Screening for large rearrangements of theBRCA1gene in German breast or ovarian cancer families using semi-quantitative multiplex PCR method

Abstract

Since the identification of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2, a large number of different germline mutations in both genes have been found by conventional PCR-based mutation detection methods. Complex germline rearrangements such as those reported in the BRCA1 gene are often not detectable by these standard diagnostic techniques. To detect large deletions or duplications encompassing one or more exons of the BRCA1 gene and in order to estimate the frequency of BRCA1 rearrangements in German breast or ovarian cancer families, a semi-quantitative multiplex PCR method was developed and applied to DNA samples of patients from families negatively tested for disease causing mutations in the BRCA1 and BRCA2 coding regions by direct sequencing. Out of 59 families analysed, one family was found to carry a rearrangement in the BRCA1 gene (duplication of exon 13). The results indicate that the semi-quantitative multiplex PCR method is useful for the detection of large rearrangements in the BRCA1 gene and therefore represents an additional valuable tool for mutation analysis of BRCA1 and BRCA2.

MLPA screening in theBRCA1gene from 1,506 German hereditary breast cancer cases: novel deletions, frequent involvement of exon 17, and occurrence in single early-onset cases

“…Including the 194 German families formerly screened for gross aberrations in the BRCA1 gene by other groups [Hofmann et al, 2002[Hofmann et al, , 2003Hartmann et al, 2004;Preisler-Adams et al, 2006], data from 1,700 families are now available to calculate reliable mutation prevalences for high-risk individuals in the German population. In total, 45 intragenic deletions, duplications, or losses of promoter region could be demonstrated in 1,700 families found to be negative for BRCA mutations after screening with PCRbased techniques.…”

“…In contrast, inconsistent results have been obtained so far for the German population. First studies indicated a low frequency [Hofmann et al, 2002[Hofmann et al, , 2003, while two subsequent studies [Hartmann et al, 2004;PreislerAdams et al, 2006] suggested a more prominent role of BRCA1 rearrangements. However, due to the low number of families included in these investigations, no conclusive recommendations for genetic counseling or testing could be drawn.…”

Detection and precise mapping of germline rearrangements inBRCA1, BRCA2, MSH2, andMLH1using zoom-in array comparative genomic hybridization (aCGH)

“…Techniques to detect these complex rearrangements include RNA/cDNA-based assays such as the protein truncation test (PTT) and DNA-based assays, such as, for example: Southern blot, color bar coding, long PCR, quantitative multiplex PCR of short fluorescent fragments (QMPSF), semiquantitative multiplex PCR, DNA microarray, and multiplex ligation-dependent probe amplification (MLPA) [Casilli et al, 2002;Frolov et al, 2002;Gad et al, 2001;Hofmann et al, 2003;Nordling et al, 1998;NystromLahti et al, 1995;Schouten et al, 2002;Swensen et al, 1997]. MLPA has become especially popular due to its ease of use, and the lesser time and sample amounts required.…”

LargeBRCA1 gene deletions are found in 3% of German high-risk breast cancer families

“…We have now used the MLPA method to test for genomic rearrangements in 75 Southern German high-risk breast and ovarian cancer families previously screened negative for point mutations by dHPLC and sequencing (Meyer et al, 2003). We also provide a follow-up to a previously published study on genomic rearrangements detected with a semi-quantitative fluorescent multiplex PCR method (Hofmann et al, 2003) in high-risk breast cancer families in Northern Germany.…”