Detection of false positive mutations in BRCA gene by next generation sequencing.

Abstract

BRCA1 and BRCA2 genes are implicated in 20-25% of hereditary breast and ovarian cancers. New age sequencing platforms have revolutionized massively parallel sequencing in clinical practice by providing cost effective, rapid, and sensitive sequencing. This study critically evaluates the false positives in multiplex panels and suggests the need for careful analysis. We employed multiplex PCR based BRCA1 and BRCA2 community Panel with ion torrent PGM machine for evaluation of these mutations. Out of all 41samples analyzed for BRCA1 and BRCA2 five were found with 950_951 insA(Asn319fs) at Chr13:32906565 position and one sample with 1032_1033 insA(Asn346fs) at Chr13:32906647, both being frame-shift mutations in BRCA2 gene. 950_951 insA(Asn319fs) mutation is reported as pathogenic allele in NCBI dbSNP. On examination of IGV for all these samples, it was seen that both mutations had 'A' nucleotide insertion at 950, and 1032 position in exon 10 of BRCA2 gene. Sanger Sequencing did not confirm these insertions. Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancer but our results indicate the need for careful sequence analysis to avoid false positive results.