Mutations in either of two recently identified genes, BRCA1 and BRCA2, are thought to be responsible for approximately two-thirds of all cases of autosomaldominantly inherited breast cancer. To examine the nature and frequency of BRCA1 and BRCA2 mutations in Japanese families exhibiting a high incidence of breast cancer, we screened 78 unrelated families in this category for mutations of these two genes. Examining the entire coding sequences as well as exon-intron boundaries of both genes by polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and multiplex-SSCP analysis, we identified possible disease-causing alterations in BRCA1 among affected members of 15 families and in BRCA2 in another 14 families. In 15 of those 29 families, the affected individuals carried missense mutations, although most germline mutations reported worldwide have been deletions or nonsense mutations. Our results, indicating that missense mutations of BRCA1 and BRCA2 tend to predominate over frameshifts or nonsense mutations in Japanese breast cancer families, will contribute significantly to an understanding of mammary tumorigenesis in Japan, and will be of vital importance for future genetic testing. pattern of inheritance of breast cancer were evaluated. For each family, a pedigree was prepared on the basis of a family member known to be affected.
Key wordsOccurrence of cancer within each pedigree was confirmed by obtaining medical records and pathology reports for all available family members whether living or deceased. The criteria for selecting "breast cancer families" for this study were as follows: (a) at least three first-degree family members with breast cancer or (b) two or more first-degree family members with breast cancer, either early-onset, bilateral, or accompanied by a history of primary cancer(s) of other organs. Blood samples were obtained from affected family members through the aforementioned Societies. Genomic DNAs were extracted from fresh blood under standard protocols (Kunkel et al. 1977).
Mutation screening
SSCP-analysis.The entire coding sequences of BRCA1 and BRCA2, and associated exon-intron boundary sequences, were examined by PCR-SSCP analysis. The PCR primers used for PCR-SSCP analysis have been described elsewhere (Katagiri et al. 1996b, Miki et al. 1996.Each genomic DNA (50␣ ng) was amplified in a reaction mixture containing 10 ml of 1 ϫ PCR buffer (25␣ mM TAPS, 50␣ mM KCl, 2␣ mM MgCl 2 , and 1␣ mM β-mercaptoethanol), 20␣ mM dNTPs, 5␣ pmol primers, 2␣ mCi of α[ 32 P]dCTP (3000␣ Ci/mmol, 10␣ mCi/ml), and 0.5 units of Taq polymerase (Boehringer Mannheim, Mannheim, Germany). PCR conditions were 1 cycle at 94°C for 2␣ min, then 30 cycles at 94°C for 30␣ s, 60°C for 30␣ s, and 72°C for 30␣ s with final elongation at 72°C for 5␣ min. Each reaction mixture was diluted with 50␣ ml of 95% formamide dye and 20␣ mM ethylenediaminetetraacetic acid (EDTA), incubated at 85°C for 5␣ min, and electrophoresed in a 6% polyacrylamide gel containing 5% glycerol and 0.5 ϫ 90␣ mM Trisborat...
