Large-scale application of ClinGen-InSiGHT APC-specific ACMG/AMP variant classification criteria leads to substantial reduction in VUS.

Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUSs), APC-specific variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) based on the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). A streamlined algorithm using the APC-specific criteria was developed and applied to assess all APC variants in ClinVar and the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) international reference APC Leiden Open Variation Database (LOVD) variant database, which included a total of 10,228 unique APC variants. Among the ClinVar and LOVD variants with an initial classification of (likely) benign or (likely) pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUSs were reclassified into clinically meaningful classes, the vast majority as (likely) benign. The total number of VUSs was reduced by 37%. In 24 out of 37 (65%) promising APC variants that remained VUS despite evidence for pathogenicity, a data-mining-driven work-up allowed their reclassification as (likely) pathogenic. These results demonstrated that the application of APC-specific criteria substantially reduced the number of VUSs in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalizable model for other gene- or disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUSs that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

Gene-specific ACMG/AMP classification criteria for germline APC variants: Recommendations from the ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel.

PURPOSE: The Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) was established by the International Society for Gastrointestinal Hereditary Tumours and the Clinical Genome Resource, who set out to develop recommendations for the interpretation of germline APC variants underlying Familial Adenomatous Polyposis, the most frequent hereditary polyposis syndrome. METHODS: Through a rigorous process of database analysis, literature review, and expert elicitation, the APC VCEP derived gene-specific modifications to the ACMG/AMP (American College of Medical Genetics and Genomics and Association for Molecular Pathology) variant classification guidelines and validated such criteria through the pilot classification of 58 variants. RESULTS: The APC-specific criteria represented gene- and disease-informed specifications, including a quantitative approach to allele frequency thresholds, a stepwise decision tool for truncating variants, and semiquantitative evaluations of experimental and clinical data. Using the APC-specific criteria, 47% (27/58) of pilot variants were reclassified including 14 previous variants of uncertain significance (VUS). CONCLUSION: The APC-specific ACMG/AMP criteria preserved the classification of well-characterized variants on ClinVar while substantially reducing the number of VUS by 56% (14/25). Moving forward, the APC VCEP will continue to interpret prioritized lists of VUS, the results of which will represent the most authoritative variant classification for widespread clinical use.

Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays.

BACKGROUND: Damaging alterations in the BRCA1 gene have been extensively described as one of the main causes of hereditary breast and ovarian cancer (HBOC). BRCA1 alterations can lead to impaired homologous recombination repair (HRR) of double-stranded DNA breaks, a process which involves the RING, BRCT and coiled-coil domains of the BRCA1 protein. In addition, the BRCA1 protein is involved in transcriptional activation (TA) of several genes through its C-terminal BRCT domain. METHODS: In this study, we have investigated the effect on HRR and TA of 11 rare BRCA1 missense variants classified as variants of uncertain clinical significance (VUS), located within or in close proximity to the BRCT domain, with the aim of generating additional knowledge to guide the correct classification of these variants. The variants were selected from our previous study "BRCA1 Norway", which is a collection of all BRCA1 variants detected at the four medical genetic departments in Norway. RESULTS: All variants, except one, showed a significantly reduced HRR activity compared to the wild type (WT) protein. Two of the variants (p.Ala1708Val and p.Trp1718Ser) also exhibited low TA activity similar to the pathogenic controls. The variant p.Trp1718Ser could be reclassified to likely pathogenic. However, for ten of the variants, the total strength of pathogenic evidence was not sufficient for reclassification according to the CanVIG-UK BRCA1/BRCA2 gene-specific guidelines for variant interpretation. CONCLUSIONS: When including the newly achieved functional evidence with other available information, one VUS was reclassified to likely pathogenic. Eight of the investigated variants affected only one of the assessed activities of BRCA1, highlighting the importance of comparing results obtained from several functional assays to better understand the consequences of BRCA1 variants on protein function. This is especially important for multifunctional proteins such as BRCA1.

Systematic large-scale application of ClinGen InSiGHT APC -specific ACMG/AMP variant classification criteria substantially alleviates the burden of variants of uncertain significance in ClinVar and LOVD databases.

Background: Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUS), APC-specific ACMG/AMP variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP). Methods: A streamlined algorithm using the APC -specific criteria was developed and applied to assess all APC variants in ClinVar and the InSiGHT international reference APC LOVD variant database. Results: A total of 10,228 unique APC variants were analysed. Among the ClinVar and LOVD variants with an initial classification of (Likely) Benign or (Likely) Pathogenic, 94% and 96% remained in their original categories, respectively. In contrast, 41% ClinVar and 61% LOVD VUS were reclassified into clinically actionable classes, the vast majority as (Likely) Benign. The total number of VUS was reduced by 37%. In 21 out of 36 (58%) promising APC variants that remained VUS despite evidence for pathogenicity, a data mining-driven work-up allowed their reclassification as (Likely) Pathogenic. Conclusions: The application of APC -specific criteria substantially reduced the number of VUS in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalisable model for other gene-/disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUS that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

Prenatal BRCA1 epimutations contribute significantly to triple-negative breast cancer development.

BACKGROUND: Normal cell BRCA1 epimutations have been associated with increased risk of triple-negative breast cancer (TNBC). However, the fraction of TNBCs that may have BRCA1 epimutations as their underlying cause is unknown. Neither are the time of occurrence and the potential inheritance patterns of BRCA1 epimutations established. METHODS: To address these questions, we analyzed BRCA1 methylation status in breast cancer tissue and matched white blood cells (WBC) from 408 patients with 411 primary breast cancers, including 66 TNBCs, applying a highly sensitive sequencing assay, allowing allele-resolved methylation assessment. Furthermore, to assess the time of origin and the characteristics of normal cell BRCA1 methylation, we analyzed umbilical cord blood of 1260 newborn girls and 200 newborn boys. Finally, we assessed BRCA1 methylation status among 575 mothers and 531 fathers of girls with (n = 102) and without (n = 473) BRCA1 methylation. RESULTS: We found concordant tumor and mosaic WBC BRCA1 epimutations in 10 out of 66 patients with TNBC and in four out of six patients with estrogen receptor (ER)-low expression (< 10%) tumors (combined: 14 out of 72; 19.4%; 95% CI 11.1-30.5). In contrast, we found concordant WBC and tumor methylation in only three out of 220 patients with 221 ER ≥ 10% tumors and zero out of 114 patients with 116 HER2-positive tumors. Intraindividually, BRCA1 epimutations affected the same allele in normal and tumor cells. Assessing BRCA1 methylation in umbilical WBCs from girls, we found mosaic, predominantly monoallelic BRCA1 epimutations, with qualitative features similar to those in adults, in 113/1260 (9.0%) of individuals, but no correlation to BRCA1 methylation status either in mothers or fathers. A significantly lower fraction of newborn boys carried BRCA1 methylation (9/200; 4.5%) as compared to girls (p = 0.038). Similarly, WBC BRCA1 methylation was found less common among fathers (16/531; 3.0%), as compared to mothers (46/575; 8.0%; p = 0.0003). CONCLUSIONS: Our findings suggest prenatal BRCA1 epimutations might be the underlying cause of around 20% of TNBC and low-ER expression breast cancers. Such constitutional mosaic BRCA1 methylation likely arise through gender-related mechanisms in utero, independent of Mendelian inheritance.

BRCA1 Norway: comparison of classification for BRCA1 germline variants detected in families with suspected hereditary breast and ovarian cancer between different laboratories.

Pathogenic germline variants in Breast cancer susceptibility gene 1 (BRCA1) predispose carriers to hereditary breast and ovarian cancer (HBOC). Through genetic testing of patients with suspected HBOC an increasing number of novel BRCA1 variants are discovered. This creates a growing need to determine the clinical significance of these variants through correct classification (class 1-5) according to established guidelines. Here we present a joint collection of all BRCA1 variants of class 2-5 detected in the four diagnostic genetic laboratories in Norway. The overall objective of the study was to generate an overview of all BRCA1 variants in Norway and unveil potential discrepancies in variant interpretation between the hospitals, serving as a quality control at the national level. For a subset of variants, we also assessed the change in classification over a ten-year period with increasing information available. In total, 463 unique BRCA1 variants were detected. Of the 126 variants found in more than one hospital, 70% were interpreted identically, while 30% were not. The differences in interpretation were mainly by one class (class 2/3 or 4/5), except for one larger discrepancy (class 3/5) which could affect the clinical management of patients. After a series of digital meetings between the participating laboratories to disclose the cause of disagreement for all conflicting variants, the discrepancy rate was reduced to 10%. This illustrates that variant interpretation needs to be updated regularly, and that data sharing and improved national inter-laboratory collaboration greatly improves the variant classification and hence increases the accuracy of cancer risk assessment.

Assessment of tumor suppressor promoter methylation in healthy individuals.

BACKGROUND: The number of tumor suppressor genes for which germline mutations have been linked to cancer risk is steadily increasing. However, while recent reports have linked constitutional normal tissue promoter methylation of BRCA1 and MLH1 to ovarian and colon cancer risk, the role of epigenetic alterations as cancer risk factors remains largely unknown, presenting an important area for future research. Currently, we lack fast and sensitive methods for assessment of promoter methylation status across known tumor suppressor genes. RESULTS: In this paper, we present a novel NGS-based approach assessing promoter methylation status across a large panel of defined tumor suppressor genes to base-pair resolution. The method omits the limitations related to commonly used array-approaches. Our panel includes 565 target regions covering the promoters of 283 defined tumor suppressors, selected by pre-specified criteria, and was applied for rapid targeted methylation-specific NGS. The feasibility of the method was assessed by analyzing normal tissue DNA (white blood cells, WBC) samples from 34 healthy postmenopausal women and by performing preliminary assessment of the methylation landscape of tumor suppressors in these individuals. The mean target coverage was 189.6x providing a sensitivity of 0.53%, sufficient for promoter methylation assessment of low-level methylated genes like BRCA1. Within this limited test-set, we detected 206 regions located in the promoters of 149 genes to be differentially methylated (hyper- or hypo-) at > 99% confidence level. Seven target regions in gene promoters (CIITA, RASSF1, CHN1, PDCD1LG2, GSTP1, XPA, and ZNF668) were found to be hyper-methylated in a minority of individuals, with a > 20 percent point difference in mean methylation across the region between individuals. In an exploratory hierarchical clustering analysis, we found that the individuals analyzed may be grouped into two main groups based on their WBC methylation profile across the 283 tumor suppressor gene promoters. CONCLUSIONS: Methylation-specific NGS of our tumor suppressor panel, with detailed assessment of differential methylation in healthy individuals, presents a feasible method for identification of novel epigenetic risk factors for cancer.

The intronic BRCA1 c.5407-25T>A variant causing partly skipping of exon 23-a likely pathogenic variant with reduced penetrance?

Rare sequence variants in the non-coding part of the BRCA genes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1 c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant's effect on mRNA splicing. Mean age at breast (n = 12) and ovarian (n = 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1 c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1 c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1 c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.