Marcelo A. Carvalho

Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis

Germ line inactivating mutations in BRCA1 confer susceptibility for breast and ovarian cancer. However, the relevance of the many missense changes in the gene for which the effect on protein function is unknown remains unclear. Determination of which variants are causally associated with cancer is important for assessment of individual risk. We used a functional assay that measures the transactivation activity of BRCA1 in combination with analysis of protein modeling based on the structure of BRCA1 BRCT domains. In addition, the information generated was interpreted in light of genetic data. We determined the predicted cancer association of 22 BRCA1 variants and verified that the common polymorphism S1613G has no effect on BRCA1 function, even when combined with other rare variants. We estimated the specificity and sensitivity of the assay, and by meta-analysis of 47 variants, we show that variants with <45% of wild-type activity can be classified as deleterious whereas variants with >50% can be classified as neutral. In conclusion, we did functional and structure-based analyses on a large series of BRCA1 missense variants and defined a tentative threshold activity for the classification missense variants. By interpreting the validated functional data in light of additional clinical and structural evidence, we conclude that it is possible to classify all missense variants in the BRCA1 COOH-terminal region. These results bring functional assays for BRCA1 closer to clinical applicability.

A guide for functional analysis of BRCA1 variants of uncertain significance

Germline mutations in the tumor suppressor gene BRCA1 confer an estimated lifetime risk of 56–80% for breast cancer and 15–60% for ovarian cancer. Since the mid 1990s when BRCA1 was identified, genetic testing has revealed over 1,500 unique germline variants. However, for a significant number of these variants, the effect on protein function is unknown making it difficult to infer the consequences on risks of breast and ovarian cancers. Thus, many individuals undergoing genetic testing for BRCA1 mutations receive test results reporting a variant of uncertain clinical significance (VUS), leading to issues in risk assessment, counseling, and preventive care. Here, we describe functional assays for BRCA1 to directly or indirectly assess the impact of a variant on protein conformation or function and how these results can be used to complement genetic data to classify a VUS as to its clinical significance. Importantly, these methods may provide a framework for genome‐wide pathogenicity assignment. Hum Mutat 33:1526–1537, 2012.

Charting the Landscape of Tandem BRCT Domain–Mediated Protein Interactions

Building a protein-protein interaction network mediated by the BRCT domain reveals players in the DNA damage response. Networking the DNA Damage Response The protein-protein interaction domain BRCT is found in proteins that participate in the DNA damage response (DDR) and has been linked to cancer. A well-known BRCT domain–containing protein is the DDR protein BRCA1, encoded by a gene that is associated with breast cancer. Woods et al. performed systems analysis to identify proteins that interacted with BRCT domains in cells exposed to DNA damage. Bioinformatics analysis of the BRCT protein-protein interaction network revealed biological processes and protein complexes that integrate the DDR with cell cycle regulation and transcription. Because of the importance of BRCT domain–containing proteins in cancer, understanding the cellular response to DDR-inducing chemotherapy and radiation therapy can aid in the development of effective treatments. Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR by combining literature curation, yeast two-hybrid screens, and tandem affinity purification coupled to mass spectrometry. We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.

Functional Impact of Missense Variants in BRCA1 Predicted by Supervised Learning

Many individuals tested for inherited cancer susceptibility at the BRCA1 gene locus are discovered to have variants of unknown clinical significance (UCVs). Most UCVs cause a single amino acid residue (missense) change in the BRCA1 protein. They can be biochemically assayed, but such evaluations are time-consuming and labor-intensive. Computational methods that classify and suggest explanations for UCV impact on protein function can complement functional tests. Here we describe a supervised learning approach to classification of BRCA1 UCVs. Using a novel combination of 16 predictive features, the algorithms were applied to retrospectively classify the impact of 36 BRCA1 C-terminal (BRCT) domain UCVs biochemically assayed to measure transactivation function and to blindly classify 54 documented UCVs. Majority vote of three supervised learning algorithms is in agreement with the assay for more than 94% of the UCVs. Two UCVs found deleterious by both the assay and the classifiers reveal a previously uncharacterized putative binding site. Clinicians may soon be able to use computational classifiers such as those described here to better inform patients. These classifiers can be adapted to other cancer susceptibility genes and systematically applied to prioritize the growing number of potential causative loci and variants found by large-scale disease association studies.

Breast Cancer Susceptibility and the DNA Damage Response

Breast cancer is a disease caused by a complex combination of genetic and environmental factors. It is one of the most common types of cancer affecting women in the Western world. In 2004 in the United States, it is estimated that more than 200,000 new cases of breast cancer will be diagnosed and over 40,000 will die of this disease. Linkage analysis of families with a high risk of breast cancer has identified two major susceptibility genes: BRCA1 and BRCA2.1,2 In the context of large, multiple-case families, the BRCA1 and BRCA2 genes are numerically the most important, accounting for more than 80% of families with six or more cases of both early-onset breast cancer and ovarian cancer.3 However, the probability of harboring a mutation is much lower in families with fewer cases of the disease, and population studies have demonstrated that these genes account for only a minority of the overall familial risk of breast cancer. In fact, as many as 60% of families with site-specific female breast cancer cannot be explained by mutations in BRCA1 and BRCA2.4,5 In addition,mutations in these genes are relatively rare in the general population. Together they account for less than 10% of all breast cancer cases4,6 (Fig 1). Therefore, the challenge is how to identify individuals at risk for the remaining cases. Conceivably, if we could identify the major genetic factors that contribute to breast cancer risk, we would be able to not only provide comprehensive early identification of individuals at risk but also tailor prevention and treatment regimens to adequately address specific molecular changes in these cancers.

Pathogenicity of the BRCA1 missense variant M1775K is determined by the disruption of the BRCT phosphopeptide-binding pocket: a multi-modal approach.

A number of germ-line mutations in the BRCA1 gene confer susceptibility to breast and ovarian cancer. However, it remains difficult to determine whether many single amino-acid (missense) changes in the BRCA1 protein that are frequently detected in the clinical setting are pathologic or not. Here, we used a combination of functional, crystallographic, biophysical, molecular and evolutionary techniques, and classical genetic segregation analysis to demonstrate that the BRCA1 missense variant M1775K is pathogenic. Functional assays in yeast and mammalian cells showed that the BRCA1 BRCT domains carrying the amino-acid change M1775K displayed markedly reduced transcriptional activity, indicating that this variant represents a deleterious mutation. Importantly, the M1775K mutation disrupted the phosphopeptide-binding pocket of the BRCA1 BRCT domains, thereby inhibiting the BRCA1 interaction with the proteins BRIP1 and CtIP, which are involved in DNA damage-induced checkpoint control. These results indicate that the integrity of the BRCT phosphopeptide-binding pocket is critical for the tumor suppression function of BRCA1. Moreover, this study demonstrates that multiple lines of evidence obtained from a combination of functional, structural, molecular and evolutionary techniques, and classical genetic segregation analysis are required to confirm the pathogenicity of rare variants of disease-susceptibility genes and obtain important insights into the underlying pathogenetic mechanisms.

Acidic pH Modulates the Interaction between Human Granulocyte-Macrophage Colony-stimulating Factor and Glycosaminoglycans

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls growth and differentiation of hematopoietic cells. Previous reports have indicated that the mitogenic activity of GM-CSF may be modulated by the glycosidic moiety of proteoglycans associated with the membrane of stromal cells. In this work, we have performed in vitrostudies of the interaction between GM-CSF and glycosaminoglycans. The addition of heparin promoted a marked blue shift in the fluorescence emission spectrum of GM-CSF as well as a 30-fold increase in the intensity of light scattering, which indicates formation of large molecular weight complexes between the two molecules. Interestingly, heparin-induced changes in the spectral properties of GM-CSF were only observed at acidic pH. The dependence on acidic pH, together with a strict dependence on glycosaminoglycan sulfation and the fact that high ionic strength destabilized the interaction, indicates that the association between GM-CSF and glycosaminoglycans is mediated by electrostatic interactions. These interactions probably involve sulfate groups in the glycosaminoglycans and positively charged histidine residues in GM-CSF. We propose that negatively charged glycolipids present on the plasma membrane of the hematopoietic and/or the stromal cell could promote an acidic microenvironment capable of triggering interaction between GM-CSF and membrane-bound proteoglycans in vivo.

Analysis of a set of missense, frameshift, and in-frame deletion variants of BRCA1

Germline mutations that inactivate BRCA1 are responsible for breast and ovarian cancer susceptibility. One possible outcome of genetic testing for BRCA1 is the finding of a genetic variant of uncertain significance for which there is no information regarding its cancer association. This outcome leads to problems in risk assessment, counseling and preventive care. The purpose of the present study was to functionally evaluate seven unclassified variants of BRCA1 including a genomic deletion that leads to the in-frame loss of exons 16/17 (Delta exons 16/17) in the mRNA, an insertion that leads to a frameshift and an extended carboxy-terminus (5673insC), and five missense variants (K1487R, S1613C, M1652I, Q1826H and V1833M). We analyzed the variants using a functional assay based on the transcription activation property of BRCA1 combined with supervised learning computational models. Functional analysis indicated that variants S1613C, Q1826H, and M1652I are likely to be neutral, whereas variants V1833M, Delta exons 16/17, and 5673insC are likely to represent deleterious variants. In agreement with the functional analysis, the results of the computational analysis also indicated that the latter three variants are likely to be deleterious. Taken together, a combined approach of functional and bioinformatics analysis, plus structural modeling, can be utilized to obtain valuable information pertaining to the effect of a rare variant on the structure and function of BRCA1. Such information can, in turn, aid in the classification of BRCA1 variants for which there is a lack of genetic information needed to provide reliable risk assessment.

Identification of filamin A as a BRCA1-interacting protein required for efficient DNA repair

The product of the breast and ovarian cancer susceptibility gene BRCA1 has been implicated in several aspects of the DNA damage response but its biochemical function in these processes has remained elusive. In order to probe BRCA1 function we conducted a yeast two-hybrid screening to identify interacting partners to a conserved motif (Motif 6) in the central region of BRCA1. Here we report the identification of the actin-binding protein Filamin A (FLNA) as BRCA1 partner and demonstrate that FLNA is required for efficient regulation of early stages of DNA repair processes. Cells lacking FLNA display a diminished BRCA1 IR-induced focus formation and a delayed kinetics of Rad51 focus formation. In addition, our data also demonstrate that FLNA is required to stabilize the interaction between components of the DNA-PK holoenzyme, DNA-PKcs and Ku86 in a BRCA1-independent fashion. Our data is consistent with a model in which absence of FLNA compromises homologous recombination and non-homologous end joining. Our findings have implications for the response to irradiation induced DNA damage.

The capacity of connective tissue stromas to sustain myelopoiesis depends both upon the growth factors and the local intercellular environment

Summry— In adults, haemopoiesis is located in the bone marrow, where it is tightly regulated by cytokines and by a physical association of haemopoietic progenitors with the stroma. However, in pathological situations, haemopoiesis can be partly or fully dislodged to peripheral tissues. It is not clear which are the requirements for a given peripheral stroma to sustain haemopoiesis. Using the growth factor‐dependent cell line FDC‐P1, we have compared the myelopoietic capacities of a murine bone marrow‐derived cell line S17, a liver inflammatory granuloma‐derived stroma (GR) that sustains haemopoiesis, and normal skin fibroblasts (SF) that sustain neither survival nor proliferation of myeloid cells. All three stromas expressed mRNA for major haemopoietins with the exception of IL‐3. Despite the incapacity of SF to sustain FDC‐P1 cells, the biologically active GM—CSF could be recovered from all the studied stromas by treatment with high‐salt buffers that release non‐covalently bound molecules from stroma cells. Glycosaminoglycans purified from stromas had distinct effect on the GM—CSF‐mediated proliferation of FDC‐P1 cells: those purified from S17 and GR cells were stimulatory, whereas those obtained from SF cells were slightly stimulatory at low concentration, but inhibitory at the higher ones. We conclude that the quality of the stroma pericellular glycoconjugates is determinant for the ability of a given stroma to sustain myelopoiesis, even when biologically active haemopoietins are locally produced.