IntroductionTwo major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling.MethodsEighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P).ResultsThree previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA).ConclusionsIn this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool.

BackgroundInherited factors predisposing individuals to breast and ovarian cancer are largely unidentified in a majority of families with hereditary breast and ovarian cancer (HBOC). We aimed to identify germline copy number variations (CNVs) contributing to HBOC susceptibility in the Finnish population.MethodsA cohort of 84 HBOC individuals (negative for BRCA1/2-founder mutations and pre-screened for the most common breast cancer genes) and 36 healthy controls were analysed with a genome-wide SNP array. CNV-affecting genes were further studied by Gene Ontology term enrichment, pathway analyses, and database searches to reveal genes with potential for breast and ovarian cancer predisposition. CNVs that were considered to be important were validated and genotyped in 20 additional HBOC individuals (6 CNVs) and in additional healthy controls (5 CNVs) by qPCR.ResultsAn intronic deletion in the EPHA3 receptor tyrosine kinase was enriched in HBOC individuals (12 of 101, 11.9%) compared with controls (27 of 432, 6.3%) (OR = 1.96; P = 0.055). EPHA3 was identified in several enriched molecular functions including receptor activity. Both a novel intronic deletion in the CSMD1 tumor suppressor gene and a homozygous intergenic deletion at 5q15 were identified in 1 of 101 (1.0%) HBOC individuals but were very rare (1 of 436, 0.2% and 1 of 899, 0.1%, respectively) in healthy controls suggesting that these variants confer disease susceptibility.ConclusionThis study reveals new information regarding the germline CNVs that likely contribute to HBOC susceptibility in Finland. This information may be used to facilitate the genetic counselling of HBOC individuals but the preliminary results warrant additional studies of a larger study group.

Background: Breast cancer has a well-established genetic component. However, genetic predisposition factors remain mostly unresolved in high-risk hereditary breast cancer families that do not carry mutations in two major susceptibility genes, BRCA1 and BRCA2. In this study, we aimed to identify rare variants contributing to disease susceptibility in the high-risk Finnish BRCA1/2-negative hereditary breast or breast-ovarian cancer families by using family-based exome sequencing approach. Methods: Germline DNA of 37 individuals from 13 high-risk hereditary breast or breast-ovarian cancer families was analyzed using targeted exome capture and massively parallel sequencing. Priority was given for rare (minor allele frequency<0.05) coding functional variants including nonsynonymous single nucleotide variants (SNVs), frameshift indels as well as stoploss/stopgain and splice-site variants. Candidate variants were selected based on the computational pathogenicity predictions by several tools and gene pathway analyses. Moreover, segregation of the variants with the disease was studied in the families. Candidate variants were confirmed by Sanger sequencing and genotyped in additional cohorts of breast cancer patients and healthy controls. Results: Altogether 21,530 rare coding functional variants (97.0% nonsynonymous SNVs, 1.6% frameshift indels, 1.4% stoploss/stopgains variants) as well as 5 splice site variants were observed in 37 individuals. Of these, 93 novel variants, that harbor genes participating in the DNA damage response pathway, were prioritized. Furthermore, only variants that were predicted to be pathogenic, were considered and they were manually studied through literature. Altogether 20 candidate variants were selected for further segregation, validation, and genotyping experiments, which are currently ongoing. Conclusions: Identified rare functional variants especially in the DNA damage response pathway genes can explain a fraction of the genetic predisposition to breast and ovarian cancer in the high-risk Finnish families. Citation Format: Kirsi M. Kuusisto, Tommi Rantapero, Minna Kankuri-Tammilehto, Matti Nykter, Satu-Leena Laasanen, Johanna Schleutker. Exome sequencing of high-risk Finnish hereditary breast and breast-ovarian cancer families. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-284. doi:10.1158/1538-7445.AM2015-LB-284