Combined genetic and high-throughput strategies for the molecular diagnosis of inherited retinal dystrophies

Most diagnostic laboratories are confronted with the increasing demand for molecular diagnosis from patients and families and the ever-increasing genetic heterogeneity of visual disorders. Concerning Retinal Dystrophies (RD), almost 200 causative genes have been reported to date, and most families carry private mutations. We aimed to approach RD genetic diagnosis using all the available genetic information to prioritize candidates for mutational screening, and then restrict the number of cases to be analyzed by massive sequencing. We constructed and optimized a comprehensive cosegregation RD-chip based on SNP genotyping and haplotype analysis. The RD-chip allows to genotype 768 selected SNPs (closely linked to 100 RD causative genes) in a single cost-, time-effective step. Full diagnosis was attained in 17/36 Spanish pedigrees, yielding 12 new and 12 previously reported mutations in 9 RD genes. The most frequently mutated genes were USH2A and CRB1. Notably, RD3-up to now only associated to Leber Congenital Amaurosis- was identified as causative of Retinitis Pigmentosa. The main assets of the RD-chip are: i) the robustness of the genetic information that underscores the most probable candidates, ii) the invaluable clues in cases of shared haplotypes, which are indicative of a common founder effect, and iii) the detection of extended haplotypes over closely mapping genes, which substantiates cosegregation, although the assumptions in which the genetic analysis is based could exceptionally lead astray. The combination of the genetic approach with whole exome sequencing (WES) greatly increases the diagnosis efficiency, and revealed novel mutations in USH2A and GUCY2D. Overall, the RD-chip diagnosis efficiency ranges from 16% in dominant, to 80% in consanguineous recessive pedigrees, with an average of 47%, well within the upper range of massive sequencing approaches, highlighting the validity of this time- and cost-effective approach whilst high-throughput methodologies become amenable for routine diagnosis in medium sized labs

A method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

Data processing; DNA sequencing; Genomic analysisTratamiento de datos; Secuenciación de ADN; Análisis genómicoTractament de dades; Seqüenciació d'ADN; Anàlisi genòmicaMethods to reconstruct the mitochondrial DNA (mtDNA) sequence using short-read sequencing come with an inherent bias due to amplification and mapping. They can fail to determine the phase of variants, to capture multiple deletions and to cover the mitochondrial genome evenly. Here we describe a method to target, multiplex and sequence at high coverage full-length human mitochondrial genomes as native single-molecules, utilizing the RNA-guided DNA endonuclease Cas9. Combining Cas9 induced breaks, that define the mtDNA beginning and end of the sequencing reads, as barcodes, we achieve high demultiplexing specificity and delineation of the full-length of the mtDNA, regardless of the structural variant pattern. The long-read sequencing data is analysed with a pipeline where our custom-developed software, baldur, efficiently detects single nucleotide heteroplasmy to below 1%, physically determines phase and can accurately disentangle complex deletions. Our workflow is a tool for studying mtDNA variation and will accelerate mitochondrial research.This research has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 824110 – EASI-Genomics (I.G.G.) and the ERC Synergy project BCLL@las under grant agreement No 810287 (I.G.G.). Institutional support was from the Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias and cofunded with ERDF funds (PI19/01772). We acknowledge the institutional support of the Spanish Ministry of Science and Innovation through the Instituto de Salud Carlos III and the 2014–2020 Smart Growth Operating Program, to the EMBL partnership and institutional co-financing with the European Regional Development Fund (MINECO/FEDER, BIO2015-71792-P). We also acknowledge the support of the Centro de Excelencia Severo Ochoa, and the Generalitat de Catalunya through the Departament de Salut, Departament d’Empresa i Coneixement and the CERCA Programme to the institute

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eightythree genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes

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

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

Haemodialysis through a cellulose membrane induces dephosphorylation of CD11b and promotes leukocyte adhesion to endothelial cells

Purpose: To explore modifications in signal mechanisms involving CD11b and leukocyte adhesion in patients under haemodialysis (HD). Methods: Samples were obtained from uremic patients at baseline, 15 and 120 min of HD from both arterial and venous lines. CD11b expression was studied by flow cytometry. To study signalling mechanisms, CD11b was immunoprecipitated using a specific antibody. Immunoprecipitates were resolved by 8% SDS-PAGE to measure phosphorylation in immunoblots. Leukocyte adhesion was measured after blood perfusion using endothelial cells (EC) as adhesive substrate. Parallel studies were performed with blood from healthy donors. Results: The percentage of CD11b+ cells increased during HD with a cellulose membrane in the venous line at 15 and 120 min (6.2±2.9% and 11.0±7.1%) and in the arterial line at 120 min (11.5±8.5 vs. 3.1±1.0% in control P < 0.05). After 120 min HD, CD11b phosphorylation decreased in leukocytes from both arterial (72.6±2.9) and venous lines (51.8±6.5) vs. basal samples (119.5±15.5 P < 0.005). Control leukocytes showed enhanced adhesion to uremic EC compared with control EC (3.0±0.3 vs. 2.3±1.0 leukocytes x100 EC-1 P < 0.05). Uremic leukocyte adhesion was enhanced after HD compared with basal samples 4.2±0.2 leukocytes/100 EC in the arterial and 4.4±0.3 in the venous line; after 120 min vs 2.3±1.0 (P < 0.005). Conclusion: Leukocyte activation during HD through a cellulose membrane occurs with decreases in CD11b phosphorylation. Activation also induces increases in CD11b expression associated with enhanced leukocyte adhesion to uremic endothelial cells

Combined genetic and high-throughput strategies for the molecular diagnosis of inherited retinal dystrophies

Most diagnostic laboratories are confronted with the increasing demand for molecular diagnosis from patients and families and the ever-increasing genetic heterogeneity of visual disorders. Concerning Retinal Dystrophies (RD), almost 200 causative genes have been reported to date, and most families carry private mutations. We aimed to approach RD genetic diagnosis using all the available genetic information to prioritize candidates for mutational screening, and then restrict the number of cases to be analyzed by massive sequencing. We constructed and optimized a comprehensive cosegregation RD-chip based on SNP genotyping and haplotype analysis. The RD-chip allows to genotype 768 selected SNPs (closely linked to 100 RD causative genes) in a single cost-, time-effective step. Full diagnosis was attained in 17/36 Spanish pedigrees, yielding 12 new and 12 previously reported mutations in 9 RD genes. The most frequently mutated genes were USH2A and CRB1. Notably, RD3-up to now only associated to Leber Congenital Amaurosis- was identified as causative of Retinitis Pigmentosa. The main assets of the RD-chip are: i) the robustness of the genetic information that underscores the most probable candidates, ii) the invaluable clues in cases of shared haplotypes, which are indicative of a common founder effect, and iii) the detection of extended haplotypes over closely mapping genes, which substantiates cosegregation, although the assumptions in which the genetic analysis is based could exceptionally lead astray. The combination of the genetic approach with whole exome sequencing (WES) greatly increases the diagnosis efficiency, and revealed novel mutations in USH2A and GUCY2D. Overall, the RD-chip diagnosis efficiency ranges from 16% in dominant, to 80% in consanguineous recessive pedigrees, with an average of 47%, well within the upper range of massive sequencing approaches, highlighting the validity of this time- and cost-effective approach whilst high-throughput methodologies become amenable for routine diagnosis in medium sized labs

Bulletin mensuel de statistique / Institut national de la statistique et des études économiques...

juillet 19801980/07 (N7,A31)

Combined Genetic and High-Throughput Strategies for Molecular Diagnosis of Inherited Retinal Dystrophies

<div><p>Most diagnostic laboratories are confronted with the increasing demand for molecular diagnosis from patients and families and the ever-increasing genetic heterogeneity of visual disorders. Concerning Retinal Dystrophies (RD), almost 200 causative genes have been reported to date, and most families carry private mutations. We aimed to approach RD genetic diagnosis using all the available genetic information to prioritize candidates for mutational screening, and then restrict the number of cases to be analyzed by massive sequencing. We constructed and optimized a comprehensive cosegregation RD-chip based on SNP genotyping and haplotype analysis. The RD-chip allows to genotype 768 selected SNPs (closely linked to 100 RD causative genes) in a single cost-, time-effective step. Full diagnosis was attained in 17/36 Spanish pedigrees, yielding 12 new and 12 previously reported mutations in 9 RD genes. The most frequently mutated genes were <i>USH2A</i> and <i>CRB1</i>. Notably, <i>RD3</i>–up to now only associated to Leber Congenital Amaurosis– was identified as causative of Retinitis Pigmentosa. The main assets of the RD-chip are: i) the robustness of the genetic information that underscores the most probable candidates, ii) the invaluable clues in cases of shared haplotypes, which are indicative of a common founder effect, and iii) the detection of extended haplotypes over closely mapping genes, which substantiates cosegregation, although the assumptions in which the genetic analysis is based could exceptionally lead astray. The combination of the genetic approach with whole exome sequencing (WES) greatly increases the diagnosis efficiency, and revealed novel mutations in <i>USH2A</i> and <i>GUCY2D</i>. Overall, the RD-chip diagnosis efficiency ranges from 16% in dominant, to 80% in consanguineous recessive pedigrees, with an average of 47%, well within the upper range of massive sequencing approaches, highlighting the validity of this time- and cost-effective approach whilst high-throughput methodologies become amenable for routine diagnosis in medium sized labs.</p></div