Germline Missense Variants in <i>BRCA1</i>: New Trends and Challenges for Clinical Annotation

Genetic testing allows for the identification of germline DNA variations, which are associated with a significant increase in the risk of developing breast cancer (BC) and ovarian cancer (OC). Detection of a BRCA1 or BRCA2 pathogenic variant triggers several clinical management actions, which may include increased surveillance and prophylactic surgery for healthy carriers or treatment with the PARP inhibitor therapy for carriers diagnosed with cancer. Thus, standardized validated criteria for the annotation of BRCA1 and BRCA2 variants according to their pathogenicity are necessary to support clinical decision-making and ensure improved outcomes. Upon detection, variants whose pathogenicity can be inferred by the genetic code are typically classified as pathogenic, likely pathogenic, likely benign, or benign. Variants whose impact on function cannot be directly inferred by the genetic code are labeled as variants of uncertain clinical significance (VUS) and are evaluated by multifactorial likelihood models that use personal and family history of cancer, segregation data, prediction tools, and co-occurrence with a pathogenic BRCA variant. Missense variants, coding alterations that replace a single amino acid residue with another, are a class of variants for which determination of clinical relevance is particularly challenging. Here, we discuss current issues in the missense variant classification by following a typical life cycle of a BRCA1 missense variant through detection, annotation and information dissemination. Advances in massively parallel sequencing have led to a substantial increase in VUS findings. Although the comprehensive assessment and classification of missense variants according to their pathogenicity remains the bottleneck, new developments in functional analysis, high throughput assays, data sharing, and statistical models are rapidly changing this scenario

BRCA1 recruitment to damaged DNA sites is dependent on CDK9

<p>Double strand break lesions, the most toxic type of DNA damage, are repaired primarily through 2 distinct pathways: homology-directed recombination (HR) and non-homologous end-joining (NHEJ). BRCA1 and 53BP1, 2 proteins containing the BRCT modular domain, play an important role in DNA damage response (DDR) by orchestrating the decision between HR and NHEJ, but the precise mechanisms regarding both pathways are not entirely understood. Previously, our group identified a putative interaction between BRCA1 and BARD1 (BRCA1-associated RING domain 1) and the cyclin-dependent kinase (CDK9). CDK9 is a component of the positive transcription elongation complex and has been implicated in genome integrity maintenance associated with the replication stress response. Here we show that CDK9 interacts with endogenous BRCA1 and BARD1 mediated by their RING finger and BRCT domains, and describe CDK9 ionizing radiation-induced foci (IRIF) formation and its co-localization with BRCA1 in DNA damage sites. Cells lacking CDK9 are characterized by an altered γ−H2AX foci dynamics after DNA damage, a reduced efficiency in HR but not in NHEJ repair, failure to form BRCA1 and RAD51 IRIF and increased sensitivity to genotoxic agents. These data indicate that CDK9 is a player in the DDR and is consistent with its participation in HR pathway by modulating BRCA1 response.</p

Network of Interactions between ZIKA Virus Non-Structural Proteins and Human Host Proteins

The Zika virus (ZIKV) is a mosquito-borne Flavivirus and can be transmitted through an infected mosquito bite or through human-to-human interaction by sexual activity, blood transfusion, breastfeeding, or perinatal exposure. After the 2015&ndash;2016 outbreak in Brazil, a strong link between ZIKV infection and microcephaly emerged. ZIKV specifically targets human neural progenitor cells, suggesting that proteins encoded by ZIKV bind and inactivate host cell proteins, leading to microcephaly. Here, we present a systematic annotation of interactions between human proteins and the seven non-structural ZIKV proteins corresponding to a Brazilian isolate. The interaction network was generated by combining tandem-affinity purification followed by mass spectrometry with yeast two-hybrid screens. We identified 150 human proteins, involved in distinct biological processes, as interactors to ZIKV non-structural proteins. Our interacting network is composed of proteins that have been previously associated with microcephaly in human genetic disorders and/or animal models. Further, we show that the protein inhibitor of activated STAT1 (PIAS1) interacts with NS5 and modulates its stability. This study builds on previously published interacting networks of ZIKV and genes related to autosomal recessive primary microcephaly to generate a catalog of human cellular targets of ZIKV proteins implicated in processes related to microcephaly in humans. Collectively, these data can be used as a resource for future characterization of ZIKV infection biology and help create a basis for the discovery of drugs that may disrupt the interaction and reduce the health damage to the fetus

BRCA1 frameshift variants leading to extended incorrect protein C termini

Summary: Carriers of BRCA1 germline pathogenic variants are at substantially higher risk of developing breast and ovarian cancer than the general population. Accurate identification of at-risk individuals is crucial for risk stratification and the implementation of targeted preventive and therapeutic interventions. Despite significant progress in variant classification efforts, a sizable portion of reported BRCA1 variants remain as variants of uncertain clinical significance (VUSs). Variants leading to premature protein termination and loss of essential functional domains are typically classified as pathogenic. However, the impact of frameshift variants that result in an extended incorrect terminus is not clear. Using validated functional assays, we conducted a systematic functional assessment of 17 previously reported BRCA1 extended incorrect terminus variants (EITs) and concluded that 16 constitute loss-of-function variants. This suggests that most EITs are likely to be pathogenic. However, one variant, c.5578dup, displayed a protein expression level, affinity to known binding partners, and activity in transcription and homologous recombination assays comparable to the wild-type BRCA1 protein. Twenty-three additional carriers of c.5578dup were identified at a US clinical diagnostic lab and assessed using a family history likelihood model providing, in combination with the functional data, a likely benign interpretation. These results, consistent with family history data in the current study and available data from ClinVar, indicate that most, but not all, BRCA1 variants leading to an extended incorrect terminus constitute loss-of-function variants and underscore the need for comprehensive assessment of individual variants