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

Hum Mutat. 2001 Aug;18(2):149-56. doi: 10.1002/humu.1164.


BRCA1 mutation detection is expensive and has sensitivity limitations, which might at least partially be overcome by RNA-based sequencing. There are claims that RNA tests are unreliable due to differential splicing, exon skipping, or nonsense-mediated mRNA decay that results in either the absence or low expression of mRNA harboring mutations. The major aim of this study was to determine if the application of specific high temperature annealing primers can assure high sensitivity of detection of BRCA1 sequence alterations by cDNA sequencing. The study group comprised 21 Polish cancer families with aggregations of breast and/or ovarian cancer. We detected mutations in 10 out of 21 unrelated patients. These were: nucleotide substitutions (c.309T>C; c.300T>G); nucleotide insertions (c.5382insC) three cases; nucleotide deletions (c.4154delA) 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 alternative 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 these were cDNA heterozygous changes. Two of these splice site changes have not been previously described. Sequencing of genomic DNA "exon by exon" did not result in the detection of any additional abnormalities. The sensitivity of our analyses was sufficient to reliably detect mutations without the necessity of tissue culturing to obtain enough template cDNA for analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing / genetics
  • Base Sequence
  • Breast Neoplasms / genetics*
  • DNA Mutational Analysis / methods*
  • DNA Primers / genetics
  • Exons / genetics
  • Female
  • Genes, BRCA1*
  • Germ-Line Mutation / genetics*
  • Humans
  • Molecular Sequence Data
  • Mutation, Missense / genetics
  • Ovarian Neoplasms / genetics*
  • Poland
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Sequence Analysis, RNA / methods*


  • DNA Primers

Associated data

  • GENBANK/U14680