The application of nonsense-mediated mRNA decay inhibition to the identification of breast cancer susceptibility genes

We applied GINI to a total of 24 LCLs, established from breast-cancer affected and unaffected women from three multiple-case non-BRCA1/2 breast cancer families. We then used Illumina gene expression microarrays to identify transcripts stabilised by the NMD inhibition.The expression profiling identified a total of eight candidate genes from these three families. One gene, PPARGC1A, was a candidate in two separate families. We performed semi-quantitative real-time reverse transcriptase PCR of all candidate genes but only PPARGC1A showed successful validation by being stabilised in individuals with breast cancer but not in many unaffected members of the same family. Sanger sequencing of all coding and splice site regions of PPARGC1A did not reveal any protein truncating mutations. Haplotype analysis using short tandem repeat microsatellite markers did not indicate the presence of a haplotype around PPARGC1A which segregated with disease in the family.The application of the GINI method to LCLs to identify transcripts harbouring germline truncating mutations is challenging due to a number of factors related to cell type, microarray sensitivity and variations in NMD efficiency.