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Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome responsible for 1% of colorectal cancers (CRCs). Up to 90% of classic FAPs are caused by inactivating mutations in APC, and mosaicism has been previously reported in 20% of de novo cases, usually linked to milder phenotypic manifestations. This study aimed to explore the prevalence of mosaicism in 11 unsolved cases of classic FAP and to evaluate the diagnostic yield of somatic testing. Paired samples of colorectal polyps, tumors, and/or mucosa were analyzed using a custom next-generation sequencing panel targeting 15 polyposis and CRC-predisposing genes. Whenever possible, the extension of mosaicism to blood or sperm was also examined. Of 11 patients with classic adenomatous polyposis, a mosaic pathogenic variant in APC was identified in 7 (64%). No other altered genes were identified. In two of seven patients (29%), mosaicism was found restricted to colonic tissues, whereas in five of seven patients (71%), it was extended to the blood. Germline affectation was confirmed in one patient. We report the first analysis at a somatic level of 15 genes associated with CRC susceptibility, which highlights the role of APC mosaicism in classic FAP etiology. The results further reinforce the importance of testing target tissues when blood test results are negative.

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Case–control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2–9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.

Background: CHEK2 variants are associated with intermediate breast cancer risk among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. Methods: First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1,848 prospective patients with HC suspicion. We refined ACMGAMP criteria and applied different combinatorial rules to classify CHEK2 variants and define risk alleles. Results: We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; 2 can also be considered “established risk-alleles” and one as “likely risk-allele”. The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary non-polyposis colorectal cancer patients). Conclusions: Here we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this work would be useful for variant classification of other genes with low effect variants

Background CHEK2 variants are associated with intermediate breast cancer risk among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. Methods First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1,848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. Results We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele". The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary non-polyposis colorectal cancer patients). Conclusions Here we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this work would be useful for variant classification of other genes with low effect variants. This article is protected by copyright. All rights reserved.

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Fanconi anemia (FA) is caused by biallelic mutations in FA genes. Monoallelic mutations in five of these genes (BRCA1, BRCA2, PALB2, BRIP1 and RAD51C) increase the susceptibility to breast/ovarian cancer and are used in clinical diagnostics as bona-fide hereditary cancer genes. Increasing evidence suggests that monoallelic mutations in other FA genes could predispose to tumor development, especially breast cancer. The objective of this study is to assess the mutational spectrum of 14 additional FA genes (FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FANCP, FANCQ, FANCR and FANCU) in a cohort of hereditary cancer patients, to compare with local cancer-free controls as well as GnomAD. A total of 1021 hereditary cancer patients and 194 controls were analyzed using our next generation custom sequencing panel. We identified 35 pathogenic variants in eight genes. A significant association with the risk of breast cancer/breast and ovarian cancer was found for carriers of FANCA mutations (odds ratio (OR) = 3.14 95% confidence interval (CI) 1.4&ndash, 6.17, p = 0.003). Two patients with early-onset cancer showed a pathogenic FA variant in addition to another germline mutation, suggesting a modifier role for FA variants. Our results encourage a comprehensive analysis of FA genes in larger studies to better assess their role in cancer risk.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification., Ohio State University Comprehensive Cancer Center Barretos Cancer Hospital. Grant Number: FINEP ‐ CT‐INFRA (02/2010) Breast Cancer Foundation of New Zealand Canadian Institutes of Health Research. Grant Number: PSR‐SIIRI‐701 Cancer Research UK. Grant Numbers: C8197/A16565, C5047/A8384, C1281/A12014, C12292/A11174, C1287/A10710, C1287/A10118, C1287/A16563, C5047/A10692, C5047/A15007 Department of Defence, USA. Grant Number: W81XWH‐10‐1‐0341 Helsinki University Hospital Research fund Scientific Foundation Asociación Española Contra el Cáncer Leiden University Medical Centre. Grant Number: Grant 30.925 Generalitat de Catalunya. Grant Numbers: PERIS_MedPerCan, URDCat, 2017SGR1282, 2017SGR496 Royal Society of New Zealand Cancer Council Victoria Netherlands Organization for Scientific Research (NWO). Grant Number: Grant 017.008.022 Breast Cancer Research Foundation Cancer Foundation of Western Australia EU H2020. Grant Number: 634935 Fundación Mutua Madrileña Seventh Framework Programme. Grant Numbers: 634935, 223175, 633784 Cancer Council South Australia Government of Galicia. Grant Number: Consolidation and structuring program: IN607B Cancer Council Tasmania Italian Association of Cancer Research. Grant Number: 15547 Queensland Cancer Fund AstraZeneca National Institute of Health (USA). Grant Numbers: 1U19 CA148065‐01, CA128978, CA192393, 1U19 CA148537, P50 CA1162091, CA116167, 1U19 CA148112 Newcastle University Dutch Cancer Society KWF. Grant Numbers: KWF/Pink Ribbon‐11704, UL2012‐5649 National Institute for Health Research. Grant Number: Manchester Biomedical Research centre (IS‐BRC‐1215 National Council of Technological and Scientific Development (CNPq) Instituto de Salud Carlos III. Grant Numbers: FIS PI15/00355, FIS PI13/01711, CIBERONC, FIS PI16/01218, PI16/00563 French National Institute of Cancer National Breast Cancer Foundation National Health and Medical Research Council. Grant Numbers: ID1061778, ID1104808 Carlos III National Health Centro de Investigación Biomédica en Red de Enferemdades Raras. Grant Number: ACCI 2016: ER17P1AC7112/2018 Cancer Council NSW Deutsche Krebshilfe. Grant Numbers: (#110837, #70111850 Fondazione Pisa. Grant Number: Grant “Clinical characterization of BRCA 1/2 Mis

Síndromes de càncer; Panell genètic; Proves genètiques Síndromes de cáncer; Panel de genes; Prueba genética Cancer syndromes; Gene panel; Genetic testing Importance Genetic testing of hereditary cancer using comprehensive gene panels can identify patients with more than one pathogenic mutation in high and/or moderate-risk-associated cancer genes. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS), which has been potentially linked to more severe clinical manifestations. Objective To determine the prevalence and clinical features of MINAS in a large cohort of adult patients with hereditary cancer homogeneously tested with the same gene panel. Patients and methods A cohort of 1023 unrelated patients with suspicion of hereditary cancer was screened using a validated panel including up to 135 genes associated with hereditary cancer and phakomatoses. Results Thirteen (1.37%) patients harbouring two pathogenic mutations in dominant cancer-predisposing genes were identified, representing 5.7% (13/226) of patients with pathogenic mutations. Most (10/13) of these cases presented clinical manifestations associated with only one of the mutations identified. One case showed mutations in MEN1 and MLH1 and developed tumours associated with both cancer syndromes. Interestingly, three of the double mutants had a young age of onset or severe breast cancer phenotype and carried mutations in moderate to low-risk DNA damage repair-associated genes; two of them presented biallelic inactivation of CHEK2. We included these two patients for the sake of their clinical interest although we are aware that they do not exactly fulfil the definition of MINAS since both mutations are in the same gene. Conclusions and relevance Genetic analysis of a broad cancer gene panel identified the largest series of patients with MINAS described in a single study. Overall, our data do not support the existence of more severe manifestations in double mutants at the time of diagnosis although they do confirm previous evidence of severe phenotype in biallelic CHEK2 and other DNA repair cancer-predisposing genes. Contract grant sponsor: Supported by the Carlos III National Health Institute and Ministerio de Educación y Ciencia funded by FEDER funds–a way to build Europe (PI16/00563, PI16/01363, SAF2015-68016-R and CIBERONC); the Government of Catalonia (Pla estratègic de recerca i innovació en salut (PERIS), 2017SGR1282 and 2017SGR496); and the scientific foundation Asociación Española Contra el Cáncer.