Absence of CALR Mutations in Idiopathic Erythrocytosis Patients with Low Serum Erythropoietin Levels

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What Singles Out the G[8-5]C Intrastrand DNA Cross-Link? Mechanistic and Structural Insights from Quantum Mechanics/Molecular Mechanics Simulations

International audienceNaturally occurring intrastrand oxidative cross-link lesions have proven to be a potent source of endogenous DNA damage. Among the variety of lesions that can be formed and have been identified, G[8-5]C damage (in which the C8 atom of a guanine is covalently bonded to the C5 atom of a nearby cytosine belonging to the same strand) occurs with a low incidence yet takes on special importance because of its high mutagenicity. Hybrid Car-Parrinello molecular dynamics simulations, rooted in density functional theory and coupled to molecular mechanics, have been performed to shed light on the cyclization process. The activation free energy of the reacting subsystem embedded in a solvated dodecamer is estimated to be 12.4 kcal/mol, which is 3 kcal/mol higher than the value for the prototypical G[8-5m]T lesion inferred employing the same theoretical framework [Garrec, J., Patel, C., Rothlisberger, U., and Dumont, E. (2012) J. Am. Chem. Soc.134, 2111-2119]. This study also situates the G[8-5m]mC lesion at an intermediate activation free energy (10.5 kcal/mol). The order of reactivity in DNA (T* > mC* > C*) is reversed compared to that in the reacting subsystems in the gas phase (C* > mC* > T*), stressing the crucial role of the solvated B-helix environment. The results of our simulations also characterize a more severe distortion for G[8-5]C than for methylene-bridged intrastrand cross-links

ADAMTS13 Gene Mutations Influence ADAMTS13 Conformation and Disease Age-Onset in the French Cohort of Upshaw-Schulman Syndrome

BACKGROUND:  Congenital thrombotic thrombocytopaenic purpura (TTP) or Upshaw-Schulman syndrome (USS) is a rare, life-threatening, inherited thrombotic microangiopathy (TMA). USS is mostly due to bi-allelic recessive sequence variations of the a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) gene inducing a severe ADAMTS13 deficiency (activity < 10 IU/dL). In healthy individuals, ADAMTS13 circulates in a folded conformation where CUB domains interact with the spacer domain. The spacer-CUB interaction is abrogated when ADAMTS13 is conformationally activated. OBJECTIVE:  This article evaluates the influence of ADAMTS13 sequence variations on both clinical/biological phenotype and ADAMTS13 conformation in USS. PATIENTS AND METHODS:  All USS patients from the French registry for TMAs (1 January 2000 to 1 June 2017) were investigated for ADAMTS13 genotype, phenotype (activity, antigen and autoantibodies) and conformation. Clinical records were analysed (inaugural acute TTP and follow-up). Child-onset USS was compared with adult-onset USS. RESULTS:  Fifty-six USS patients from 51 families (34 child-onset and 22 adult-onset cases) were enrolled. Child-onset USS was characterized by a large panel of ADAMTS13 sequence variations (n = 43), spread all over ADAMTS13 gene and not correlated with either clinical features or plasmatic ADAMTS13 parameters. In contrast, adult-onset USS, consisting exclusively in pregnancy-induced TTP, included a smaller and distinct panel of ADAMTS13 sequence variations (n = 20) because of one mutation (p.Arg1060Trp) present in 82% of patients. ADAMTS13 conformation was studied in 16 USS patients (5 child-onset and 11 adult-onset USS, encompassing 16 distinct ADAMTS13 sequence variations) whose ADAMTS13 antigen levels were detectable: 14 of 16 patients (87.5%) exhibited abnormalities of ADAMTS13 conformation. CONCLUSION:  In USS, age-onset defines two entities and ADAMTS13 sequence variations modify ADAMTS13 conformation.status: publishe

Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicom's BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy

Deep Learning for Detecting BRCA Mutations in High-Grade Ovarian Cancer based on an Innovative Tumor Segmentation Method from Whole-Slide Images

International audienceBRCA1/2 genes play a crucial role in repairing DNA double-strand breaks through homologous recombination. Their mutations represent a significant proportion of homologous recombination deficiency and are a reliable effective predictor of sensitivity of high-grade ovarian cancer (HGOC) to poly(ADP-ribose) polymerase inhibitors. However, their testing by next-generation sequencing is costly, time-consuming, and can be affected by various preanalytical factors. In this study, we present a deep learning classifier for BRCA mutational status prediction from HES-stained whole-slide images (WSI) of HGOC. We constituted the OvarIA cohort composed of 867 HGOC patients with known BRCA somatic mutational status coming from two different pathology departments. We first developed a tumor segmentation model according to dynamic sampling and then trained a visual representation encoder with momentum contrastive learning on the predicted tumor tiles. We finally trained a BRCA classifier on over a million tumor tiles in multiple-instance learning with an attention-based mechanism. The tumor segmentation model trained on 8 WSI obtained a dice score of 0.915 and an intersection-over-union of 0.847 on a test set of 50 WSI while the BRCA classifier achieved the state-of-the-art AUC of 0.739 in 5-fold cross-validation and 0.681 on the testing set. An additional multiscale approach indicates that the relevant information for predicting BRCA mutations is more located in the tumor context than in the cell morphology. Our results suggest that BRCA somatic mutations have a discernible phenotypic effect which could be detected by deep learning and could be used as a pre-screening tool in the future

Regional and latitudinal patterns of soft-bottom macrobenthic invertebrates along French coasts: Results from the RESOMAR database

International audienceThis study aims to describe the patterns of soft bottom macrozoobenthic richness along French coasts. It is based on a collaborative database developed by the “Réseau des Stations et Observatoires Marins” (RESOMAR). We investigated patterns of species richness in sublittoral soft bottom habitats (EUNIS level 3) at two different spatial scales: 1) seaboards: English Channel, Bay of Biscay and Mediterranean Sea and 2) 0.5° latitudinal and longitudinal grid. Total observed richness, rarefaction curves and three incidence-based richness estimators (Chao2, ICE and Jacknife1) were used to compare soft bottom habitats species richness in each seaboard. Overall, the Mediterranean Sea has the highest richness and despite higher sampling effort, the English Channel hosts the lowest number of species. The distribution of species occurrence within and between seaboards was assessed for each major phylum using constrained rarefaction curves. The Mediterranean Sea hosts the highest number of exclusive species. In pairwise comparisons, it also shares a lower proportion of taxa with the Bay of Biscay (34.1%) or the English Channel (27.6%) than that shared between these two seaboards (49.7%). Latitudinal species richness patterns along the Atlantic and English Channel coasts were investigated for each major phylum using partial LOESS regression controlling for sampling effort. This showed the existence of a bell-shaped latitudinal pattern, highlighting Brittany as a hotspot for macrobenthic richness at the confluence of two biogeographic provinces

BRCA Share: A Collection of Clinical BRCA Gene Variants

International audienceAs next-generation sequencing increases access to human genetic variation, the challenge of determining clinical significance of variants becomes ever more acute. Germline variants in the BRCA1 and BRCA2 genes can confer substantial lifetime risk of breast and ovar-ian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database Additional Supporting Information may be found in the online version of this article. † These authors contributed equally to this work. ‡ This author is deceased. of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share TM , a database created by a unique international alliance of academic centers and commercial testing laboratories. By integrating the content of the Universal Mutation Database generated by the French Unicancer Genetic Group with the testing results of two large commercial laboratories, Quest Diagnostics and Laboratory Corporation of America (LabCorp), BRCA Share TM has assembled one of the largest publicly accessible collections of BRCA variants currently available. Although access is available to academic researchers without charge, commercial participants in the project are required to pay a support fee and contribute their data. The fees fund the ongoing cu-ration effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share TM databases can therefore be considered as models of successful data sharing between private companies and the academic world

Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk

WOS:000454834200006International audienceBRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. Implications: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection

Classification of 101 BRCA1 and BRCA2 variants of uncertain significance by cosegregation study: A powerful approach

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