Quantifying prediction of pathogenicity for within-codon concordance (PM5) using 7541 functional classifications of BRCA1 and MSH2 missense variants.

PURPOSE: Conditions and thresholds applied for evidence weighting of within-codon concordance (PM5) for pathogenicity vary widely between laboratories and expert groups. Because of the sparseness of available clinical classifications, there is little evidence for variation in practice. METHODS: We used as a truthset 7541 dichotomous functional classifications of BRCA1 and MSH2, spanning 311 codons of BRCA1 and 918 codons of MSH2, generated from large-scale functional assays that have been shown to correlate excellently with clinical classifications. We assessed PM5 at 5 stringencies with incorporation of 8 in silico tools. For each analysis, we quantified a positive likelihood ratio (pLR, true positive rate/false positive rate), the predictive value of PM5-lookup in ClinVar compared with the functional truthset. RESULTS: pLR was 16.3 (10.6-24.9) for variants for which there was exactly 1 additional colocated deleterious variant on ClinVar, and the variant under examination was equally or more damaging when analyzed using BLOSUM62. pLR was 71.5 (37.8-135.3) for variants for which there were 2 or more colocated deleterious ClinVar variants, and the variant under examination was equally or more damaging than at least 1 colocated variant when analyzed using BLOSUM62. CONCLUSION: These analyses support the graded use of PM5, with potential to use it at higher evidence weighting where more stringent criteria are met

Functional analyses of BRCA1 variants of unknown significance found in hereditary breast or ovarian cancer families in Norway: A tool for improved diagnosis

Tumorsuppressorgenet BRCA1 speler mange viktige roller for å hindre tumorutvikling, og endringar i BRCA1-genet er ei av hovudårsakene til arveleg bryst- og eggstokk-kreft (HBOC). I artikkel I vart det laga ei oversikt over BRCA1-variantar påvist i familiar der ein mistenkjer HBOC ved dei fire medisinsk genetiske avdelingane i Noreg. Dei interne variantklassifiseringane vart samanlikna, og resultatet viste diskrepans i 30% av klassifiseringa mellom dei ulike laboratoria. Etter fleire digitale møter vart diskrepansen redusert til 10%, noko som viser at varianttolking bør bli oppdatert jamleg og at datadeling og nært samarbeid mellom laboratoria gir meir nøyaktige tolkingar. I artikkel II vart 14 sjeldne BRCA1-missensvariantar frå artikkel I, alle av usikker klinisk betyding (VUSar) og fordelt langs heile genet, analysert med fleire ulike funksjonelle analyser, inkludert undersøking av proteinuttrykking og proteinstabilitet, subcellulær lokasjon og proteininteraksjon med BARD1 og PALB2. I motsetning til fleire tidlegare studiar som berre har studert separate proteindomener av BRCA1, har vi her brukt full-lengdeprotein for å betre kunne gjenskape heile den naturlege tilstanden til proteinet. Målet var å undersøke hypotesa som seier at BRCA1-missensvariantar lokalisert utanfor proteindomener med kjend funksjon ikkje er funksjonelt viktige. Totalt fire variantar lokalisert utanfor kjende domener gjorde BRCA1 proteinet meir utsett for proteasom-mediert degradering, eller ga lågare proteinstabilitet samanlikna med villtypeproteinet. Desse funna indikerer at også variantar utanfor dei kjende domenene RING, BRCT og coiled-coil kan påverke BRCA1-proteinfunksjon. I artikkel III undersøkte vi effekten av 11 sjeldne BRCA1 VUSar frå artikkel I som var lokalisert enten i eller nær BRCT-domenet med hensyn til homologi-retta reparasjon (HRR) av dobbeltråda DNA-brot og transkripsjonell aktivering (TA). Berre ein av dei analyserte variantane viste same HRR-aktivitet som villtypeproteinet, medan alle dei andre viste signifikant redusert aktivitet. To av dei analyserte variantane viste redusert TA-aktivitet lik dei patogene kontrollane. Resultatet indikerer at fleire av variantane potensielt kan påverke BRCA1-proteinfunksjonen, men fleire studiar er nødvendige for å avklare patogeniteten til variantane. Vi framhevar også viktigheita av å samanlikne resultat frå fleire ulike funksjonelle analysemetodar. Sidan ein variant potensielt kan påverke berre ein eller nokre av funksjonane til eit proteinet er dette spesielt viktig for multifunksjonelle protein slik som BRCA1. Dei funksjonelle analysane i artikkel II og III ga ny kunnskap, noko som bidrog til reklassifisering av sju variantar frå VUS til truleg benign, og ein variant til truleg patogen.The tumour suppressor gene BRCA1 plays multiple roles in preventing tumour development, and alterations in the BRCA1 gene are one of the main causes of hereditary breast and ovarian cancer (HBOC). In paper I, an overview of the BRCA1 variant spectrum found in families with suspected HBOC at the four diagnostic genetic laboratories in Norway was made. The internal variant classifications were compared, which revealed discrepancies in 30% of the classification between the laboratories. The discrepancies were reduced to 10% through a series of digital meetings, which illustrates that variant interpretation needs to be regularly updated, and that data sharing and inter-laboratory collaboration improves the accuracy of variant interpretation. In paper II, 14 rare missense BRCA1 variants from Paper II, all of uncertain clinical significance (VUSs) and distributed throughout the gene, were assessed by multiple functional analyses, i.e. protein expression levels and stability, subcellular localisation, and protein interactions with BARD1 and PALB2. In contrast to several previous studies focusing on separate domains, the full-length protein was utilised to better mimic the native state of the protein, and we aimed to investigate the hypothesis stating that BRCA1 missense variants located outside protein domains with known function are of no functional importance. In total, four variants located outside the known domains were found to make the BRCA1 protein more prone to proteasome-mediated degradation, or showed reduced protein stability compared to the wild type (WT) protein. These findings indicate that also variants located outside the RING, BRCT and coiled-coiled domains could affect the BRCA1 protein function. In paper III, we investigated the effect of 11 rare BRCA1 VUSs selected from paper I, located either within or in close proximity to the BRCT domain, with respect to homologous recombination repair (HRR) of double stranded DNA breaks and transcriptional activation (TA). Only one variant exhibited HRR activity comparable to the WT protein, whereas all other variants showed a significantly lower activity. Two of the variants exhibited TA activity similar to the pathogenic controls. Our results thus indicate that several of the variants of interest could potentially impair BRCA1 protein function, but further studies are needed to clarify their pathogenicity. We highlight the importance of comparing results obtained from several functional assays for multifunctional proteins such as BRCA1, as a variant could potentially affect only one or some of the proteins’ multiple activities. The functional assays performed in paper II and III provided new knowledge, which contributed to reclassification of seven of the BRCA1 variants from VUS to likely benign, and one variant to likely pathogenic.Doktorgradsavhandlin

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1

BackgroundGermline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases.MethodsA training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered.ResultsGene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign.ConclusionsOur recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications

A Unified Framework for the Prioritization of Variants of Uncertain Significance in Hereditary Breast and Ovarian Cancer Patients

A significant proportion of hereditary breast and ovarian cancer (HBOC) patients receive uninformative genetic testing results, an issue exacerbated by the overwhelming quantity of variants of uncertain significance identified. This thesis describes a framework where, aside from protein coding changes, information theory (IT)-based sequence analysis identifies and prioritizes pathogenic variants occurring within sequence elements predicted to be recognized by proteins involved in mRNA splicing, transcription, and untranslated region binding and structure. To support the utilization of IT analysis, we established IT-based variant interpretation accuracy by performing a comprehensive review of mutations altering mRNA splicing in rare and common diseases. Custom probes targeting 20 complete HBOC genes for sequencing in 379 BRCA-uninformative patients identified 47,501 unique variants and we prioritized 429 variants in both BRCA and non-BRCA genes. Our approach focuses attention on a limited set of variants from a spectrum of functional mutation types for downstream functional and co-segregation analysis

Clinical likelihood ratios and balanced accuracy for 44 in silico tools against multiple large-scale functional assays of cancer susceptibility genes.

PURPOSE: Where multiple in silico tools are concordant, the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework affords supporting evidence toward pathogenicity or benignity, equivalent to a likelihood ratio of ~2. However, limited availability of "clinical truth sets" and prior use in tool training limits their utility for evaluation of tool performance. METHODS: We created a truth set of 9,436 missense variants classified as deleterious or tolerated in clinically validated high-throughput functional assays for BRCA1, BRCA2, MSH2, PTEN, and TP53 to evaluate predictive performance for 44 recommended/commonly used in silico tools. RESULTS: Over two-thirds of the tool-threshold combinations examined had specificity of 0.7. For Meta-SNP, the equivalent PLR = 42.9 (14.4-406) and NLR = 19.4 (15.6-24.9). CONCLUSION: Against these clinically validated "functional truth sets," there was wide variation in the predictive performance of commonly used in silico tools. Overall, REVEL and Meta-SNP had best balanced accuracy and might potentially be used at stronger evidence weighting than current ACMG/AMP prescription, in particular for predictions of benignity

Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5 breakpoint in intron 4; 3 breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G>C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies

Unravelling genetic predisposition to familial breast and ovarian cancer: new susceptibility genes and variant interpretation by in silico approaches

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut d'Oncologia Vall d’Hebron (VHIO)Patients with hereditary breast and ovarian cancer (HBOC) in whom a causative pathogenic variant is not identified after genetic analysis may not benefit from prevention, early detection, or precision treatment measures. This negative or inconclusive results are due, among other causes, to the detection of variants of uncertain significance (VUS).The main objective of this thesis is to increase the capacity of genetic diagnosis of patients with HBOC, by focusing on i) the optimisation in the interpretation of exonic and intronic variants that might affect RNA quality or quantity but remain as variants of uncertain significance (VUS) and ii) the identification of new susceptibility genes for HBOC. The article included in this thesis, Moles-Fernández et al., 2018 (DOI: 10.3389/fgene.2018.00366) explains an optimization in the identification of potentially spliceogenic variants located near to splicing sites, and provides recommendations to use for analysing donor and acceptor sites. Moreover, the creation or activation of cryptic sites along deep intronic regions could alter splicing causing the inclusion of intronic sequences in RNA. In the article, Moles-Fernández et al., 2021 (DOI: 10.3390/cancers13133341), a framework for the identification of deep intronic spliceogenic is provided, after the performance analysis of SpliceAI in silico tool in a dataset of spliceogenic and non-spliceogenic deep intronic variants. In addition, the importance of the splicing regulatory elements balance in the pseudoexon creation is described. The American College of Medical Genetics (ACMG) variant interpretation guidelines provide general recommendations to classify variants. In the included article Feliubadalò et al., 2021 (DOI: 10.1093/clinchem/hvaa250), ACMG guidelines were adapted to ATM gene. We focused on in silico splicing evidence (PP3/BP4). After reclassification of variants following the adapted guidelines, a reduction of VUS was obtained. On the other hand, in patients without pathogenic variants identified in HBOC related genes, the phenotype could be due to deleterious variants in genes still not known associated with the disease. For this reason, in Moles-Fernández et al., (article in preparation), the aim was to identify candidate genes through exomes and extended panel analysis and validate their risk association by performing a case-control study. The significant identification of loss-of-function variants in ALKBH3, BLM, CAMKK1, FANCD2, FANCM, NEIL3, PER1, RBL1, RECQL4, WRN and XRCC4 genes in patients with HBOC suggests that they might be breast/ovarian cancer susceptibility genes

Interpreting protein variant effects with computational predictors and deep mutational scanning

Computational predictors of genetic variant effect have advanced rapidly in recent years. These programs provide clinical and research laboratories with a rapid and scalable method to assess the likely impacts of novel variants. However, it can be difficult to know to what extent we can trust their results. To benchmark their performance, predictors are often tested against large datasets of known pathogenic and benign variants. These benchmarking data may overlap with the data used to train some supervised predictors, which leads to data re-use or circularity, resulting in inflated performance estimates for those predictors. Furthermore, new predictors are usually found by their authors to be superior to all previous predictors, which suggests some degree of computational bias in their benchmarking. Large-scale functional assays known as deep mutational scans provide one possible solution to this problem, providing independent datasets of variant effect measurements. In this Review, we discuss some of the key advances in predictor methodology, current benchmarking strategies and how data derived from deep mutational scans can be used to overcome the issue of data circularity. We also discuss the ability of such functional assays to directly predict clinical impacts of mutations and how this might affect the future need for variant effect predictors