Detection of germline mutations in the BRCA1 gene by RNA-based sequencing

BRCA1 mutation detection is expensive and has sensitivity limitations, whic h might at least partially be overcome by RNA based sequencing. There are c laims that RNA tests are unreliable due to differential splicing, exon skip ping, or nonsense-mediated mRNA decay that results in either the absence or low expression of mRNA harboring mutations. The major aim of this study wa s to determine if the application of specific high temperature annealing pr imers can assure high sensitivity of detection of BRCA1 sequence alteration s by cDNA sequencing. The study group comprised 21 Polish cancer families w ith aggregations of breast and/or ovarian cancer. We detected mutations in 10 out of 21 unrelated patients. These were: nucleotide substitutions (c.30 9T>C; c.300T>G); nucleotide insertions (c.5382insC) three cases; nucleotide deletions (c.4154deIA) one case, (c. 185delAG) one case, (c.3819delGTAAA) two cases; and the deletion of the entire sequence of exon 22, one case. In addition, we identified three transcript variants resulting from alternati ve splice sites affecting the last six nucleotides of exon 1a (GTAAAG), and the first three nucleotides (CAG) of exon 8 and exon 14, In all cases thes e were cDNA heterozygous changes. Two of these splice site changes have not been previously described. Sequencing of genomic DNA "exon by exon" did no t result in the detection of any additional abnormalities. The sensitivity of our analyses was sufficient to reliably detect mutations without the nec essity of tissue culturing to obtain enough template cDNA for analysis. Hum Mutat 18:149-156,2001. (C) 2001 Wiley-Liss, Inc.