Novel de novo BRCA2 mutation in a patient with a family history of breast cancer

Abstract

Background: BRCA2 germ-line mutations predispose to breast and ovarian cancer. Mutations are widespread and unclassified splice variants are frequently encountered. We describe the parental origin and functional characterization of a novel de novo BRCA2 splice site mutation found in a patient exhibiting a ductal carcinoma at the age of 40.

Methods: Variations were identified by denaturing high performance liquid chromatography (dHPLC) and sequencing of the BRCA1 and BRCA2 genes. The effect of the mutation on splicing was examined by exon trapping in COS-7 cells and by RT-PCR on RNA isolated from whole blood. The paternity was determined by single nucleotide polymorphism (SNP) microarray analysis. Parental origin of the de novo mutation was determined by establishing mutation-SNP haplotypes by variant specific PCR, while de novo and mosaic status was investigated by sequencing of DNA from leucocytes and carcinoma tissue.

Results: A novel BRCA2 variant in the splice donor site of exon 21 (nucleotide 8982+1 G-->A/c.8754+1 G-->A) was identified. Exon trapping showed that the mutation activates a cryptic splice site 46 base pairs 3' of exon 21, resulting in the inclusion of a premature stop codon and synthesis of a truncated BRCA2 protein. The aberrant splicing was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. The mutation was not found in any of the patient's parents or in the mother's carcinoma, showing it is a de novo mutation. Variant specific PCR indicates that the mutation arose in the male germ-line.

Conclusion: We conclude that the novel BRCA2 splice variant is a de novo mutation introduced in the male spermatozoa that can be classified as a disease causing mutation.

Figure 1

Family pedigree. Breast cancers are indicated as well as the age at diagnosis. Acc, accident; BRC, breast cancer. The number following the cancer gives the age at diagnosis. Moreover, the genotypes from variant specific PCR are indicated. Diagonal slash indicates deceased. The proband is indicated with an arrow. Proband = individual 1, Sister = individual 2, Mother = individual 3, Father = individual 4.

Figure 2

Identification of the BRCA2 nucleotide 8982+1 G→A/c.8754+1 G→A variant. DNA was purified from whole blood and BRCA2 exon 21 was amplified using the primers 5'-CTTTGGGTGTTTTATGCTTGT-3' and 5'-CTGGCACATCACTGAAAATC-3' and sequenced. The analysis revealed a nucleotide 8982+1 G→A/c.8754+1 G→A mutation in BRCA2 (sense strand). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the cryptic splice site are underlined.

Figure 3

Exon trapping analysis. (A) Structure of the exon trapping vector pSPL3 containing the BRCA2 exon 21 and 449 bp of intron 20 and 408 bp of intron 21, respectively containing the wild-type or the nucleotide 8982+1 G→A/c.8754+1 G→A variant. (B) COS-7 cells were transfected with pSPL3-BRCA2-exon 21 wild-type or pSPL3-BRCA2-exon 21 mutant plasmids. Total RNA was isolated, RT-PCR analysis was performed and the PCR products (in duplicates) were resolved on a 2% agarose gel. The 299 bp product corresponds to wild-type exon 21 (unaltered splicing), while the 345 bp product corresponds to exon 21 and the inclusion of 46 bp of intron 21. The sizes of the DNA marker are indicated to the right. (C) Sequence of exon 21 (345 bp band). The nucleotide 8982+1 G→A/c.8754+1 G→A mutation and the TAA stop codon are underlined (sense strand).

Figure 4

RT-PCR was performed on RNA purified from whole blood from the proband. 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 (503 bp and 549 bp) were obtained from the patient (Lane 1). The sizes of the DNA marker are indicated to the left. The PCR products were cloned and sequence analysis revealed that the 549 bp band contained the inclusion of 46 bp of intron 21 (data not shown).