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

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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Update of the UMD-VHL database: classification of 164 challenging variants based on genotype-phenotype correlation among 605 entries.

BACKGROUND: The von Hippel-Lindau (VHL) disease is a hereditary tumour syndrome caused by germline mutations in VHL tumour suppressor gene. The identification of VHL variants requires accurate classification which has an impact on patient management and genetic counselling. METHODS: The TENGEN (French oncogenetics network of neuroendocrine tumors) and PREDIR (French National Cancer Institute network for Inherited predispositions to kidney cancer) networks have collected VHL genetic variants and clinical characteristics of all VHL-suspected patients analysed from 2003 to 2021 by one of the nine laboratories performing VHL genetic testing in France. Identified variants were registered in a locus-specific database, the Universal Mutation Database-VHL database (http://www.umd.be/VHL/). RESULTS: Here we report the expert classification of 164 variants, including all missense variants (n=124), all difficult interpretation variants (n=40) and their associated phenotypes. After initial American College of Medical Genetics classification, first-round classification was performed by the VHL expert group followed by a second round for discordant and ambiguous cases. Overall, the VHL experts modified the classification of 87 variants including 30 variants of uncertain significance that were as (likely)pathogenic variants for 19, and as likely benign for 11. CONCLUSION: Consequently, this work has allowed the diagnosis and influenced the genetic counselling of 45 VHL-suspected families and can benefit to the worldwide VHL community, through this review.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

Highlighting the Dystonic Phenotype Related to GNAO1.

BACKGROUND: Most reported patients carrying GNAO1 mutations showed a severe phenotype characterized by early-onset epileptic encephalopathy and/or chorea. OBJECTIVE: The aim was to characterize the clinical and genetic features of patients with mild GNAO1-related phenotype with prominent movement disorders. METHODS: We included patients diagnosed with GNAO1-related movement disorders of delayed onset (>2 years). Patients experiencing either severe or profound intellectual disability or early-onset epileptic encephalopathy were excluded. RESULTS: Twenty-four patients and 1 asymptomatic subject were included. All patients showed dystonia as prominent movement disorder. Dystonia was focal in 1, segmental in 6, multifocal in 4, and generalized in 13. Six patients showed adolescence or adulthood-onset dystonia. Seven patients presented with parkinsonism and 3 with myoclonus. Dysarthria was observed in 19 patients. Mild and moderate ID were present in 10 and 2 patients, respectively. CONCLUSION: We highlighted a mild GNAO1-related phenotype, including adolescent-onset dystonia, broadening the clinical spectrum of this condition. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Loss of Calmodulin- and Radial-Spoke-Associated Complex Protein CFAP251 Leads to Immotile Spermatozoa Lacking Mitochondria and Infertility in Men.

Flagella and motile cilia share a 9 + 2 microtubule-doublet axoneme structure, and asthenozoospermia (reduced spermatozoa motility) is found in 76% of men with primary ciliary dyskinesia (PCD). Nevertheless, causal genetic variants in a conserved axonemal component have been found in cases of isolated asthenozoospermia: 30% of men with multiple morphological anomalies of sperm flagella (MMAF) carry bi-allelic mutations in DNAH1, encoding one of the seven inner-arm dynein heavy chains of the 9 + 2 axoneme. To further understand the basis for isolated asthenozoospermia, we used whole-exome and Sanger sequencing to study two brothers and two independent men with MMAF. In three men, we found bi-allelic loss-of-function mutations in WDR66, encoding cilia- and flagella-associated protein 251 (CFAP251): the two brothers were homozygous for the frameshift chr12: g.122359334delA (p.Asp42Metfs∗4), and the third individual was compound heterozygous for chr12: g.122359542G>T (p.Glu111∗) and chr12: g.122395032_122395033delCT (p.Leu530Valfs∗4). We show that CFAP251 is normally located along the flagellum but is absent in men carrying WDR66 mutations and reveal a spermatozoa-specific isoform probably generated during spermatozoon maturation. CFAP251 is a component of the calmodulin- and radial-spoke- associated complex, located adjacent to DNAH1, on the inner surface of the peripheral microtubule doublets of the axoneme. In Tetrahymena, the CFAP251 ortholog is necessary for efficient coordinated ciliary beating. Using immunofluorescent and transmission electron microscopy, we provide evidence that loss of CFAP251 affects the formation of the mitochondrial sheath. We propose that CFAP251 plays a structural role during biogenesis of the spermatozoon flagellum in vertebrates.

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

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.

Proposition of adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants.

In 2015, the ACMG-AMP guidelines provided a general procedure for the objective and reproducible classification of genomic variants. While the benefits of this framework are of major importance, its adaptation for locus-specific use is needed. Multiple Endocrine Neoplasia type 1 (MEN1) occurs due to inactivating mutations in the tumour suppressor gene MEN1, including 20% of missense variants. The classification of these variants may be extremely challenging. Here, we compared the interpretation of the 122 MEN1 missense variants, identified in the French population over the past 15 years by the TENGEN network (French oncogenetics network of neuroendocrine tumors) versus by using the ACMG-AMP guidelines, and analyzed the causes of discordance. A total of 59.8% of missense variants were termed as (likely)-pathogenic variants by TENGEN versus only 28.7% using ACMG-AMP guidelines. Actually, 53.4% (39/73) of TENGEN (likely)-pathogenic variants were declassified in variant of uncertain significance (VUS) by using ACMG-AMP guidelines, thereby affecting the clinical management of patients and their families. Twenty of these ACMG-AMP VUS were found in patients with a clinically authentic MEN1 disease. Here, TENGEN proposes adjustments to the ACMG-AMP framework for the interpretation of MEN1 missense variants. These propositions merge both the classification systems, and are particularly interesting, as MEN1 is included in the ACMG secondary findings list for reporting in clinical genomic sequencing.

Tricho-Hepato-Enteric Syndrome mutation update: Mutations spectrum of TTC37 and SKIV2L, clinical analysis and future prospects.

Tricho-Hepato-Enteric syndrome (THES) is a very rare autosomal recessive syndromic enteropathy caused by mutations of either TTC37 or SKIV2L genes. Very little is known of these two gene products in mammals nor of the pathophysiology of the disease. Since the identification of the genes, we have set up the molecular diagnostic of THES in routine, gathering a large cohort with clinical and molecular data. Here, we report the phenotype and genotype analysis of this cohort together with an extensive literature review of THES cases worldwide, that is, 96 individuals harboring mutations in one gene or the other. We set up locus-specific databases for both genes and reviewed the type of mutation as well as their localization in the proteins. No hot spot is evidenced for any type of mutation. The phenotypic analysis was first made on the whole cohort but is limited due to heterogeneity in clinical descriptions. We then examined the lab diagnostic cohort in detail for clinical manifestations. For the first time, we are able to suggest that patients lacking SKIV2L seem more severely affected than those lacking TTC37, in terms of liver damage and prenatal growth impairment.

Muscular MRI-based algorithm to differentiate inherited myopathies presenting with spinal rigidity.

OBJECTIVES: Inherited myopathies are major causes of muscle atrophy and are often characterized by rigid spine syndrome, a clinical feature designating patients with early spinal contractures. We aim to present a decision algorithm based on muscular whole body magnetic resonance imaging (mWB-MRI) as a unique tool to orientate the diagnosis of each inherited myopathy long before the genetically confirmed diagnosis. METHODS: This multicentre retrospective study enrolled 79 patients from referral centres in France, Brazil and Chile. The patients underwent 1.5-T or 3-T mWB-MRI. The protocol comprised STIR and T1 sequences in axial and coronal planes, from head to toe. All images were analyzed manually by multiple raters. Fatty muscle replacement was evaluated on mWB-MRI using both the Mercuri scale and statistical comparison based on the percentage of affected muscle. RESULTS: Between February 2005 and December 2015, 76 patients with genetically confirmed inherited myopathy were included. They were affected by Pompe disease or harbored mutations in RYR1, Collagen VI, LMNA, SEPN1, LAMA2 and MYH7 genes. Each myopathy had a specific pattern of affected muscles recognizable on mWB-MRI. This allowed us to create a novel decision algorithm for patients with rigid spine syndrome by segregating these signs. This algorithm was validated by five external evaluators on a cohort of seven patients with a diagnostic accuracy of 94.3% compared with the genetic diagnosis. CONCLUSION: We provide a novel decision algorithm based on muscle fat replacement graded on mWB-MRI that allows diagnosis and differentiation of inherited myopathies presenting with spinal rigidity. KEY POINTS: • Inherited myopathies are rare, diagnosis is challenging and genetic tests require specialized centres and often take years. • Inherited myopathies are often characterized by spinal rigidity. • Whole body magnetic resonance imaging is a unique tool to orientate the diagnosis of each inherited myopathy presenting with spinal rigidity. • Each inherited myopathy in this study has a specific pattern of affected muscles that orientate diagnosis. • A novel MRI-based algorithm, usable by every radiologist, can help the early diagnosis of these myopathies.

Exome Sequencing Identifies Two Variants of the Alkylglycerol Monooxygenase Gene as a Cause of Relapses in Visceral Leishmaniasis in Children, in Sudan.

Background: Visceral leishmaniasis (kala-azar, KA) is the most severe form of leishmaniasis, characterized by fever, weight loss, hepatosplenomegaly, and lymphadenopathy. During an outbreak of KA in Babar El Fugara (Sudan), 5.7% of cured patients displayed relapses, with familial clustering in half the cases. Methods: We performed whole-exome sequencing on 10 relapsing individuals and 11 controls from 5 nuclear families. Results: Rare homozygous and compound-heterozygous nonsense (c.1213C > T, rs139309795, p.Arg405*) and missense (c.701A > G, rs143439626, p.Lys234Arg) mutations of the alkylglycerol monooxygenase (AGMO) gene were associated with KA relapse in 3 families. Sequencing in additional family members confirmed the segregation of these mutations with relapse and revealed an autosomal dominant mode of transmission. These mutations were detected heterozygous in 2 subjects among 100 unrelated individuals with KA who never relapsed after cure, suggesting incomplete penetrance of AGMO deficiency. AGMO is expressed in hematopoietic cells, and is strongly expressed in the liver. AGMO modulates PAF production by mouse macrophages, suggesting that it may act through the PAF/PAF receptor pathway previously shown to have anti-Leishmania activity. Conclusions: This is the first demonstration that relapses after a first episode of KA are due to differences in human genetic susceptibility and not to modifications of parasite pathogenicity.

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

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 TERT, RTEL1, PARN or SFTPC coding regions. The burden test, based on 81 RA-ILD patients and 1010 controls of European ancestry, revealed an excess of TERT, RTEL1, PARN or SFTPC mutations in RA-ILD patients (OR 3.17, 95% CI 1.53-6.12; p=9.45×10-4). Telomeres were shorter in RA-ILD patients with a TERT, RTEL1 or PARN mutation than in controls (p=2.87×10-2).Our results support the contribution of FPF-linked genes to RA-ILD susceptibility.

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

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.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2, TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma.

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 BCOR (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, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) 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.

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

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.

Clinical Interpretation of Variants from Next-Generation Sequencing: The 2016 Scientific Meeting of the Human Genome Variation Society.

The 2016 scientific meeting of the Human Genome Variation Society (HGVS; http://www.hgvs.org) was held on the 20th of May in Barcelona, Spain, with the theme of "Clinical Interpretation of Variants from Next-Generation Sequencing."

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

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.

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

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.

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

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.

BRCA Share: A Collection of Clinical BRCA Gene Variants.

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.