An investigation of DNA sequence variants of unknown significance in hereditary breast cancer

• Main Author: Copson, Ellen Roxane
• Published: University of Southampton 2007
• Subjects: 616.994
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485037

Inheritance of a protein truncating mutation of the tumour suppressor gene BRCA1 causes approximately 5% of breast tumours. Over 450 distinct BRCA1 missense mutations have also been found in patients with a family history of breast cancer and the functional significance of most of these is unclear. Increasing evidence suggests that DNA missense mutations can affect RNA stability or sequence by disrupting splicing regulators. I have used a variety of techniques to investigate the effect of genomic BRCA1 missense mutations on transcript expression. BRCA1 monoallelic expression has previously been reported in association with missense mutations. I therefore initially used the technique of Pyrosequencing™ to identify imbalances in BRCA1 cDNA allele expression. Analysis of patients with known BRCA1 truncating mutations, missense mutations and controls identified no examples of monoallelic expression and indicated that a previous report of monoallelic BRCA1 expression was erroneous. I subsequently developed a series of multiplex RT-PCR reactions using overlapping primer pairs to identify alternative BRCA1 transcripts in the same groups of subjects. This technique effectively demonstrated the common BRCA1 isoforms and confirmed that the c4185A>G silent mutation is associated with deletion of exon 12. To provide a systematic analysis of the effect of BRCA1 mutations on splicing regulation I designed minigenes centred on 4 different BRCA1 exons; 5,6,10 and 18. All mutations within these exons reported to the Breast Cancer Information Core database were introduced into the appropriate minigenes, and wildtype and mu'tant miriigel1es were transfected into HEK 293 cells. The resulting transcripts were examined to identify aberrant splicing. Only one of twenty-one missense mutations investigated resulted in alternative transcripts, suggesting that only a small proportion of BRCA1 missense mutations do affect splicing. Additional work was also performed to investigate whether the MDM2 SNP 309 acts as a disease modifying gene in BRCA1 associated hereditary cancer.