX1. Five families were chosen with 6 affected and 6 healthy controls. Lymphoblastoid cell lines were derived from whole blood and treated with emetine to block NMD pathway. Total RNA was extracted from harvested cells (treated and untreated) and labeled with Cy5 and Cy3. Labeled samples were hybridized to the Agilent 44K Whole Human Genome Oligonucleotide Microarrays. After hybridization microarrays were scanned and data was extracted using Feature Extraction software. The candidate genes (n=17) for subsequent sequence analysis were selected according to the microarray analysis.