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The use of multigene panel testing for patients with a predisposition to Hereditary Breast and Ovarian Cancer syndrome (HBOC) is increasing as the identification of mutations is useful for diagnosis and disease management. Here, we conducted a retrospective analysis of BRCA1/2 and non-BRCA gene sequencing in 4630 French HBOC suspected patients. Patients were investigated using a germline cancer panel including the 13 genes defined by The French Genetic and Cancer Group (GGC)—Unicancer. In the patients analyzed, 528 pathogenic and likely pathogenic variants (P/LP) were identified, including BRCA1 (n = 203, 38%), BRCA2 (n = 198, 37%), PALB2 (n = 46, 9%), RAD51C (n = 36, 7%), TP53 (n = 16, 3%), and RAD51D (n = 13, 2%). In addition, 35 novel (P/LP) variants, according to our knowledge, were identified, and double mutations in two distinct genes were found in five patients. Interestingly, retesting a subset of BRCA1/2-negative individuals with an expanded panel produced clinically relevant results in 5% of cases. Additionally, combining in silico (splicing impact prediction tools) and in vitro analyses (RT-PCR and Sanger sequencing) highlighted the deleterious impact of four candidate variants on splicing and translation. Our results present an overview of pathogenic variations of HBOC genes in the southeast of France, emphasizing the clinical relevance of cDNA analysis and the importance of retesting BRCA-negative individuals with an expanded panel.

The PMS2 gene is one of the DNA mismatch repair genes (MMR) implicated in Lynch syndrome (LS). A subset of PMS2 pathogenic variants (PVs) are splice variants mostly affecting canonical GT/AG splicing sequences. However, the majority of the intronic variants outside canonical splice sites remained as variants of unknown significance, even though some of them would alter the splicing process. In this report, we describe the analysis of such an intronic variant (c.251-5T C) detected in an 82-year-old patient diagnosed with endometrial cancer displaying microsatellite instability and the loss of PMS2 expression displayed. RNA analysis demonstrated that this variant lead to the complete exon 4 skipping, resulting in the synthesis of a truncated protein. This finding shows the relevance of functional RNA analysis in the non-canonical intronic variant assessment and the importance of systematic evaluation of MSI/loss of expression of MMR genes for LS screening in patients with endometrial cancers.

PALB2 (partner and localizer of BRCA2), as indicated by its name, is a BRCA2-interacting protein that plays an important role in homologous recombination (HR) and DNA double-strand break (DSB) repair. While pathogenic variants of PALB2 have been well proven to confer an increased risk of breast cancer, data on its involvement in prostate cancer (PrC) have not been clearly demonstrated. We investigated, using targeted next generation sequencing (NGS), a 59-year-old Caucasian man who developed synchronous breast and prostate cancers. This genetic investigation allowed to identify an intragenic germline heterozygous duplication in PALB2, implicating intronic repetitive sequences spanning exon 11. This variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints have been identified and characterized at the nucleotide level (c.3114-811_3202-1756dup) using an approach based on walking PCR, long range PCR, and Sanger sequencing. RT-PCR using mRNA extracted from lymphocytes and followed by Sanger sequencing revealed a tandem duplication r.3114_3201dup; p.(Gly1068Glufs * 14). This duplication results in the synthesis of a truncated, and most-likely, non-functional protein. These findings expand the phenotypic spectrum of PALB2 variants and may improve the yield of genetic diagnoses in this field.

Up to 80% of BRCA1 and BRCA2 genetic variants remain of uncertain clinical significance (VUSs). Only variants classified as pathogenic or likely pathogenic can guide breast and ovarian cancer prevention measures and treatment by PARP inhibitors. We report the first results of the ongoing French national COVAR (cosegregation variant) study, the aim of which is to classify BRCA1/2 VUSs. The classification method was a multifactorial model combining different associations between VUSs and cancer, including cosegregation data. At this time, among the 653 variants selected, 101 (15%) distinct variants shared by 1,624 families were classified as pathogenic/likely pathogenic or benign/likely benign by the COVAR study. Sixty-six of the 101 (65%) variants classified by COVAR would have remained VUSs without cosegregation data. Of note, among the 34 variants classified as pathogenic by COVAR, 16 remained VUSs or likely pathogenic when following the ACMG/AMP variant classification guidelines. Although the initiation and organization of cosegregation analyses require a considerable effort, the growing number of available genetic tests results in an increasing number of families sharing a particular variant, and thereby increases the power of such analyses. Here we demonstrate that variant cosegregation analyses are a powerful tool for the classification of variants in the BRCA1/2 breast-ovarian cancer predisposition genes.

Context: Cirrhosis impacts the coagulation cascade since the first stage of the liver disease. For years, a procoagulant imbalance, related to the severity of cirrhosis, has been demonstrated. This procoagulant imbalance is detected when coagulation assays are sensibilised to the protein C pathway. A resistance to thrombomodulin (TM) is consistently found in thrombin generation assays. TM and endothelial protein C receptor (EPCR) are two transmembrane proteins involved in the anticoagulant protein C pathway. A soluble form of EPCR (sEPCR) is release of the endothelial surface upon cleavage by mettaloprotease. Soluble EPCR have a procoagulant and proinflammatory effects: bound to PC or activated PC (APC), PC is unavailable for activation by thrombin-TM complex and APC is unable to inactivate FVa and FVIIIa. The aim of this study was to evaluate the plasma levels of sEPCR in non-complicated cirrhotic patients as a potential marker for procoagulant imbalance, compared to healthy controls. Materials and Methods: Patients prospectively included were confirmed cirrhotic patients (prothrombin time 20 kPa and/or histology and/or association of portal hypertension and liver failure). Patients were free of hepatocellular carcinoma and were not anticoagulated. Patients with on-going infection or inflammatory complication were excluded. None of them had a thromboembolic event or a familial history of thromboembolism. Controls were free of anticoagulation, coagulation disorders and without oral contraceptives. All ethical requirments were obtained and patients and controls gave their written informed consent. Kruskal-Wallis test and Dunn post test were used for statistical analysis. Results are expressed as mean (Q1 - Q3) values of sEPCR. Results: Fifty two cirrhotic patients (24 Child-Pugh A, 21 Child-Pugh B and 7 Child-Pugh C) and 32 healthy controls were prospectively included in our hospital. The sEPCR values for healthy controls were 100 ng/mL (81 - 135 ng/mL). Comparatively to controls, Child-Pugh A patients and Child-Pugh B patients had statistically significant higher values of sEPCR : 169 ng/mL (117-225 ng/mL, p < 0.01) and 176 (135 - 230 ng/mL, p < 0.001) respectively (Figure 1). Compared to healthy controls, Child-Pugh C patients had an increased level of sEPCR : 174 ng/mL (78 - 254 ng/mL, not significant but probably underpowered). These data are in lines with the thrombin generation profiles showing a resistance to TM in cirrhotic patients increasing from Child-Pugh A to C. No patient have developed thrombosis during the follow-up period. Conclusion: Patients with Child-Pugh A and B cirrhosis had significantly higher values of sEPCR compared to healthy controls (p < 0.01 for both). The putative role of sEPCR as a marker of the procoagulant imbalance associated to cirrhosis remains to be accurately evaluated. Figure 1. sEPCR levels in plasma of cirrhotic patients. Figure 1. sEPCR levels in plasma of cirrhotic patients. Disclosures No relevant conflicts of interest to declare.

The active form of human immunodeficiency virus type 1 protease (HIV-1 PR) is a homodimeric structure in which two subunits are linked through a two-stranded antiparallel beta-sheet consisting of the N- and C-termini of each monomer. To inhibit the dimerization process or disrupt the dimeric interface leading to inactive enzyme, conformationally constrained "molecular tongs" have been designed and synthesized to interfere with one monomer end in a beta-sheet fashion. These molecules are based on two peptidic strands attached to an aromatic scaffold. Inhibitions (submicromolar range) were obtained with molecular tongs containing tripeptidic or tetrapeptidic arms attached to a pyridinediol- or naphthalenediol-based scaffold (Kid = 0.56-4.5 microM at pH 4.7 and 30 degrees C). Kinetic studies are in agreement with an interface inhibition mechanism.