Significant Contribution of Germline BRCA2 Rearrangements in Male Breast Cancer Families

Abstract

Although screening for large deletions or duplications of the BRCA1 gene is becoming a routine component of the molecular diagnosis of familial breast cancer, little is known about the occurrence of such rearrangements in the BRCA2 gene. Because of the high frequency of BRCA2 mutations in breast cancer families with at least one case of male breast cancer, we selected a cohort of 39 such families, tested negative for mutations in the coding regions of BRCA1 and BRCA2, and developed an assay for BRCA2 rearrangements, based on quantitative multiplex PCR of short fluorescent fragments (QMPSF). We found three rearrangements: (1) a deletion of exons 12 and 13; (2) a duplication of exons 1 and 2; and (3) a complete deletion of BRCA2. We determined the boundaries of the deletion of exons 12 and 13, showing that it resulted from an unequal recombination between Alu sequences. We mapped the complete BRCA2 deletion, which extends over at least 298 kb and showed that it does not affect APRIN/AS3, previously characterized as a tumor suppressor gene, but it comprises several loci corresponding to proven or putative transcripts of unknown functional significance. These data suggest that screening for BRCA2 rearrangements should be done, especially in male breast cancer families tested negative for BRCA1 and BRCA2 mutations.

Partial duplications of the MSH2 and MLH1 genes in hereditary nonpolyposis colorectal cancer

“…QMPSF analysis allowed us in this large series of suspected HNPCC patients to detect MSH2 and MLH1 duplications S Baert-Desurmont et al 10 distinct MSH2 or MLH1 partial genomic duplications, which were all confirmed by another independent method. This shows, as previously illustrated for the BRCA1, 27 BRCA2, 28 and APP genes, 29 that QMPSF is a reliable method for the detection of genomic duplications. Although we cannot provide in this study an accurate estimation of the contribution of MSH2 and MLH1 duplications to HNPCC, as the population analysed was heterogeneous including both AMS þ patients and AMSÀ patients with an MSI phenotype in the tumour, the frequency of MSH2 and MLH1 duplications appears low.…”

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements

“…Thus, the present study indicates that primers previously designed for fluorescent multiplex PCR tests can be adapted for NFMP-HPLC. This could simplify the application of the method to other genes when the corresponding fluorescent tests have already been developed, such as for BRCA1 [Casilli et al, 2002], p53 [Bougeard et al, 2003], BRCA2 [Tournier et al, 2004], RB1 , and CFTR [Audrézet et al, 2004]. We also adjusted the number of cycles to optimize nonfluorescent detection.…”

“…Semiquantitative PCR protocols, which are more practical than FISH and Southern blot for routine applications, include semiquantitative multiplex PCR of short fluorescent fragments (QMPSF) and multiplex ligation-dependent probe amplification (MLPA) assays successfully adapted to the analysis of different genes [Charbonnier et al, 2000;Casilli et al, 2002;Schouten et al, 2002;Sellner and Taylor, 2004]. QMPSF is based on the simultaneous amplification of short genomic sequences that correspond to the different exons by fluorescently labeled primers, and several multiplex PCRs may be necessary to cover one gene [Charbonnier et al, 2000[Charbonnier et al, , 2002Wang et al, 2002;Casilli et al, 2002;Audrézet et al, 2004;Tournier et al, 2004]. MLPA has the advantage of being available as a commercial kit.…”

“…Similar applications of the instrument to other nonfluorescent semiquantitative analyses have been recently developed. For example, DHPLC was employed under denaturing conditions for semiquantitative analysis of allele specific expression [Tournier et al, 2004] and to measure relative dosages of highly homologous genes [Su et al, 2005]. In the present study we developed and validated a simple and versatile method based on nonfluorescent multiplex-PCR coupled to HPLC analysis (NFMP-HPLC) that uses a DHPLC instrument under nondenaturing conditions to assess relative copy number of multiple exons for detection of genomic rearrangements.…”

Comparison of a quantitative PCR method with FISH for the assessment of the four aneuploidies commonly evaluated in CLL patients

“…These probes target the 13q-deleted region (D13S319, D13S25), the ATM gene, the TP53 gene and chromosome 12 centromere. As probes are expensive and as FISH is a time-consuming method, we aimed to evaluate the application of a quantitative PCR method (quantitative multiplex PCR of short fluorescent fragments [6][7][8] or QMPSFs) initially developed for the evaluation of constitutional aneuploidies to the somatic aneuploidies acquired by CLL patients.…”