BRCA2 germline mutations among early onset breast cancer patients unselected for family history of the disease.

EDITOR—Germline mutations in the BRCA1 and BRCA2 genes are associated with an increased risk of breast and ovarian cancer. DiVerent patient populations, predominantly with at least some familial aggregation of breast/ ovarian cancer, have been screened to estimate the frequency of mutations and identify women for whom mutation analysis should be considered. The prevalence of BRCA1 and BRCA2 gene mutations has been studied in only a few population based series of patients. We tested for BRCA2 germline mutations in 40 patients diagnosed with breast cancer before the age of 40, unselected for family history of the disease. These patients were drawn from breast cancer patients recruited between 1992 and 1995 in a population based genetic epidemiological case-control study conducted in a region around Heidelberg in Germany. The mean age of onset of the disease in the tested population was 33.8 years (range 25-39 years). Written informed consent was obtained from all patients. The 26 coding exons and flanking intronic regions were analysed from genomic DNA using published 11 or newly designed primers (available on request). Polymerase chain reactions contained 50 ng DNA, 200 mmol/l of each dinucleotide, 1.5-3.0 mmol/l MgCl, 200 nmol/l of each primer, and 1 unit of AmpliTaq (PE Applied Biosystems, Weiterstadt, Germany) in a total volume of 25 μl. Conditions were one minute at 94°C, one minute at 55°C-62°C, and one or two minutes at 72°C for 35 cycles with five minutes at 94°C before and seven minutes at 72°C after cycling. Amplified fragments were excised from agarose gels, eluted in sterile water, and sequenced directly, applying the Thermo SequenaseTM Fluorescent Cycle Sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany) according to the manufacturer’s protocol. Sequencing primers were those used for exon amplification or additional internal primers (44 sequencing reactions/patient), and were Cy5TM labelled, allowing sequence analysis on ALF express devices (Amersham Pharmacia Biotech). Electrophoresis was performed according to standard protocols. Five mutations predicted to lead to a truncated protein and three rare missense variants were identified (table 1). A pathogenic role of the rare missense variants remains to be clarified. The conservation of the isoleucine residue at codon 729 extends to monkey and dog, but not to hamster and mouse homologues. The threonine residue at codon 2515 is replaced in mouse and rat by a similar serine, and is changed in one of our patients to a non-conserved isoleucine, whereas the valine residue at codon 2728 is conserved among the three species and is changed to a similar isoleucine in the patient. Neither of the latter variants has so far been found in control populations but only in patients. 15 To our knowledge, the amino acid replacement at codon 729 and the 4 bp deletion at nucleotide 4875 (fig 1)