Oncogene (1997) 15, 1773 ± 1779 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 Novel BRCA1 mutations and more frequent intron-20 alteration found among 236 women from Western Poland Krzysztof Sobczak1, Piotr Kozl owski1, Marek Napieral a1, Jakub Czarny1, Marcin WozÂniak1, Mal gorzata Kapus cin ska1,2, Mal gorzataL os ko1, Magdalena Koziczak1, Anna Jasin ska1, Jolanta Powierska1, Ryszard Braczkowski3, Jan Br borowicz4, Dariusz Godlewski2, . e˛ Andrzej Mackiewicz5 and Wl odzimierz Krzyzosiak1 1Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61 ± 704 PoznanÂ; 2Cancer Epidemiology and Prevention Union, Wielkopolska Cancer Center, Garbary 15, 61 ± 866 PoznanÂ; 35-th Department of Internal Medicine, Silesian University School of Medicine, Bytom; 4Chair of Oncology, University School of Medical Sciences, La˛kowa 1/2 61-878 PoznanÂ; 5Department of Cancer Immunology, University School of Medical Sciences at Wielkopolska Cancer Center, Garbary 15, 61-866 PoznanÂ, Poland Three dierent novel BRCA1 mutations, ®ve independent most frequently form a favorable genetic background cases of the same 12 bp insertion-duplication in intron-20 for the disease. Also at increased risk are the carriers of and two novel rare BRCA1 sequence variants were one abnormal allele of the ataxia telangiectasia gene identi®ed among 122 Polish women with positive, in most (Swift et al., 1991), the carriers of rare minisatelite cases moderate family history of breast and/or ovarian allele at the HRAS1 locus (Krontiris et al., 1993) and cancer, 80 controls and 34 unselected breast cancer the members of Li Fraumeni families with germline tissue specimens. All mutations and variants were mutation in the p53 gene (Srivastava et al., 1990). germline. The 4153 delA frameshift mutation, the The BRCA1 gene located on chromosome 17q21 Tyr105Cys missense mutation and two cases of the (Hall et al., 1990) is also linked to hereditary ovarian alteration in intron-20 were found in the group of healthy cancer (Narod et al., 1991). More than 400 BRCA1 women with positive family history. Two other cases of sequence variants have been identi®ed thus far and the intronic insertion were found in unselected controls. deposited in Breast Cancer Information Core Database Their carriers had no family history of breast or ovarian (Friend et al., 1995) which now contains about 150 cancer but other cancers occurred in their families. The dierent cancer predisposing mutations. Most of this 1782 Trp/STOP nonsense mutation and one case of the knowledge comes from studies of breast and breast- insertion in intron-20 were ®rst found in tissue specimens ovarian cancer families. Therefore the known muta- of breast cancer patient and breast/ovarian cancer tions are probably biased towards those with more patient, respectively. Their carriers also had no family severe eects on phenotype. It is expected that less history of breast or ovarian cancer. The distribution of expressive mutations will be identi®ed when studies the insertion in intron-20 in analysed groups and results expand and include women with less profound family of RT ± PCR experiments suggest a less prominent role history and women from the general population. It is for this variant considered earlier a splicing mutation. also very likely that the incidence of BRCA1 mutations This study shows also, that more population-oriented in dierent ethnic groups will vary and that dierent research is needed, involving women with less profound mutations will occur in populations from dierent or even no family history of breast and ovarian cancer, geographical regions. First indications of that come to better understand the role and signi®cance of dierent from the studies of Jewish women of Eastern European BRCA1 variants and mutations. descent (Friedman et al., 1995) and from the analysis of Swedish (Johannsson et al., 1996) and Austrian Keywords: BRCA1; mutations and variants; SSCP- (Wagner et al., 1996) breast cancer families. heteroduplex analysis; Polish women; positive family As most women have no close relative with breast or history ovarian cancer, a positive family history of these diseases is one of the primary risk factors. Thus, for women with one aected ®rst-degree relative, the risk of developing the disease is increased by 1.5-3 times Introduction (Thompson, 1994). In this paper we describe the results of BRCA1 gene analysis in a group of Polish women Approximately one in ten women in western countries selected for increased risk of breast and ovarian cancer will develop breast cancer during their lifetime based on their family history. We have also examined a (Wooster & Stratton, 1995). In most cases the disease substantial number of unselected controls and a set of is sporadic; however, some 5 ± 10% of women with breast cancer tissue samples. breast cancer inherit increased susceptibility to the disease (Claus et al., 1991). Within this fraction of breast cancer the germline mutations in BRCA1 (Miki Results et al., 1994) and BRCA2 (Wooster et al., 1995) genes BRCA1 mutations Correspondence: W Krzyz⋅osiak Received 21 August 1996; revised 17 June 1997; accepted 19 June All 24 BRCA1 exons, together with ¯anking intron 1997 fragments containing splice donor and splice acceptor BRCA1 mutations in Polish women K Sobczak et al 1774 sites, were ampli®ed from individual genomic DNA and controls was ampli®ed with primers located in samples and screened for sequence variations by BRCA1 exons 19 and 22, to see whether any shorter or combined single strand conformation polymorphism longer fragment would appear from carrier's cDNA. and heteroduplex analysis ± SSCP ± HDX. The ob- That would re¯ect either exon skipping, cryptic site served variants were sequenced, each along with the activation or intron retention during splicing of RNA corresponding normal fragment. The sequencing results transcript from the mutant allele. However, there was for most interesting variants are shown in Figure 1. no indication of any additional product besides the Three novel BRCA1 mutations identi®ed in this study normal 244 bp fragment. and the characteristics of women in whom these The nonsense Trp4Stop mutation in exon 22 found mutations were found are shown in Table 1. Five in patient 118 truncates the protein of amino acid 1781. independent cases of the same intron-20 alteration are The protein does not contain terminal 82 amino acids also included in the table. Every type of mutation is including 12 residues of the conserved C-terminal represented: frameshift, nonsense, missense and the domain. The carrier was diagnosed with breast cancer potential splicing mutation. The latter is very interesting at the age of 40 and underwent surgery. The mutation as the 12 bp duplication-insertion in intron-20 is was ®rst detected in the tumor, then con®rmed in the observed in our study at unusually high frequency. proband's blood DNA as well as in four out of ®ve In an attempt to clarify the molecular outcome of family members tested. The SSCP analysis of this intronic insertion we have performed the following proband's tumor DNA shows also LOH at the RT ± PCR experiment. The blood cDNA of the carriers BRCA1 locus (Figure 2). Interestingly, in the same a b 433 A/G – Tyr105Cys 4153 del A – frameshift A C G T A C G T A C G T A C G T normal mutant normal mutant c d 5465 G/A – Trp1782Stop Insertion 12 bp in intron 20 A C G T A C G T A C G T A C G T C C T C A C C T T A T G normal mutant normal mutant Figure 1 Sequencing results for BRCA1 mutations: (a) missense mutation in case 91; (b) frameshift mutation in case 98; (c) nonsense mutation in patient 118 and (d) for 12 bp insertion in intron-20, representative for cases 58, 69, 75, 82 and patient 271 BRCA1 mutations in Polish women K Sobczak et al 1775 Table 1 Characteristics of BRCA1 mutations or variants exon/ codon/ Case intron nucleotide Mutation Age Medical history Family history M: BrCa (47/48) frameshift MA1: BrCa (52/55), 98 exon 11 1345/4153 delA 38 None MA2: BrCa (45/66) STOP 1465 MA3:OvCa (62/66) GM: OvCa (59/60) nonsense 118 exon 22 1782/5465 TGG/TGA 43 BrCa (40/7) F: CoCa (60/62) Tyr/STOP F:LuCa (68/70) missense M: UtCa (42/43) 91 exon 7 105/433 TAT/TGT 36 None MA: BrCa (54/56) Tyr/Cys B: LuCa (47/48) MU: BCa (59/60) F: Mel (57/59) 58 36 None M: UtCa (61/ ± ) MA: LuCa (46/47) 69 45 None M: BrCa (62/65) 12bp 48 bp from M: UtCa (60/7) 75 intron 20 end of duplication/insertion 46 None F: StCa (65/65) exon 20 GTATTCCACTCC M: BrCa (67/75) 82 51 None S: BrCa (44/7), B: OsSa (40/7) FA: Leu (35/35) 271 BrCa D37 none OvCa (39/39) BrCa ± breast cancer, OvCa ± ovarian cancer, UtCa ± uterine cancer, LuCa ± lung cancer, StCa ± stomach cancer, OsSa ± osteosarcoma, Leu ± leukemia, Mel ± melanoma, BCa ± brain cancer (age of diagnosis/age of death) M ± mother, F ± father, S ± sister, B ± brother, GM ± grandmother, MA ± maternal aunt, MU ± maternal uncle tumor sample the p53 gene mutation Pro278A1a was . earlier found (Krzyzosiak et al., unpublished) and con®rmed to be somatic. The frameshift mutation at codon 1345 which triggers the protein synthesis termination at codon 1465 was found in the DNA of a healthy 38-year-old woman, case 98. Her mother and the mother's two sisters had breast cancer diagnosed respectively at the age of 47, 52 and 45. Another mother's sister and a grandmother had ovarian cancer, diagnosed respec- tively at the ages of 62 and 59.