Figure 1

Identification and characterization of the RAD51C deletion. Identification of the RAD51C deletion: (A), (B) probands were recruited at the hereditary breast cancer (BC) and ovarian cancer (OC) centers in Cologne and Munich, Germany. Screening for genomic rearrangements was performed by employing a multiplex ligation-dependent probe amplification (MLPA) assay covering all nine coding exons of RAD51C as well as PALB2 and partially RAD50 (P260 MLPA probemix; MRC Holland, Amsterdam, the Netherlands). Deletions of exons 5 to 9 of the RAD51C gene were identified in two families (#1-IV-2; #2-III-6, #2-III-7) using blood-derived genomic DNA (gDNA). In both families, non-BC/OC entities were reported. BCL, B-cell lymphoma; RCC, renal cell carcinoma; CC, colon cancer; NHL, non-Hodgkin lymphoma. (C) MLPA data analysis was carried out using Coffalyser.Net software (MRC Holland). Characterization of the RAD51C deletion: (D) a deletion-specific junction fragment polymerase chain reaction (PCR) was performed using the primers 5′-TCTCTGTGTCCTCATATGATAGG-3′ and 5′-CTAGGATCACACTATTGCACTC-3′. A 681 base pair (bp) fragment was observed using gDNA derived from individuals #1-IV-2, #2-III-6 and #2-III-7, but was absent in individual #1-IV-1, indicating maternal inheritance in family #1. NTC, no template control. (E) Sequencing of the junction fragment in all cases revealed a recurrent 36,637 bp deletion, which is flanked by Alu repeats. The genomic breakpoint within intron 4 is located in a 7 bp region identical between both flanking Alu sequences (indicated). Hence, the deletion probably originates from an Alu repeat-mediated nonhomologous recombination event. (F) Blood-derived RAD51C transcripts from individual #1-IV-2 and a control were analyzed by real-time PCR as described previously [6]. Primer sequences are available on request. Amplicons spanning exons 2/3 and 3/4 were detected at similar levels, while those spanning exons 7/8/9 and 8/9 were less abundant in patient #1-IV-2 compared with the respective control (***P <0.001, t test). Detection levels in the control sample were set to 100%. Results given as mean ± standard deviation.