Publications by authors named "David Salgado"

44 Publications

The lncRNA 44s2 Study Applicability to the Design of 45-55 Exon Skipping Therapeutic Strategy for DMD.

Biomedicines 2021 Feb 20;9(2). Epub 2021 Feb 20.

SU, INSERM UMRS974, AIM, Center of Research in Myology, Pitié-Salpêtrière Hospital, 75013 Paris, France.

In skeletal muscle, long noncoding RNAs (lncRNAs) are involved in dystrophin protein stabilization but also in the regulation of myocytes proliferation and differentiation. Hence, they could represent promising therapeutic targets and/or biomarkers for Duchenne and Becker muscular dystrophy (DMD/BMD). DMD and BMD are X-linked myopathies characterized by a progressive muscular dystrophy with or without dilatative cardiomyopathy. Two-thirds of gene mutations are represented by deletions, and 63% of patients carrying deletions are eligible for 45 to 55 multi-exons skipping (MES), becoming BMD patients (BMDΔ45-55). We analyzed the genomic lncRNA presence in 38 BMDΔ45-55 patients and characterized the lncRNA localized in introns 44 and 55 of the gene. We highlighted that all four lncRNA are differentially expressed during myogenesis in immortalized and primary human myoblasts. In addition, the lncRNA44s2 was pointed out as a possible accelerator of differentiation. Interestingly, lncRNA44s expression was associated with a favorable clinical phenotype. These findings suggest that lncRNA44s2 could be involved in muscle differentiation process and become a potential disease progression biomarker. Based on these results, we support MES45-55 therapy and propose that the design of the CRISPR/Cas9 MES45-55 assay consider the lncRNA sequences bordering the exonic 45 to 55 deletion.
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http://dx.doi.org/10.3390/biomedicines9020219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924625PMC
February 2021

TGFβ signalling acts as a molecular brake of myoblast fusion.

Nat Commun 2021 02 2;12(1):749. Epub 2021 Feb 2.

Institut NeuroMyoGène (INMG), University Claude Bernard Lyon1, CNRS UMR 5310, INSERM U1217, Lyon, France.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace.
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http://dx.doi.org/10.1038/s41467-020-20290-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854724PMC
February 2021

Identification of a peripheral blood gene signature predicting aortic valve calcification.

Physiol Genomics 2020 12 12;52(12):563-574. Epub 2020 Oct 12.

Intercellular Signaling in Cardiovascular Development and Disease Laboratory, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.

Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. Identification of early predictive markers could help optimize patient management. RNA-sequencing was carried out on human fetal aortic valves at gestational , , and and on a case-control study with adult noncalcified and calcified bicuspid and tricuspid aortic valves. In dimension reduction and clustering analyses, diseased valves tended to cluster with fetal valves at rather than normal adult valves, suggesting that part of the disease program might be due to reiterated developmental processes. The analysis of groups of coregulated genes revealed predominant immune-metabolic signatures, including innate and adaptive immune responses involving lymphocyte T-cell metabolic adaptation. Cytokine and chemokine signaling, cell migration, and proliferation were all increased in CAVD, whereas oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at fetal stages and increased in adult controls, suggesting that these processes intensify throughout life and heighten in disease. Cellular stress response and neurodegeneration gene signatures were aberrantly expressed in CAVD, pointing to a mechanistic link between chronic inflammation and biological aging. Comparison of the valve RNA-sequencing data set with a case-control study of whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a highly predictive gene signature of CAVD and of moderate aortic valve calcification in overtly healthy individuals. These data deepen and broaden our understanding of the molecular basis of CAVD and identify a peripheral blood gene signature for the early detection of aortic valve calcification.
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http://dx.doi.org/10.1152/physiolgenomics.00034.2020DOI Listing
December 2020

-dependent coordination of mouse cardiac progenitor cell patterning and differentiation.

Elife 2020 08 17;9. Epub 2020 Aug 17.

Aix Marseille Univ, INSERM, Marseille Medical Genetics, Marseille, France.

Perturbation of addition of second heart field (SHF) cardiac progenitor cells to the poles of the heart tube results in congenital heart defects (CHD). The transcriptional programs and upstream regulatory events operating in different subpopulations of the SHF remain unclear. Here, we profile the transcriptome and chromatin accessibility of anterior and posterior SHF sub-populations at genome-wide levels and demonstrate that Hoxb1 negatively regulates differentiation in the posterior SHF. Spatial mis-expression of in the anterior SHF results in hypoplastic right ventricle. Activation of in embryonic stem cells arrests cardiac differentiation, whereas -deficient mouse embryos display premature cardiac differentiation. Moreover, ectopic differentiation in the posterior SHF of embryos lacking both and its paralog results in atrioventricular septal defects. Our results show that Hoxb1 plays a key role in patterning cardiac progenitor cells that contribute to both cardiac poles and provide new insights into the pathogenesis of CHD.
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http://dx.doi.org/10.7554/eLife.55124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462617PMC
August 2020

Transgenesis and web resources in quail.

Elife 2020 05 27;9. Epub 2020 May 27.

Australian Regenerative Medicine Institute (ARMI), Monash University, Clayton, Australia.

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail () is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.
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http://dx.doi.org/10.7554/eLife.56312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286689PMC
May 2020

Piezo1 is required for outflow tract and aortic valve development.

J Mol Cell Cardiol 2020 06 3;143:51-62. Epub 2020 Apr 3.

Institut de Génomique Fonctionnelle, Université de Montpellier, CNRS, INSERM LabEx ICST, Montpellier, France. Electronic address:

Aims: During embryogenesis, the onset of circulatory blood flow generates a variety of hemodynamic forces which reciprocally induce changes in cardiovascular development and performance. It has been known for some time that these forces can be detected by as yet unknown mechanosensory systems which in turn promote cardiogenic events such as outflow tract and aortic valve development. PIEZO1 is a mechanosensitive ion channel present in endothelial cells where it serves to detect hemodynamic forces making it an ideal candidate to play a role during cardiac development. We sought to determine whether PIEZO1 is required for outflow tract and aortic valve development.

Methods And Results: By analysing heart development in zebrafish we have determined that piezo1 is expressed in the developing outflow tract where it serves to detect hemodynamic forces. Consequently, disrupting Piezo1 signalling leads to defective outflow tract and aortic valve development and indicates this gene may be involved in the etiology of congenital heart diseases. Based on these findings, we analysed genomic data generated from patients who suffer from left ventricular outflow tract obstructions (LVOTO) and identified 3 probands who each harboured potentially pathogenic variants in PIEZO1. Subsequent in vitro and in vivo assays indicates that these variants behave as dominant negatives leading to an inhibition of normal PIEZO1 mechanosensory activity. Expressing these dominant negative PIEZO1 variants in zebrafish endothelium leads to defective aortic valve development.

Conclusion: These data indicate that the mechanosensitive ion channel piezo1 is required for outflow tract and aortic valve development.
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http://dx.doi.org/10.1016/j.yjmcc.2020.03.013DOI Listing
June 2020

Leveraging European infrastructures to access 1 million human genomes by 2022.

Nat Rev Genet 2019 11 27;20(11):693-701. Epub 2019 Aug 27.

Global Alliance for Genomics and Health, Toronto, Ontario, Canada.

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.
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http://dx.doi.org/10.1038/s41576-019-0156-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115898PMC
November 2019

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

Nat Commun 2019 04 26;10(1):1929. Epub 2019 Apr 26.

INSERM U-1251, MMG, Aix-Marseille University, Marseille, 13885, France.

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.
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http://dx.doi.org/10.1038/s41467-019-09459-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486645PMC
April 2019

VarAFT: a variant annotation and filtration system for human next generation sequencing data.

Nucleic Acids Res 2018 07;46(W1):W545-W553

Aix Marseille Univ, INSERM, MMG, 13005, Marseille, France.

With the rapidly developing high-throughput sequencing technologies known as next generation sequencing or NGS, our approach to gene hunting and diagnosis has drastically changed. In <10 years, these technologies have moved from gene panel to whole genome sequencing and from an exclusively research context to clinical practice. Today, the limit is not the sequencing of one, many or all genes but rather the data analysis. Consequently, the challenge is to rapidly and efficiently identify disease-causing mutations within millions of variants. To do so, we developed the VarAFT software to annotate and pinpoint human disease-causing mutations through access to multiple layers of information. VarAFT was designed both for research and clinical contexts and is accessible to all scientists, regardless of bioinformatics training. Data from multiple samples may be combined to address all Mendelian inheritance modes, cancers or population genetics. Optimized filtration parameters can be stored and re-applied to large datasets. In addition to classical annotations from dbNSFP, VarAFT contains unique features at the disease (OMIM), phenotypic (HPO), gene (Gene Ontology, pathways) and variation levels (predictions from UMD-Predictor and Human Splicing Finder) that can be combined to optimally select candidate pathogenic mutations. VarAFT is freely available at: http://varaft.eu.
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http://dx.doi.org/10.1093/nar/gky471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030844PMC
July 2018

Clinical Outcomes in Duchenne Muscular Dystrophy: A Study of 5345 Patients from the TREAT-NMD DMD Global Database.

J Neuromuscul Dis 2017;4(4):293-306

Kanchi Kamakoti CHILDS Trust and Apollo Children's Hospitals, Chennai, India.

Background: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population.

Objective: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients.

Methods: In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age.

Results: Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions.

Conclusions: This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.
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http://dx.doi.org/10.3233/JND-170280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701764PMC
July 2019

Exome sequencing identifies recurrent alterations and the absence of , and mutations in splenic diffuse red pulp small B-cell lymphoma.

Haematologica 2017 10 27;102(10):1758-1766. Epub 2017 Jul 27.

Cancer Research Center of Lyon, INSERM 1052 CNRS 5286, Team "Clinical and Experimental Models of Lymphomagenesis", Faculté de Médecine et de Maïeutique Lyon-Sud Charles Mérieux, Oulins, France.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, , and , common mutations in splenic marginal zone lymphoma, were rare (one mutant in 42 samples; 2%) or absent ( and ) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.
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http://dx.doi.org/10.3324/haematol.2016.160192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622860PMC
October 2017

[OISO, automatic treatment of patients management in oncogenetics].

Bull Cancer 2017 Jul - Aug;104(7-8):602-607. Epub 2017 Jul 8.

Aix-Marseille université, Inserm, GMGF UMR_S 910, 27, boulevard Jean-Moulin, 13385 Marseille, France; RGDS, hôpital Clairval, 317, boulevard du Redon, 13009 Marseille, France; AP-HM Timone, département de génétique médicale, 264, rue Saint-Pierre, 13005 Marseille, France. Electronic address:

Introduction: Oncogenetics is a long-term process, which requires a close relation between patients and medical teams, good familial links allowing lifetime follow-up. Numerous documents are exchanged in between the medical team, which has to frequently interact. We present here a new tool that has been conceived specifically for this management.

Methods: The tool has been developed according to a model-view-controler approach with the relational system PostgreSQL 9.3. The web site used PHP 5.3, HTML5 and CSS3 languages, completed with JavaScript and jQuery-AJAX functions and two additional modules, FPDF and PHPMailer.

Results: The tool allows multiple interactions, clinical data management, mailing and emailing, follow-up plannings. Requests are able to follow all patients and planning automatically, to send information to a large number of patients or physicians, and to report activity.

Discussion: The tool has been designed for oncogenetics and adapted to its different aspects. The CNIL delivered an authorization for use. Secured web access allows the management at a regional level. Its simple concept makes it evolutive according to the constant updates of genetic and clinical management of patients.
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http://dx.doi.org/10.1016/j.bulcan.2017.06.003DOI Listing
August 2017

Shared genetic predisposition in rheumatoid arthritis-interstitial lung disease and familial pulmonary fibrosis.

Eur Respir J 2017 05 11;49(5). Epub 2017 May 11.

APHP, Hôpital Bichat, Service de Rhumatologie, DHU FIRE, Paris, France.

Despite its high prevalence and mortality, little is known about the pathogenesis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Given that familial pulmonary fibrosis (FPF) and RA-ILD frequently share the usual pattern of interstitial pneumonia and common environmental risk factors, we hypothesised that the two diseases might share additional risk factors, including FPF-linked genes. Our aim was to identify coding mutations of FPF-risk genes associated with RA-ILD.We used whole exome sequencing (WES), followed by restricted analysis of a discrete number of FPF-linked genes and performed a burden test to assess the excess number of mutations in RA-ILD patients compared to controls.Among the 101 RA-ILD patients included, 12 (11.9%) had 13 WES-identified heterozygous mutations in the , , or coding regions The burden test, based on 81 RA-ILD patients and 1010 controls of European ancestry, revealed an excess of , , or mutations in RA-ILD patients (OR 3.17, 95% CI 1.53-6.12; p=9.45×10). Telomeres were shorter in RA-ILD patients with a , or mutation than in controls (p=2.87×10).Our results support the contribution of FPF-linked genes to RA-ILD susceptibility.
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http://dx.doi.org/10.1183/13993003.02314-2016DOI Listing
May 2017

New advances in DPYD genotype and risk of severe toxicity under capecitabine.

PLoS One 2017 8;12(5):e0175998. Epub 2017 May 8.

Aix-Marseille University, INSERM UMR S910, GMGF, Marseille, France.

Background: Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine.

Methods: Two-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. DPYD exome, along with flanking intronic regions 3'UTR and 5'UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement.

Results: Among the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient (among which, F100L and A26T, both pathogenic in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3-4 toxicity (sensitivity 16.7%, positive predictive value (PPV) 71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3-4 toxicity (PPV 72.7%, RR 7.6, p<0.001), and was significantly associated with grade 4 toxicity (sensitivity 60%, PPV 27.3%, RR 31.4, p = 0.001), suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR.

Conclusions: Exploring an extended set of deleterious DPYD variants improves the performance of DPYD genotyping for predicting both grade 3-4 and grade 4 toxicities (digestive and/or neurologic and/or hematotoxicities) related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421769PMC
September 2017

WES/WGS Reporting of Mutations from Cardiovascular "Actionable" Genes in Clinical Practice: A Key Role for UMD Knowledgebases in the Era of Big Databases.

Hum Mutat 2016 12 10;37(12):1308-1317. Epub 2016 Oct 10.

Aix Marseille Univ,  INSERM, GMGF, Marseille, France.

High-throughput next-generation sequencing such as whole-exome and whole-genome sequencing are being rapidly integrated into clinical practice. The use of these techniques leads to the identification of secondary variants for which decisions about the reporting or not to the patient need to be made. The American College of Medical Genetics and Genomics recently published recommendations for the reporting of these variants in clinical practice for 56 "actionable" genes. Among these, seven are involved in Marfan Syndrome And Related Disorders (MSARD) resulting from mutations of the FBN1, TGFBR1 and 2, ACTA2, SMAD3, MYH11 and MYLK genes. Here, we show that mutations collected in UMD databases for MSARD genes (UMD-MSARD) are rarely reported, including the most frequent ones, in global scale initiatives for variant annotation such as the NHLBI GO Exome Sequencing Project (ESP), the Exome Aggregation Consortium (ExAC), and ClinVar. The predicted pathogenic mutations reported in global scale initiatives but absent in locus-specific databases (LSDBs) mainly correspond to rare events. UMD-MSARD databases are therefore the only resources providing access to the full spectrum of known pathogenic mutations. They are the most comprehensive resources for clinicians and geneticists to interpret MSARD-related variations not only primary variants but also secondary variants.
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http://dx.doi.org/10.1002/humu.23119DOI Listing
December 2016

BRCA Share: A Collection of Clinical BRCA Gene Variants.

Hum Mutat 2016 12 28;37(12):1318-1328. Epub 2016 Sep 28.

Institut Claudius Régaud, Toulouse, France.

As 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 ovarian cancer. Assessment of variant pathogenicity is a vital part of clinical genetic testing for these genes. A database of clinical observations of BRCA variants is a critical resource in that process. This article describes BRCA Share™, 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™ 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 curation effort, as well as planned experiments to functionally characterize variants of uncertain significance. BRCA Share™ databases can therefore be considered as models of successful data sharing between private companies and the academic world.
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http://dx.doi.org/10.1002/humu.23113DOI Listing
December 2016

Actionable Genes, Core Databases, and Locus-Specific Databases.

Hum Mutat 2016 12 26;37(12):1299-1307. Epub 2016 Sep 26.

Aix Marseille Univ, INSERM, GMGF, Marseille, France.

Adoption of next-generation sequencing (NGS) in a diagnostic context raises numerous questions with regard to identification and reports of secondary variants (SVs) in actionable genes. To better understand the whys and wherefores of these questioning, it is necessary to understand how they are selected during the filtering process and how their proportion can be estimated. It is likely that SVs are underestimated and that our capacity to label all true SVs can be improved. In this context, Locus-specific databases (LSDBs) can be key by providing a wealth of information and enabling classifying variants. We illustrate this issue by analyzing 318 SVs in 23 actionable genes involved in cancer susceptibility syndromes identified through sequencing of 572 participants selected for a range of atherosclerosis phenotypes. Among these 318 SVs, only 43.4% are reported in Human Gene Mutation Database (HGMD) Professional versus 71.4% in LSDB. In addition, 23.9% of HGMD Professional variants are reported as pathogenic versus 4.8% for LSDB. These data underline the benefits of LSDBs to annotate SVs and minimize overinterpretation of mutations thanks to their efficient curation process and collection of unpublished data.
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http://dx.doi.org/10.1002/humu.23112DOI Listing
December 2016

How to Identify Pathogenic Mutations among All Those Variations: Variant Annotation and Filtration in the Genome Sequencing Era.

Hum Mutat 2016 12 26;37(12):1272-1282. Epub 2016 Sep 26.

Aix Marseille University, INSERM, GMGF, Marseille, France.

High-throughput sequencing technologies have become fundamental for the identification of disease-causing mutations in human genetic diseases both in research and clinical testing contexts. The cumulative number of genes linked to rare diseases is now close to 3,500 with more than 1,000 genes identified between 2010 and 2014 because of the early adoption of Exome Sequencing technologies. However, despite these encouraging figures, the success rate of clinical exome diagnosis remains low due to several factors including wrong variant annotation and nonoptimal filtration practices, which may lead to misinterpretation of disease-causing mutations. In this review, we describe the critical steps of variant annotation and filtration processes to highlight a handful of potential disease-causing mutations for downstream analysis. We report the key annotation elements to gather at multiple levels for each mutation, and which systems are designed to help in collecting this mandatory information. We describe the filtration options, their efficiency, and limits and provide a generic filtration workflow and highlight potential pitfalls through a use case.
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http://dx.doi.org/10.1002/humu.23110DOI Listing
December 2016

Overview of the interactive task in BioCreative V.

Database (Oxford) 2016 1;2016. Epub 2016 Sep 1.

Microbial Genomics and Bioinformatics Group, Max Planck Institute for Marine Microbiology, Bremen, Germany.

Fully automated text mining (TM) systems promote efficient literature searching, retrieval, and review but are not sufficient to produce ready-to-consume curated documents. These systems are not meant to replace biocurators, but instead to assist them in one or more literature curation steps. To do so, the user interface is an important aspect that needs to be considered for tool adoption. The BioCreative Interactive task (IAT) is a track designed for exploring user-system interactions, promoting development of useful TM tools, and providing a communication channel between the biocuration and the TM communities. In BioCreative V, the IAT track followed a format similar to previous interactive tracks, where the utility and usability of TM tools, as well as the generation of use cases, have been the focal points. The proposed curation tasks are user-centric and formally evaluated by biocurators. In BioCreative V IAT, seven TM systems and 43 biocurators participated. Two levels of user participation were offered to broaden curator involvement and obtain more feedback on usability aspects. The full level participation involved training on the system, curation of a set of documents with and without TM assistance, tracking of time-on-task, and completion of a user survey. The partial level participation was designed to focus on usability aspects of the interface and not the performance per se In this case, biocurators navigated the system by performing pre-designed tasks and then were asked whether they were able to achieve the task and the level of difficulty in completing the task. In this manuscript, we describe the development of the interactive task, from planning to execution and discuss major findings for the systems tested.Database URL: http://www.biocreative.org.
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http://dx.doi.org/10.1093/database/baw119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009325PMC
November 2017

Comparing targeted exome and whole exome approaches for genetic diagnosis of neuromuscular disorders.

Appl Transl Genom 2015 Dec 4;7:26-31. Epub 2015 Sep 4.

Aix Marseille University, INSERM, GMGF, UMR_S 910, 13385, Marseille, France.

Massively parallel sequencing is rapidly becoming a widely used method in genetic diagnostics. However, there is still no clear consensus as to which approach can most efficiently identify the pathogenic mutations carried by a given patient, while avoiding false negative and false positive results. We developed a targeted exome approach (MyoPanel2) in order to optimize genetic diagnosis of neuromuscular disorders. Using this approach, we were able to analyse 306 genes known to be mutated in myopathies as well as in related disorders, obtaining 98.8% target sequence coverage at 20 ×. Moreover, MyoPanel2 was able to detect 99.7% of 11,467 known mutations responsible for neuromuscular disorders. We have then used several quality control parameters to compare performance of the targeted exome approach with that of whole exome sequencing. The results of this pilot study of 140 DNA samples suggest that targeted exome sequencing approach is an efficient genetic diagnostic test for most neuromuscular diseases.
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http://dx.doi.org/10.1016/j.atg.2015.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803780PMC
December 2015

UMD-Predictor: A High-Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution.

Hum Mutat 2016 May 22;37(5):439-46. Epub 2016 Feb 22.

Aix-Marseille Université, GMGF, Marseille 13385, France.

Whole-exome sequencing (WES) is increasingly applied to research and clinical diagnosis of human diseases. It typically results in large amounts of genetic variations. Depending on the mode of inheritance, only one or two correspond to pathogenic mutations responsible for the disease and present in affected individuals. Therefore, it is crucial to filter out nonpathogenic variants and limit downstream analysis to a handful of candidate mutations. We have developed a new computational combinatorial system UMD-Predictor (http://umd-predictor.eu) to efficiently annotate cDNA substitutions of all human transcripts for their potential pathogenicity. It combines biochemical properties, impact on splicing signals, localization in protein domains, variation frequency in the global population, and conservation through the BLOSUM62 global substitution matrix and a protein-specific conservation among 100 species. We compared its accuracy with the seven most used and reliable prediction tools, using the largest reference variation datasets including more than 140,000 annotated variations. This system consistently demonstrated a better accuracy, specificity, Matthews correlation coefficient, diagnostic odds ratio, speed, and provided the shortest list of candidate mutations for WES. Webservices allow its implementation in any bioinformatics pipeline for next-generation sequencing analysis. It could benefit to a wide range of users and applications varying from gene discovery to clinical diagnosis.
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http://dx.doi.org/10.1002/humu.22965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067603PMC
May 2016

A mutation in the Gardos channel is associated with hereditary xerocytosis.

Blood 2015 Sep 6;126(11):1273-80. Epub 2015 Jul 6.

Aix-Marseille Université, INSERM, Unité Mixte de Recherche S910, Marseille, France; Assistance Publique-Hopitaux de Marseille, Département de Génétique Médicale, Hôpital d'Enfants de la Timone, Marseille, France; Assistance Publique-Hopitaux de Marseille, Laboratoire de Biochimie, Hôpital Conception, Marseille, France;

The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel.
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http://dx.doi.org/10.1182/blood-2015-04-642496DOI Listing
September 2015

The CHEMDNER corpus of chemicals and drugs and its annotation principles.

J Cheminform 2015 19;7(Suppl 1 Text mining for chemistry and the CHEMDNER track):S2. Epub 2015 Jan 19.

Information Technology Supporting Center, Institute of Scientific and Technical Information of China, Beijing, PR China.

The automatic extraction of chemical information from text requires the recognition of chemical entity mentions as one of its key steps. When developing supervised named entity recognition (NER) systems, the availability of a large, manually annotated text corpus is desirable. Furthermore, large corpora permit the robust evaluation and comparison of different approaches that detect chemicals in documents. We present the CHEMDNER corpus, a collection of 10,000 PubMed abstracts that contain a total of 84,355 chemical entity mentions labeled manually by expert chemistry literature curators, following annotation guidelines specifically defined for this task. The abstracts of the CHEMDNER corpus were selected to be representative for all major chemical disciplines. Each of the chemical entity mentions was manually labeled according to its structure-associated chemical entity mention (SACEM) class: abbreviation, family, formula, identifier, multiple, systematic and trivial. The difficulty and consistency of tagging chemicals in text was measured using an agreement study between annotators, obtaining a percentage agreement of 91. For a subset of the CHEMDNER corpus (the test set of 3,000 abstracts) we provide not only the Gold Standard manual annotations, but also mentions automatically detected by the 26 teams that participated in the BioCreative IV CHEMDNER chemical mention recognition task. In addition, we release the CHEMDNER silver standard corpus of automatically extracted mentions from 17,000 randomly selected PubMed abstracts. A version of the CHEMDNER corpus in the BioC format has been generated as well. We propose a standard for required minimum information about entity annotations for the construction of domain specific corpora on chemical and drug entities. The CHEMDNER corpus and annotation guidelines are available at: http://www.biocreative.org/resources/biocreative-iv/chemdner-corpus/.
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http://dx.doi.org/10.1186/1758-2946-7-S1-S2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331692PMC
March 2015

The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.

Hum Mutat 2015 Apr 17;36(4):395-402. Epub 2015 Mar 17.

The John Walton Muscular Dystrophy Research Centre, MRC Centre for Neuromuscular Diseases Institute of Genetic Medicine, University of Newcastle, Central Parkway, Newcastle upon Tyne, UK.

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).
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http://dx.doi.org/10.1002/humu.22758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405042PMC
April 2015

Identification of splicing defects caused by mutations in the dysferlin gene.

Hum Mutat 2014 Dec;35(12):1532-41

Aix Marseille Université, GMGF, Marseille, 13385, France; Inserm, UMR_S 910, Marseille, 13385, France.

Missense, iso-semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5' or 3' splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5' or 3' splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.
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http://dx.doi.org/10.1002/humu.22710DOI Listing
December 2014