A Yeast Recombination Assay to Characterize Human BRCA1 Missense Variants of Unknown Pathological Significance

The BRCA1 tumor suppressor gene is found mutated in familial breast cancer. Although many of the mutations are clearly pathological because they give rise to truncated proteins, several missense variants of uncertain pathological consequences have been identified. A novel functional assay to screen for BRCA1 missense variants in a simple genetic system could be very useful for the identification of potentially deleterious mutations. By using two prediction computer programs, Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen), seven nonsynonymous missense BRCA1 variants likely disrupting the gene function were selected as potentially deleterious. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) was used to test these cancer-related missense mutations for their ability to affect cell growth and homologous recombination (HR) at the HIS3 and ADE2 loci. The variants localized in the BRCA1 C-Terminus (BRCT) domain did not show any growth inhibition when overexpressed in agreement with previous results. Overexpression of either wild-type BRCA1 or two neutral missense variants did not increase yeast HR but when cancer-related variants were overexpressed a significant increase in recombination was observed. Results clearly showed that this genetic system can be useful to discriminate between neutral and deleterious BRCA1 missense variants

A recombination-based method to characterize human BRCA1 missense variants

Purpose. Many missense variants in BRCA1 are of unclear clinical significance. Functional and genetic approaches have been proposed for elucidating the clinical significance of such variants. The purpose of the present study was to evaluate BRCA1 missense variants for their effect on both Homologous Recombination (HR) and Non Homologous End Joining (NHEJ). Methods. HR frequency evaluation: HeLaG1 cells, containing a stably integrated plasmid that allows to measure HR events by gene conversion events were transfected with the pcDNA3β expression vector containing the BRCA1-wild type (BRCA1-WT) or the BRCA1-Unclassified Variants (BRCA1-UCVs). The NHEJ was measured by a random plasmid integration assay. Results. This assays suggested a BRCA1 involvement mainly in the NHEJ. As a matter of fact, the Y179C and the A1789T variant altered significantly the NHEJ activity as compared to the wild type, suggesting that they may be related to BRCA1 associated pathogenicity by affecting this function. The variants N550H and I1766S, and the mutation M1775R did not alter the NHEJ frequency. Conclusions. These data, beside proposing a method for the study of BRCA1 variants effect on HR and NHEJ, highlighted the need for a range of functional assays to be performed in order to identify variants with altered function

Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

BACKGROUND: BRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer. METHODS: We compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain. RESULTS: 201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair. CONCLUSIONS: Our results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup

In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 "new" unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a "clinical warning"; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure)

Characterisation of gene expression profiles of yeast cells expressing BRCA1 missense variants

Germline mutations in breast cancer susceptibility gene 1 (BRCA1) confer high risk of developing breast and ovarian cancers. Even though most BRCA1 cancer-predisposing mutations produce a non-functional truncated protein, 5-10% of them cause single amino acid substitutions. This second type of mutations represents a useful tool for examining BRCA1 molecular functions. Human BRCA1 inhibits cell proliferation in transformed Saccharomyces cerevisiae cells and this effect is abolished by disease-associated mutations in the BRCT domain. Moreover, BRCA1 mutations located both inside and outside the BRCT domain may induce an increase in the homologous recombination frequency in yeast cells. Here we present a microarray analysis of gene expression induced in yeast cells transformed with five BRCA1 missense variants, in comparison with gene expression induced by wildtype BRCA1. Data analysis was performed by grouping the BRCA1 variants into three sets: Recombination (R)-set (Y179C and S1164I), Recombination and Proliferation (RP)-set(I1766S and M1775R) and Proliferation (P)-set (A1789T), according to their effects on yeast cell phenotype. We found 470, 740 and 1136 differentially expressed genes in R-, P- and RP-set, respectively. Our results point to some molecular mechanisms critical for the control of cell proliferation and of genome integrity providing support to a possible pathogenic role of the analysed mutations. They also confirm that yeast, despite the absence of a BRCA1 homologue, represents a valid model system to examine BRCA1 molecular functions, as the molecular pathways activated by BRCA1 variants are conserved in humans

A Multisystem Mitochondrial Disease Caused by a Novel MT-TL1 mtDNA Variant: A Case Report

Background: Mitochondrial tRNA (MTT) genes are hotspot for mitochondrial DNA mutation and are responsible of half mitochondrial disease. MTT mutations are associated with a broad spectrum of phenotype often with complex multisystem involvement and complex genotype-phenotype correlations. MT-TL1 mutations, among which the m.3243A>G mutation is the most frequent, are associated with myopathy, maternal inherited diabetes and deafness, MELAS, cardiomyopathy, and focal segmental glomerulosclerosis.Case study: Here we report the case of an Italian 49-years old female presenting with encephalomyopathy, chronic proteinuric kidney disease and a new heteroplasmic m.3274 3275delAC MT-TL1 gene mutation.Conclusions: Our case demonstrates a systemic mitochondrial disease caused by the heteroplasmic m.3274 3275delAC MT-TL1 gene mutation, not yet described in the literature. A mitochondrial disease should be suspected in case of complex multisystem phenotypes, including steroid-resistant nephrotic syndrome with multisystemic involvement

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer.

Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM -/- patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

Ovarian cancer pathology characteristics as predictors of variant pathogenicity in BRCA1 and BRCA2

Background: The distribution of ovarian tumour characteristics differs between germline BRCA1 and BRCA2 pathogenic variant carriers and non-carriers. In this study, we assessed the utility of ovarian tumour characteristics as predictors of BRCA1 and BRCA2 variant pathogenicity, for application using the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) variant classification system. Methods: Data for 10,373 ovarian cancer cases, including carriers and non-carriers of BRCA1 or BRCA2 pathogenic variants, were collected from unpublished international cohorts and consortia and published studies. Likelihood ratios (LR) were calculated for the association of ovarian cancer histology and other characteristics, with BRCA1 and BRCA2 variant pathogenicity. Estimates were aligned to ACMG/AMP code strengths (supporting, moderate, strong). Results: No histological subtype provided informative ACMG/AMP evidence in favour of BRCA1 and BRCA2 variant pathogenicity. Evidence against variant pathogenicity was estimated for the mucinous and clear cell histologies (supporting) and borderline cases (moderate). Refined associations are provided according to tumour grade, invasion and age at diagnosis. Conclusions: We provide detailed estimates for predicting BRCA1 and BRCA2 variant pathogenicity based on ovarian tumour characteristics. This evidence can be combined with other variant information under the ACMG/AMP classification system, to improve classification and carrier clinical management.</p

BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc. The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5′ noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency \u3c 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C\u3eT and PAX5 binding to BRCA2:c.-296C\u3eT. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC