Identification of a Danish breast/ovarian cancer family double heterozygote for BRCA1 and BRCA2 mutations

Abstract

Mutations in the two breast cancer susceptibility genes BRCA1 and BRCA2 are associated with increased risk of breast and ovarian cancer. Patients with mutations in both genes are rarely reported and often involve Ashkenazi founder mutations. Here we report the first identification of a Danish breast and ovarian cancer family heterozygote for mutations in the BRCA1 and BRCA2 genes. The BRCA1 nucleotide 5215G > A/c.5096G > A mutation results in the missense mutation Arg1699Gln, while the BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G is novel. Exon trapping experiments and reverse transcriptase (RT)-PCR analysis revealed that the BRCA2 mutation results in skipping of exon 7, thereby introducing a frameshift and a premature stop codon. We therefore classify the mutation as disease causing. Since the BRCA1 Arg1699Gln mutation is also suggested to be disease-causing, we consider this family double heterozygote for BRCA1 and BRCA2 mutations.

Fig. 1

Family pedigree. Breast and ovarian cancer are indicated as well as the age at diagnosis. Diagonal slash indicates deceased, while the proband is indicated with an arrow. Mutation positive individuals are indicated with +. +/+ indicates individuals with both mutations

Fig. 2

Identification of the BRCA1 nucleotide 5215G > A/c.5096G > A mutation, and the BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G mutation. DNA was purified from the patient and the BRCA1 and BRCA2 genes were amplified using intronic primer pairs flanking each exon. The PCR products were pre-screened by dHPLC (denaturing high performance liquid chromatography) and sequenced. The analysis revealed a a BRCA1 nucleotide 5215G > A/c.5096G > A mutation and a b BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G mutation

Fig. 3

Exon trapping and RT–PCR analysis. a COS-7 cells were transfected with pSPL3-BRCA2-exon 7 wild-type or pSPL3-BRCA2-exon 7 mutant plasmids in duplicates. Total RNA was isolated, RT–PCR analysis was performed and the PCR products were resolved on a 2% agarose gel. The 292 bp product corresponds to inclusion of exon 7 (unaltered splicing), while the 172 bp product corresponds to the exclusion of exon 7. The sizes of the DNA marker (M) are indicated to the right. b RT–PCR was performed on RNA purified from whole blood. The cDNA was amplified with specific BRCA2 primers. The sample was separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Two RT–PCR products (368 and 253 bp) were obtained from the patient (Lane 2). The PCR products were cloned and sequence analysis revealed that the 253 bp band lacked exon 7 (data not shown)