HUMAN MUTATION

MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief 31: E 1200-E1240 (2010) Online

© 2010 WILEY-LISS, INC. DOI: 10.1002/humu.21202

Received 22 April 2009; accepted revised manuscript 17 December 2009.

Characterization of BRCA1 and BRCA2 Deleterious Mutations and Variants of Unknown Clinical Significance in Unilateral and Bilateral Breast Cancer: The WECARE Study Åke Borg1*, Robert W. Haile2, Kathleen E. Malone4, Marinela Capanu3, Ahn Diep2, Therese Törngren1,Sharon Teraoka5, Colin B. Begg3, Duncan C. Thomas2, Patrick Concannon5, Lene Mellemkjaer6, Leslie Bernstein7,Lina Tellhed1, Shanyan Xue2, Eric R. Olson8, Xiaolin Liang3, Jessica Dolle4, Anne-Lise Børresen-Dale9,The WECARE Study Collaborative Group10, and Jonine L. Bernstein3

1Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden; 2Department of Preventive Medicine, University of Southern California, Los Angeles, CA, USA; 3Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 4Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 5Department of Biochemistry and Molecular Genetics and Center for Public Health Genomics, University of Virginia, Charlottesville, VA, USA; 6Institute of Cancer Epidemiology, Danish Cancer Society, Copenhagen, Denmark; 7Department of Population Sciences, Division of Cancer Etiology, City of Hope National Medical Center, Duarte CA USA; 8Molecular Genetics Program, Benaroya Research Institute, Seattle, WA, USA; 9Departments of Genetics and Medicine, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Norway; 9Faculty division The Norwegian Radium Hospital, Faculty of Medicine, University of Oslo, Norway; 10 WECARE Study Collaborative Group members are listed before references

*Correspondence to: Åke Borg, Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden. Telephone: +46 46 177502; Fax: +46 46 147327; E-mail: ake.borg@med.lu.se

Contract grant sponsor: National Cancer Institute, awards CA097397, CA131010 and CA098438.

Communicated by David E. Goldgar

ABSTRACT: BRCA1 and BRCA2 screening in women at high-risk of breast cancer results in the identification of both unambiguously defined deleterious mutations and sequence variants of unknown clinical significance (VUS). We examined a population-based sample of young women with contralateral breast cancer (CBC, n=705) or unilateral breast cancer (UBC, n=1398). We identified 470 unique sequence variants, of which 113 were deleterious mutations. The remaining 357 VUS comprised 185 unique missense changes, 60% were observed only once, while 3% occurred with a frequency of >10%. Deleterious mutations occurred three times more often in women with CBC (15.3%) than in women with UBC (5.2%), whereas combined, VUS were observed in similar frequencies in women with CBC and UBC. A protein alignment algorithm defined 16 rare VUS, occurring at highly conserved residues and/or conferring a considerable biochemical difference, the majority located in the BRCA2 DNA-binding domain. We confirm a multiplicity of BRCA1 and BRCA2 VUS that occur at a wide range of allele frequencies. Although some VUS inflict chemical differences at conserved residues, suggesting a deleterious effect, the majority are not associated with an increased risk of CBC. ©2010 Wiley-Liss, Inc.

KEY WORDS: Breast cancer, BRCA1, BRCA2, contralateral, risk, deleterious mutation, unclassified variants

OFFICIAL JOURNAL

www.hgvs.org

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INTRODUCTION

Women with a BRCA1 (MIM# 113705) or BRCA2 (MIM# 600185) germline mutation are at high risk of developing breast and ovarian cancer (Stratton and Rahman, 2008, Begg et al., 2008). Mutation screening is widely available from health care, research, and commercial laboratories. Thousands of different disease-associated mutations have been identified (Breast Cancer Information Core, BIC). Most deleterious mutations introduce premature termination codons through small frameshift deletions or insertions, nonsense or splice junction alterations, or large deletions or duplications. Some splice site mutations and large rearrangements do not change the reading frame, but result in a loss or gain of one to several exons, which is thought to compromise the gene function. Deleterious missense mutations are typically confined within specific residues of functional motifs. However, the risk contribution of numerous other sequence variants remains unclear. These ‘variants of unknown significance’ (VUS) include missense changes and small in-frame deletions and insertions, coding synonymous nucleotide substitutions that do not lead to amino acid shifts, as well as alterations in non-coding intervening sequences (IVS) or in untranslated exonic regions (UTRs).

Classifying VUS remains a great challenge for designing tailored genetic counseling and disease prevention strategies. First, VUS greatly outnumber known deleterious mutations and include both unidentified deleterious mutations as well as neutral variants with no clinical importance. As individual VUS are rare, the usual genetic approach of linkage/segregation or association analysis typically used to classify sequence variants is difficult to apply. As homozygosity or compound heterozygosity for deleterious mutations are embryonically lethal or associated with severe syndromes, VUS that co-occur with a known disease-associated mutation in trans can be classified as not being high-risk alleles. However, this approach relies on the correct determination of allelic phases (Judkins et al., 2005; Tavtigian et al., 2006). A variety of functional assays have been developed for BRCA1 including transcription activation small colony phenotype rescue of radiation resistance and ubiquitin ligase activity; and for BRCA2, assays for homologous recombination, crosslink dependent survival, centrosome amplification or rescue of mouse embryonic stem cell viability (Carvalho et al., 2007; Kuznetsov et al., 2008). Although potentially excellent strategies, they are mostly restricted to variants of certain domains and require laborious optimization. In the absence of a universal functional assay, in silico predictions of missense variants by assessment of phylogenetic conservation and severity in modification of biophysical characteristics of amino acids have also been utilized (Abkevich et al., 2004; Tavtigian et al., 2006). Moreover, all coding sequence alterations, including synonymous variants, can be assessed for potential to disrupt exonic splicing enhancer and silencer elements, or introduce cryptic splice sites, and should be considered along with variants in the exon/intron splice junction consensus sites as potentially affecting transcript processing. Multifactorial likelihood-ratio models have been developed to integrate information from these various sources, including tumor histopathological characteristics, providing likelihood ratios for or against disease causality for a considerable number of VUS (Goldgar et al., 2004; Chenevix-Trench et al., 2006; Easton et al., 2007). Nevertheless, the majority of variants studied remain of unknown clinical importance.

The Women’s Environment, Cancer, and Radiation Epidemiology (WECARE) Study was established to examine the combined roles of postoperative radiation exposure and genetic susceptibility in the etiology of contralateral breast cancer (CBC). A large multi-center, population-based series of asynchronous CBC and a reference group of matched unilateral breast cancer (UBC) was designed (Bernstein et al., 2004). We previously reported results from this series regarding estimates of risk variation in carriers of deleterious BRCA gene mutations (Begg et al., 2008). In the present study, we report results on the frequency and pattern of all types of BRCA1 and BRCA2 sequence variants. We used in silico analysis to predict the functional consequence of missense variants and CBC-UBC comparisons of groupings of variants to explore their association with breast cancer risk.

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MATERIALS AND METHODS

Study population The WECARE Study population included 705 women with CBC and 1398 women with UBC who were

ascertained through five population-based cancer registries. Controls (women with UBC) were individually matched to the cases (women with CBC) on age, date of diagnosis, race, and registry (Bernstein et al., 2004). All were diagnosed with their first primary breast cancer at age 54 years or younger (mean age 46 years) and the mean interval from first to second breast cancer in the CBC group was 5 years (range 1-16 years). UBC controls were also counter-matched on radiation treatment (Bernstein et al., 2004). The proportion of women of Jewish ancestry was similar by CBC-UBC status (7.7%). A first-degree (affected mother, sister or daughter) family history of breast cancer was reported more frequently in women with CBC (32%) than in women with UBC (21%).

BRCA gene analysis and quality controls The complete coding sequences of BRCA1 and BRCA2 were screened for variations by denaturing high-

performance liquid chromatography (DHPLC) and sequence analysis, including a stepwise laboratory quality control (QC) scheme as previously described (Begg et al., 2008). In total, 139 samples were tested in the inter-lab QC step. Of the 9,869 fragments tested, discrepant results were observed for 52 (0.53%) of the fragments. For the intra-lab QC, of the 21,158 fragments tested, from 298 samples, discrepant results were observed for 111 (0.52%) fragments. In terms of the consistency of classifying deleterious mutation carrier status, among the 298 women whose specimens were tested twice within the same screening laboratory, only two (0.67%) had discrepant results, and among the 139 women whose samples were re-screened by the centralized laboratory, three (2.16%) had discrepant results. When QC results were un-blinded, discrepant results were resolved.

Sequence variation nomenclature All sequence variants were named and are referred to in the text according to the nomenclature used by Human

Genome Variation Society (HGVS) (http://www.hgvs.org) recommendation guidelines, using the A of the ATG-translation initiation codon as nucleotide +1 (den Dunnen and Antonarakis, 2000). Mutations are also provided using the BIC (http://research.nhgri.nih.gov/bic) nomenclature, with nucleotide numbering starting at the first transcribed base of BRCA1 (GenBank U14680.1) and BRCA2 (NM_000059.1). In Supp. Tables S1 and S2, all sequence variants are listed according to both BIC and HGVS.

In silico analyses BRCA missense variants were analyzed using web-based algorithms, using information that is entirely external

to the WECARE Study data. Align-GVGD (http://agvgd.iarc.fr/) combines the biophysical characteristics (side-chain composition, polarity and volume) of amino acids and protein multiple sequence alignments (Grantham Variation (GV) and Grantham Deviation (GD) scores) to predict where amino acid substitutions fall in a spectrum from enriched deleterious to enriched neutral, providing GV and GD scores (0 to >200) and graded classifiers (C0 to C65) (Tavtigian et al., 2006). Alignments were done with up to 13 BRCA1 sequences and 12 BRCA2 sequences to the depth of Xenopus laevis (frog), Tetraodon nigroviridis (pufferfish - green spotted) or Strongylocentrotus purpuratus (purple sea urchin).

Definition of deleterious mutations Sequence variants were categorized based on their predicted effect on the mRNA and amino acid level and

defined as deleterious mutations according to the following established (BIC) criteria: (1) all frameshift and nonsense variants with the exception of the neutral stop codon BRCA2 c.9976A>T (BIC: K3326X) (Mazoyer et al., 1996), and other variants located 3’ thereof; (2) all IVS variants occurring in the consensus splice acceptor or donor sequence sites, either within 2 bp of exon-intron junctions or when experimentally demonstrated to result in abnormal mRNA transcript processing; (3) missense variants that have been conclusively demonstrated, on the

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basis of data from linkage analysis of high risk families, functional assays or biochemical evidence, to have a deleterious effect on known functional regions. In this study, all variants not previously identified as being deleterious by these criteria were considered “VUS”.

Statistical Analyses Age-adjusted conditional logistic regression was used to estimate the relative risks (RR) and corresponding

95% confidence intervals (95% CI) of common VUS as well as of rare variants grouped by type (deleterious, missense VUS, synonymous VUS, IVS VUS). Models included an “offset” term to account for the counter-matching. All analyses were conducted using SAS TPHREG (SAS Institute, Cary, NC).

RESULTS

Deleterious mutations identified The mutation screening of 2103 participants (705 CBC and 1398 UBC) resulted in the discovery of 113 unique

deleterious mutations, 57 in BRCA1 and 56 in BRCA2 (Figure 1, Supp. Table S1). These variants, and the corresponding familial aggregations of breast cancer in their relatives, were used in an earlier article to calculate penetrance estimators (Begg et al., 2008). The variants included 73 small frameshift deletions or insertions, 26 nonsense mutations, 7 missense and 7 splice site mutations. Five of the 7 deleterious splice site mutations were located within 2 bp from the exon/intron boundaries. BRCA2 c.9501+3A>T (BIC: IVS25+3A>T) was classified as deleterious because segregation/clinical data are supportive and cDNA analysis confirms aberrant transcript splicing, i.e. exon 25 skipping, resulting in a frameshift and an almost immediate premature stop codon. BRCA1 c.211A>G (BIC: R71G) is located in exon 5, two nucleotides from the 3´-exon/intron junction, and shown to introduce an aberrant cryptic splice site (Vega et al., 2001). All 7 splice site mutations were predicted to give rise to out-of-frame transcripts leading to premature stop codons. The 7 missense mutations defined as deleterious included BRCA1 c.130T>A (BIC: C44S) and c.181T>G (BIC: C61G), both occurring at zinc-ligating residues in the RING domain (Morris et al., 2006), and repeatedly found to segregate with disease in breast-ovarian cancer families in Scandinavia or globally. BRCA1 c.5095C>T (BIC: R1699W), c.5123C>A (BIC: A1708E), c.5213G>A (BIC: G1738E) and c.5324T>G (BIC: M1775R) all affect structurally and functionally critical residues in the BRCT domains and are mutations described previously in breast cancer families (Vallon-Christersson et al., 2001; Olopade et al., 2003; Williams et al., 2003). BRCA2 c.3G>A (BIC: M1I) disrupts the translation initiation codon and was classified as deleterious since a possible re-initiation at Met-124 would result in an amino-terminal truncated protein lacking important regulatory regions (Xia et al., 2006). Finally, three sequence variants – BRCA2c.9976A>T (BIC: K3326X), c.10095delCins11 (BIC: 10323delCins11) and c.10150C>T (BIC: R3384X) – predicted to result in protein truncation were ruled as exceptions that could not be classified because of their location near the 3´-end and possibly dispensable part of the gene. Thus, the most carboxy-terminal deleterious mutation in BRCA2 was c.9541_9554del14 (BIC: 9769del14) and c.5558dupA (BIC: 5677insA) in BRCA1, the latter predicted to result in a protein truncated by only the last 11 amino acids. Our approach to classifying mutations as deleterious follows the approach currently used in oncogenetic clinics and is compatible with classifications in the BIC. Our screening method did not target larger genomic deletions or duplications. Thus, it is possible that some deleterious mutations may have been missed.

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Figure 1. Spectrum of deleterious BRCA1 (n=57) and BRCA2 (n=56) mutations detected in 705 women with CBC (red) and 1398 women with UBC (yellow), displayed by gene 5’ to 3’ location and number of observations. Mutation nomenclature according to BIC.

Frequency of deleterious mutations in women with CBC and UBC A total of 181 (8.6%) of 2103 participants were found to carry one of the 113 unique deleterious BRCA

mutations (Figure 1, Supp. Table S1). No woman carried more than one deleterious mutation. Mutations were more common in BRCA1 (n=109) than in BRCA2 (n=72). Multiple women carried known founder mutations such as BRCA1 c.68_69delAG (BIC: 185delAG) (n=16), c.181T>G (BIC: C61G) (n=10), c.5266dupC (BIC: 5382insC) (n=10), and BRCA2 c.5946delT (BIC: 6174delT) (n=5) and c.2808_2811delACAA (BIC: 3036del4) (n=5). Eighty-nine of the 113 mutations were detected only once (Figure 1). Deleterious mutations were nearly three times more common in women with CBC (15.3%) than in women with UBC (5.2%). Carrying a mutation in either BRCA1 or BRCA2 was associated with a 4-fold increased risk of CBC (95% CI 2.8-5.7) as compared to not having a deleterious mutation (Table 1). BRCA1 mutation carriers had a 4.5-fold increased risk (95% CI 2.8-7.1) and carriers of a BRCA2 mutation had a 3.4-fold increased risk (95% CI 2.0-5.8) of CBC. The increased risk of CBC was evident irrespective of mutation type, i.e. frameshift, nonsense, splice site or missense mutation (Table 1) (Malone et al., submitted 2009).

Effect of position of deleterious mutations

A lower frequency of CBC patients with mutations located in the 3´ part of BRCA1 as compared with the 5´ and middle-region of BRCA1 was noted (Figure 1). The RR of CBC in the 13 women carrying the most 3´ located truncating mutations (here including c.5266dupC (BIC: 5382insC), c.5444G>A (BIC: W1815X), c.5503C>T (BIC: R1835X) and c.5558dupA (BIC: 5677insA)) was 0.7, while the RR was 5.3 in the 80 women carrying any other truncating BRCA1 mutation (Table 1). No such effect for corresponding 3´ located truncating BRCA2 mutations remained after excluding c.9976A>T (BIC: K3326X), c.10095delCins11 (BIC: 10323delCins11) and c.10150C>T (BIC: R3384X). There was no significant difference regarding the risk of CBC in women with

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premature stop codons occurring within or outside (either N- or C-terminal) the ‘ovarian cancer-cluster region’ (OCCR, nucleotides 3059-6629; GenBank NM_000059.1) of BRCA2 (Table 1) (Gayther et al., 1997).

Table 1. Risk of CBC compared to UBC, in relation to known deleterious mutations in BRCA1 and BRCA2. CBCa UBCa Carrier Status N % N % Adjb % RRc 95% CI No known deleterious mutation in BRCA1/2 d 597 84.7 1325 94.8 95.1 1.0 Ref. Deleterious mutation in BRCA1 or BRCA2 108 15.3 73 5.2 4.9 4.0 2.8-5.7 Deleterious mutation in BRCA1 67 9.5 42 3.0 2.6 4.5 2.8-7.1 Deleterious mutation in BRCA2 41 5.8 31 2.2 2.4 3.4 2.0-5.8 Frameshift, nonsense, splice mutation in BRCA1 56 7.9 37 2.6 2.4 4.2 2.5-7.0 Deleterious missense mutation in BRCA1 11 1.6 5 0.4 0.2 6.3 2.0-19.9 Frameshift, nonsense, splice mutation in BRCA2 41 5.8 30 2.1 2.3 3.6 2.1-6.1 Deleterious missense mutation in BRCA2 0 0 1 0.1 0.1 - - BRCA1, 3´end mutations e 3 0.4 10 0.7 0.5 0.7 0.2-3.2 BRCA1, all other truncating mutations 53 7.5 27 1.9 1.8 5.3 3.0-9.3 BRCA2, OCCR mutations f 11 1.6 11 1.1 1.1 3.2 1.3-8.0 BRCA2, all other deleterious truncating mutation 30 4.3 19 1.1 1.2 3.8 1.9-7.3

a Women with UBC (N=1398) served as reference group for women with CBC (N=705). b UBC proportions computed as if UBCs were randomly selected. c Relative risk was adjusted for counter-matching and age at first primary; 95%CI: 95% confidence interval. d Wildtype and all variants of unknown significance that have not earlier been classified as deleterious (see Supp. Table S2). e 5382insC, W1815X, R1835X and 5677insA of BRCA1 (GenBank U14680.1), nomenclature according to BIC. f OCCR (Ovarian Cancer Cluster Region) premature stop codon occurring within nt. 3059-6629 of BRCA2 (GenBank NM_000059.1). Mutation nomenclature according to HGVS is found in Supp. Table S1.

Classification of VUS Screening of 2103 women resulted in the identification of an additional 357 unique sequence variants that were

not classified as deleterious (complete list in Supp. Table S2). These 357 variants, including both VUS and suspected (BIC) neutral variants, were broadly categorized according to type into: non-synonymous (n=185), synonymous (n=69), and IVS alterations (n=91). The results from these 345 variants are summarized in Tables 2a and 2b. In addition we found 4 rare in-frame deletions, 5 variants in UTRs, 1 neutral frameshift (BRCA2 c.10095delCins11 (BIC: 10323delCins11)) and 2 neutral nonsense (BRCA2 c.9976A>T (BIC: K3326X) and c.10150C>T (BIC: R3384X)) variants. The majority (n=210) of the 357 variants were observed only once while 81 other variants had a minor allele frequency (MAF) less than 0.1% (observed 4 times or less among the 4206 alleles). In contrast there were 16 common variants with MAF>10%. A substantial number (n=133) of variants found once or a few times have not been reported earlier (in BIC) (highlighted in blue in Supp. Table S2). This observation should, however, be interpreted cautiously since VUS are probably less frequently reported and are likely under-represented in public databases. Interestingly, the numbers of coding variants found in the BRCA genes are high considering that the average gene contains only a handful (~4) of coding SNPs with an allele frequency of at least a few percent in the population. Typically, non-synonymous SNPs not only occur less often but also have lower MAF, often less than 5% (Cargill et al., 1999).

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Table 2a. MAF of BRCA1 sequence VUSa, identified in women with CBC or UBC.

Typeb MAF CBC UBC Relative Risk (95% CI) c,d

Missense 53 unique variants <0.1% 21 46 1.0 (0.6-1.7) 4 unique variants 0.1-0.5% 9 35 0.6 (0.3-1.2) 2 unique variants 0.5-2.5% 42 81 1.1 (0.8-1.7) Q356R 6.0% 65 165 0.8 (0.6-1.2) D693N 7.1% 87 180 1.1 (0.8-1.4) E1038G 25% 271 646 0.9 (0.7-1.0) K1183R 26% 272 642 0.9 (0.7-1.1) P871L 26% 278 668 0.8 (0.7-1.0) S1613G 29% 291 693 0.8 (0.7-1.0)

Synonymous 13 unique variants <0.1% 4 15 0.6 (0.2-1.7) 2 unique variants 0.1-0.5% 2 9 0.5 (0.1-2.2) no variant 0.5-2.5% - - - L771L 25% 268 640 0.8 (0.7-1.0) S1436S 26% 269 653 0.8 (0.7-1.0) S694S 28% 278 676 0.8 (0.7-1.0) IVS 32 unique variants <0.1% 11 21 1.4 (0.6-3.0) 4 unique variants 0.1-0.5% 10 19 1.2 (0.6-2.7) no variant 0.5-2.5% - - - IVS17-53C>T 2.7% 24 77 0.7 (0.4-1.1) IVS7-34C>T 19% 222 495 1.0 (0.8-1.2) IVS8-58delT 28% 285 672 0.9 (0.7-1.1) IVS7+36del14 33% 314 745 0.8 (0.7-1.0)

a Individuals with rare variants are aggregated within MAF categories. Common variants (MAF>2.5%) are listed separately. Frequencies correspond to combined frequencies of heterozygotes or homozygote variants. b In addition to the variant types listed, 2 women with CBC and 7 women with UBC had rare variants that were either in-frame deletions or variants in the untranslated regions. BRCA1 (GenBank U14680.1) mutation nomenclature according to BIC. IVS=intervening sequence (noncoding). Mutation nomenclature according to HGVS is found in Supp. Table S1. c 95% confidence interval. d The reference group for each RR consists of women carrying all other types of VUSs and excludes carriers of known deleterious BRCA mutations.

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Table 2b. MAF of BRCA2 sequence VUSa, identified in women with CBC or UBC. Typeb MAF CBC UBC Relative Risk (95%CI) c,d

Missense 102 unique variants <0.1% 46 72 1.4 (0.9-2.0) 10 unique variants 0.1-0.5% 24 57 0.9 (0.5-1.5) 4 unique variants 0.5-2.5% 22 64 0.7 (0.4-1.2) T1915M 2.6% 26 74 0.8 (0.5-1.2) N289H 3.5% 44 92 1.1 (0.7-1.6) N991D 3.5% 43 92 1.0 (0.7-1.5) N372H 28% 310 627 1.2 (1.0-1.4) Synonymous 40 unique variants <0.1% 18 34 1.2 (0.7-2.2) 6 unique variants 0.1-0.5% 19 37 1.1 (0.6-2.0) no variant 0.5-2.5% - - - H743H 3.4% 42 90 1.0 (0.7-1.5) S455S 3.5% 44 89 1.1 (0.7-1.6) V1269V 16% 177 419 0.9 (0.7-1.1) S2414S 19% 207 477 1.0 (0.8-1.2) K1132K 23% 267 581 1.1 (0.9-1.3) IVS 41 unique variants <0.1% 20 33 1.4 (0.8-2.5) 8 unique variants 0.1-0.5% 20 48 0.9 (0.5-1.6) 1 unique variants 0.5-2.5% 8 23 0.7 (0.3-1.6) IVS16-14T>C 28% 325 689 1.1 (0.9-1.3)

a Individuals with rare variants are aggregated within MAF categories. Common variants (MAF>2.5%) are listed separately. Frequencies correspond to combined frequencies of heterozygotes or homozygote variants. b In addition to the variants listed, 3 women with CBC and 3 women with UBC had rare variants that were either in-frame deletions, frameshifts, nonsense mutations or variants in the untranslated regions (UTR). In addition there is a 5’ UTR polymorphism (203G>A) that was observed in 269 CBC and 528 UBC (OR=1.0). BRCA2 (GenBank NM_000059.1) mutation nomenclature according to BIC. IVS=intervening sequence (noncoding). Mutation nomenclature according to HGVS is found in Supp. Table S1. c 95% confidence interval. d The reference group for each RR consists of women carrying all other types of VUSs and excludes carriers of known deleterious BRCA mutations.

Frequency of VUS in women with CBC and UBC Combined, rare VUS occurred in similar proportions in women with CBC and UBC (Tables 2a and 2b).

Broadly, VUS, regardless of their MAFs’, occurred in similar frequencies in women with CBC and UBC for both BRCA1 variants (Table 2a) and BRCA2 variants (Table 2b). In these tables, rare variants have been grouped according to both type and MAF, while more common variants (MAF>2.5%) are listed individually. None of the sub-groups demonstrate a statistically significant effect on risk of CBC. Since these comparisons aggregate many individual variants we cannot on this basis rule out the possibility that there are a few rare variants that are actually associated with risk.

BRCA1 has four very common missense variants (c.2612C>T (BIC: P871L), c.3113A>G (BIC: E1038G), c.3548A>G (BIC: K1183R), c.4837A>G (BIC: S1613G)) with MAF>20%, and another two (c.1067A>G (BIC: Q356R and c.2077G>A (BIC: D693N)) with MAFs of 6% and 7% respectively. In BRCA2, one missense variant

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(c.1114A>C (BIC: N372H)) has a MAF>20% and three others (c.865A>C (BIC: N289H), c.2971A>G (BIC: N991D) and c.5744C>T (BIC: T1915M)) have MAFs of 2-4%. It is expected that these 10 are neutral variants of negligible functional and clinical significance. The observed relative frequencies of these variants in CBC versus UBC are consistent with the hypothesis that these variants do not affect the risk of breast cancer. However, homozygous status for BRCA2 His372 has previously been associated with a slight increased risk of breast cancer (Healey et al., 2000). We saw no statistically significant increased risk of CBC in homozygous or heterozygous His-372 carriers as compared to WT (RR for heterozygous 1.19 95%CI: 0.97-1.47, and RR for homozygous 1.12 95%CI: 0.77-1.63).

Functional assessment of non-synonymous VUS We used Align-GVGD to further assess the functional effect of missense variants, with alignment to 13 BRCA1

and 12 BRCA2 ortholog sequences down to sea urchin. Six VUS (BRCA1 c.2596C>T (BIC: R866C), BRCA2 c.4585G>A (BIC: G1529R), c.7878G>C (BIC: W2626C), c.7988A>T (BIC: E2663V), c.7994A>G (BIC: D2665G) and c.9154C>T (BIC: R3052W)) occurred at strongly conserved residues (GV=0) and had a GD�65. Thus, these were inferred to belong to the class (C65) of substitutions most likely to interfere with function and were in this respect comparable to five of the missense mutations a priori classified as deleterious (the remaining, BRCA1 c.5324T>G (BIC: M1775R) had a GV=14.30 and was inferred to class C45, while BRCA2 c.3G>A (BIC: M1I) had a GV=0 but GD as low as 10.12). Two other BRCA2 variants (c.9104A>G (BIC: Y3035C) and c.10045A>G (BIC: T3349A)) were defined as interfering with function (A-GVGD class C55), an additional 8 VUS had either a low GV or high GD score which lifted them above class C0 and the remaining 169 amino acid substitutions were less likely to compromise function. Details of 16 variants are presented in Table 3 with corresponding details of all VUS missense variants in Supp. Table S3. Lowering the sequence alignment stringency to the level of pufferfish and frog, respectively, led to another 7 and 16 VUS predicted to be functionally defective (C15-C55, Supp. Table S3).

All 8VUS with the highest likelihood of being deleterious (A-GVGD class C55-C65 in alignment down to sea urchin) were rare (found once or twice), while another 8VUS reaching class C15-C35 in alignment to sea urchin were observed a total of 18 times. However, the aggregated ratio of CBC (11) to UBC (18) only slightly exceeds the reference ratio of one CBC to every two UBCs in the study. The 15 VUS assigned to class C15-C65 in alignments to pufferfish or frog, but to C0 in alignment to sea urchin, were observed 78 times. The latter group included BRCA1 c.134A>C (BIC: K45T) in the RING domain (see below), as well as BRCA2 c.8851G>A (BIC: A2951T) (class C55), which was observed 22 times and had an elevated frequency in women with CBC (10 CBC versus 12 UBC). Interestingly, this variant has earlier been suggested as a moderate-risk predisposing allele (Hammet et al., 2008).

Functional domains affected by non-synonymous VUS None of the missense or in frame deletion variants identified in the present study affected the numerous putative

phosphorylation sites in BRCA1 or BRCA2, nor were they found in the nuclear localization signals of BRCA1 (amino acids 503-508 and 606-615) nor of BRCA2 (amino acids 3266-3269 and 3311-3315). Besides the two deleterious mutations at Cys44 and Cys61, a third variant (c.134A>C (BIC: K45T), observed once) affected the BRCA1 RING domain (amino acids 1-100). Although not as strongly conserved as the RING domain cysteine residues, Lys45 is part of a beta-sheet and K45T disrupts a salt bridge to Glu75, likely to weaken the folding and interaction with the E2 ubiquitin-conjugating enzyme UbcH5a (Morris et al., 2006). Several known deleterious missense mutations were found in the BRCA1 carboxy terminal region including the two BRCT domains (amino acids 1625-1825), but only one (c.5401G>T (BIC: G1801C)) of five other missense VUS in this region had an A-GVGD score suggesting a possible functional interference. The majority of BRCA2 VUS with an A-GVGD score suggesting a deleterious effect were located in the DNA binding domain (amino acids 2500-3098). Moreover, c.4585G>A (BIC: G1529R) affects a highly conserved residue in the Rad51 binding BRC4 motif of BRCA2 (Bignell et al., 1997). BRCA1 c.2596C>T (BIC: R866C) is located in a highly conserved region ‘motif 6’, still of unknown function, while BRCA1 c.4172T>C (BIC: I1391T) affects the coiled coil region (Abkevich et al., 2004).

E 1210 Borg et al.

Table 3. (a) BRCA1 and BRCA2 VUS identified in CBC and UBC as potentially deleterious by A-GVGD protein alignment algorithm (to the depth of Sea Urchin), yielding a GV=0 and/or a GD�65. (b) A-GVGD scores, CBC and UBC numbers of BRCA1 and BRCA2 missense variants earlier classified as being deleterious mutations. (a) A-GVGD Gene Exon NTa Base Ch BICb Class GV GD CBC UBC BRCA1 7 433 A>G Y105C c,d C15 120 131 0 1 BRCA1 8 655 A>G Y179C C35 40 182 0 2 BRCA1 11 2640 C>T R841W C15 111 78 2 6 BRCA1 11 2715 C>T R866C c,d C65 0 180 0 1 BRCA1 12 4291 T>C I1391T C25 30 70 1 0 BRCA1 22 5520 G>T G1801C C15 157 107 0 1 BRCA2 11 4813 G>A G1529R c,d C65 0 125 2 0 BRCA2 17 8106 G>C W2626C C65 0 214 1 0 BRCA2 17 8107 A>T I2627F C15 0 21 0 1 BRCA2 18 8216 A>T E2663V C65 0 121 0 1 BRCA2 18 8222 A>G D2665G c,d C65 0 94 1 1 BRCA2 18 8267 A>G D2680G C15 44 75 0 1 BRCA2 22 9078 G>T K2950N d C35 26 79 3 0 BRCA2 23 9332 A>G Y3035C C55 21 192 1 1 BRCA2 24 9382 C>T R3052W C65 0 101 0 1 BRCA2 27 10273 A>G T3349A c,d C55 0 58 0 1 Total 11 18 (b) BRCA1 3 249 T>A C44S e C65 0 112 2 0 BRCA1 5 300 T>G C61G e C65 0 158 8 2 BRCA1 18 5214 C>T R1699W e C65 0 101 0 1 BRCA1 18 5242 C>A A1708E e C65 0 107 1 0 BRCA1 20 5332 G>A G1738E e C65 0 98 0 1 BRCA1 21 5443 T>G M1775R e C45 14 91 0 1 BRCA2 2 231 G>A M1I e C0 0 10 0 1 Total 11 6 a NT: nucleotide. BRCA1 (GenBank U14680.1), BRCA2 (GenBank NM_000059.1), according to BIC. b BIC: nomenclature according to Breast Cancer Information Core (see Supp. Table S2 for HGV nomenclature). c Classified as of no clinical interest in BIC. d Classified as neutral in the likelihood-ratio model developed by Easton et al. (2007). e BRCA1 and BRCA2 missense variants a priori classified as deleterious mutations in the present study.

DISCUSSION

Predisposition to breast cancer can be attributed to several levels of genetic susceptibility: rare high-risk alleles, rare moderate-risk alleles and common low-risk alleles (Stratton and Rahman, 2008). Deleterious mutations in BRCA1 and BRCA2 account for a considerable proportion of dominantly inherited breast cancer and have received wide acceptance in diagnostic testing and prevention. BRCA1 and BRCA2 mutation screening results in the identification of sequence variants that cannot be unequivocally referred to as deleterious or neutral. This is emphasized by the results from the present study of a high-risk population of young (<55 years) women with breast cancer. Using a carefully optimized DHPLC screening approach to analyze all coding exons and flanking intronic regions of BRCA1 and BRCA2, we identified 470 unique sequence variants in 2103 women. The majority (n=299) of variants were observed only once, however, there were some that were very common (MAF>10%).

BRCA1 and BRCA2 Sequence Variants in Unilateral and Bilateral Breast Cancer E 1211

Based on documented knowledge on effects of variants that give rise to premature stop codons (via frameshift insertions or deletions, nonsense or consensus splice site sequence changes) or missense alterations at critical residues in functional domains, we defined 113 unique BRCA1 or BRCA2 variants as deleterious mutations. These were observed in 181 (8.6%) of the 2103 women. We confirmed earlier observations (BIC; Thomassen et al., 2008), of global or local founder effects for certain recurrent mutations and also describe a set of unique mutations as previously reported (Begg et al., 2008; Malone et al., submitted 2009). Deleterious mutations were three times as common in women with CBC (15.3%) than in women with UBC (5.2%), and carrying a deleterious mutation in either BRCA1 or BRCA2 conferred an approximately 4-fold increased risk of CBC among survivors of a first breast cancer (Begg et al., 2008; Malone et al., submitted 2009).

Previous studies have suggested a genotype-phenotype correlation within BRCA1 in which women with truncating mutations 3´ of the exon 12/13 boundary show a significantly decreased ovarian to breast cancer ratio (Gayther et al., 1995; Holt et al., 1996; Thompson et al., 2002). Our study design precluded assessment of the breast-ovarian cancer mutation location question but allowed us to assess whether the occurrence of CBC was related to location. We observed a lower frequency of CBC patients with mutations located in the 3´ part of BRCA1 as compared with the 5´ and middle-region of BRCA1. This was an unexpected finding considering that most transcripts with premature termination codons are targeted for degradation by the nonsense-mediated mRNA decay (NMD) surveillance pathway, resulting in no or low expression of nonsense transcripts and truncated protein products (Perrin-Vidoz et al., 2002; Conti and Izaurralde, 2005). However, transcripts bearing termination codons located <50 nucleotides from the last exon-exon junction (EEJ) or lacking a downstream EEJ (i.e., premature stop codons occurring in the last exon) may escape NMD. Thus, transcripts with premature stop codons occurring at BRCA1 codon 1807 or later may be translated and partly functional. Indeed, decreased but detectable expression of mutant protein has been found in HCC1937 cells carrying the BRCA1 5382insC mutation (Scully et al., 1999). However, despite the noted differences in the frequencies of women with CBC and UBC in the 5´ and 3´ parts of BRCA1, the numbers are small and results must be carefully interpreted. Truncating mutations in a central region of BRCA2 have been associated with an increased risk of developing ovarian cancer relative to breast cancer as compared to mutations occurring outside this OCCR bounds (Gayther et al., 1997; Thompson et al., 2001). It is less likely that escape from the NMD pathway explains this BRCA2 genotype-phenotype correlation (Ware et al., 2006), and we detected only a small, non-significant difference in the incidence of CBC in women with truncating mutations in a region overlapping with the OCCR. However, it cannot be excluded that expression of truncated BRCA proteins in variable degree interferes with normal BRCA function in tumorigenesis or retains some functions that influence tumor biology and DNA damage control in response to therapy in the absence of wildtype BRCA protein.

An even more complicated circumstance concerns the significance of expression of full-length BRCA proteins with missense mutations or small in-frame amino acid deletions/insertions. We identified seven missense variants that have been previously unambiguously defined as high-risk alleles due to known interference with function or translation initiation, or based on segregation analysis in affected families. These deleterious mutations were located at residues in the BRCA1 RING domain, important for ubiquitin-ligase activity, or in the BRCA1 BRCT-domains that interact with numerous proteins involved in transcription and DNA repair. However, we identified an additional 185 unique missense variants (and four in-frame single amino acid deletions) that could not straightforwardly be classified as risk-alleles. Again, the majority of these types of variants were rare and occurred in single individuals, while the minority were common. While it might be argued that the common variants are unlikely to be functional and should be referred to as neutral variants or polymorphisms, there is some suggestion that common BRCA missense variants may have a role as low-risk alleles (Healey et al., 2000; Hammet et al., 2008). We saw no evidence for increased risk associated with any of the common missense variants.

Theoretically, to further examine the role of each missense variant, one could take into account the multiplicity of functions and regions in both BRCA1 and BRCA2 that are involved in protein or DNA binding or constitute sites for enzymatic modification; however, the limited number of individual variants and lack of experimental validation would make such comparisons speculative and inconclusive. Therefore, we used publicly available protein alignment algorithms to more objectively evaluate the possible consequence of each amino acid substitution. Using Align-GVGD, we identified a set of 16 missense VUS (Table 3) that occurred at highly conserved (Grantham Variation=0) residues or conferred a considerable biochemical difference (Grantham Deviation�65). Six of these occurred in BRCA1 and regions of unclear function such as the highly conserved motif 6 (Arg-866) or the coiled coil region (Ile-1391) (Abkevich et al., 2004), while ten were found in BRCA2.

E 1212 Borg et al.

Interestingly, the majority (8/10) of the latter were located in the BRCA2 DNA binding domain (amino acids 2500-3098), an observation also made by others (Wu et al., 2005; Easton et al., 2007).

It is notable that the relative frequencies of CBC and UBC among the 16 VUS suggested by A-GVGD as candidate risk variants show no collective evidence of increased breast cancer risk (11/29 (38%) CBC, as compared with 705/1398 (34%) in the entire study). Furthermore, four (BRCA1 c.314A>G (BIC: Y105C), c.2596C>T (BIC: R866C), BRCA2 c.4585G>A (BIC: G1529R) and c.7994A>G (BIC: D2665G)) of the 16 VUS suggested by A-GVGD as candidate risk variants have been reported in BIC as of no clinical interest primarily based on co-occurrence (also in trans) with deleterious mutations and lack of segregation with disease in families, and six (BRCA1 c.314A>G (BIC: Y105C), c.2596C>T (BIC: R866C), BRCA2 c.4585G>A (BIC: G1529R), c.7994A>G (BIC: D2665G), c.8850G>T (BIC: K2950N) and c.10045A>G (BIC: T3349A)) were also classified as neutral in the likelihood-ratio model developed by Easton et al. (2007). However, two VUS scored as possibly deleterious were in agreement with Easton et al. (2007) (BRCA2 c.7878G>C (BIC: W2626C) and c.7988A>T (BIC: E2663V)) and two (BRCA2 c.7988A>T (BIC: E2663V) and c.9154C>T (BIC: R3052W)) were confirmed as deleterious in a recent study using a mouse embryonic stem cell assay (Kuznetsov et al., 2008). Thus, there is little evidence from this admittedly small list of candidates that tools based on evolutionary conservation are effective at identifying risk variants reliably.

In general, approximately two-thirds of randomly occurring point mutations in coding sequence would alter an amino acid, and the fact that non-synonymous coding SNPs comprise less than one half of coding SNPs in the genome implies a strong selection against amino acid altering changes (Cargill et al., 1999). A major interest in human genetics is to distinguish mutations that are functionally neutral from those that contribute to disease. Amino acid substitutions currently account for approximately half of the known gene lesions responsible for human inherited disease. It has been suggested that most rare missense variants are probably at least mildly deleterious and the accumulation of such low-risk variants may be the basis for complex diseases such as breast cancer (Kryukov et al., 2007; Stratton and Rahman, 2008). In the WECARE Study, we could only define a small fraction of all identified BRCA1 and BRCA2 missense variants as likely to have had a deleterious effect on breast cancer risk. The absence of an aggregate association between the remaining missense VUS and risk may indicate that there are very few additional clearly deleterious missense variants. However, this does not exclude the possibility that a few additional BRCA missense variants are functionally perturbed and associated with an increased breast cancer risk. For example, in this study we made no attempt to investigate the potential effect of sequence variants with respect to altering, adding or disrupting motifs of exonic splicing enhancers or silencers, or splice donor, acceptor or branch point sites. This analysis would include also synonymous and noncoding variants and add additional levels of complexity in interpreting the significance of BRCA sequence variants found in women with breast cancer.

In conclusion, this WECARE Study comprises a comprehensive effort in genetic screening of a large and uniform sample set, and resulted in a careful characterization of the landscape of BRCA1 and BRCA2 sequence variants in young women with breast cancer. One striking observation was the high frequency of rare missense variants that could not be unequivocally classified. We identified a number of new potentially deleterious missense mutations for further analysis and emphasize the large BRCA2 DNA binding domain as a possible target for additional candidates. Our overall conclusion, however, is that the majority of VUSs found in BRCA screening of affected women represent neutral alleles of no or little significance in the etiology of breast cancer.

WECARE STUDY COLLABORATIVE GROUP

Memorial Sloan Kettering Cancer Center (New York, NY): Jonine L. Bernstein Ph.D. (Study P.I.), Colin B. Begg. Ph.D., Marinela Capanu Ph.D., Xiaolin Liang M.D., Anne S. Reiner M.P.H., Tracy M. Layne M.P.H.; City of Hope (Duarte, CA): Leslie Bernstein Ph.D., Laura Donnelly-Allen; Danish Cancer Society (Copenhagen, Denmark): Jørgen H. Olsen M.D., D.M.Sc., Michael Andersson M.D., D.M.Sc., Lisbeth Bertelsen M.D.Ph.D., Per Guldberg Ph.D., Lene Mellemkjær Ph.D.;

Fred Hutchinson Cancer Research Center (Seattle, WA): Kathleen E. Malone Ph.D., Noemi Epstein; International Epidemiology Institute (Rockville, MD) and Vanderbilt University (Nashville, TN): John D. Boice

BRCA1 and BRCA2 Sequence Variants in Unilateral and Bilateral Breast Cancer E 1213

Jr. Sc.D.; Lund University (Lund, Sweden): Åke Borg Ph.D., Therese Törngren M.Sc., Lina Tellhed, B.Sc.; National Cancer Institute (Bethesda, MD): Daniela Seminara Ph.D. M.P.H.; New York University (New York, NY): Roy E. Shore Ph.D., Dr.PH.; Norwegian Radium Hospital (Oslo, Norway): Anne-Lise Børresen-Dale Ph.D., Laila Jansen; University of California at Irvine (Irvine, CA): Hoda Anton-Culver, Ph.D., Joan Largent Ph.D. M.P.H.; University of Iowa (Iowa City, IA): Charles F. Lynch M.D., Ph.D., Jeanne DeWall M.A.; University of Southern California (Los Angeles, CA): Robert W. Haile DrPH., Bryan M. Langholz Ph.D., Duncan C. Thomas Ph.D., Shanyan Xue M.D., Nianmin Zhou, M.D, Anh T. Diep, Evgenia Ter-Karapetova; University of Southern Maine (Portland, ME):W. Douglas Thompson Ph.D.; University of Texas, M.D. Anderson Cancer Center (Houston, TX): Marilyn Stovall Ph.D., Susan Smith M.P.H.; University of Virginia (Charlottesville, VA): Patrick Concannon, Ph.D., Sharon Teraoka, Ph.D., Eric R. Olson, Nirasha Ramchurren, Ph.D.

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c.15

29C

>A

p.Se

r510

X

N

1

1 B

RC

A1

11

1675

de

l A

Stop

531

16

75de

lA

c.15

56de

lA

p.Ly

s519

fs

F 2

2

BR

CA

1 11

18

06

C to

T

Gln

to S

top

Q56

3X

c.16

87C

>T

p.G

ln56

3X

N

1 1

B

RC

A1

11

1921

de

l 248

St

op 6

03

1921

del2

48

c.18

02_2

049d

el24

8 p.

His

601f

s F

1 1

B

RC

A1

11

2154

A

to T

Ly

s to

Stop

K

679X

c.

2035

A>T

p.

Lys6

79X

N

1

1

BR

CA

1 11

24

33

del G

St

op 7

91

2433

delG

c.

2314

delG

p.

Val

772f

s F

1

1 B

RC

A1

11

2508

de

l G

Stop

802

25

08de

lG

c.23

89de

lG

p.G

lu79

7fs

F 1

1

BR

CA

1 11

25

52

del C

St

op 8

14

2552

delC

c.

2433

delC

p.

Lys8

12fs

F

1

1 B

RC

A1

11

2594

de

l C

Stop

845

25

94de

lC

c.24

75de

lC

p.A

sp82

5fs

F 3

1 2

BR

CA

1 11

26

34

del C

St

op 8

45

2634

delC

c.

2515

delC

p.

His

839f

s F

1 1

B

RC

A1

11

2754

G

to T

G

lu to

Sto

p E8

79X

c.

2635

G>T

p.

Glu

879X

N

1

1

BR

CA

1 11

27

98

del G

AA

A

Stop

998

27

98de

l4

c.26

79_2

682d

elG

AA

A

p.Ly

s893

fs

F 1

1

BR

CA

1 11

28

41

G to

T

Glu

to S

top

E908

X

c.27

22G

>T

p.G

lu90

8X

N

1 1

B

RC

A1

11

2983

C

to A

Se

r to

Stop

S9

55X

c.

2864

C>A

p.

Ser9

55X

N

1

1

BR

CA

1 11

31

24

del A

St

op 1

023

3124

delA

c.

3005

delA

p.

Asn

1002

fs

F 1

1

BR

CA

1 11

31

48

del C

T St

op 1

016

3148

delC

T c.

3029

_303

0del

CT

p.Pr

o101

0fs

F 1

1

BR

CA

1 11

31

71

ins T

GA

GA

St

op 1

025

3171

ins5

c.

3048

_305

2dup

5 p.

Asn

1018

fs

F 1

1

BR

CA

1 11

33

07

del C

C in

s G

Stop

106

3 33

07de

lCC

insG

c.

3188

_318

9del

CC

insG

p.

Ser1

063X

F

1

1 B

RC

A1

11

3347

de

l AG

St

op 1

084

3347

delA

G

c.32

28_3

229d

elA

G

p.G

ly10

77fs

F

1 1

B

RC

A1

11

3438

G

to T

G

lu to

Sto

p E1

107X

c.

3319

G>T

p.

Glu

1107

fs

N

2 2

B

RC

A1

11

3450

de

l CA

AG

St

op 1

115

3450

del4

c.

3331

_333

4del

CA

AG

p.

Gln

1111

fs

F 1

1

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

17

Gen

eE

xon

NT

1B

ase

Cha

nge

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A1

11

3508

C

to G

Se

r to

Stop

S1

130X

c.

3389

C>G

p.

Ser1

130X

N

1

1

BR

CA

1 11

36

00

del 1

1 St

op 1

163

3600

del1

1 c.

3481

_349

1del

11

p.G

lu11

61fs

F

2 2

B

RC

A1

11

3726

C

to T

A

rg to

Sto

p R

1203

X

c.36

07C

>T

p.A

rg12

03X

N

3

1 2

BR

CA

1 11

38

29

del T

St

op 1

263

3829

delT

c.

3710

delT

p.

Ile12

37fs

F

1 1

B

RC

A1

11

3867

G

to T

G

lu to

Sto

p E1

250X

c.

3748

G>T

p.

Glu

1250

X

N

4 2

2 B

RC

A1

11

3875

de

l GTC

T St

op 1

262

3875

del4

c.

3756

_375

9del

GTC

T p.

Ser1

253f

s F

4 4

B

RC

A1

11

4184

de

l TC

AA

St

op 1

364

4184

del4

c.

4065

_406

8del

TCA

A

p.A

sn13

55fs

F

2

2 B

RC

A1

12

4302

C

to T

G

ln to

Sto

p Q

1395

X

c.41

83C

>T

p.G

ln13

95X

N

1

1

BR

CA

1 13

43

41

C to

T

Gln

to S

top

Q14

08X

c.

4222

C>T

p.

Gln

1408

X

N

1

1 B

RC

A1

13

4370

de

l GT

Stop

142

6 43

70de

lGT

c.42

51_4

252d

elG

T p.

Leu1

418f

s F

1 1

B

RC

A1

13

4446

C

to T

A

rg to

Sto

p R

1443

X

c.43

27C

>T

p.A

rg14

43X

N

2

1 1

BR

CA

1 13

44

76+1

G

to T

-

IVS1

3+1G

>T

c.43

57+1

G>T

S 2

1 1

BR

CA

1 16

51

05+2

T

to C

-

IVS1

6+2T

>C

c.49

86+2

T>C

S 1

1

BR

CA

1 18

52

14

C>T

A

rg to

Trp

R

1699

W

c.50

95C

>T

p.A

rg16

99Tr

p M

1

1

BR

CA

1 18

52

42

C>A

A

la to

Glu

A

1708

E c.

5123

C>A

p.

Ala

1708

Glu

M

1

1

BR

CA

1 20

53

32

G to

A

Gly

to G

lu

G17

38E

c.52

13G

>A

p.G

ly17

38G

lu

M

1

1

BR

CA

1 20

53

49

del A

St

op 1

764

5349

delA

c.

5230

delA

p.

Arg

1744

fs

F 1

1

BR

CA

1 20

53

82

ins C

St

op 1

829

5382

insC

c.

5266

dupC

p.

Gln

1756

fs

F 10

2

8 B

RC

A1

21

5443

T

to G

M

et to

Arg

M

1775

R

c.53

24T>

G

p.M

et17

75A

rg

M

1

1 B

RC

A1

23

5563

G

to A

Tr

p to

Sto

p W

1815

X

c.54

44G

>A

p.Tr

p181

5X

N

1

1 B

RC

A1

24

5622

C

to T

A

rg to

Sto

p R

1835

X

c.55

03C

>T

p.A

rg18

35X

N

1

1

BR

CA

1 24

56

77

ins A

St

op 1

853

5677

insA

c.

5558

dupA

p.

Tyr1

853X

F

1 1

B

RC

A1

Tota

l

10

9 67

42

B

RC

A2

2 23

1 G

to A

M

et to

Ile

M1I

c.

3G>A

p.

Met

1Ile

M

1

1

BR

CA

2 2

279

del A

C

Stop

29

279d

elA

C

c.51

_52d

elA

C

p.A

rg18

fs

F 1

1

BR

CA

2 4

592

del A

St

op 1

35

592d

elA

c.

364d

elA

p.

Thr1

22fs

F

1 1

B

RC

A2

5 69

7 de

l AA

St

op 1

81

697d

elA

A

c.46

9_47

0del

AA

p.

Lys1

57fs

F

1

1 B

RC

A2

7 83

8 de

l C

Stop

210

83

8del

C

c.61

0del

C

p.Le

u204

fs

F 1

1

BR

CA

2 9

983

del A

CA

G

Stop

275

98

3del

4 c.

755_

758d

elA

CA

G

p.A

sp25

2fs

F 2

1 1

BR

CA

2 10

14

61

ins A

St

op 4

20

1461

insA

c.

1233

dupA

p.

Pro4

12fs

F

1

1 B

RC

A2

10

1538

de

l AA

GA

St

op 4

58

1538

del4

c.

1310

_131

3del

AA

GA

p.

Lys4

37fs

F

3 3

B

RC

A2

10

1898

de

l 14

Stop

557

18

98de

l14

c.16

70_1

683d

el14

p.

Leu5

57X

F

1 1

B

RC

A2

10

2034

de

l A

Stop

613

20

34de

lA

c.18

13de

lA

p.Ile

605f

s F

1 1

B

RC

A2

10

2041

in

s A

Stop

615

20

41in

sA

c.18

13du

pA

p.Ile

605f

s F

3 2

1 B

RC

A2

11

2482

de

l GA

CT

Stop

770

24

82de

l4

c.22

54_2

257d

elG

AC

T p.

Asp

752f

s F

1 1

B

RC

A2

11

2600

C

to A

Se

r to

Stop

S7

91X

c.

2372

C>A

p.

Ser7

91X

N

1

1

E 12

18 B

org

et al

.

Gen

eE

xon

NT

1B

ase

Cha

nge

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

11

3036

de

l AC

AA

St

op 9

58

3036

del4

c.

2808

_281

1del

AC

AA

p.

Ala

938f

s F

5 4

1 B

RC

A2

11

3058

A

to T

Ly

s to

Stop

K

944X

c.

2830

A>T

p.

Lys9

44X

N

1

1

BR

CA

2 11

33

37

C to

T

Gln

to S

top

Q10

37X

c.

3109

C>T

p.

Gln

1037

X

N

1 1

B

RC

A2

11

3492

in

s T

Stop

109

8 34

92in

sT

c.32

64du

pT

p.G

ln10

89fs

F

1

1 B

RC

A2

11

3773

de

l TT

Stop

118

2 37

73de

lTT

c.35

45_3

546d

elTT

p.

Phe1

182X

F

1

1 B

RC

A2

11

3827

de

l GT

Stop

120

0 38

27de

lGT

c.35

99_3

600d

elG

T p.

Cys

1200

X

F 1

1

BR

CA

2 11

40

75

del G

T St

op 1

284

4075

delG

T c.

3847

_384

8del

GT

p.V

al12

83fs

F

1 1

B

RC

A2

11

4093

de

l AA

AT

Stop

129

1 40

93de

l4

c.38

65_3

868d

elA

AA

T p.

Lys1

289f

s F

1

1 B

RC

A2

11

4206

in

s TG

CT

Stop

133

0 42

06in

s4

c.39

75_3

978d

upTT

GC

p.

Ala

1327

fs

F 1

1

BR

CA

2 11

46

77

del A

St

op 1

485

4677

delA

c.

4449

delA

p.

Asp

1484

fs

F 1

1

BR

CA

2 11

51

64

del G

AA

A

Stop

166

8 51

64de

l4

c.49

36_4

939d

elG

AA

A

p.G

lu16

46fs

F

1 1

B

RC

A2

11

5804

de

l TTA

A

Stop

186

1 58

04de

l4

c.55

76_5

579d

elTT

AA

p.

Ile18

59fs

F

1

1 B

RC

A2

11

5844

de

l AG

TAA

St

op 1

873

5844

del5

c.

5616

_562

0del

5 p.

Lys1

872f

s F

1

1 B

RC

A2

11

5873

C

to A

Se

r to

Stop

S1

882X

c.

5645

C>A

p.

Ser1

882X

N

1

1

BR

CA

2 11

59

10

C to

G

Tyr t

o St

op

Y18

94X

c.

5682

C>G

p.

Tyr1

894X

N

1

1

BR

CA

2 11

59

50

del C

T St

op 1

909

5950

delC

T c.

5722

_572

3del

CT

p.Le

u190

8fs

F 2

1 1

BR

CA

2 11

61

74

del T

St

op 2

003

6174

delT

c.

5946

delT

p.

Ser1

982f

s F

5 2

3 B

RC

A2

11

6286

G

to T

G

lu to

Sto

p E2

020X

c.

6058

G>T

p.

Glu

2020

X

N

1

1 B

RC

A2

11

6503

de

l TT

Stop

209

8 65

03de

lTT

c.62

75_6

276d

elTT

p.

Leu2

092f

s F

1

1 B

RC

A2

11

6633

de

l CTT

AA

St

op 2

137

6633

del5

c.

6405

_640

9del

5 p.

Asn

2135

fs

F 1

1

BR

CA

2 11

66

71

del C

T St

op 2

149

6671

delC

T c.

6443

_644

4del

CT

p.Se

r214

8fs

F 1

1

BR

CA

2 11

67

14

del A

CA

A

Stop

216

6 67

14de

l4

c.64

86_6

489d

elA

CA

A

p.Ly

s216

2fs

F 1

1

BR

CA

2 11

68

84

C to

G

Ser t

o St

op

S221

9X

c.66

56C

>G

p.Se

r221

9X

N

1

1 B

RC

A2

13

7169

de

l C

Stop

231

5 71

69de

lC

c.69

41de

lC

p.Th

r231

4fs

F 1

1

BR

CA

2 14

72

53

del A

A

Stop

235

8 72

53de

lAA

c.

7025

_702

6del

AA

p.

Gln

2342

fs

F 1

1

BR

CA

2 14

72

95

del T

T St

op 2

358

7295

delT

T c.

7067

_706

8del

TT

p.Ph

e235

6fs

F 1

1

BR

CA

2 14

72

97

del C

T St

op 2

358

7297

delC

T c.

7069

_707

0del

CT

p.Le

u235

7fs

F 2

1 1

BR

CA

2 14

74

38

del 7

insT

G

Stop

241

0 74

38de

l7in

s2

c.72

10_7

216d

el7i

nsTG

p.

Lys2

404f

s F

1

1 B

RC

A2

15

7786

C

to T

A

rg to

Sto

p R

2520

X

c.75

58C

>T

p.A

rg25

20X

N

1

1

BR

CA

2 16

79

90

del A

TA in

s TT

Stop

264

7 79

90de

l3in

s2

c.77

62_7

764d

elA

TAin

sTT

p.Ile

2588

fs

F 1

1

BR

CA

2 17

81

41

del T

TCC

T St

op 2

638

8141

del5

c.

7913

_791

7del

5 p.

Phe2

638X

F

1 1

B

RC

A2

18

8550

in

s T

Stop

278

1 85

50in

sT

c.83

22du

pT

p.M

et27

75fs

F

1 1

B

RC

A2

18

8558

de

l A

Stop

282

0 85

58de

lA

c.83

30de

lA

p.Ly

s277

7fs

F 1

1

BR

CA

2 20

88

03

del C

St

op 2

862

8803

delC

c.

8575

delC

p.

Gln

2859

fs

F 1

1

BR

CA

2 20

88

23

ins T

St

op 2

868

8823

insT

c.

8594

dupT

p.

Leu2

865f

s F

1 1

B

RC

A2

21

8958

de

l T

Stop

292

6 89

58de

lT

c.87

30de

lT

p.A

sn29

10fs

F

1 1

B

RC

A2

22

9181

+1

G to

T

- IV

S22+

1G>T

c.

8953

+1G

>T

S

1 1

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

19

Gen

eE

xon

NT

1B

ase

Cha

nge

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

23

9197

G

to A

Tr

p to

Sto

p W

2990

X

c.89

69G

>A

p.Tr

p299

0fs

N

1

1 B

RC

A2

23

9326

in

s A

Stop

304

3 93

26in

sA

c.90

97du

pA

p.Th

r303

3fs

F 1

1

BR

CA

2 24

94

81

ins A

St

op 3

110

9481

insA

c.

9253

dupA

p.

Thr3

085f

s F

1 1

B

RC

A2

25

9610

C

to T

A

rg to

Sto

p R

3128

X

c.93

82C

>T

p.A

rg31

28X

N

1

1

BR

CA

2 25

97

29+3

A

to T

-

IVS2

5+3A

>T

c.95

01+3

A>T

S 2

2

BR

CA

2 26

97

69

del 1

4 St

op 3

183

9769

del1

4 c.

9541

_955

4del

14

p.M

et31

81fs

F

1 1

B

RC

A2

Tota

l

72

41

31

1 BR

CA

1: n

ucle

otid

e nu

mbe

r acc

ordi

ng to

Gen

Ban

k U

1468

0.1.

http

://w

ww

.ncb

i.nlm

.nih

.gov

/ent

rez/

view

er.fc

gi?d

b=nu

cleo

tide&

val=

5559

31

1 BR

CA

2: n

ucle

otid

e nu

mbe

r acc

ordi

ng to

Gen

Bank

NM

_000

059.

1. h

ttp://

ww

w.n

cbi.n

lm.n

ih.g

ov/e

ntre

z/vi

ewer

.fcgi

?db=

nucl

eotid

e&va

l=45

0245

0 2 B

IC (B

reas

t Can

cer I

nfor

mat

ion

Cor

e). h

ttp://

rese

arch

.nhg

ri.ni

h.go

v/bi

c/

3 HG

VS

(Hum

an G

enom

e V

aria

tion

Soci

ety)

. http

://w

ww

.hgv

s.org

/mut

nom

en/re

cs.h

tml

4 Typ

e: F

=fra

me

shift

, M=m

isse

nse,

S=s

plic

e si

te, N

=non

sens

e 5 N

umbe

r of o

bser

vatio

ns

mut

atio

ns n

ot e

arlie

r des

crib

ed in

BIC

(by

Nov

embe

r 200

8)

E 12

20 B

org

et al

.

Supp

. Tab

le S2

. BRC

A1 an

d BR

CA2 u

nclas

sified

varia

nts i

dent

ified

in 70

5 con

tralat

eral

(CBC

) and

1398

uni

later

al br

east

canc

er p

atien

ts (U

BC).

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A1

2 10

1-10

T

to C

-

IV

S1-1

0T>C

c.

-29T

>C

IV

S 1

10

BR

CA

1 3

200-

129

G to

A

- IV

S2-1

29G

>A

c.81

-129

G>A

IVS

10

1 B

RC

A1

3 20

0-55

T

to G

-

IVS2

-55T

>G

c.81

-55T

>G

IV

S 1

01

BR

CA

1 3

233

G to

A

Lys t

o Ly

s K

38K

c.

114T

>A

p.=

Syn

33

0 B

RC

A1

3 25

3 A

to C

Ly

s to

Thr

K45

T c.

134A

>C

p.Ly

s45T

hr

M

10

1 B

RC

A1

5 25

4-53

T

to C

-

IVS4

-53T

>C

c.13

5-53

T>C

IVS

10

1 B

RC

A1

5 25

4-23

C

to T

-

IVS4

-23C

>T

c.13

5-23

C>T

IVS

10

1 B

RC

A1

5 33

1+23

T

to A

-

IVS5

+23T

>A

c.21

2+23

T>A

IVS

41

3 B

RC

A1

7 42

1-70

G

to A

-

IVS6

-70G

>A

c.30

2-70

G>A

IVS

10

1 B

RC

A1

7 43

3 A

to G

Ty

r to

Cys

Y

105C

c.

314A

>G

p.Ty

r105

Cys

M

1

01

BR

CA

1 7

544

C to

A

Pro

to H

is

P142

H

c.42

5C>A

p.

Pro1

42H

is

M

42

2 B

RC

A1

7 56

0+36

de

l 14

- IV

S7+3

6del

14

c.44

1+36

del1

4

IVS

1154

377

777

BR

CA

1 8

561-

34

C to

T

- IV

S7-3

4C>T

c.

442-

34C

>T

IV

S 77

925

452

5 B

RC

A1

8 62

8 G

to A

A

rg to

Gln

R

170Q

c.

509G

>A

p.A

rg17

0Gln

M

2

11

BR

CA

1 8

655

A to

G

Tyr t

o C

ys

Y17

9C

c.53

6A>G

p.

Tyr1

79C

ys

M

20

2 B

RC

A1

9 66

7-80

T>

C

- IV

S8-8

0T>C

c.

548-

80T>

C

IV

S 5

32

BR

CA

1 9

667-

58

delT

-

IVS8

-58d

elT

c.54

8-58

delT

IVS

1044

342

702

BR

CA

1 9

667-

17

G to

T

- IV

S8-1

7G>T

c.

548-

17G

>T

IV

S 2

11

BR

CA

1 9

667-

3 de

l T

- IV

S8-3

delT

c.

548-

3del

T

IVS

10

1 B

RC

A1

9 71

0 C

to T

C

ys to

Cys

C

197C

c.

591C

>T

p.=

Syn

31

2 B

RC

A1

10

713-

72

A>G

-

IVS9

-72A

>G

c.59

4-72

A>G

IVS

11

0 B

RC

A1

10

713-

34

T to

C

- IV

S9-3

4T>C

c.

594-

34T>

C

IV

S 1

01

BR

CA

1 10

76

0 A

to G

A

sp to

Gly

D

214G

c.

641A

>G

p.A

sp21

4Gly

M

1

10

BR

CA

1 11

81

2 G

to A

Th

r to

Thr

T231

T c.

693G

>A

p.=

Syn

10

1 B

RC

A1

11

855

T to

G

Leu

to V

al

L246

V

c.73

6T>G

p.

Leu2

46V

al

M

32

1 B

RC

A1

11

944

C to

T

Gly

to G

ly

G27

5G

c.82

5C>T

p.

= Sy

n 1

01

BR

CA

1 11

10

67

C to

G

Ser t

o A

rg

S316

R

c.94

8C>G

p.

Ser3

16A

rg

M

10

1 B

RC

A1

11

1100

A

to G

Th

r to

Thr

T327

T c.

981A

>G

p.=

Syn

40

4 B

RC

A1

11

1152

G

to T

A

sp to

Tyr

D

345Y

c.

1033

G>T

p.

Asp

345T

yr

M

11

0 B

RC

A1

11

1184

G

to A

Ly

s to

Lys

K35

5K

c.10

65G

>A

p.=

Syn

10

1 B

RC

A1

11

1186

A

to G

G

ln to

Arg

Q

356R

c.

1067

A>G

p.

Gln

356A

rg

M

251

8117

0 B

RC

A1

11

1224

de

l GA

T de

l Asp

D

369d

el

c.11

05de

lGA

T p.

Asp

369d

el

IFD

1

01

BR

CA

1 11

12

56

T to

G

Ile to

Met

I3

79M

c.

1137

T>G

p.

Ile37

9Met

M

1

01

BR

CA

1 11

15

75

T to

C

Phe

to L

eu

F486

L c.

1456

T>C

p.

Phe4

86Le

u M

4

13

BR

CA

1 11

16

05

C to

T

Arg

to C

ys

R49

6C

c.14

86C

>T

p.A

rg49

6Cys

M

2

11

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

21

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A1

11

1606

G

to A

A

rg to

His

R

496H

c.

1487

G>A

p.

Arg

496H

is

M

40

4 B

RC

A1

11

1728

A

to G

A

sn to

Asp

N

537D

c.

1609

A>G

p.

Asn

537A

sp

M

10

1 B

RC

A1

11

1767

A

to C

A

sn to

His

N

550H

c.

1648

A>C

p.

Asn

550H

is

M

31

2 B

RC

A1

11

1831

T

to C

Ile

to T

hr

I571

T c.

1712

T>C

p.

Ile57

1Thr

M

1

01

BR

CA

1 11

19

08

G to

A

Glu

to L

ys

E597

K

c.17

89G

>A

p.G

lu59

7Lys

M

3

03

BR

CA

1 11

19

65

del T

CT

del S

er

S616

del

c.18

46de

lTC

T p.

Ser6

16de

l IF

D

10

1 B

RC

A1

11

1984

C

to T

A

la to

Val

A

622V

c.

1865

C>T

p.

Ala

622V

al

M

11

0 B

RC

A1

11

2030

T

to C

Th

r to

Thr

T637

T c.

1911

T>C

p.

= Sy

n 1

01

BR

CA

1 11

20

90

A to

G

Gln

to G

ln

Q65

7Q

c.19

71A

>G

p.=

Syn

30

3 B

RC

A1

11

2196

G

to A

A

sp to

Asn

D

693N

c.

2077

G>A

p.

Asp

693A

sn

M

296

106

190

BR

CA

1 11

22

01

C to

T

Ser t

o Se

r S6

94S

c.20

82C

>T

p.=

Syn

1040

337

703

BR

CA

1 11

24

18

A to

G

Ser t

o G

ly

S767

G

c.22

99A

>G

p.Se

r767

Gly

M

1

01

BR

CA

1 11

24

30

T to

C

Leu

to L

eu

L771

L c.

2311

T>C

p.

= Sy

n 98

732

166

6 B

RC

A1

11

2471

G

to A

Se

r to

Ser

S784

S c.

2352

G>A

p.

= Sy

n 1

10

BR

CA

1 11

25

35

G to

A

Ala

to T

hr

A80

6T

c.24

16G

>A

p.A

la80

6Thr

M

1

10

BR

CA

1 11

25

77

A to

G

Lys t

o G

lu

K82

0E

c.24

58A

>G

p.Ly

s820

Glu

M

5

14

BR

CA

1 11

25

92

G to

T

Asp

to T

yr

D82

5Y

c.24

73G

>T

p.A

sp82

5Tyr

M

1

10

BR

CA

1 11

25

96

C to

A

Thr t

o Ly

s T8

26K

c.

2477

C>A

p.

Thr8

26Ly

s M

1

01

BR

CA

1 11

26

02

del G

CT

del G

ly 8

28

G82

8del

c.

2483

delG

CT

p.G

ly82

8del

IF

D

10

1 B

RC

A1

11

2637

A

to G

Se

r to

Gly

S8

40G

c.

2518

A>G

p.

Ser8

40G

ly

M

10

1 B

RC

A1

11

2640

C

to T

A

rg to

Trp

R

841W

c.

2521

C>T

p.

Arg

841T

rp

M

82

6 B

RC

A1

11

2641

G

to A

A

rg to

Gln

R

841Q

c.

2522

G>A

p.

Arg

841G

ln

M

10

1 B

RC

A1

11

2685

T

to C

Ty

r to

His

Y

856H

c.

2566

T>C

p.

Tyr8

56H

is

M

11

0 B

RC

A1

11

2715

C

to T

A

rg to

Cys

R

866C

c.

2596

C>T

p.

Arg

866C

ys

M

10

1 B

RC

A1

11

2727

G

to C

A

la to

Pro

A

870P

c.

2608

G>C

p.

Ala

870P

ro

M

11

0 B

RC

A1

11

2731

C

to T

Pr

o to

Leu

P8

71L

c.26

12C

>T

p.Pr

o871

Leu

M

1029

333

696

BR

CA

1 11

28

47

C to

G

Gln

to G

lu

Q91

0E

c.27

28C

>G

p.G

ln91

0Glu

M

1

01

BR

CA

1 11

28

52

A to

G

Gly

to G

ly

G91

1G

c.27

33A

>G

p.=

Syn

50

5 B

RC

A1

11

2933

A

to G

Pr

o to

Pro

P9

38P

c.28

14A

>G

p.=

Syn

20

2 B

RC

A1

11

3003

G

to A

G

lu to

Lys

E9

62K

c.

2884

G>A

p.

Glu

962L

ys

M

10

1 B

RC

A1

11

3119

G

to A

G

lu to

Glu

E1

000E

c.

3000

G>A

p.

= Sy

n 1

10

BR

CA

1 11

31

43

G to

A

Met

to Il

e M

1008

I c.

3024

G>A

p.

Met

1008

Ile

M

40

4 B

RC

A1

11

3232

A

to G

G

lu to

Gly

E1

038G

c.

3113

A>G

p.

Glu

1038

Gly

M

99

632

467

2 B

RC

A1

11

3238

G

to A

Se

r to

Asn

S1

040N

c.

3119

G>A

p.

Ser1

040A

sn

M

7328

45

BR

CA

1 11

33

40

G to

C

Arg

to T

hr

R10

74T

c.32

21G

>C

p.A

rg10

74Th

r M

1

10

BR

CA

1 11

34

15

C to

T

Pro

to L

eu

P109

9L

c.32

96C

>T

p.Pr

o109

9Leu

M

1

01

BR

CA

1 11

34

21

G to

A

Ser t

o A

sn

S110

1N

c.33

02G

>A

p.Se

r110

1Asn

M

2

02

BR

CA

1 11

35

35

G to

T

Ser t

o Ile

S1

139I

c.

3416

G>T

p.

Ser1

139I

le

M

11

0

E 12

22 B

org

et al

.

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A1

11

3537

A

to G

Se

r to

Gly

S1

140G

c.

3418

A>G

p.

Ser1

140G

ly

M

20

2 B

RC

A1

11

3573

G

to A

A

sp to

Asn

D

1152

N

c.34

54G

>A

p.A

sp11

52A

sn

M

10

1 B

RC

A1

11

3667

A

to G

Ly

s to

Arg

K

1183

R

c.35

48A

>G

p.Ly

s118

3Arg

M

99

832

867

0 B

RC

A1

11

3719

G

to C

G

ln to

His

Q

1200

H

c.36

00G

>C

p.G

ln12

00H

is

M

22

0 B

RC

A1

11

3727

G

to A

A

rg to

Gln

R

1203

Q

c.36

08G

>A

p.A

rg12

03G

ln

M

10

1 B

RC

A1

11

3827

T

to G

A

sn to

Lys

N

1236

K

c.37

08T>

G

p.A

sn12

36Ly

s M

1

01

BR

CA

1 11

38

36

T to

A

Ser t

o Se

r S1

239S

c.

3717

T>A

p.

= Sy

n 1

10

BR

CA

1 11

38

64

A to

C

Thr t

o Pr

o T1

249P

c.

3745

A>C

p.

Thr1

249P

ro

M

10

1 B

RC

A1

11

3916

G

to C

Se

r to

Thr

S126

6T

c.37

97G

>C

p.Se

r126

6Thr

M

2

11

BR

CA

1 11

39

42

A to

G

Ile to

Val

I1

275V

c.

3823

A>G

p.

Ile12

75V

al

M

10

1 B

RC

A1

11

4158

A

to G

A

rg to

Gly

R

1347

G

c.40

39A

>G

p.A

rg13

47G

ly

M

165

11

BR

CA

1 11

41

66

G to

T

Thr t

o Th

r T1

349T

c.

4047

G>T

p.

= Sy

n 1

01

BR

CA

1 11

42

15+3

A

to G

-

IVS1

1+3A

>G

c.40

96+3

A>G

IVS

11

0 B

RC

A1

11

4215

+39

T to

C

- IV

S11+

39T>

C

c.40

96+3

9T>C

IVS

10

1 B

RC

A1

12

4216

-28

A to

G

- IV

S11-

28A

>G

c.40

97-2

8A>G

IVS

11

0 B

RC

A1

12

421

6-11

T

to C

-

IVS1

1-11

T>C

c.

4097

-11T

>C

IV

S 1

01

BR

CA

1 12

42

30

G to

C

Gly

to A

rg

G13

71R

c.

4111

G>C

p.

Gly

1371

Arg

M

1

10

BR

CA

1 12

42

91

T to

C

Ile to

Thr

I1

391T

c.

4172

T>C

p.

Ile13

91Th

r M

1

10

BR

CA

1 12

43

04+2

0 de

l 18

- IV

S12+

20de

l18

c.41

85+2

0del

18

IV

S 1

01

BR

CA

1 12

43

04+2

1 de

l TG

-

IVS1

2+21

delT

G

c.41

85+2

1del

TG

IV

S 1

10

BR

CA

1 12

43

04+3

7 de

l TG

-

IVS1

2+37

delT

G

c.41

85+3

7del

TG

IV

S 2

20

BR

CA

1 13

44

27

C to

T

Ser t

o Se

r S1

436S

c.

4308

C>T

p.

= Sy

n 10

0532

368

2 B

RC

A1

13

4476

+17

A to

G

- IV

S13+

17A

>G

c.43

57+1

7A>G

IVS

10

1 B

RC

A1

14

4477

-37

G to

A

- IV

S13-

37G

>A

c.43

58-3

7G>A

IVS

11

0 B

RC

A1

14

4477

-28

T to

C

- IV

S13-

28T>

C

c.43

58-2

8T>C

IVS

11

0 B

RC

A1

14

447

7-10

C

to T

-

IVS1

3-10

C>T

c.

4358

-10C

>T

IV

S 2

11

BR

CA

1 14

45

29

A to

T

Glu

to A

sp

E147

0D

c.44

10A

>T

p.G

lu14

70A

sp

M

10

1 B

RC

A1

14

4603

+14

A to

G

- IV

S14+

14A

>G

c.44

84+1

4A>G

IVS

20

2 B

RC

A1

14

4603

+61

G to

T

- IV

S14+

61G

>T

c.44

84+6

1G>T

IVS

10

1 B

RC

A1

15

4654

G

to T

Se

r to

Ile

S151

2I

c.45

35G

>T

p.Se

r151

2Ile

M

18

315

B

RC

A1

15

4755

G

to A

A

sp to

Asn

D

1546

N

c.46

36G

>A

p.A

sp15

46A

sn

M

20

2 B

RC

A1

16

4869

G

to T

A

la to

Ser

A

1584

S c.

4750

G>T

p.

Ala

1584

Ser

M

11

0 B

RC

A1

16

4890

G

to A

G

ly to

Ser

G

1591

S c.

4771

G>A

p.

Gly

1591

Ser

M

11

0 B

RC

A1

16

4931

A

to G

G

ln to

Gln

Q

1604

Q

c.48

12A

>G

p.=

Syn

72

5 B

RC

A1

16

4956

A

to G

Se

r to

Gly

S1

613G

c.

4837

A>G

p.

Ser1

613G

ly

M

1069

348

721

BR

CA

1 16

50

02

T to

C

Met

to T

hr

M16

28T

c.48

83T>

C

p.M

et16

28Th

r M

1

01

BR

CA

1 16

50

75

G to

A

Met

to Il

e M

1652

I c.

4956

G>A

p.

Met

1652

Ile

M

6118

43

BR

CA

1 17

51

06-2

0 A

to G

-

IVS1

6-20

A>G

c.

4987

-20A

>G

IV

S 1

01

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

23

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A1

17

5124

G

to T

A

la to

Ser

A

1669

S c.

5005

G>T

p.

Ala

1669

Ser

M

10

1 B

RC

A1

18

5194

-53

C to

T

- IV

S17-

53C

>T

c.50

75C

>T

IV

S 11

431

83

BR

CA

1 20

53

13-4

3 T

to C

-

IVS1

9-43

T>C

c.

5194

-43T

>C

IV

S 1

01

BR

CA

1 20

53

96+6

0 in

s 12

- IV

S20+

60in

s12

c.52

77+6

0ins

12

IV

S 11

29

BR

CA

1 20

53

96+7

8 G

to A

-

IVS2

0+78

G>A

c.

5277

+78G

>A

IV

S 8

44

BR

CA

1 20

53

96+1

02

A to

G

- IV

S20+

102A

>G

c.52

77+1

02A

>G

IV

S 1

01

BR

CA

1 21

54

51+7

8 C

to T

-

IVS2

1+78

C>T

c.

5332

+78C

>T

IV

S 2

11

BR

CA

1 21

54

51+8

1 T

to C

-

IVS2

1+81

T>C

c.

5332

+81T

>C

IV

S 1

10

BR

CA

1 22

55

20

G to

T

Gly

to C

ys

G18

01C

c.

5401

G>T

p.

Gly

1801

Cys

M

1

01

BR

CA

1 22

55

25+3

3 A

to T

-

IVS2

2+33

A>T

c.

5406

+33A

>T

IV

S 2

02

BR

CA

1 22

55

25+6

8 T

to C

-

IVS2

2+68

T>C

c.

5406

+68T

>C

IV

S 6

24

BR

CA

1 23

55

30

T to

A

Val

to A

sp

V18

04D

c.

5411

T>A

p.

Val

1804

Asp

M

1

01

BR

CA

1 24

.5

587-

10

C to

A

- IV

S23-

10C

>A

c.54

68-1

0C>A

IVS

10

1 B

RC

A1

24

.558

7-8

G to

T

- IV

S23-

8G>T

c.

5468

-8G

>T

IV

S 1

01

BR

CA

1 24

56

88

C to

G

Gln

to G

lu

Q18

57E

c.55

69C

>G

p.G

ln18

57G

lu

M

11

0 B

RC

A1

24

5747

C

to G

-

U

GA

+36C

>G

c.*3

6C>G

UTR

5

14

BR

CA

1 24

58

07

C to

T

- U

GA

+96C

>T

c.*9

6C>T

UTR

1

10

BR

CA

2 2

203

G to

A

- 20

3G>A

c.

-26G

>A

U

TR

797

269

528

BR

CA

2 2

218

C to

T

- 21

8C>T

c.

-11C

>T

U

TR

10

1 B

RC

A2

2 29

5+62

T

to G

-

IVS2

+62T

>G

c.67

+62T

>G

IV

S 13

67

BR

CA

2 3

296-

31

A to

G

- IV

S2-3

1A>G

c.

68-3

1A>G

IVS

10

1 B

RC

A2

3 29

6-7

T to

A

- IV

S2-7

T>A

c.

68-7

T>A

IVS

101

9 B

RC

A2

3 35

3 A

to G

Ty

r to

Cys

Y

42C

c.

125A

>G

p.Ty

r42C

ys

M

113

8 B

RC

A2

3 40

7 A

to G

A

sn to

Ser

N

60S

c.17

9A>G

p.

Asn

60Se

r M

1

10

BR

CA

2 3

451

G to

C

Ala

to P

ro

A75

P c.

223G

>C

p.A

la75

Pro

M

22

0 B

RC

A2

3 49

5 G

to A

Pr

o to

Pro

P8

9P

c.26

7G>A

p.

= Sy

n 1

10

BR

CA

2 4

545-

37

insT

ATG

-

IVS3

-37i

nsTA

TG

c.31

7-37

insT

ATG

IVS

11

0 B

RC

A2

4 65

3+33

A

to G

-

IVS4

+33A

>G

c.42

5+33

A>G

IVS

11

0 B

RC

A2

5 65

4-37

T

to A

-

IVS4

-37T

>A

c.42

6-37

T>A

IVS

10

1 B

RC

A2

5 66

1 de

l GTT

de

l Val

V

145d

el

c.43

3del

GTT

p.

Val

145d

el

IFD

1

10

BR

CA

2 5

683

C to

A

Thr t

o Ly

s T1

52K

c.

455C

>A

p.Th

r152

Lys

M

10

1 B

RC

A2

5 69

5 A

to G

A

sp to

Gly

D

156G

c.

467A

>G

p.A

sp15

6Gly

M

1

10

BR

CA

2 6

744+

21

A to

T

- IV

S6+2

1A>T

c.

516+

21A

>T

IV

S 4

13

BR

CA

2 6

744+

54

T to

C

- IV

S6+5

4T>C

c.

516+

54T>

C

IV

S 3

21

BR

CA

2 6

744+

58

G to

A

- IV

S6+5

8G>A

c.

516+

58G

>A

IV

S 1

01

BR

CA

2 7

745-

19

C to

T

- IV

S6-1

9C>T

c.

517-

19C

>T

IV

S 1

10

BR

CA

2 7

823

G to

A

Ala

to T

hr

A19

9T

c.59

5G>A

p.

Ala

199T

hr

M

21

1

E 12

24 B

org

et al

.

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

7 85

9+25

C

to T

-

IVS7

+25C

>T

c.63

1+25

C>T

IVS

42

2 B

RC

A2

8 86

0-69

T

to C

-

IVS7

-69T

>C

c.63

2-69

T>C

IVS

62

4 B

RC

A2

8 86

0-64

G

to A

-

IVS7

-64G

>A

c.63

2-64

G>A

IVS

10

1 B

RC

A2

8 86

0-27

C

to G

-

IVS7

-27C

>G

c.63

2-27

C>G

IVS

10

1 B

RC

A2

9 91

0-32

A

to G

-

IVS8

-32A

>G

c.68

2-32

A>G

IVS

21

1 B

RC

A2

9 91

0-12

de

l TA

-

IVS8

-12d

elTA

c.

682-

12de

lTA

IVS

11

0 B

RC

A2

9 99

7 A

to C

A

sn to

His

N

257H

c.

769A

>C

p.A

sn25

7His

M

1

10

BR

CA

2 10

10

93

A to

C

Asn

to H

is

N28

9H

c.86

5A>C

p.

Asn

289H

is

M

148

5197

B

RC

A2

10

1184

A

to G

A

sn to

Ser

N

319S

c.

956A

>G

p.A

sn31

9Ser

M

1

01

BR

CA

2 10

11

92

A to

C

Lys t

o G

ln

K32

2Q

c.96

4A>C

p.

Lys3

22G

ln

M

10

1 B

RC

A2

10

1206

C

to A

Se

r to

Arg

S3

26R

c.

978C

>A

p.Se

r326

Arg

M

5

14

BR

CA

2 10

12

61

A to

G

Lys t

o G

lu

K34

5E

c.10

33A

>G

p.Ly

s345

Glu

M

1

01

BR

CA

2 10

12

68

A to

G

Gln

to A

rg

Q34

7R

c.10

40A

>G

p.G

ln34

7Arg

M

1

10

BR

CA

2 10

13

42

A to

C

Asn

to H

is

N37

2H

c.11

14A

>C

p.A

sn37

2His

M

10

2236

066

2 B

RC

A2

10

1379

C

to T

Se

r to

Phe

S384

F c.

1151

C>T

p.

Ser3

84Ph

e M

5

23

BR

CA

2 10

13

95

G to

A

Pro

to P

ro

P389

P c.

1167

G>A

p.

= Sy

n 1

10

BR

CA

2 10

14

60

T to

C

Ile to

Thr

I4

11T

c.12

32T>

C

p.Ile

411T

hr

M

10

1 B

RC

A2

10

1503

A

to G

G

lu to

Glu

E4

25E

c.12

75A

>G

p.=

Syn

11

0 B

RC

A2

10

1590

A

to G

Ly

s to

Lys

K45

4K

c.13

62A

>G

p.=

Syn

30

3 B

RC

A2

10

1593

A

to G

Se

r to

Ser

S455

S c.

1365

A>G

p.

= Sy

n 14

651

95

BR

CA

2 10

16

13

A to

G

Glu

to G

ly

E462

G

c.13

85A

>G

p.G

lu46

2Gly

M

1

01

BR

CA

2 10

16

75

G to

C

Ala

to P

ro

A48

3P

c.14

47G

>C

p.A

la48

3Pro

M

1

01

BR

CA

2 10

16

88

C to

A

Ala

to G

lu

A48

7E

c.14

60C

>A

p.A

la48

7Glu

M

3

03

BR

CA

2 10

16

94

C to

G

Ser t

o C

ys

S489

C

c.14

66C

>G

p.Se

r489

Cys

M

3

03

BR

CA

2 10

17

00

C to

G

Thr t

o Se

r T4

91S

c.14

72C

>G

p.Th

r491

Ser

M

10

1 B

RC

A2

10

1742

T

to C

Ile

to T

hr

I505

T c.

1514

T>C

p.

Ile50

5Thr

M

2

02

BR

CA

2 10

20

14

G to

C

Asp

to H

is

D59

6H

c.17

86G

>C

p.A

sp59

6His

M

3

21

BR

CA

2 10

20

16

T to

C

Asp

to A

sp

D59

6D

c.17

88T>

C

p.=

Syn

43

1 B

RC

A2

10

2020

A

to G

Th

r to

Ala

T5

98A

c.

1792

A>G

p.

Thr5

98A

la

M

115

6 B

RC

A2

10

2032

G

to A

G

ly to

Arg

G

602R

c.

1804

G>A

p.

Gly

602A

rg

M

11

0 B

RC

A2

10

2046

G

to A

Pr

o to

Pro

P6

06P

c.18

18G

>A

p.=

Syn

10

1 B

RC

A2

10

2117

C

to T

Th

r to

Ile

T630

I c.

1889

C>T

p.

Thr6

30Ile

M

1

01

BR

CA

2 10

21

37+2

3 in

s T

-

IVS1

0+23

insT

c.

1909

+23i

nsT

IV

S 5

05

BR

CA

2 11

21

39

T to

C

Gly

to G

ly

G63

7G

c.19

11T>

C

p.=

Syn

21

1 B

RC

A2

11

2166

C

to T

Se

r to

Ser

S646

S c.

1938

C>T

p.

= Sy

n 6

15

BR

CA

2 11

21

92

C to

G

Pro

to A

rg

P655

R

c.19

64C

>G

p.Pr

o655

Arg

M

7

25

BR

CA

2 11

23

10

T to

C

Asn

to A

sn

N69

4N

c.20

82T>

C

p.=

Syn

11

0 B

RC

A2

11

2457

T

to C

H

is to

His

H

743H

c.

2229

T>C

p.

= Sy

n 14

449

95

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

25

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

11

2578

A

to G

M

et to

Val

M

784V

c.

2350

A>G

p.

Met

784V

al

M

20

2 B

RC

A2

11

2640

A

to G

G

lu to

Glu

E8

04E

c.24

12A

>G

p.=

Syn

11

0 B

RC

A2

11

2778

A

to G

G

ln to

Gln

Q

850Q

c.

2550

A>G

p.

= Sy

n 1

01

BR

CA

2 11

29

37

A to

G

Leu

to L

eu

L903

L c.

2709

A>G

p.

= Sy

n 1

01

BR

CA

2 11

30

31

G to

A

Asp

to A

sn

D93

5N

c.28

03G

>A

p.A

sp93

5Asn

M

1

10

BR

CA

2 11

30

69

G to

T

Leu

to P

he

L947

F c.

2841

G>T

p.

Leu9

47Ph

e M

1

10

BR

CA

2 11

31

11

G to

A

Gln

to G

ln

Q96

1Q

c.28

83G

>A

p.=

Syn

41

3 B

RC

A2

11

3147

G

to A

Se

r to

Ser

S973

S c.

2919

G>A

p.

= Sy

n 1

01

BR

CA

2 11

31

85

A to

G

Asn

to S

er

N98

6S

c.29

57A

>G

p.A

sn98

6Ser

M

1

10

BR

CA

2 11

31

91

A to

C

Asp

to A

la

D98

8A

c.29

63A

>C

p.A

sp98

8Ala

M

1

10

BR

CA

2 11

31

99

A to

G

Asn

to A

sp

N99

1D

c.29

71A

>G

p.A

sn99

1Asp

M

14

851

97

BR

CA

2 11

32

83

C to

G

Leu

to V

al

L101

9V

c.30

55C

>G

p.Le

u101

9Val

M

1

01

BR

CA

2 11

34

14

T to

C

Pro

to P

ro

P106

2P

c.31

86T>

C

p.=

Syn

10

1 B

RC

A2

11

3444

A

to G

Le

u to

Leu

L1

072L

c.

3216

A>G

p.

= Sy

n 1

01

BR

CA

2 11

34

92

T to

C

Pro

to P

ro

P108

8P

c.32

64T>

C

p.=

Syn

40

4 B

RC

A2

11

3624

A

to G

Ly

s to

Lys

K11

32K

c.

3396

A>G

p.

= Sy

n 92

631

860

8 B

RC

A2

11

3645

G

to A

Ly

s to

Lys

K11

39K

c.

3417

G>A

p.

= Sy

n 1

01

BR

CA

2 11

36

48

T to

C

Ser t

o Se

r S1

140S

c.

3420

T>C

p.

= Sy

n 2

02

BR

CA

2 11

37

44

G to

A

Ser t

o Se

r S1

172S

c.

3516

G>A

p.

= Sy

n 4

04

BR

CA

2 11

39

45

A to

G

Lys t

o Ly

s K

1239

K

c.37

17A

>G

p.=

Syn

11

0 B

RC

A2

11

4035

T

to C

V

al to

Val

V

1269

V

c.38

07T>

C

p.=

Syn

651

212

439

BR

CA

2 11

40

42

A to

G

Met

to V

al

M12

72V

c.

3814

A>G

p.

Met

1272

Val

M

1

01

BR

CA

2 11

40

62

T to

C

His

to H

is

H12

78H

c.

3834

T>C

p.

= Sy

n 1

01

BR

CA

2 11

40

76

T to

C

Val

to A

la

V12

83A

c.

3848

T>C

p.

Val

1283

Ala

M

1

10

BR

CA

2 11

40

97

G to

A

Cys

to T

yr

C12

90Y

c.

3869

G>A

p.

Cys

1290

Tyr

M

41

3 B

RC

A2

11

4144

G

to A

V

al to

Ile

V13

06I

c.39

16G

>A

p.V

al13

06Ile

M

1

10

BR

CA

2 11

42

89

C to

T

Thr t

o M

et

T135

4M

c.40

61C

>T

p.Th

r135

4Met

M

2

11

BR

CA

2 11

42

96

G to

A

Leu

to L

eu

L135

6L

c.40

68G

>A

p.=

Syn

156

9 B

RC

A2

11

4318

A

to C

Ile

to L

eu

I136

4L

c.40

90A

>C

p.Ile

1364

Leu

M

20

2 B

RC

A2

11

4415

A

to G

G

ln to

Arg

Q

1396

R

c.41

87A

>G

p.G

ln13

96A

rg

M

11

0 B

RC

A2

11

4452

G

to A

G

ln to

Gln

Q

1408

Q

c.42

24G

>A

p.=

Syn

11

0 B

RC

A2

11

4469

C

to T

Th

r to

Met

T1

414M

c.

4241

C>T

p.

Thr1

414M

et

M

10

1 B

RC

A2

11

4486

G

to T

A

sp to

Tyr

D

1420

Y

c.42

58G

>T

p.A

sp14

20Ty

r M

31

625

B

RC

A2

11

4587

A

to G

Ly

s to

Lys

K14

53K

c.

4359

A>G

p.

= Sy

n 1

01

BR

CA

2 11

46

64

G to

C

Ser t

o Th

r S1

479T

c.

4436

G>C

p.

Ser1

479T

hr

M

20

2 B

RC

A2

11

4789

C

to T

Le

u to

Leu

L1

521L

c.

4561

C>T

p.

= Sy

n 1

10

BR

CA

2 11

47

91

G to

A

Leu

to L

eu

L152

1L

c.45

63G

>A

p.=

Syn

52

3 B

RC

A2

11

4813

G

to A

G

ly to

Arg

G

1529

R

c.45

85G

>A

p.G

ly15

29A

rg

M

22

0

E 12

26 B

org

et al

.

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

11

4842

T

to C

Se

r to

Ser

S153

8S

c.46

14T>

C

p.=

Syn

10

1 B

RC

A2

11

4909

C

to A

H

is to

Asn

H

1561

N

c.46

81C

>A

p.H

is15

61A

sn

M

21

1 B

RC

A2

11

4995

A

to G

Pr

o to

Pro

P1

589P

c.

4767

A>G

p.

= Sy

n 1

10

BR

CA

2 11

50

42

T to

C

Val

to A

la

V16

05A

c.

4814

T>C

p.

Val

1605

Ala

M

1

01

BR

CA

2 11

50

58

G to

A

Val

to V

al

V16

10V

c.

4830

G>A

p.

= Sy

n 1

10

BR

CA

2 11

50

93

G to

C

Arg

to T

hr

R16

22T

c.48

65G

>C

p.A

rg16

22Th

r M

1

01

BR

CA

2 11

51

55

G to

A

Val

to Il

e V

1643

I c.

4927

G>A

p.

Val

1643

Ile

M

11

0 B

RC

A2

11

5248

A

to G

Se

r to

Gly

S1

674G

c.

5020

A>G

p.

Ser1

674G

ly

M

11

0 B

RC

A2

11

5279

C

to G

Th

r to

Ser

T168

4S

c.50

51C

>G

p.Th

r168

4Ser

M

1

01

BR

CA

2 11

54

19

C to

T

His

to T

yr

H17

31Y

c.

5191

C>T

p.

His

1731

Tyr

M

10

1 B

RC

A2

11

5426

C

to T

Se

r to

Phe

S173

3F

c.51

98C

>T

p.Se

r173

3Phe

M

1

10

BR

CA

2 11

54

27

C to

T

Ser t

o Se

r S1

733S

c.

5199

C>T

p.

= Sy

n 18

513

B

RC

A2

11

5547

G

to A

G

lu to

Glu

E1

773E

c.

5319

G>A

p.

= Sy

n 1

01

BR

CA

2 11

56

06

A to

G

Asn

to S

er

N17

93S

c.53

78A

>G

p.A

sn17

93Se

r M

1

01

BR

CA

2 11

56

46

A to

G

Glu

to G

lu

E180

6E

c.54

18A

>G

p.=

Syn

30

3 B

RC

A2

11

5723

C

to A

Se

r to

Tyr

S183

2Y

c.54

95C

>A

p.Se

r183

2Tyr

M

1

01

BR

CA

2 11

58

06

A to

G

Lys t

o G

lu

K18

60E

c.55

78A

>G

p.Ly

s186

0Glu

M

1

01

BR

CA

2 11

58

62

C to

G

Asn

to L

ys

N18

78K

c.

5634

C>G

p.

Asn

1878

Lys

M

22

0 B

RC

A2

11

5863

G

to A

G

lu to

Lys

E1

879K

c.

5635

G>A

p.

Glu

1879

Lys

M

11

0 B

RC

A2

11

5868

T

to G

A

sn to

Lys

N

1880

K

c.56

40T>

G

p.A

sn18

80Ly

s M

3

21

BR

CA

2 11

58

87

A to

G

Thr t

o A

la

T188

7A

c.56

59A

>G

p.Th

r188

7Ala

M

1

01

BR

CA

2 11

58

96

A to

G

Met

to V

al

M18

90V

c.

5668

A>G

p.

Met

1890

Val

M

1

01

BR

CA

2 11

59

11

G to

A

Glu

to L

ys

E189

5K

c.56

83G

>A

p.G

lu18

95Ly

s M

1

01

BR

CA

2 11

59

32

G to

A

Asp

to A

sn

D19

02N

c.

5704

G>A

p.

Asp

1902

Asn

M

1

01

BR

CA

2 11

59

72

C to

T

Thr t

o M

et

T191

5M

c.57

44C

>T

p.Th

r191

5Met

M

10

830

78

BR

CA

2 11

59

96

A to

C

Asp

to A

la

D19

23A

c.

5768

A>C

p.

Asp

1923

Ala

M

1

01

BR

CA

2 11

60

13

A to

G

Ile to

Val

I1

929V

c.

5785

A>G

p.

Ile19

29V

al

M

10

1 B

RC

A2

11

6107

G

to A

C

ys to

Tyr

C

1960

Y

c.58

79G

>A

p.C

ys19

60Ty

r M

1

10

BR

CA

2 11

61

72

A to

G

Ser t

o G

ly

S198

2G

c.59

44A

>G

p.Se

r198

2Gly

M

1

01

BR

CA

2 11

63

28

C to

T

Arg

to C

ys

R20

34C

c.

6100

C>T

p.

Arg

2034

Cys

M

21

615

B

RC

A2

11

6347

T

to A

Ile

to L

ys

I204

0K

c.61

19T>

A

p.Ile

2040

Lys

M

11

0 B

RC

A2

11

6545

T

to C

Le

u to

Pro

L2

106P

c.

6317

T>C

p.

Leu2

106P

ro

M

10

1 B

RC

A2

11

6550

C

to T

A

rg to

Cys

R

2108

C

c.63

22C

>T

p.A

rg21

08C

ys

M

21

1 B

RC

A2

11

6551

G

to A

A

rg to

His

R

2108

H

c.63

23G

>A

p.A

rg21

08H

is

M

72

5 B

RC

A2

11

6575

A

to G

H

is to

Arg

H

2116

R

c.63

47A

>G

p.H

is21

16A

rg

M

21

1 B

RC

A2

11

6640

G

to T

V

al to

Phe

V

2138

F c.

6412

G>T

p.

Val

2138

Phe

M

21

1 B

RC

A2

11

6683

C

to A

Se

r to

Tyr

S215

2Y

c.64

55C

>A

p.Se

r215

2Tyr

M

1

01

BR

CA

2 11

66

93

C to

T

Leu

to L

eu

L215

5L

c.64

65C

>T

p.=

Syn

11

0

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

27

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

11

6717

A

to G

G

ln to

Gln

Q

2163

Q

c.64

89A

>G

p.=

Syn

11

0 B

RC

A2

11

6741

C

to G

V

al to

Val

V

2171

V

c.65

13C

>G

p.=

Syn

42

2 B

RC

A2

11

6841

G

to A

V

al to

Met

V

2205

M

c.66

13G

>A

p.V

al22

05M

et

M

10

1 B

RC

A2

11

6976

A

to G

Th

r to

Ala

T2

250A

c.

6748

A>G

p.

Thr2

250A

la

M

21

1 B

RC

A2

11

7049

G

to T

G

ly to

Val

G

2274

V

c.68

21G

>T

p.G

ly22

74V

al

M

10

1 B

RC

A2

11

7069

+11

A to

G

-

IVS1

1+11

A>G

c.

6841

+11A

>G

IV

S 1

01

BR

CA

2 11

70

69+3

3 A

to G

-

IVS1

1+33

A>G

c.

6841

+33A

>G

IV

S 1

10

BR

CA

2 11

70

69+3

6 A

to G

-

IVS1

1+36

A>G

c.

6841

+36A

>G

IV

S 1

10

BR

CA

2 12

70

70-7

3 T

to A

-

IVS1

1-73

T>A

c.

6842

-73T

>A

IV

S 1

10

BR

CA

2 12

70

70-2

2 T

to A

-

IVS1

1-22

T>A

c.

6842

-22T

>A

IV

S 1

10

BR

CA

2 12

70

81

A to

G

Ile to

Val

I2

285V

c.

6853

A>G

p.

Ile22

85V

al

M

40

4 B

RC

A2

12

7149

A

to G

Se

r to

Ser

S230

7S

c.69

21A

>G

p.=

Syn

10

1 B

RC

A2

13

7171

A

to C

Ile

to L

eu

I231

5L

c.69

43A

>C

p.Ile

2315

Leu

M

10

1 B

RC

A2

13

7235

+4

A to

G

- IV

S13+

4A>G

c.

7007

+4A

>G

IV

S 1

01

BR

CA

2 13

72

35+5

2 C

to A

-

IVS1

3+52

C>A

c.

7007

+52C

>A

IV

S 1

01

BR

CA

2 14

72

36-6

2 A

to G

-

IVS1

3-62

A>G

c.

7008

-62A

>G

IV

S 1

10

BR

CA

2 14

72

45

G to

C

Lys t

o A

sn

K23

39N

c.

7017

G>C

p.

Lys2

339A

sn

M

20

2 B

RC

A2

14

7275

T

to A

Ph

e to

Leu

F2

349L

c.

7047

T>A

p.

Phe2

349L

eu

M

11

0 B

RC

A2

14

7280

C

to G

A

la to

Gly

A

2351

G

c.70

52C

>G

p.A

la23

51G

ly

M

11

0 B

RC

A2

14

7285

G

to C

G

ly to

Arg

G

2353

R

c.70

57G

>C

p.G

ly23

53A

rg

M

11

0 B

RC

A2

14

7378

C

to A

G

ln to

Lys

Q

2384

K

c.71

50C

>A

p.G

ln23

84Ly

s M

1

01

BR

CA

2 14

74

60

A to

C

Lys t

o Th

r K

2411

T c.

7232

A>C

p.

Lys2

411T

hr

M

10

1 B

RC

A2

14

7470

A

to G

Se

r to

Ser

S241

4S

c.72

42A

>G

p.=

Syn

743

245

498

BR

CA

2 14

75

47

A to

G

His

to A

rg

H24

40R

c.

7319

A>G

p.

His

2440

Arg

M

3

12

BR

CA

2 14

76

25

C to

T

Ala

to V

al

A24

66V

c.

7397

C>T

p.

Ala

2466

Val

M

3

12

BR

CA

2 14

76

41

A to

G

Thr t

o Th

r T2

471T

c.

7413

A>G

p.

= Sy

n 1

01

BR

CA

2 14

76

63+5

T

to C

-

IVS1

4+5T

>C

c.74

35+5

T>C

IVS

10

1 B

RC

A2

14

7663

+23

A to

G

- IV

S14+

23A

>G

c.74

35+2

3A>G

IVS

10

1 B

RC

A2

14

7663

+53

C to

T

-

IVS1

4+53

C>T

c.

7435

+53C

>T

IV

S 2

20

BR

CA

2 15

76

97

T to

C

Ile to

Thr

I2

490T

c.

7469

T>C

p.

Ile24

90Th

r M

11

56

BR

CA

2 15

77

78

C to

G

Thr t

o Se

r T2

517S

c.

7550

C>G

p.

Thr2

517S

er

M

10

1 B

RC

A2

15

7845

+15

A to

G

- IV

S15+

15A

>G

c.76

17+1

5A>G

IVS

10

1 B

RC

A2

15

7845

+17

C to

T

- IV

S15+

17C

>T

c.76

17+1

7C>T

IVS

10

1 B

RC

A2

16

7854

G

to A

Th

r to

Thr

T254

2T

c.76

26G

>A

p.=

Syn

20

2 B

RC

A2

16

8033

+6

C to

G

- IV

S16+

6C>G

c.

7805

+6C

>G

IV

S 2

11

BR

CA

2 17

80

34-4

0 A

to G

-

IVS1

6-40

A>G

c.

7806

-40A

>G

IV

S 4

13

BR

CA

2 17

80

34-3

9 A

to G

-

IVS1

6-39

A>G

c.

7806

-39A

>G

IV

S 1

10

BR

CA

2 17

80

34-1

4 T

to C

-

IVS1

6-14

T>C

c.

7806

-14T

>C

IV

S 11

2239

173

1

E 12

28 B

org

et al

.

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

17

8106

G

to C

Tr

p to

Cys

W

2626

C

c.78

78G

>C

p.Tr

p262

6Cys

M

1

10

BR

CA

2 17

81

07

A to

T

Ile to

Phe

I2

627F

c.

7879

A>T

p.

Ile26

27Ph

e M

1

01

BR

CA

2 17

81

42

T to

G

Phe

to L

eu

F263

8L

c.79

14T>

G

p.Ph

e263

8Leu

M

1

01

BR

CA

2 17

82

04+3

5 C

to A

-

IVS1

7+35

C>A

c.

7976

+35C

>A

IV

S 2

02

BR

CA

2 17

82

04+3

6 G

to A

-

IVS1

7+36

G>A

c.

7976

+36G

>A

IV

S 1

01

BR

CA

2 18

82

16

A to

T

Glu

to V

al

E266

3V

c.79

88A

>T

p.G

lu26

63V

al

M

10

1 B

RC

A2

18

8222

A

to G

A

sp to

Gly

D

2665

G

c.79

94A

>G

p.A

sp26

65G

ly

M

21

1 B

RC

A2

18

8255

T

to C

M

et to

Thr

M

2676

T c.

8027

T>C

p.

Met

2676

Thr

M

10

1 B

RC

A2

18

8267

A

to G

A

sp to

Gly

D

2680

G

c.80

39A

>G

p.A

sp26

80G

ly

M

10

1 B

RC

A2

18

8318

G

to A

Se

r to

Asn

S2

697N

c.

8090

G>A

p.

Ser2

697A

sn

M

11

0 B

RC

A2

18

8362

G

to A

A

sp to

Asn

D

2712

V

c.81

34G

>A

p.A

sp27

12A

sn

M

11

0 B

RC

A2

18

8377

G

to T

A

la to

Ser

A

2717

S c.

8149

G>T

p.

Ala

2717

Ser

M

63

3 B

RC

A2

18

8410

G

to A

V

al to

Ile

V27

28I

c.81

82G

>A

p.V

al27

28Ile

M

10

28

BR

CA

2 19

86

88

A to

C

Val

to V

al

V28

20V

c.

8460

A>C

p.

= Sy

n 7

34

BR

CA

2 20

87

95

A to

C

Glu

to A

la

E285

6A

c.85

67A

>C

p.G

lu28

56A

la

M

154

11

BR

CA

2 20

87

97

G to

A

Ala

to T

hr

A28

57T

c.85

69G

>A

p.A

la28

57Th

r M

1

01

BR

CA

2 20

88

60+1

4 T

to C

-

IVS2

0+14

T>C

c.

8632

+14T

>C

IV

S 1

01

BR

CA

2 20

88

60+1

43

insC

-

IVS2

0+14

3ins

C

c.86

32+1

43in

sC

IV

S 1

10

BR

CA

2 21

88

61-1

6 C

to G

-

IVS2

0-16

C>G

c.

8633

-16C

>G

IV

S 2

02

BR

CA

2 21

88

75

C to

T

Pro

to S

er

P288

3S

c.86

47C

>T

p.Pr

o288

3Ser

M

1

01

BR

CA

2 21

89

82+7

5 A

to G

-

IVS2

1+75

A>G

c.

8754

+75A

>G

IV

S 1

01

BR

CA

2 22

90

58

A to

T

Ile to

Phe

I2

944F

c.

8830

A>T

p.

Ile29

44Ph

e M

2

11

BR

CA

2 22

90

78

G to

T

Lys t

o A

sn

K29

50N

c.

8850

G>T

p.

Lys2

950A

sn

M

33

0 B

RC

A2

22

9079

G

to A

A

la to

Thr

A

2951

T c.

8851

G>A

p.

Ala

2951

Thr

M

2210

12

BR

CA

2 22

91

30

A to

C

Thr t

o Pr

o T2

968P

c.

8902

A>C

p.

Thr2

968P

ro

M

21

1 B

RC

A2

22

9133

G

to A

V

al to

Met

V

2969

M

c.89

05G

>A

p.V

al29

69M

et

M

10

1 B

RC

A2

23

9332

A

to G

Ty

r to

Cys

Y

3035

C

c.91

04A

>G

p.Ty

r303

5Cys

M

2

11

BR

CA

2 24

93

82

C to

T

Arg

to T

rp

R30

52W

c.

9154

C>T

p.

Arg

3052

Trp

M

10

1 B

RC

A2

24

9463

G

to A

V

al to

Ile

V30

79I

c.92

35G

>A

p.V

al30

79Ile

M

1

01

BR

CA

2 24

94

84+2

8 de

l T

- IV

S24+

28de

lT

c.92

56+2

8del

T

IVS

22

0 B

RC

A2

25

9485

-113

T

to G

-

IVS2

4-11

3T>G

c.

9257

-113

T>G

IVS

50

5 B

RC

A2

25

9485

-110

A

to G

-

IVS2

4-11

0A>G

c.

9257

-110

A>G

IVS

10

1 B

RC

A2

25

9485

-83

G to

A

- IV

S24-

83G

>A

c.92

57-8

3G>A

IVS

188

10

BR

CA

2 25

94

85-4

9 T

to C

-

IVS2

4-49

T>C

c.

9257

-49T

>C

IV

S 1

10

BR

CA

2 25

94

85-1

6 T

to C

-

IVS2

4-16

T>C

c.

9257

-16T

>C

IV

S 34

826

B

RC

A2

25

9520

T

to C

Ty

r to

His

Y

3098

H

c.92

92T>

C

p.Ty

r309

8His

M

1

01

BR

CA

2 25

95

29

C to

T

Leu

to L

eu

L310

1L

c.93

01C

>T

p.=

Syn

10

1 B

RC

A2

25

9594

A

to G

A

la to

Ala

A

3122

A

c.93

66A

>G

p.=

Syn

10

1

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

29

Gen

eE

xon

NT

1B

ase

Ch

AA

Cha

nge

BIC

2H

GV

3 DN

A

HG

V p

rote

in

Typ

e4T

otal

5C

BC

UB

CB

RC

A2

25

9639

T

to G

Th

r to

Thr

T313

7T

c.94

11T>

G

p.=

Syn

11

0 B

RC

A2

26

9741

A

to G

Ile

to M

et

I317

1M

c.95

13A

>G

p.Ile

3171

Met

M

1

10

BR

CA

2 26

98

34

G to

C

Pro

to P

ro

P320

2P

c.96

06G

>C

p.=

Syn

11

0 B

RC

A2

26

9876

+54

G to

A

- IV

S26+

54G

>A

c.96

48+5

4G>A

IVS

21

1 B

RC

A2

26

9876

+84

G to

A

- IV

S26+

84G

>A

c.96

48+8

4G>A

IVS

123

9 B

RC

A2

26

9876

+106

de

l T

- IV

S26+

106d

elT

c.96

48+1

06de

lT

IV

S 5

23

BR

CA

2 27

98

77-6

7 de

l TTA

C

- IV

S26-

67de

l4

c.96

49-6

7del

TTA

C

IV

S 1

01

BR

CA

2 27

98

77-1

9 G

to A

-

IVS2

6-19

G>A

c.

9649

-19G

>A

IV

S 2

02

BR

CA

2 27

99

58

G to

A

Val

to Il

e V

3244

I c.

9730

G>A

p.

Val

3244

Ile

M

21

1 B

RC

A2

27

1010

3 C

to T

Pr

o to

Leu

P3

292L

c.

9875

C>T

p.

Pro3

292L

eu

M

10

1 B

RC

A2

27

1020

4 A

to T

Ly

s to

stop

K

3326

X

c.99

76A

>T

p.Ly

s332

6X

N

409

31

BR

CA

2 27

10

273

A to

G

Thr t

o A

la

T334

9A

c.10

045A

>G

p.Th

r334

9Ala

M

1

01

BR

CA

2 27

10

323

del C

ins

11

Stop

336

9 10

323d

elC

ins1

1 c.

1009

5del

Cin

s11

p.Se

r336

6fs

F 2

11

BR

CA

2 27

10

338

G to

A

Arg

to A

rg

R33

70R

c.

1011

0G>A

p.

= Sy

n 8

35

BR

CA

2 27

10

339

A to

G

Thr t

o A

la

T337

1A

c.10

111A

>G

p.Th

r337

1Ala

M

3

03

BR

CA

2 27

10

378

C to

T

Arg

to S

top

R33

84X

c.

1015

0C>T

p.

Arg

3384

X

N

11

0 B

RC

A2

27

1043

1 G

to A

Th

r to

Thr

T340

1T

c.10

203G

>A

p.=

Syn

10

1 B

RC

A2

27

1046

2 A

to G

Ile

to V

al

I341

2V

c.10

234A

>G

p.Ile

3412

Val

M

28

1018

B

RC

A2

27

1051

6 C

to T

3'

UTR

U

AA

+31C

>T

c.*3

1C>T

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1

01

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CA

1: n

ucle

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Gen

Ban

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http

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rez/

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1 BR

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2: n

ucle

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ng to

Gen

Ban

k N

M_0

0005

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://w

ww

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i.nlm

.nih

.gov

/ent

rez/

view

er.fc

gi?d

b=nu

cleo

tide&

val=

4502

450

2 BIC

(Bre

ast C

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r Inf

orm

atio

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ore)

. http

://re

sear

ch.n

hgri.

nih.

gov/

bic/

3 H

GV

S (H

uman

Gen

ome

Var

iatio

n So

ciet

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ttp://

ww

w.h

gvs.o

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l4 T

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ear

lier d

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org

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GV

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Puf

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1B

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K

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Sy

n

B

RC

A1

3 25

3 A

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K

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M

C

098

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30.7

5

C25

26.0

0 66

.45

C

2526

.00

66.4

5 B

RC

A1

5 25

4-53

T

to C

IV

S4-5

3T>C

IV

S

B

RC

A1

5 25

4-23

C

to T

IV

S4-2

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IV

S

B

RC

A1

5 33

1+23

T

to A

IV

S5+2

3T>A

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S

B

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A1

7 42

1-70

G

to A

IV

S6-7

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S

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RC

A1

7 43

3 A

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Y

105C

M

C15

120.

91

131.

30

C15

120.

91

131.

30

C

1511

7.31

13

1.30

B

RC

A1

7 54

4 C

to A

P1

42H

M

C0

353.

86

0.00

C0

226.

93

11.1

5

C0

97.5

9 11

.15

BR

CA

1 7

560+

36

del 1

4 IV

S7+3

6del

14

IVS

BR

CA

1 8

561-

34

C to

T

IVS7

-34C

>T

IVS

BR

CA

1 8

628

G to

A

R17

0Q

M

C

057

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0.00

C0

57.0

3 0.

00

C

056

.64

0.00

B

RC

A1

8 65

5 A

to G

Y

179C

M

C35

40.2

2 18

2.30

C35

40.2

2 18

2.30

C45

35.6

8 18

4.08

B

RC

A1

9 66

7-80

T>

C

IVS8

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IVS

BR

CA

1 9

667-

58

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IV

S8-5

8del

T IV

S

B

RC

A1

9 66

7-17

G

to T

IV

S8-1

7G>T

IV

S

B

RC

A1

9 66

7-3

del T

IV

S8-3

delT

IV

S

B

RC

A1

9 71

0 C

to T

C

197C

Sy

n

B

RC

A1

10

713-

72

A>G

IV

S9-7

2A>G

IV

S

B

RC

A1

10

713-

34

T to

C

IVS9

-34T

>C

IVS

BR

CA

1 10

76

0 A

to G

D

214G

M

C0

353.

86

0.00

C0

353.

86

0.00

C0

271.

19

0.00

B

RC

A1

11

812

G to

A

T231

T Sy

n

B

RC

A1

11

855

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G

L246

V

M

C

025

1.61

0.

00

C

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5.27

0.

00

C

030

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0.00

B

RC

A1

11

944

C to

T

G27

5G

Syn

BR

CA

1 11

10

67

C to

G

S316

R

M

C

022

0.50

63

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C

021

7.19

68

.90

C

2546

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83.2

3 B

RC

A1

11

1100

A

to G

T3

27T

Syn

BR

CA

1 11

11

52

G to

T

D34

5Y

M

C

010

6.33

70

.69

C

010

6.33

70

.69

C

010

6.33

70

.69

BR

CA

1 11

11

84

G to

A

K35

5K

Syn

BR

CA

1 11

11

86

A to

G

Q35

6R

M

C

025

5.29

19

.25

C

023

8.22

28

.37

C

010

1.52

28

.37

BR

CA

1 11

12

24

del G

AT

D36

9del

IF

D

BRCA

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BRC

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eque

nce V

arian

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later

al an

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later

al Br

east

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cer

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31

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GV

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A

GV

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NT

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Ch

BIC

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Cla

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GV

GD

Cla

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DC

lass

GV

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BR

CA

1 11

12

56

T to

G

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M

M

C

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00

C

092

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8 0.

00

BR

CA

1 11

15

75

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C

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L M

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33

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C0

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92

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C0

212.

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1605

C

to T

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95

56.6

4

C0

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95

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4

C15

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47

112.

77

BR

CA

1 11

16

06

G to

A

R49

6H

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C

024

4.95

0.

00

C

024

4.95

0.

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C

013

9.47

0.

00

BR

CA

1 11

17

28

A to

G

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7D

M

C

096

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C0

96.2

0 0.

00

C

096

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B

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11

1767

A

to C

N

550H

M

C0

151.

68

0.00

C0

118.

33

4.81

C0

79.7

9 26

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BR

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1 11

18

31

T to

C

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T M

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171.

12

0.00

C0

142.

37

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C0

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37

0.00

B

RC

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1908

G

to A

E5

97K

M

C0

152.

38

0.00

C0

152.

38

0.00

C0

128.

69

0.00

B

RC

A1

11

1965

de

l TC

T S6

16de

l IF

D

BR

CA

1 11

19

84

C to

T

A62

2V

M

C

022

4.84

2.

03

C

022

4.84

2.

03

C

022

4.84

2.

03

BR

CA

1 11

20

30

T to

C

T637

T Sy

n

B

RC

A1

11

2090

A

to G

Q

657Q

Sy

n

B

RC

A1

11

2196

G

to A

D

693N

M

C0

176.

25

0.00

C0

176.

25

0.00

C0

170.

55

0.00

B

RC

A1

11

2201

C

to T

S6

94S

Syn

BR

CA

1 11

24

18

A to

G

S767

G

M

C

016

7.35

29

.38

C

016

7.35

29

.38

C

016

7.35

29

.38

BR

CA

1 11

24

30

T to

C

L771

L Sy

n

B

RC

A1

11

2471

G

to A

S7

84S

Syn

BR

CA

1 11

25

35

G to

A

A80

6T

M

C

025

4.20

0.

00

C

025

4.20

0.

00

C

015

5.03

0.

00

BR

CA

1 11

25

77

A to

G

K82

0E

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

026

2.85

0.

00

BR

CA

1 11

25

92

G to

T

D82

5Y

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

025

1.13

51

.67

BR

CA

1 11

25

96

C to

A

T826

K

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

021

5.31

73

.23

BR

CA

1 11

26

02

del G

CT

G82

8del

IF

D

BR

CA

1 11

26

37

A to

G

S840

G

M

C

014

4.64

28

.37

C

014

4.64

28

.37

C

012

3.92

28

.37

BR

CA

1 11

26

40

C to

T

R84

1W

M

C

1511

1.49

78

.29

C

1511

1.49

78

.29

C

1510

9.21

85

.03

BR

CA

1 11

26

41

G to

A

R84

1Q

M

C

011

1.49

0.

00

C

011

1.49

0.

00

C

010

9.21

0.

00

BR

CA

1 11

26

85

T to

C

Y85

6H

M

C

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D X

enop

us

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eE

xon

NT

1B

ase

Ch

BIC

2T

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Cla

ss5

GV

GD

Cla

ssG

VG

DC

lass

GV

GD

BR

CA

2 10

20

16

T to

C

D59

6D

Syn

BR

CA

2 10

20

20

A to

G

T598

A

M

C

017

0.63

0.

00

C

016

7.89

0.

00

C

014

4.64

0.

00

BR

CA

2 10

20

32

G to

A

G60

2R

M

C

017

4.09

13

.17

C

017

4.09

13

.17

C

017

0.79

13

.17

BR

CA

2 10

20

46

G to

A

P606

P Sy

n

B

RC

A2

10

2117

C

to T

T6

30I

M

C

035

3.86

0.

00

C

024

8.25

28

.78

C

020

2.47

39

.32

BR

CA

2 10

21

37+2

3 in

s T

IVS1

0+23

insT

IV

S

B

RC

A2

11

2139

T

to C

G

637G

Sy

n

B

RC

A2

11

2166

C

to T

S6

46S

Syn

BR

CA

2 11

21

92

C to

G

P655

R

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

026

1.33

13

.17

BR

CA

2 11

23

10

T to

C

N69

4N

Syn

BR

CA

2 11

24

57

T to

C

H74

3H

Syn

BR

CA

2 11

25

78

A to

G

M78

4V

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

025

8.32

0.

00

BR

CA

2 11

26

40

A to

G

E804

E Sy

n

B

RC

A2

11

2778

A

to G

Q

850Q

Sy

n

B

RC

A2

11

2937

A

to G

L9

03L

Syn

BR

CA

2 11

30

31

G to

A

D93

5N

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

027

1.19

0.

00

BR

CA

2 11

30

69

G to

T

L947

F M

C0

353.

86

0.00

C0

353.

86

0.00

C0

353.

86

0.00

B

RC

A2

11

3111

G

to A

Q

961Q

Sy

n

B

RC

A2

11

3147

G

to A

S9

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Syn

BR

CA

2 11

31

85

A to

G

N98

6S

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C

035

3.86

0.

00

C

021

7.19

0.

00

C

021

7.19

0.

00

BR

CA

2 11

31

91

A to

C

D98

8A

M

C

035

3.86

0.

00

C

027

6.16

0.

00

C

027

6.16

0.

00

BR

CA

2 11

31

99

A to

G

N99

1D

M

C

035

3.86

0.

00

C

026

8.84

0.

00

C

026

6.51

0.

00

BR

CA

2 11

32

83

C to

G

L101

9V

M

C

024

6.16

0.

00

C

024

6.16

0.

00

C

024

5.67

11

.33

BR

CA

2 11

34

14

T to

C

P106

2P

Syn

BR

CA

2 11

34

44

A to

G

L107

2L

Syn

BR

CA

2 11

34

92

T to

C

P108

8P

Syn

BR

CA

2 11

36

24

A to

G

K11

32K

Sy

n

B

RC

A2

11

3645

G

to A

K

1139

K

Syn

BR

CA

2 11

36

48

T to

C

S114

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Syn

BR

CA

2 11

37

44

G to

A

S117

2S

Syn

BR

CA

2 11

39

45

A to

G

K12

39K

Sy

n

B

RC

A2

11

4035

T

to C

V

1269

V

Syn

BR

CA

2 11

40

42

A to

G

M12

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M

C0

353.

86

0.00

C0

353.

86

0.00

C0

235.

10

0.00

B

RC

A2

11

4062

T

to C

H

1278

H

Syn

BR

CA

2 11

40

76

T to

C

V12

83A

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C0

353.

86

0.00

C0

353.

86

0.00

C0

246.

16

35.6

0 B

RC

A2

11

4097

G

to A

C

1290

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M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

024

8.25

53

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E 12

36 B

org

et al

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A

GV

GD

4 Sea

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A

GV

GD

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BR

CA

2 11

41

44

G to

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V13

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C

035

3.86

0.

00

C

024

0.36

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00

C

096

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0.00

B

RC

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to T

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86

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353.

86

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C0

246.

14

46.9

3 B

RC

A2

11

4296

G

to A

L1

356L

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n

B

RC

A2

11

4318

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to C

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353.

86

0.00

C0

353.

86

0.00

C0

260.

21

4.86

B

RC

A2

11

4415

A

to G

Q

1396

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M

C

035

3.86

0.

00

C

023

3.41

39

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C

010

7.73

39

.51

BR

CA

2 11

44

52

G to

A

Q14

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n

B

RC

A2

11

4469

C

to T

T1

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M

C0

261.

51

0.00

C0

261.

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0.00

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261.

51

0.00

B

RC

A2

11

4486

G

to T

D

1420

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M

C

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9.25

62

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9.25

62

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C

1587

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87.6

7 B

RC

A2

11

4587

A

to G

K

1453

K

Syn

BR

CA

2 11

46

64

G to

C

S147

9T

M

C

025

4.15

0.

00

C

025

4.15

0.

00

C

024

4.14

0.

00

BR

CA

2 11

47

89

C to

T

L152

1L

Syn

BR

CA

2 11

47

91

G to

A

L152

1L

Syn

BR

CA

2 11

48

13

G to

A

G15

29R

M

C65

0.00

12

5.13

C65

0.00

12

5.13

C65

0.00

12

5.13

B

RC

A2

11

4842

T

to C

S1

538S

Sy

n

B

RC

A2

11

4909

C

to A

H

1561

N

M

C

018

1.60

0.

00

C

016

5.75

0.

00

C

016

5.75

0.

00

BR

CA

2 11

49

95

A to

G

P158

9P

Syn

BR

CA

2 11

50

42

T to

C

V16

05A

M

C0

244.

82

0.00

C0

144.

08

1.01

C0

144.

08

1.01

B

RC

A2

11

5058

G

to A

V

1610

V

Syn

BR

CA

2 11

50

93

G to

C

R16

22T

M

C

035

3.86

0.

00

C

035

3.86

0.

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C

035

3.86

0.

00

BR

CA

2 11

51

55

G to

A

V16

43I

M

C

035

3.86

0.

00

C

035

3.86

0.

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C

024

2.23

0.

00

BR

CA

2 11

52

48

A to

G

S167

4G

M

C

013

8.80

0.

00

C

013

8.80

0.

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C

013

8.80

0.

00

BR

CA

2 11

52

79

C to

G

T168

4S

M

C

017

1.12

0.

00

C

014

2.37

0.

00

C

010

3.68

0.

00

BR

CA

2 11

54

19

C to

T

H17

31Y

M

C0

353.

86

0.00

C0

353.

86

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C0

231.

11

63.2

4 B

RC

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11

5426

C

to T

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353.

86

0.00

C15

209.

54

110.

53

C

1599

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110.

53

BR

CA

2 11

54

27

C to

T

S173

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Syn

BR

CA

2 11

55

47

G to

A

E177

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Syn

BR

CA

2 11

56

06

A to

G

N17

93S

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

035

3.86

0.

00

BR

CA

2 11

56

46

A to

G

E180

6E

Syn

BR

CA

2 11

57

23

C to

A

S183

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M

C

035

3.86

0.

00

C

035

3.86

0.

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C

025

7.76

12

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BR

CA

2 11

58

06

A to

G

K18

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M

C

024

6.80

16

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C

024

4.26

16

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C

024

4.26

16

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BR

CA

2 11

58

62

C to

G

N18

78K

M

C0

262.

60

0.00

C0

170.

08

0.00

C0

170.

08

0.00

B

RC

A2

11

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G

to A

E1

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8 0.

00

C

061

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61.8

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BR

CA

2 11

58

68

T to

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9

C0

112.

83

34.6

9

C0

111.

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36.7

1 B

RC

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to G

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C0

353.

86

0.00

C0

255.

29

0.00

C0

144.

99

0.00

B

RC

A2

11

5896

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to G

M

1890

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M

C

035

3.86

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3.86

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C

023

6.57

0.

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BR

CA

2 11

59

11

G to

A

E189

5K

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C

035

3.86

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C

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6.16

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C

017

6.53

0.

00

BRCA

1 and

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arian

ts in

Uni

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BR

CA

2 11

59

32

G to

A

D19

02N

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C0

353.

86

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C0

353.

86

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C0

213.

16

22.7

5 B

RC

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11

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C

to T

T1

915M

M

C0

353.

86

0.00

C0

258.

55

0.00

C0

162.

56

0.00

B

RC

A2

11

5996

A

to C

D

1923

A

M

C

035

3.86

0.

00

C

025

1.13

0.

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C

015

8.16

1.

52

BR

CA

2 11

60

13

A to

G

I192

9V

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

024

2.23

0.

00

BR

CA

2 11

61

07

G to

A

C19

60Y

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C0

353.

86

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C0

255.

29

4.05

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225.

33

4.05

B

RC

A2

11

6172

A

to G

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C0

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C

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50.1

5

C0

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BR

CA

2 11

63

28

C to

T

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C0

353.

86

0.00

C0

353.

86

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C0

262.

81

11.3

3 B

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11

6347

T

to A

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M

C0

353.

86

0.00

C0

353.

86

0.00

C0

353.

86

0.00

B

RC

A2

11

6545

T

to C

L2

106P

M

C0

353.

86

0.00

C0

353.

86

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C0

251.

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B

RC

A2

11

6550

C

to T

R

2108

C

M

C

035

3.86

0.

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C

035

3.86

0.

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C

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7.27

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BR

CA

2 11

65

51

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A

R21

08H

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C0

353.

86

0.00

C0

353.

86

0.00

C0

267.

27

0.00

B

RC

A2

11

6575

A

to G

H

2116

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M

C

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3.86

0.

00

C

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8.33

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C

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6.00

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62

BR

CA

2 11

66

40

G to

T

V21

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C

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1.33

21

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C

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1.33

21

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C

026

1.33

21

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BR

CA

2 11

66

83

C to

A

S215

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C

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6.57

85

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C

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5.31

85

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C

1576

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92.2

5 B

RC

A2

11

6693

C

to T

L2

155L

Sy

n

B

RC

A2

11

6717

A

to G

Q

2163

Q

Syn

BR

CA

2 11

67

41

C to

G

V21

71V

Sy

n

B

RC

A2

11

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G

to A

V

2205

M

M

C

035

3.86

0.

00

C

023

8.68

0.

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C

013

4.86

0.

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BR

CA

2 11

69

76

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G

T225

0A

M

C

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2.38

0.

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C

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4.06

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C

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4.06

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BR

CA

2 11

70

49

G to

T

G22

74V

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C0

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76

0.00

C0

160.

70

0.00

C0

160.

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0.00

B

RC

A2

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B

RC

A2

11

7069

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A to

G

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1+33

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S

B

RC

A2

11

7069

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A to

G

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S

B

RC

A2

12

7070

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A

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B

RC

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12

7070

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A

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28.6

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28

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BR

CA

2 12

71

49

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G

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BR

CA

2 13

71

71

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C

I231

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C

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9.50

0.

00

C

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0.

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C

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9.50

0.

00

BR

CA

2 13

72

35+4

A

to G

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B

RC

A2

13

7235

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A

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B

RC

A2

14

7236

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A to

G

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3-62

A>G

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S

B

RC

A2

14

7245

G

to C

K

2339

N

M

C

024

8.62

0.

00

C

096

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28.6

1

C0

96.4

4 28

.61

BR

CA

2 14

72

75

T to

A

F234

9L

M

C

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1.25

0.

00

C

012

4.40

0.

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C

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8.52

0.

00

BR

CA

2 14

72

80

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G

A23

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M

C0

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C0

154.

93

28.3

7

C0

154.

93

28.3

7 B

RC

A2

14

7285

G

to C

G

2353

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M

C

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1.53

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07

C

017

0.54

5.

07

C

017

0.54

5.

07

BR

CA

2 14

73

78

C to

A

Q23

84K

M

C0

353.

86

0.00

C0

266.

79

0.00

C0

263.

90

0.00

E 12

38 B

org

et al

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A

GV

GD

4 Sea

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GV

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DC

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GV

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BR

CA

2 14

74

60

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77.7

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650.

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77.7

4 B

RC

A2

14

7470

A

to G

S2

414S

Sy

n

B

RC

A2

14

7547

A

to G

H

2440

R

M

C

026

8.84

13

.17

C

026

3.50

13

.17

C

026

3.50

13

.17

BR

CA

2 14

76

25

C to

T

A24

66V

M

C0

353.

86

0.00

C0

353.

86

0.00

C0

353.

86

0.00

B

RC

A2

14

7641

A

to G

T2

471T

Sy

n

B

RC

A2

14

7663

+5

T to

C

IVS1

4+5T

>C

IVS

BR

CA

2 14

76

63+2

3 A

to G

IV

S14+

23A

>G

IVS

BR

CA

2 14

76

63+5

3 C

to T

IV

S14+

53C

>T

IVS

BR

CA

2 15

76

97

T to

C

I249

0T

M

C

096

.19

49.4

2

C45

14.3

0 81

.04

C

4514

.30

81.0

4 B

RC

A2

15

7778

C

to G

T2

517S

M

C0

230.

85

0.00

C0

113.

34

46.7

0

C0

90.0

0 48

.76

BR

CA

2 15

78

45+1

5 A

to G

IV

S15+

15A

>G

IVS

BR

CA

2 15

78

45+1

7 C

to T

IV

S15+

17C

>T

IVS

BR

CA

2 16

78

54

G to

A

T254

2T

Syn

BR

CA

2 16

80

33+6

C

to G

IV

S16+

6C>G

IV

S

B

RC

A2

17

8034

-40

A to

G

IVS1

6-40

A>G

IV

S

B

RC

A2

17

8034

-39

A to

G

IVS1

6-39

A>G

IV

S

B

RC

A2

17

8034

-14

T to

C

IVS1

6-14

T>C

IV

S

B

RC

A2

17

8106

G

to C

W

2626

C

M

C

650.

00

214.

36

C

650.

00

214.

36

C

650.

00

214.

36

BR

CA

2 17

81

07

A to

T

I262

7F

M

C

150.

00

21.2

8

C15

0.00

21

.28

C

150.

00

21.2

8 B

RC

A2

17

8142

T

to G

F2

638L

M

C0

21.6

1 21

.82

C

150.

00

21.8

2

C15

0.00

21

.82

BR

CA

2 17

82

04+3

5 C

to A

IV

S17+

35C

>A

IVS

BR

CA

2 17

82

04+3

6 G

to A

IV

S17+

36G

>A

IVS

BR

CA

2 18

82

16

A to

T

E266

3V

M

C

650.

00

121.

34

C

650.

00

121.

34

C

650.

00

121.

34

BR

CA

2 18

82

22

A to

G

D26

65G

M

C65

0.00

93

.77

C

650.

00

93.7

7

C65

0.00

93

.77

BR

CA

2 18

82

55

T to

C

M26

76T

M

C

092

.35

0.00

C0

92.3

5 0.

00

C

092

.35

0.00

B

RC

A2

18

8267

A

to G

D

2680

G

M

C

1544

.60

75.3

3

C15

44.6

0 75

.33

C

1544

.60

75.3

3 B

RC

A2

18

8318

G

to A

S2

697N

M

C0

82.6

7 0.

00

C

082

.67

0.00

C0

82.6

7 0.

00

BR

CA

2 18

83

62

G to

A

D27

12V

M

C0

152.

33

55.0

5

C0

114.

13

71.4

1

C0

112.

66

71.4

4 B

RC

A2

18

8377

G

to T

A

2717

S M

C0

134.

90

2.75

C0

133.

94

2.75

C0

133.

94

2.75

B

RC

A2

18

8410

G

to A

V

2728

I M

C0

28.6

8 0.

00

C

028

.68

0.00

C0

28.6

8 0.

00

BR

CA

2 19

86

88

A to

C

V28

20V

Sy

n

B

RC

A2

20

8795

A

to C

E2

856A

M

C0

171.

12

0.00

C0

133.

09

0.00

C0

106.

71

0.00

B

RC

A2

20

8797

G

to A

A

2857

T M

C0

245.

92

0.00

C0

241.

18

0.00

C0

241.

18

0.00

B

RC

A2

20

8860

+14

T to

C

IVS2

0+14

T>C

IV

S

B

RC

A2

20

8860

+143

in

sC

IVS2

0+14

3ins

C

IVS

BR

CA

2 21

88

61-1

6 C

to G

IV

S20-

16C

>G

IVS

BRCA

1 and

BRC

A2 S

eque

nce V

arian

ts in

Uni

later

al an

d Bi

later

al Br

east

Can

cer

E 12

39

A

GV

GD

4 Sea

urc

hin

A

GV

GD

Puf

ferf

ish

AG

VG

D X

enop

us

Gen

eE

xon

NT

1B

ase

Ch

BIC

2T

ype3

Cla

ss5

GV

GD

Cla

ssG

VG

DC

lass

GV

GD

BR

CA

2 21

88

75

C to

T

P288

3S

M

C

013

9.14

36

.40

C

011

3.69

36

.40

C

011

3.69

36

.40

BR

CA

2 21

89

82+7

5 A

to G

IV

S21+

75A

>G

IVS

BR

CA

2 22

90

58

A to

T

I294

4F

M

C

04.

86

21.2

8

C0

4.86

21

.28

C

04.

86

21.2

8 B

RC

A2

22

9078

G

to T

K

2950

N

M

C

3526

.00

79.2

4

C35

26.0

0 79

.24

C

650.

00

93.8

8 B

RC

A2

22

9079

G

to A

A

2951

T M

C0

99.1

3 29

.38

C

550.

00

58.0

2

C55

0.00

58

.02

BR

CA

2 22

91

30

A to

C

T296

8P

M

C

037

.56

0.00

C0

37.5

6 0.

00

C

037

.56

0.00

B

RC

A2

22

9133

G

to A

V

2969

M

M

C

030

.92

0.00

C15

0.00

20

.52

C

150.

00

20.5

2 B

RC

A2

23

9332

A

to G

Y

3035

C

M

C

5521

.61

191.

71

C

5521

.61

191.

71

C

5521

.61

191.

71

BR

CA

2 24

93

82

C to

T

R30

52W

M

C65

0.00

10

1.29

C65

0.00

10

1.29

C65

0.00

10

1.29

B

RC

A2

24

9463

G

to A

V

3079

I M

C0

28.6

8 0.

00

C

028

.68

0.00

C0

28.6

8 0.

00

BR

CA

2 24

94

84+2

8 de

l T

IVS2

4+28

delT

IV

S

B

RC

A2

25

9485

-113

T

to G

IV

S24-

113T

>G

IVS

BR

CA

2 25

94

85-1

10

A to

G

IVS2

4-11

0A>G

IV

S

B

RC

A2

25

9485

-83

G to

A

IVS2

4-83

G>A

IV

S

B

RC

A2

25

9485

-49

T to

C

IVS2

4-49

T>C

IV

S

B

RC

A2

25

9485

-16

T to

C

IVS2

4-16

T>C

IV

S

B

RC

A2

25

9520

T

to C

Y

3098

H

M

C

015

2.53

0.

00

C

012

0.91

0.

00

C

010

5.60

0.

00

BR

CA

2 25

95

29

C to

T

L310

1L

Syn

BR

CA

2 25

95

94

A to

G

A31

22A

Sy

n

B

RC

A2

25

9639

T

to G

T3

137T

Sy

n

B

RC

A2

26

9741

A

to G

I3

171M

M

C0

185.

64

0.00

C0

181.

93

0.00

C0

181.

93

0.00

B

RC

A2

26

9834

G

to C

P3

202P

Sy

n

B

RC

A2

26

9876

+54

G to

A

IVS2

6+54

G>A

IV

S

B

RC

A2

26

9876

+84

G to

A

IVS2

6+84

G>A

IV

S

B

RC

A2

26

9876

+106

de

l T

IVS2

6+10

6del

T IV

S

B

RC

A2

27

9877

-67

del T

TAC

IV

S26-

67de

l4

IVS

BR

CA

2 27

98

77-1

9 G

to A

IV

S26-

19G

>A

IVS

BR

CA

2 27

99

58

G to

A

V32

44I

M

C

023

4.51

0.

00

C

016

9.82

0.

00

C

016

9.82

0.

00

BR

CA

2 27

10

103

C to

T

P329

2L

M

C

075

.14

62.6

8

C0

75.1

4 62

.68

C

075

.14

62.6

8 B

RC

A2

27

1020

4 A

to T

K

3326

X

N

BR

CA

2 27

10

273

A to

G

T334

9A

M

C

550.

00

58.0

2

C55

0.00

58

.02

C

550.

00

58.0

2 B

RC

A2

27

1032

3 de

l C in

s 11

1032

3del

Cin

s11

F

B

RC

A2

27

1033

8 G

to A

R

3370

R

Syn

BR

CA

2 27

10

339

A to

G

T337

1A

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

021

5.24

0.

00

BR

CA

2 27

10

378

C to

T

R33

84X

N

B

RC

A2

27

1043

1 G

to A

T3

401T

Sy

n

E 12

40 B

org

et al

.

A

GV

GD

4 Sea

urc

hin

A

GV

GD

Puf

ferf

ish

AG

VG

D X

enop

us

Gen

eE

xon

NT

1B

ase

Ch

BIC

2T

ype3

Cla

ss5

GV

GD

Cla

ssG

VG

DC

lass

GV

GD

BR

CA

2 27

10

462

A to

G

I341

2V

M

C

035

3.86

0.

00

C

035

3.86

0.

00

C

024

4.82

0.

00

BR

CA

2 27

10

516

C to

T

UTR

U

TR

1 BR

CA

1: n

ucle

otid

e nu

mbe

r acc

ordi

ng to

Gen

Ban

k U

1468

0.1.

http

://w

ww

.ncb

i.nlm

.nih

.gov

/ent

rez/

view

er.fc

gi?d

b=nu

cleo

tide&

val=

5559

31

1 BR

CA

2: n

ucle

otid

e nu

mbe

r acc

ordi

ng to

Gen

Bank

NM

_000

059.

1. h

ttp://

ww

w.n

cbi.n

lm.n

ih.g

ov/e

ntre

z/vi

ewer

.fcgi

?db=

nucl

eotid

e&va

l=45

0245

0 2 B

IC (B

reas

t Can

cer I

nfor

mat

ion

Cor

e). h

ttp://

rese

arch

.nhg

ri.ni

h.go

v/bi

c/ .

HG

VS

nom

encl

atur

e in

Sup

p. T

able

S2.

3 T

ype:

F=f

ram

e sh

ift, M

=mis

sens

e, N

=non

sens

e, S

yn=S

ynon

ymou

s, IF

D=i

n fr

ame

dele

tion,

IVS=

inte

rven

ing

sequ

ence

, UTR

=unt

rans

late

d re

gion

4 A

lign

Gra

ntha

m V

aria

tion

and

Gra

ntha

m D

evia

tion

(AG

VG

D)

scor

es (

of O

ctob

er 2

008)

http

://ag

vgd.

iarc

.fr,

alig

ned

dow

n to

sea

urc

hin

(Stro

ngyl

ocen

trotu

s pu

rpur

atus

), pu

ffer

fish

(Tet

raod

on n

igro

virid

is) o

r fro

g (X

enop

us la

evis

) 5 A

-GV

GD

gra

ded

clas

sifie

rs, o

rder

ed fr

om m

ost l

ikel

y to

inte

rfer

e w

ith fu

nctio

n to

leas

t lik

ely:

GD

>=65

+Tan

(10)

x(G

V^2

.5) =

> C

lass

C65

<=>

mos

t lik

ely

GD

>=55

+Tan

(10)

x(G

V^2

.0) =

> C

lass

C55

G

D>=

45+T

an(1

5)x(

GV

^1.7

) =>

Cla

ss C

45

GD

>=35

+Tan

(50)

x(G

V^1

.1) =

> C

lass

C35

G

D>=

25+T

an(5

5)x(

GV

^0.9

5) =

> C

lass

C25

G

D>=

15+T

an(7

5)x(

GV

^0.6

) =>

Cla

ss C

15

Else

(GD

<15+

Tan(

75)x

(GV

^0.6

)) =

> C

lass

C0

<=>

less

like

ly