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ClinVar variant interpretations from May 2019 for variants identified in discovery cohorts

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60–3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants.Significance:Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Supplementary methods; FS1: BRIP1 nonsense and frameshift mutations; FS2: BRIP1 expression in CRISPR cell lines; FS3: hydroxyurea and TMPyP4 challenge; FS4: HEK293TN CRISPR cell lines; FS5: HeLa stable clone rescues; FS6: Transient rescue screening assay; FS7: Degradation of missense mutant proteins; TS1: List of BRIP1 missense variants identified in cohorts

Germline loss-of-function mutations in BRCA1 interacting protein C-terminal helicase 1 (BRIP1) are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand cross-link (ICL) repair and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants, for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next-generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 patients with ovarian cancer revealed nearly 2% of patients carry a very rare missense variant (minor allele frequency < 0.0001) in BRIP1. This is 3-fold higher than the frequency of all rare BRIP1 missense alleles reported in more than 60,000 individuals of the general population (P < 0.0001, χ2 test). Using CRISPR-Cas9 gene editing technology and rescue assays, we functionally characterized 20 of these missense variants, focusing on the altered protein's ability to repair ICL damage. A total of 75% of the characterized variants rendered the protein hypomorph or null. In a clinical cohort of >117,000 patients with breast and ovarian cancer who underwent panel testing, the combined OR associated with BRIP1 hypomorph or null missense carriers compared with the general population was 2.30 (95% confidence interval, 1.60–3.30; P < 0.0001). These findings suggest that novel missense variants within the helicase domain of BRIP1 may confer risk for both breast and ovarian cancer and highlight the importance of functional testing for additional variants. Significance: Functional characterization of rare variants of uncertain significance in BRIP1 revealed that 75% demonstrate loss-of-function activity, suggesting rare missense alleles in BRIP1 confer risk for both breast and ovarian cancer.

Background Variants at the Oculocutaneous albinism 2 (OCA2)/ HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2) locus have been associated with pigmentation phenotypes as well as risk of developing multiple types of skin cancer. Objectives The goal of this study was to evaluate OCA2/HERC2 locus variants for impact on time to develop cutaneous squamous cell carcinoma (cSCC) in organ transplant recipients (OTRs) who are at elevated risk of developing cSCC. Methods Participants were solid organ transplant recipients ascertained from two centers (n=125 and 261) with an average of 13.1 years follow-up post-transplant. DNA was available for genotyping for all participants in addition to medical records and questionnaire data. The Ohio State University (OSU) study design was a case-control with prospective follow-up, and the University of California San Francisco (UCSF) study design was a national cross-sectional survey with retrospective chart review. Results OCA2 variants rs12913832 and rs916977 were significantly associated with time to first cSCC post-transplant. OTRs homozygous for the brown eye alleles of rs916977 (GG) and rs12913832 (AA) had significant delays of time to first cSCC post-transplant compared to individuals homozygous for the blue eye alleles [HR=0.34, p

Desmoid fibromatosis is a locally aggressive clonal fibroblastic proliferation with high recurrence rates and no metastatic potential. Implicated molecular aberrations occur within the Wnt/β-catenin pathway (APC and β-catenin gene mutations). Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are profibrotic growth factors, downstream from nuclear translocation of β-catenin, that lead to increased fibrogenesis. CTGF (a downstream effector of TGF-β) is a matricellular protein that modulates the activity of growth factors, adhesion molecules, integrins, and extracellular matrix thus playing a central role in tissue remodeling and fibrosis. Recently there has been growing interest in use of extracellular matrix inhibitors for treatment of various fibrogenic diseases. Desmoid fibromatosis samples (n=15) were evaluated for expression of β-catenin, TGF-β, and CTGF using immunohistochemistry on formalin paraffin-embedded material. A control group comprising scar tissue and adjacent normal skin (n=10) were simultaneously immunostained with above mentioned markers. Real-time polymerase chain reaction was performed on frozen specimens of desmoid fibromatosis (n=6) and normal skin (n=2). All 15 desmoid tumors were positive for β-catenin (surrogate marker of Wnt/β-catenin pathway dysregulation) which was negative in control normal skin and scar samples. TGF-β and CTGF were negative in 9 of 10 normal skin controls. TGF-β and CTGF were positive in all cases of scar tissue. All 15 cases of desmoid tumors were positive for TGF-β and CTGF. The real-time polymerase chain reaction showed higher expression levels of TGF-β and CTGF in desmoid fibromatosis compared with normal skin. The high constitutive expression of β-catenin downstream effectors; TGF-β, CTGF has the potential for enabling targeted therapy.

Germline loss-of-function mutations in BRIP1 are associated with ovarian carcinoma and may also contribute to breast cancer risk, particularly among patients who develop disease at an early age. Normal BRIP1 activity is required for DNA interstrand crosslink repair, and is thus central to the maintenance of genome stability. Although pathogenic mutations have been identified in BRIP1, genetic testing more often reveals missense variants for which the impact on molecular function and subsequent roles in cancer risk are uncertain. Next generation sequencing of germline DNA in 2,160 early-onset breast cancer and 1,199 ovarian cancer patients revealed nearly 2% of patients carry a very rare missense variant (MAF117,000 breast and ovarian cancer patients who underwent panel testing, the combined odds ratio associated with BRIP1 hypomorph or null missense carriers compared to the general population was 2.30 (95%CI=1.60-3.30, p

BACKGROUND Exome and targeted sequencing studies have identified potential driver mutations for a variety of tumor types. Cutaneous squamous cell carcinoma (cSCC) is one of the most highly mutated cancers but typically is associated with low rates of metastasis and high survival rates. Nevertheless, metastatic cSCC is a significant health threat; up to 8800 individuals die each year of this disease. METHODS Because it is difficult to predict which cSCCs are more likely to metastasize, and because to the best of the authors' knowledge there are no targeted therapies specifically designated for patients with metastatic cSCC, exome and/or targeted sequencing of 18 metastatic and 10 primary cSCCs was performed to identify mutations that were more frequent in metastatic tumors and might be targeted for therapeutic benefit. The authors compared their results with published sequencing results of an additional 223 primary tumors and 68 metastatic cSCCs. RESULTS The authors identified genes demonstrating higher mutation frequencies in metastatic cSCC compared with primary tumors, including the chromatin remodeling gene lysine methyltransferase 2D (KMT2D) and the classic skin tumor suppressor tumor protein p53 (TP53), which was found to be mutated in 54% of primary tumors compared with 85% of metastatic tumors (P

Genes associated with hereditary breast and ovarian cancer (HBOC) are often sequenced in search of mutations that are predictive of susceptibility to these cancer types, but the sequence results are frequently ambiguous because of the detection of missense substitutions for which the clinical impact is unknown. The BARD1 protein is the heterodimeric partner of BRCA1 and is included on clinical gene panels for testing for susceptibility to HBOC. Like BRCA1, it is required for homology-directed DNA repair (HDR). We measured the HDR function of 29 BARD1 missense variants, 27 culled from clinical test results and two synthetic variants. Twenty-three of the assayed variants were functional for HDR; of these, four are known neutral variants. Three variants showed intermediate function, and three others were defective in HDR. When mapped to BARD1 domains, residues crucial for HDR were located in the N- and C- termini of BARD1. In the BARD1 RING domain, critical residues mapped to the zinc-coordinating amino acids and to the BRCA1-BARD1 binding interface, highlighting the importance of interaction between BRCA1 and BARD1 for HDR activity. Based on these results, we propose that the HDR assay is a useful complement to genetic analyses to classify BARD1 variants of unknown clinical significance.

Background Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. Methods MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Results Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. Conclusion These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.

Mus spretus mice are highly resistant to several types of cancer compared to Mus musculus mice. To determine whether differences in microRNA (miRNA) expression account for some of the differences in observed skin cancer susceptibility between the strains, we performed miRNA expression profiling of skin RNA for over 300 miRNAs. Five miRNAs, miR-1, miR-124a-3, miR-133a, miR-134, miR-206, were differentially expressed by array and/or qPCR. miR-1 was previously shown to have tumor suppressing abilities in multiple tumor types. We found miR-1 expression to be lower in mouse cutaneous squamous cell carcinomas (cSCCs) compared to normal skin. Based on the literature and our expression data, we performed detailed studies on predicted miR-1 targets and evaluated the effect of miR-1 expression on two murine cSCC cell lines, A5 and B9. Following transfection of miR-1, we found decreased mRNA expression of three validated miR-1 targets, Met, Twf1 and Ets1 and one novel target Bag4. Decreased expression of Ets1 was confirmed by Western analysis and by 3’ reporter luciferase assays containing wildtype and mutated Ets1 3’UTR. We evaluated the effect of miR-1 on multiple tumor phenotypes including apoptosis, proliferation, cell cycle and migration. In A5 cells, expression of miR-1 led to decreased proliferation compared to a control miR. miR-1 expression also led to increased apoptosis at later time points (72 and 96 h) and to a decrease in cells in S-phase. In summary, we identified five miRNAs with differential expression between cancer resistant and cancer susceptible mice and found that miR-1, a candidate tumor suppressor, has targets with defined roles in tumorigenesis.

Although only a small proportion of cutaneous squamous cell carinomas (cSCC) metastasize, approximately 3900-8800 individuals in the United States die of metastatic cSCC each year. Little is known about the molecular events that lead approximately 2-6% of cSCCs to metastasize. Treatment efforts are hampered by a lack of ability to predict which lesions are more likely to recur or metastasize as these known correlates do not have high specificity for prediction. Furthermore, there is no FDA approved therapy that is specifically indicated for metastatic cSCC. Deep-sequencing efforts have identified targetable mutations and pathways for several cancers. Exome and targeted sequencing of primary cSCCs led to the discovery that inactivating mutations in NOTCH pathway genes occur in over 75% of tumors; however NOTCH mutations are not indicative of metastasis. Published studies identified frequent mutations in KMT2C and KMT2D in cSCCs with aggressive features. To identify mutations that are unique to or enriched in metastatic samples which may help to identify novel therapeutics for treating aggressive cSCCs, we performed exome sequencing of 37 samples consisting of matched normal DNAs, primary tumors and metastatic cSCCs. Many of the mutations found in the metastatic samples were also observed in the matched primary tumors. We found an enrichment of mutations in chromatin remodeling genes. Epigenetic regulatory genes mutated in the metastatic samples included KMT2D (64%), KMT2C (44%), SETD2 (45%), KAT6A (36%), EP300 (18%), and KDM6A/UTX (9%). We performed additional targeted deep-sequencing to evaluate whether candidate genes were frequently mutated in non-aggressive cSCCs. Initial analyses of targeted-sequencing of 10 non-aggressive primary cSCCs and 7 additional metastatic cSCCs as well as data from published exome studies on non-aggressive cSCCs suggest that chromatin remodeling genes are mutated at a much higher rate in metastatic cSCCs. These data provide potential pathways for therapeutic intervention for this deadly cancer. Citation Format: Amanda Ewart Toland, Dawn Allain, Jessica Gillespie, Priyaharsini Nagarajan, Sara Peters, Hatice Gulcin Ozer, Thomas Olencki, Ayse Selen Yilmaz, Theodoros Teknos. Exome sequencing identifies frequent histone methyltransferase mutations in metastatic SCC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 147.