BRCA1 and BRCA2 genetic testing-pitfalls and recommendations for managing variants of uncertain clinical significance.

BACKGROUND: Increasing use of BRCA1/2 testing for tailoring cancer treatment and extension of testing to tumour tissue for somatic mutation is moving BRCA1/2 mutation screening from a primarily prevention arena delivered by specialist genetic services into mainstream oncology practice. A considerable number of gene tests will identify rare variants where clinical significance cannot be inferred from sequence information alone. The proportion of variants of uncertain clinical significance (VUS) is likely to grow with lower thresholds for testing and laboratory providers with less experience of BRCA. Most VUS will not be associated with a high risk of cancer but a misinterpreted VUS has the potential to lead to mismanagement of both the patient and their relatives. DESIGN: Members of the Clinical Working Group of ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) global consortium (www.enigmaconsortium.org) observed wide variation in practices in reporting, disclosure and clinical management of patients with a VUS. Examples from current clinical practice are presented and discussed to illustrate potential pitfalls, explore factors contributing to misinterpretation, and propose approaches to improving clarity. RESULTS AND CONCLUSION: Clinicians, patients and their relatives would all benefit from an improved level of genetic literacy. Genetic laboratories working with clinical geneticists need to agree on a clinically clear and uniform format for reporting BRCA test results to non-geneticists. An international consortium of experts, collecting and integrating all available lines of evidence and classifying variants according to an internationally recognized system, will facilitate reclassification of variants for clinical use.

Modes of delivery of genetic testing services and the uptake of cancer risk management strategies in BRCA1 and BRCA2 carriers.

BRCA testing services are now offered by various healthcare providers, thus it is important to evaluate whether the implementation of cancer risk management (CRM) strategies varies by service provider. Using a registry-based sample of 795 female BRCA mutation carriers, we explored the association between uptake of CRM strategies with duration of genetic counseling (GC) sessions, provider type, and other demographic and clinical variables. All participants completed a baseline questionnaire. Information about uptake of CRM strategies was collected for a subset of 438 participants who completed additional questions. Summary statistics and Pearson chi-squared analysis were used to examine the associations between demographic and clinical variables with service delivery factors and with the uptake of various CRM strategies. Overall uptake of CRM strategies was high across all provider types. However, GC sessions were longer when provided by a genetics professional than by another provider (p < 0.001). Furthermore, higher frequencies of uptake of most CRM strategies were associated with longer GC sessions and when testing was performed by a genetics professional. Identification of factors to optimize delivery of these specialized GC services is important to maximize implementation of CRM strategies in BRCA carriers.

Lessons from postgenome-wide association studies: functional analysis of cancer predisposition loci.

In the last few years, genome-wide association studies (GWASs) have identified hundreds of predisposition loci for several types of human cancers. Recent progress has been made in determining the underlying mechanisms through which different single-nucleotide polymorphisms (SNPs) affect predisposition to cancer. Although there has been much debate about the clinical utility of GWASs, less attention has been paid to how GWASs and post-GWASs functional analysis have contributed to understanding the aetiology of cancer. Most common variants associated with cancer risk are localized in nonprotein-coding regions highlighting transcriptional regulation as a common theme in the mechanism of cancer predisposition. Here, we outline strategies to functionally dissect predisposition loci and discuss their limitations as well as challenges for future studies.

Evolution of the triplet BRCT domain.

Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C-Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

Effects of dicopper oxide and copper sulfate on growth performance and gut microbiota in broilers.

An experiment was conducted to determine the effects of two sources of copper (Cu) from copper sulfate (CuSO4) and dicopper oxide (Cu2O, CoRouge) at three levels of inclusion (15, 75, and 150 mg/kg) on growth performance and gut microbiota of broilers. A total of 840 one-d-old male chickens (Ross 308) were weighed and randomly allocated to seven dietary treatments: negative control (NC, a basal diet without Cu addition), and the NC supplemented with 15, 75, or 150 mg Cu/kg from CuSO4 or Cu2O (12 replicate pens/treatment, 10 chicks per pen). Broilers were challenged by reusing an old litter with high concentrations in Clostridium perfringens to promote necrotic enteritis. Broiler performance was registered at d 21, 35, and 42. Excreta samples were collected at d 14, 28, and 42 for antimicrobial resistance (AMR) analyses. At d 43, one broiler per pen was euthanized to obtain ileal content for microbial characterization. Body weight d 35 and daily gain d 42 improved (P < 0.05) in Cu2O as Cu dose inclusion increased from 15 mg/kg to 150 mg/kg. Supplementation of 150 mg/kg of Cu from Cu2O decreased the abundance (P < 0.01) of some families such as Streptococcaceae and Corynebacteriaceae and increased the abundance (P < 0.05) of some commensal bacteria like Clostridiaceae and Peptostreptococcaceae. Phenotypic AMR was not different among treatments on d 14 and 28. Isolated Enterococcus spp. from broilers fed the NC diet on d 42 showed higher (P < 0.05) resistance to enrofloxacin, gentamicin, and chloramphenicol compared with Cu treatments. By contrast, the isolated Escherichia coli from broilers fed 150 mg/kg of Cu, either from CuSO4 or Cu2O, showed higher (P < 0.05) resistance to streptomycin and chloramphenicol compared to the NC. This study suggests that supplementing 150 mg/kg of Cu from Cu2O establishes changes in the gut microbiota by regulating the bacterial population in the ileum, which may explain the positive impact on broilers' growth performance.

In situ protein expression of RRM1, ERCC1, and BRCA1 in metastatic breast cancer patients treated with gemcitabine-based chemotherapy.

Ribonucleotide reductase 1 (RRM1) is a determinant of gemcitabine efficacy in non-small-cell lung cancer and pancreatic cancer. We investigated the protein levels of RRM1 and two other DNA repair enzymes, ERCC1 and BRCA1, in 55 metastatic breast cancer (MBC) patients undergoing gemcitabine-based chemotherapy. With automated in situ protein quantification (AQUA v1.6), the average scores for RRM1, ERCC1, and BRCA1 ranged from 245.6-2774.1, 74.0-410.3, and 54.4-1833.1, respectively. They were significantly associated with each other (Spearman's rho > or = .36; p < or = .007). Given their pattern of distribution, RRM1 and BRCA1 are potentially suitable markers for clinical decision making in MBC.

BRCA1: exploring the links to transcription.

Progress on determining the function of the breast and ovarian cancer susceptibility gene BRCA1 suggests it might be involved in two fundamental cellular processes: DNA repair and transcriptional regulation. Recent developments indicate that BRCA1 is a multifunctional protein, and disruption of its transcriptional activity could be crucial for tumor development.

Cerebral and cardiac congenital malformations in neonatal West Indian manatees (Trichechus manatus).

Strandings of live new-born West Indian manatees (WIMs; Trichechus manatus) are one of the main challenges for the conservation of this species in Brazil, particularly in the northeastern states. Congenital malformations (CMs) are rare in sirenians. We identified CMs in two of 19 stranded WIMs that were rescued, rehabilitated and subjected to complete pathological examinations in Ceará and Rio Grande do Norte States between 1992 and 2017. In case 1, dilation of the cerebral lateral and fourth ventricles with abundant cerebrospinal fluid (internal hydrocephalus), was diagnosed. Furthermore, this animal developed necrotizing enterocolitis associated with pneumatosis intestinalis and aspiration pneumonia late during rehabilitation. Cardiac malformations in case 2 included: right ventricle hypoplasia with marked stenosis of the tricuspid outflow, high ventricular septal defect, segmental pulmonary artery aneurysm, mitral valve haemocyst and left ventricular hypertrophy. Herein, we provide the first description of a neural tube defect, specifically a developmental internal hydrocephalus, and multiple cardiac congenital anomalies, together with their respective clinicopathological features in manatees. Although the aetiology of the CMs remains unknown in these cases, a genetic basis is plausible given the low genetic variability in this population. These cases add to the body of knowledge on health and disease aspects of manatees and may provide scientific basis for future medical and conservation efforts on neonatal WIMs.

Cancer risks in first degree relatives of BRCA1 mutation carriers: effects of mutation and proband disease status.

BACKGROUND: Mutations in the BRCA1 (MIM 113705) gene are found in many families with multiple cases of breast and ovarian cancer, and women with a BRCA1 mutation are at significantly higher risk of developing breast and ovarian cancer than are the general public. METHODS: We obtained blood samples and pedigree information from 3568 unselected cases of early-onset breast cancer and 609 unselected patients with ovarian cancer from hospitals throughout Poland. Genetic testing was performed for three founder BRCA1 mutations. We also calculated the risk of breast and ovarian cancer to age 75 in the first degree relatives of carriers using Kaplan-Meier methods. RESULTS: The three founder BRCA1 mutations were identified in 273 samples (187 with 5382insC, 22 with 4153delA, and 64 with C61G). A mutation was present in 4.3% of patients with breast cancer and 12.3% of patients with ovarian cancer. The overall risk of breast cancer to age 75 in relatives was 33% and the risk of ovarian cancer was 15%. The risk for breast cancer was 42% higher among first degree relatives of carriers of the C61G missense mutation compared to other mutations (HR = 1.42; p = 0.10) and the risk for ovarian cancer was lower than average (OR = 0.26; p = 0.03). Relatives of women diagnosed with breast cancer had a higher risk of breast cancer than relatives of women diagnosed with ovarian cancer (OR = 1.7; p = 0.03). CONCLUSIONS: The risk of breast cancer in female relatives of women with a BRCA1 mutation depends on whether the proband was diagnosed with breast or ovarian cancer.

Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases.

When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.

Classification of BRCA1 missense variants of unknown clinical significance.

BACKGROUND: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. OBJECTIVE: To investigate a panel of missense variants. METHODS AND RESULTS: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. CONCLUSIONS: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.

Functional assay for BRCA1: mutagenesis of the COOH-terminal region reveals critical residues for transcription activation.

The breast and ovarian cancer susceptibility gene product BRCA1 is a tumor suppressor, but its precise biochemical function remains unknown. The BRCA1 COOH terminus acts as a transcription activation domain, and germ-line cancer- predisposing mutations in this region abolish transcription activation, whereas benign polymorphisms do not. These results raise the possibility that loss of transcription activation by BRCA1 is crucial for oncogenesis. Therefore, identification of residues involved in transcription activation by BRCA1 will help understand why particular germ-line missense mutations are deleterious and may provide more reliable presymptomatic risk assessment. The BRCA1 COOH terminus (amino acids 1560-1863) consists of two BRCTs preceded by a region likely to be nonglobular. We combined site-directed and random mutagenesis, followed by a functional transcription assay in yeast: (a) error-prone PCR-induced random mutagenesis generated eight unique missense mutations causing loss of function, six of which targeted hydrophobic residues conserved in canine, mouse, rat, and human BRCA1; (b) random insertion of a variable pentapeptide cassette generated 21 insertion mutants. All pentapeptide insertions NH2-terminal to the BRCTs retained wild-type activity, whereas insertions in the BRCTs were, with few exceptions, deleterious; and (c) site-directed mutagenesis was used to characterize five known germ-line mutations and to perform deletion analysis of the COOH terminus. Deletion analysis revealed that the integrity of the most COOH-terminal hydrophobic cluster (I1855, L1854, and Y1853) is necessary for activity. We conclude that the integrity of the BRCT domains is crucial for transcription activation and that hydrophobic residues may be important for BRCT function. Therefore, the yeast-based assay for transcription activation can be used successfully to provide tools for structure-function analysis of BRCA1 and may form the basis of a BRCA1 functional assay.

Effect of feeding strategy on environmental impacts of pig fattening in different contexts of production: evaluation through life cycle assessment.

Life cycle assessment (LCA) has been used in many studies to evaluate the effect of feeding strategy on the environmental impact of pig production. However, because most studies have been conducted in European conditions, the question of possible interactions with the context of production is still under debate. The objective of this study was to evaluate these effects in 2 contrasted geographic contexts of production, South America (Brazil) and Europe (France). The LCA considered the process of pig fattening, including production and transport of feed ingredients and feed, raising of fattening pigs, and manure storage, transport, and spreading. Impacts were calculated at the farm gate, and the functional unit considered was 1 kg of BW gain over the fattening period. The performances of pigs were simulated for each scenario using the InraPorc population model (2,000 pigs per scenario considering between-animal variability). The LCA calculations were performed for each pig according to its own performance and excretion, and the results were subjected to variance analysis. The results indicate that for some impacts there are clear interactions between the effects of the feeding program, the origin of soybean, and the location of production. For climate change, interest in phase feeding and incorporation of crystalline AA (CAA) is limited and even counterproductive in Brazil with soybeans from the South (without deforestation), whereas they appear to be efficient strategies with soybeans from the Center West (with deforestation), especially in France. Rather similar effects, as those for climate change, were observed for cumulative energy demand. Conversely, potential eutrophication and acidification impacts were reduced by phase feeding and CAA addition in a rather similar way in all situations. Individual daily feeding, the only strategy that took into account between-animal variability, was the most effective approach for reducing the life cycle impact of pig fattening in all situations, whereas the potential of phase feeding programs and CAA was dependent on soybean origin and the geographical context of pig production, in contrast with previous results.

A nuclear function for the tumor suppressor BRCA1.

The breast and ovarian cancer susceptibility gene BRCA1 has been recently cloned and revealed an open reading frame of 1863 amino acids, but a lack of significant homology to any known protein in the database has led to few clues about its functions. One of the first steps to investigate the function of BRCA1 was to define its subcellular localization. Several reports have led to contradictory findings that include: nuclear localization in normal cells and cytoplasmic in breast and ovarian cancer cells; nuclear in both normal and cancer cells; cytoplasmic and secreted to the extracellular space; present in tube-like invaginations of the nucleus; and colocalizing with the centrosome. As is apparent, the subcellular localization has been the most controversial aspect of BRCA1 biology and is a key point to uncover its functions. In this paper we review the published data on subcellular localization of BRCA1 with special emphasis on the antibodies and techniques used. We conclude that there is now overwhelming evidence to support a nuclear localization for BRCA1, both in normal and cancer cells. In addition, several BRCA1-interacting proteins have been isolated and they are preferentially located in the nucleus. Evidence supporting a physiological function for BRCA1 during DNA repair and transcriptional activation is also discussed.

Yeast-based assays for detection and characterization of mutations in BRCA1.

Germ-line mutations in BRCA1 account for the majority of families with breast and ovarian cancer predisposition. BRCA1 encodes a 1,863 amino acid protein with no ascribed function. Due to its size and the fact that mutations are evenly scattered along the sequence, screening for mutations is particularly challenging. Here we review recently published yeast-based assays that may form the basis of an alternative diagnostic test for BRCA1. Although individually limited, these assays may, when combined, become a useful method to screen for cancer predisposing mutations. In any event, the yeast-based assays could complement results from direct sequencing providing functional information about unique mutations.

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro.

Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.

In vitro collagen synthesis by liver connective tissue cells isolated from schistosomal granulomas.

Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.

Clonal heterogeneity in murine liver myofibroblasts.

GR primary cells cultures were isolated from hepatic granulomas induced in C3H mice livers by Schistosoma mansoni infection; the GRX continuous cell line was derived from GR cells after long-term culture and a progressive drift towards a rapidly proliferating cell population. These cells were analyzed and compared in terms of their clonal heterogeneity. Clones were classified on the basis of cell substrate, cell-cell adhesion (growth morphology of the clone) and fat droplet accumulation. GR cells were composed of two slow-growing clone types, while GRX cells gave rise to clones with several phenotypes, including the two found in the GR cells. The overall proportion of different clones in the GRX cell population was stable in long-term cultures, as well as after recloning of the highly proliferating, but not the slowly proliferating, clones. We propose that the slow-growing clones are maintained in the overall population by continuous contribution of new slow-growing cells from the rapidly growing ones. The slow-growing clones may represent the basal population of liver connective tissue cells that can be mobilized into injured tissues and that are involved in tissue repair. The highly proliferating clones with a broad capacity of phenotype expression that arise after long-term growth stimulation of the local cell population may represent the hypertrophic connective tissue cells, such as those observed in progressive fibrotic reactions associated with chronic liver tissue inflammation.

Effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6 in a serum-free medium.

The effect of various factors and substrates on the growth of a human hepatoblastoma cell line, HuH-6, which was inoculated at low density in a serum-free medium was examined. Several supplements were required to enhance cell growth of HuH-6. These included cholera toxin (CT), glucagon (Glu) and selenium (Se). Type IV collagen (C-IV) provided the most conductive environment tested for cell growth. These results suggest that CT, Glu, Se, and C-IV are important stimulators for the continuous growth of HuH-6 in a serum-free medium at low density.