A DGGE system for comprehensive mutation screening ofBRCA1andBRCA2: application in a Dutch cancer clinic setting

Abstract

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.

Robust homology-directed repair within mouse mammary tissue is not specifically affected by Brca2 mutation

“…The Brca2 Δ27 allele encodes a peptide that retains binding to several HDR proteins, but lacks the C terminus of the protein that includes a RAD51 interaction site527. A similar frameshift mutation has been found in multiple Dutch families with breast and ovarian cancer28. Brca2 Δ27/Δ27 mice are viable and fertile, but show an increased incidence of tumours at long latency compared with wild-type littermates, including mammary27.…”

“…The mouse Brca2 Δ27 hypomorphic mutation models a truncation mutation found in multiple families with breast and ovarian cancer28. Mammary tissue during puberty and pregnancy from these mice have a similar two–three-fold HDR defect as in other proliferative tissue types, including small intestine epithelium, suggesting that an HDR defect per se may not confer tissue specificity for BRCA2-associated breast cancers.…”

Lack of large genomic deletions in BRIP1, PALB2, and FANCD2 genes in BRCA1/2 negative familial breast cancer

“…In the current study, we analyzed a total of 716 unrelated Dutch BRCA1/2 negative familial breast cancer patients (inclusion criteria have described elsewhere [30] for large genomic deletions in, BRIP1/FANCJ, PALB2/FANCN, and FANCD2 using Multiplex Ligation-dependent Probe Amplification (MLPA; [25]. We failed to detect deletions in any of these genes.…”

Behavioral and psychosocial effects of rapid genetic counseling and testing in newly diagnosed breast cancer patients: Design of a multicenter randomized clinical trial

“…In 5%-10% of the cases, breast cancer has a hereditary basis. In 15%-30% of patients from high-risk families, breast cancer is caused by a germline mutation in the BRCA1 or BRCA2 gene [1-4]. A similar percentage of breast cancers is expected to have a genetic basis due to mutations in (combinations of) other low penetrance breast cancer susceptibility genes [5].…”