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

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

Risk estimation as a decision-making tool for genetic analysis of the breast cancer susceptibility genes. EC Demonstration Project on Familial Breast Cancer.

For genetic counselling of a woman on familial breast cancer, an accurate evaluation of the probability that she carries a germ-line mutation is needed to assist in making decisions about genetic-testing. We used data from eight collaborating centres comprising 618 families (346 breast cancer only, 239 breast or ovarian cancer) recruited as research families or counselled for familial breast cancer, representing a broad range of family structures. Screening was performed in affected women from 618 families for germ-line mutations in BRCA1 and in 176 families for BRCA2 mutations, using different methods including SSCP, CSGE, DGGE, FAMA and PTT analysis followed by direct sequencing. Germ-line BRCA1 mutations were detected in 132 families and BRCA2 mutations in 16 families. The probability of being a carrier of a dominant breast cancer gene was calculated for the screened individual under the established genetic model for breast cancer susceptibility, first, with parameters for age-specific penetrances for breast cancer only [7] and, second, with age-specific penetrances for ovarian cancer in addition [20]. Our results indicate that the estimated probability of carrying a dominant breast cancer gene gives a direct measure of the likelihood of detecting mutations in BRCA1 and BRCA2. For breast/ovarian cancer families, the genetic model according to Narod et al. [20] is preferable for calculating the proband's genetic risk, and gives detection rates that indicate a 50% sensitivity of the gene test. Due to the incomplete BRCA2 screening of the families, we cannot yet draw any conclusions with respect to the breast cancer only families

Ethical, social and economic issues in familial breast cancer: a compilation of views from the EC biomed II demonstration project

ABSTRACT: Demand for clinical services for familial breast cancer is continuing to rise across Europe. Service provision is far from uniform and, in most centres, its evolution has been determined by local conditions, specifically by local research interests, rather than by central planning. However, in a number of countries there is evidence of progress towards co-ordinated development and audit of clinics providing risk assessment, counselling, screening and, in some cases, prophylactic intervention. Much important information should emerge from continued observation and comparative assessment of these developments. In most countries for which relevant data are available, there is a distinct bias towards higher social class among those who avail themselves of clinic facilities (in line with findings from many other health-promotion initiatives). This should be addressed when considering future organisation of clinical services. Molecular genetic studies designed to identify the underlying mutations responsible for familial breast cancer are not generally regarded as part of the clinical service and are funded through research grants (if at all). Economic considerations suggest that there is a case for keeping this policy under review. Familial cancers throw into sharp relief certain ethical and legal issues that have received much recent attention from government advisory bodies, patients ’ representatives, professional commentators and the popular media. Two are of particular importance; first, the right to gain access to medical records of relatives, in order to provide accurate risk assessment for a given family member, versus the right to privacy in respect of personal medical information and, second, the obligation (or otherwise) to inform family members of their risk status if they have not actively sought that knowledge. The legal position seems to vary from country to country and, in many cases, is unclear. In view of pressures to establish uniform approaches to medical confidentiality across the EC, it is important to evaluate the experience of participants in this Demonstration Programme and to apply the principle of “ non-malfeasance ” in formulating regu- lations that should govern future practice in this field. Data on economic aspects of familial breast cancer are remarkably sparse and outdated. As evidence accrues on the influence of screening and intervention programmes on morbidity and mortality, there is a strong case for evaluating the cost-effectiveness of different models of service provisi

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

Inherited mutations in breast cancer susceptibility genes among a triple negative breast cancer color unselected for family history of breast cancer

Abstract not availabl

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)