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Presents the abstract of the article 'The Czech Republic: Genetic Counseling in High-Risk Breast/Ovarian Cancer Families,' by L. Foretova, E. Machackova, D. Valik and L. Messiaen.

Background: The polygenic risk score (PRS) allows the quantification of the polygenic effect of many low-penetrance alleles on the risk of breast cancer (BC). This study aimed to evaluate the performance of two sets comprising 77 or 313 low-penetrance loci (PRS77 and PRS313) in patients with BC in the Czech population., Methods: In a retrospective case-control study, variants were genotyped from both the PRS77 and PRS313 sets in 1329 patients with BC and 1324 noncancer controls, all women without germline pathogenic variants in BC predisposition genes. Odds ratios (ORs) were calculated according to the categorical PRS in individual deciles. Weighted Cox regression analysis was used to estimate the hazard ratio (HR) per standard deviation (SD) increase in PRS., Results: The distributions of standardized PRSs in patients and controls were significantly different (p < 2.2 × 10 -16 ) with both sets. PRS313 outperformed PRS77 in categorical and continuous PRS analyses. For patients in the highest 2.5% of PRS313, the risk reached an OR of 3.05 (95% CI, 1.66-5.89; p = 1.76 × 10 -4 ). The continuous risk was estimated as an HR per SD  of 1.64 (95% CI, 1.49-1.81; p < 2.0 × 10 -16 ), which resulted in an absolute risk of 21.03% at age 80 years for individuals in the 95th percentile of PRS313. Discordant categorization into PRS deciles was observed in 248 individuals (9.3%)., Conclusions: Both PRS77 and PRS313 are able to stratify individuals according to their BC risk in the Czech population. PRS313 shows better discriminatory ability. The results support the potential clinical utility of using PRS313 in individualized BC risk prediction., (© 2024 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118&#95;1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Germline mutations in checkpoint kinase 2 (CHEK2), a multiple cancer-predisposing gene, increase breast cancer (BC) risk; however, risk estimates differ substantially in published studies. We analyzed germline CHEK2 variants in 1,928 high-risk Czech breast/ovarian cancer (BC/OC) patients and 3,360 population-matched controls (PMCs). For a functional classification of VUS, we developed a complementation assay in human nontransformed RPE1-CHEK2-knockout cells quantifying CHK2-specific phosphorylation of endogenous protein KAP1. We identified 10 truncations in 46 (2.39%) patients and in 11 (0.33%) PMC (p = 1.1 × 10 -14 ). Two types of large intragenic rearrangements (LGR) were found in 20/46 mutation carriers. Truncations significantly increased unilateral BC risk (OR = 7.94; 95%CI 3.90-17.47; p = 1.1 × 10 -14 ) and were more frequent in patients with bilateral BC (4/149; 2.68%; p = 0.003), double primary BC/OC (3/79; 3.80%; p = 0.004), male BC (3/48; 6.25%; p = 8.6 × 10 -4 ), but not with OC (3/354; 0.85%; p = 0.14). Additionally, we found 26 missense VUS in 88 (4.56%) patients and 131 (3.90%) PMC (p = 0.22). Using our functional assay, 11 variants identified in 15 (0.78%) patients and 6 (0.18%) PMC were scored deleterious (p = 0.002). Frequencies of functionally intermediate and neutral variants did not differ between patients and PMC. Functionally deleterious CHEK2 missense variants significantly increased BC risk (OR = 3.90; 95%CI 1.24-13.35; p = 0.009) and marginally OC risk (OR = 4.77; 95%CI 0.77-22.47; p = 0.047); however, carriers low frequency will require evaluation in larger studies. Our study highlights importance of LGR detection for CHEK2 analysis, careful consideration of ethnicity in both cases and controls for risk estimates, and demonstrates promising potential of newly developed human nontransformed cell line assay for functional CHEK2 VUS classification., (© 2019 UICC.)

Background: Deleterious mutations in the BRCA1 and BRCA2 genes account for a considerable proportion of dominantly inherited breast and ovarian cancer susceptibility. The laboratory interpretation has always been dependent on the information available at the time of the report conclusion. The aim of this study has been to review the results from the BRCA testing at Masaryk Memorial Cancer Institute (MMCI)., Patients and Methods: Patients with suspected hereditary predisposition to breast/ovarian cancer, belonging to 7,400 families, were referred by genetic counsellors for BRCA1 and BRCA2 mutation testing at the MMCI from 1999 to the beginning of 2018. Various methods have been used over 20 years of laboratory practice - starting with the Protein Truncation Test and Heteroduplex Analysis via the High Resolution Melting analysis and Sanger sequencing up to Next Generation Sequencing., Results: BRCA1 and BRCA2 mutation screening resulted in the identification of 1,021 families with a germline high-risk BRCA1 mutation and 497 families carrying a high-risk BRCA2 mutation, representing a mutation detection rate of 20.5%. A broad spectrum of unique mutations classified as pathogenic or likely pathogenic has been detected in both genes - 124 in the BRCA1 and 123 in the BRCA2 gene. Other sequence variants (96 unique variants in the BRCA1 and 126 in the BRCA2 gene) have been revised and classified as benign or likely benign. The other 82 unique variants remain classified as of uncertain significance mainly due to a lack of information for inclusion in other groups. All the results are summarised in the tables, including the reasons for their classification., Conclusion: The clinical classification of rare sequence variants identified in the high-risk breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling. Here we present an overview of BRCA mutation frequencies in our region and the retrospective evaluation and eventually reclassification of previously reported rare variants in light of recent findings.

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is a rare variant of familial adenomatous polyposis. It is an autosomal-dominant cancer-predisposition syndrome with massive polyposis of the stomach and a significant risk of gastric adenocarcinoma. Li et al., 2016, described point mutations in the Ying Yang 1 binding site of the APC gene 1B promoter associated with GAPPS syndrome. The first GAPPS syndrome in a Czech family was described in 2016. At Masaryk Memorial Cancer Institute, GAPPS syndrome was diagnosed in eight families using Sanger sequencing. In all families, one mutation in promoter 1B of APC gene NM&#95;001127511: c.-191T&gt;C was detected. This mutation was not found in any patient with multiple colon polyposis without a detected classic mutation in the APC gene. In total, 24 carriers of this mutation in promoter 1B of the APC gene were detected. Out of those 24 carriers, 20 had massive gastric polyposis with more than 100 fundic glandular polyps diagnosed between the age of 22 and 65, 5 had already died of adenocarcinoma of the stomach (at the ages of 29, 40, 59, 60 and 64, respectively) and another woman was treated at the age of 29. Two female carriers do not yet have polyposis of the stomach at the ages of 31 and 65, respectively; one female carrier has incipient polyposis at the age of 58. A male carrier does not have any clinical symptoms, gastroscopy was not indicated because of his age. Prophylactic total gastrectomy with D2 lymphadenectomy has already been performed 6 times at Masaryk Memorial Cancer Institute, in 5 cases without adenocarcinoma at the ages of 27, 34, 44, 51 and 66, respectively; in one female carrier adenocarcinoma of the stomach was detected in a histology specimen. Two prophylactic gastrectomies with D1 lymphadenectomy were performed at University Hospital Brno at the ages of 42 and 50, respectively. In the Czech Republic point mutation c.-191T&gt;C (rs879253783) in the 1B promoter of the APC gene is a frequent cause of gastric polyposis with a high risk of gastric adenocarcinoma, even at a young age. Positively tested individuals are recommended to high-risk oncology clinic. A necessary part of the discussion with the patient is information about a preventive gastrectomy.

Heterozygous germline BRCA2 mutations predispose to breast, ovarian, pancreatic and other types of cancer. The presence of a pathogenic mutation in patients or their family members warrants close surveillance or prophylactic surgery. Besides clearly pathogenic mutations, variants leading only to a single amino acid substitution are often identified. The influence of such variants on cancer risk is often unknown, making their presence a major clinical problem. When genetic methods are insufficient to classify these variants, functional assays with various cellular models are performed. We developed and applied a new syngeneic model of human cancer cells to test all variants of unknown significance in exon 18 identified by genetic testing of high-risk cancer patients in the Czech Republic, via introduction of constructs containing each of these variants into the wild-type allele of BRCA2-heterozygous DLD1 cells (BRCA2 wt/Δex11 ). We found unaffected DNA repair function of BRCA2 in cell lines BRCA2 7997G>C/Δex11 , BRCA2 8111C>T/Δex11 , BRCA2 8149G>T/Δex11 , BRCA2 8182G>A/Δex11 , and BRCA2 8182G>T/Δex11 , whereas the cell line BRCA2 8168A>G/Δex11 and the nonsense mutation carrying line BRCA2 8305G>T/ Δex11 did affect protein function. Targeting the BRCA2 wild-type allele with a construct carrying the variant c.7988A> G resulted in incorporation exclusively into the already defective allele in all viable clones, strongly suggesting a detrimental phenotype. Our model thus offers a valuable tool for the functional evaluation of unclassified variants in the BRCA2 gene and provides a stable and distributable cellular resource for further research.

Purpose: Carriers of BRCA1/2 mutations are at high lifetime risk of breast cancer (BC); however, the BC onset broadly vary in individual patients. Recently, polyglutamine (poly-Q) repeat length polymorphism of the amplified in breast cancer 1 (AIB1) gene was analyzed as a risk factor influencing BC onset in BRCA1/2 mutation carriers with contradictory results., Methods: We genotyped AIB1 poly-Q repeat in 243 BRCA1/2 mutation carriers, 61 patients with familial BC (negatively tested for the presence of BRCA1/2 mutation), 221 patients with sporadic BC, and 176 non-cancer controls using denaturing high-performance liquid chromatography and statistically evaluated the effect of AIB1 poly-Q repeat length polymorphism on BC onset., Results: Having used previously published statistical analyses of AIB1 poly-Q repeat length (≥28 and ≥29 repeat cutpoints or analysis of AIB1 poly-Q repeat length as continuous variable), we did not find any association between AIB1 poly-Q repeat length and BC development in analyzed BC groups. However, the analysis of individual genotypes revealed that AIB1 genotype consisting of 28/28 glutamine repeats served as a protective factor in BRCA1 mutation carriers (HR = 0.64; 95% CI 0.41-0.99; P = 0.045) and as a risk factor in carriers of mutation in exon 11 of the BRCA2 gene (HR = 3.50; 95% CI 1.25-9.78; P = 0.017)., Conclusions: Our results confirm that AIB1 poly-Q repeat length polymorphism does not influence the BC risk in general but suggest that the specific AIB1 genotypes should be considered in patients with BC carrying mutation in the BRCA1/2 genes.

Background: The incidence of breast cancer has doubled over the past 20 years in the Czech Republic. Hereditary factors may be a cause of young onset, bilateral breast or ovarian cancer, and familial accumulation of the disease. BRCA1 and BRCA2 mutations account for an important fraction of hereditary breast and ovarian cancer cases. One thousand and ten unrelated high-risk probands with breast and/or ovarian cancer were analysed for the presence of a BRCA1 or BRCA2 gene mutation at the Masaryk Memorial Cancer Institute (Czech Republic) during 1999-2006., Methods: The complete coding sequences and splice sites of both genes were screened, and the presence of large intragenic rearrangements in BRCA1 was verified. Putative splice-site variants were analysed at the cDNA level for their potential to alter mRNA splicing., Results: In 294 unrelated families (29.1% of the 1,010 probands) pathogenic mutations were identified, with 44 different BRCA1 mutations and 41 different BRCA2 mutations being detected in 204 and 90 unrelated families, respectively. In total, three BRCA1 founder mutations (c.5266dupC; c.3700&#95;3704del5; p.Cys61Gly) and two BRCA2 founder mutations (c.7913&#95;7917del5; c.8537&#95;8538del2) represent 52% of all detected mutations in Czech high-risk probands. Nine putative splice-site variants were evaluated at the cDNA level. Three splice-site variants in BRCA1 (c.302-3C>G; c.4185G>A and c.4675+1G>A) and six splice-site variants in BRCA2 (c.475G>A; c.476-2>G; c.7007G>A; c.8755-1G>A; c.9117+2T>A and c.9118-2A>G) were demonstrated to result in aberrant transcripts and are considered as deleterious mutations., Conclusion: This study represents an evaluation of deleterious genetic variants in the BRCA1 and 2 genes in the Czech population. The classification of several splice-site variants as true pathogenic mutations may prove useful for genetic counselling of families with high risk of breast and ovarian cancer.

Background: Alterations in the highly penetrant cancer susceptibility gene BRCA1 are responsible for the majority of hereditary breast and/or ovarian cancers. However, the number of detected germline mutations has been lower than expected based upon genetic linkage data. Undetected deleterious mutations in the BRCA1 gene in some high-risk families could be due to the presence of intragenic rearrangements as deletions, duplications or insertions spanning whole exons. Standard PCR-based screening methods are mainly focused on detecting point mutations and small insertions/deletions, but large rearrangements might escape detection.The purpose of this study was to determine the type and frequency of large genomic rearrangements in the BRCA1 gene in hereditary breast and ovarian cancer cases in the Czech Republic., Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to examine BRCA1 rearrangements in 172 unrelated patients with hereditary breast and/or ovarian cancer syndrome without finding deleterious mutation after complete screening of whole coding regions of BRCA1/2 genes. Positive MLPA results were confirmed and located by long-range PCR. The breakpoints of detected rearrangements were characterized by sequencing., Results: Six different large deletions in the BRCA1 gene were identified in 10 out of 172 unrelated high-risk patients: exons 1A/1B and 2 deletion; partial deletion of exon 11 and exon 12; exons 18 and 19 deletion; exon 20 deletion; exons 21 and 22 deletion; and deletion of exons 5 to 14. The breakpoint junctions were localized and further characterized. Destabilization and global unfolding of the mutated BRCT domains explain the molecular and genetic defects associated with the exon 20 in-frame deletion and the exon 21 and 22 in-frame deletion, respectively., Conclusion: Using MLPA, mutations were detected in 6% of high-risk patients previously designated as BRCA1/2 mutation-negative. The breakpoints of five out of six large deletions detected in Czech patients are novel. Screening for large genomic rearrangements in the BRCA1 gene in the Czech high-risk patients is highly supported by this study.

Germline mutations in BRCA1 and BRCA2 account for majority of hereditary breast and ovarian cancer. The complete coding sequence analysis of both genes was carried out in 197 breast/ovarian cancer patients from high-risk families and 53 patients with sporadic breast/ovarian cancer. In summary, 59 mutations (16 different) in BRCA1 and 29 mutations (17 different) in BRCA2 were identified in unrelated breast and/or ovarian index cases. Using the BIC Database numbering, the most frequently found mutations in BRCA1 were c.5385dupC (22 cases), c.3819&#95;3823delGTAAA (8 cases) and c.300T>G (6 cases). The most frequently found mutations in BRCA2 were c.8138&#95;8142delCCTTT (7 cases) and c.8765&#95;8766delAG (7 cases). Altogether, these 5 mutations represented 56.8% of all detected mutations. A broad spectrum of other mutations was detected including four novel mutations (c.2881delA in BRCA1; and c. 6677&#95;6678delAA, c.6982dupT and c.8397&#95;8400dupTGGG in BRCA2). Deleterious mutations were found in 80 (40.6%) of 197 high risk-families, in 6 (37.5%) of 16 patients with sporadic bilateral breast, ovarian or both cancers and in 2 (6.2%) of 32 women with sporadic early-onset unilateral breast cancer. No mutation was detected in 5 cases of sporadic early-onset unilateral ovarian cancer., (Copyright 2004 Wiley-Liss, Inc.)

Germline mutations in breast cancer susceptibility genes, BRCA1 and BRCA2, are responsible for a substantial proportion of high-risk breast and breast/ovarian cancer families. To characterize the spectrum of BRCA1 and BRCA2 mutations, we screened Czech families with breast/ovarian cancer using the non-radioactive protein truncation test, heteroduplex analysis and direct sequencing. In a group of 100 high-risk breast and breast/ovarian cancer families, four novel frame shift mutations were identified in BRCA1 and BRCA2 genes. In BRCA1, two novel frame shift mutations were identified as 3761-3762delGA and 2616-2617ins10; in BRCA2, two novel frame shift mutations were identified as 5073-5074delCT and 6866delC. Furthermore, a novel missense substitution M18K in BRCA1 gene in a breast/ovarian cancer family was identified which lies adjacent just upstream of the most highly conserved C3HC4 RING zinc finger motif. To examine the tertiary structure of the RING zinc finger domain and possible effects of M18K substitution on its stability, we used threading techniques according to the crystal structure of RAG1 dimerization domain of the DNA-binding protein., (Copyright 2000 Wiley-Liss, Inc.)