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Fulltext Clinical, splicing, and functional analysis to classify BRCA2 exon 3 variants: Application of a points-based ACMG/AMP approach

Thomassen M, Mesman RLS, Hansen TVO, Menendez M, Rossing M, Esteban-Sánchez A, et al.

Hum Mutat, 2022 Dec;43(12):1921-1944.
PMID: 35979650 DOI: 10.1002/humu.24449

Skipping of BRCA2 exon 3 (∆E3) is a naturally occurring splicing event, complicating clinical classification of variants that may alter ∆E3 expression. This study used multiple evidence types to assess pathogenicity of 85 variants in/near BRCA2 exon 3. Bioinformatically predicted spliceogenic variants underwent mRNA splicing analysis using minigenes and/or patient samples. ∆E3 was measured using quantitative analysis. A mouse embryonic stem cell (mESC) based assay was used to determine the impact of 18 variants on mRNA splicing and protein function. For each variant, population frequency, bioinformatic predictions, clinical data, and existing mRNA splicing and functional results were collated. Variant class was assigned using a gene-specific adaptation of ACMG/AMP guidelines, following a recently proposed points-based system. mRNA and mESC analysis combined identified six variants with transcript and/or functional profiles interpreted as loss of function. Cryptic splice site use for acceptor site variants generated a transcript encoding a shorter protein that retains activity. Overall, 69/85 (81%) variants were classified using the points-based approach. Our analysis shows the value of applying gene-specific ACMG/AMP guidelines using a points-based approach and highlights the consideration of cryptic splice site usage to appropriately assign PVS1 code strength.
Fulltext Exome genotyping arrays to identify rare and low frequency variants associated with epithelial ovarian cancer risk

Permuth JB, Pirie A, Ann Chen Y, Lin HY, Reid BM, Chen Z, et al.

Hum Mol Genet, 2016 08 15;25(16):3600-3612.
PMID: 27378695 DOI: 10.1093/hmg/ddw196

Rare and low frequency variants are not well covered in most germline genotyping arrays and are understudied in relation to epithelial ovarian cancer (EOC) risk. To address this gap, we used genotyping arrays targeting rarer protein-coding variation in 8,165 EOC cases and 11,619 controls from the international Ovarian Cancer Association Consortium (OCAC). Pooled association analyses were conducted at the variant and gene level for 98,543 variants directly genotyped through two exome genotyping projects. Only common variants that represent or are in strong linkage disequilibrium (LD) with previously-identified signals at established loci reached traditional thresholds for exome-wide significance (P P≥5.0 ×10 - 7) were detected for rare and low-frequency variants at 16 novel loci. Four rare missense variants were identified (ACTBL2 rs73757391 (5q11.2), BTD rs200337373 (3p25.1), KRT13 rs150321809 (17q21.2) and MC2R rs104894658 (18p11.21)), but only MC2R rs104894668 had a large effect size (OR = 9.66). Genes most strongly associated with EOC risk included ACTBL2 (PAML = 3.23 × 10 - 5; PSKAT-o = 9.23 × 10 - 4) and KRT13 (PAML = 1.67 × 10 - 4; PSKAT-o = 1.07 × 10 - 5), reaffirming variant-level analysis. In summary, this large study identified several rare and low-frequency variants and genes that may contribute to EOC susceptibility, albeit with possible small effects. Future studies that integrate epidemiology, sequencing, and functional assays are needed to further unravel the unexplained heritability and biology of this disease.
Fulltext Characterization of the Cancer Spectrum in Men With Germline BRCA1 and BRCA2 Pathogenic Variants: Results From the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA)

Silvestri V, Leslie G, Barnes DR, CIMBA Group, Agnarsson BA, Aittomäki K, et al.

JAMA Oncol, 2020 Aug 01;6(8):1218-1230.
PMID: 32614418 DOI: 10.1001/jamaoncol.2020.2134

IMPORTANCE: The limited data on cancer phenotypes in men with germline BRCA1 and BRCA2 pathogenic variants (PVs) have hampered the development of evidence-based recommendations for early cancer detection and risk reduction in this population.
OBJECTIVE: To compare the cancer spectrum and frequencies between male BRCA1 and BRCA2 PV carriers.
DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study of 6902 men, including 3651 BRCA1 and 3251 BRCA2 PV carriers, older than 18 years recruited from cancer genetics clinics from 1966 to 2017 by 53 study groups in 33 countries worldwide collaborating through the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Clinical data and pathologic characteristics were collected.
MAIN OUTCOMES AND MEASURES: BRCA1/2 status was the outcome in a logistic regression, and cancer diagnoses were the independent predictors. All odds ratios (ORs) were adjusted for age, country of origin, and calendar year of the first interview.
RESULTS: Among the 6902 men in the study (median [range] age, 51.6 [18-100] years), 1634 cancers were diagnosed in 1376 men (19.9%), the majority (922 of 1,376 [67%]) being BRCA2 PV carriers. Being affected by any cancer was associated with a higher probability of being a BRCA2, rather than a BRCA1, PV carrier (OR, 3.23; 95% CI, 2.81-3.70; P
Fulltext Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

Hakkaart C, Pearson JF, Marquart L, Dennis J, Wiggins GAR, Barnes DR, et al.

Commun Biol, 2022 Oct 06;5(1):1061.
PMID: 36203093 DOI: 10.1038/s42003-022-03978-6

The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.
Fulltext Fine-mapping of 150 breast cancer risk regions identifies 191 likely target genes

Fachal L, Aschard H, Beesley J, Barnes DR, Allen J, Kar S, et al.

Nat Genet, 2020 01;52(1):56-73.
PMID: 31911677 DOI: 10.1038/s41588-019-0537-1

Genome-wide association studies have identified breast cancer risk variants in over 150 genomic regions, but the mechanisms underlying risk remain largely unknown. These regions were explored by combining association analysis with in silico genomic feature annotations. We defined 205 independent risk-associated signals with the set of credible causal variants in each one. In parallel, we used a Bayesian approach (PAINTOR) that combines genetic association, linkage disequilibrium and enriched genomic features to determine variants with high posterior probabilities of being causal. Potentially causal variants were significantly over-represented in active gene regulatory regions and transcription factor binding sites. We applied our INQUSIT pipeline for prioritizing genes as targets of those potentially causal variants, using gene expression (expression quantitative trait loci), chromatin interaction and functional annotations. Known cancer drivers, transcription factors and genes in the developmental, apoptosis, immune system and DNA integrity checkpoint gene ontology pathways were over-represented among the highest-confidence target genes.