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To characterize the phenotype observed in a case series with macular disease and determine the cause.Multicenter case series.Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration.Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing.Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients.All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor.Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.

The Cancer Programme of the 100,000 Genomes Project was a transformational UK government initiative that aimed to bring whole genome sequencing (WGS) to cancer patients and evaluate the opportunities for precision cancer care. Genomics England, in partnership with NHS England, generated whole genome analyses for 13,880 solid tumours across 33 different cancer types, and genomic data were linked with real-world health data within a secure national research environment. Here, we report the overall findings of the programme, focusing on clinical actionability and potential wider clinical significance. We found variation between cancer types in the incidence of somatic mutations of different types in genes currently recommended for standard-of-care testing. For example, 94% of glioblastoma multiforme cases had small variants and 54% had copy number aberrations (CNAs) in at least one gene recommended for clinical testing, whereas sarcoma was found to have the highest proportion of actionable structural variants (13%). We confirmed the importance of utilising pan- genomic markers, such as mutational signatures, with 51% of high grade serous ovarian cancer cases showing homologous recombination deficiency, 13% of which were associated with pathogenic germline variants, indicating the value of combined somatic and germline analyses. We also observed a significant co-occurrence of somatic small variants and CNAs in several known oncogenes including EGFR, GNAS, BRAF, and KRAS. Our findings demonstrate the value of combining genomic testing with real world clinical and treatment data to inform clinical recommendations for genomic testing in cancer, to enable survival analysis and improve understanding of the long-term effects of clinical cancer genomics on patient outcomes.

Purpose: This study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants. Methods: Individuals with protein altering variants in SOX11 were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of SOX11 in developing human brain was defined using RNAscope. Results: We reported 38 new patients with SOX11 variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of SOX11 syndrome. A distinctive pattern of blood DNA methylation was identified in SOX11 syndrome, separating SOX11 syndrome from other BAFopathies. Conclusion: SOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies. (C) 2022 The Authors. Published by Elsevier Inc. on behalf of American College of Medical Genetics and Genomics.

Background Sequencing of miRNAs isolated from exosomes has great potential to identify novel disease biomarkers, but exosomes have low amount of RNA, hindering adequate analysis and quantification. Here, we have assessed several steps in developing an optimized small RNA (sRNA) library preparation protocol for next-generation sequencing (NGS) miRNA analysis from urinary exosomes. Methods A total of 24 urinary exosome samples from donors were included in this study. RNA was extracted by column-based methods. The quality of extracted RNA was assessed by spectrophotometric quantification and Bioanalyzer software analysis. All libraries were prepared using the CleanTag small RNA library preparation protocol and the effect of our additional modifications on adapter-dimer presence, sequencing data and tagged small RNA library population was also analyzed. Results Our results show that good quality sequencing libraries can be prepared following our optimized small RNA library preparation protocol from urinary exosomes. When the size selection by gel purification step was included within the workflow, adapter-dimer was totally removed from cDNA libraries. Furthermore, the inclusion of this modification step within small RNA library protocol augmented the small RNA mapped reads, with an especially significant 37% increase in miRNA reads, and the gel purification step made no difference to the tagged miRNA population. Conclusions This study provides researchers with an optimized small RNA library preparation workflow for next generation sequencing based exosome-associated miRNA analysis that yields a high amount of miRNA mapped reads without skewing the tagged miRNA population significantly.

BACKGROUND: Repeat expansion disorders affect about 1 in 3000 individuals and are clinically heterogeneous diseases caused by expansions of short tandem DNA repeats. Genetic testing is often locus-specific, resulting in underdiagnosis of people who have atypical clinical presentations, especially in paediatric patients without a previous positive family history. Whole genome sequencing is increasingly used as a first-line test for other rare genetic disorders, and we aimed to assess its performance in the diagnosis of patients with neurological repeat expansion disorders.METHODS: We retrospectively assessed the diagnostic accuracy of whole genome sequencing to detect the most common repeat expansion loci associated with neurological outcomes (AR, ATN1, ATXN1, ATXN2, ATXN3, ATXN7, C9orf72, CACNA1A, DMPK, FMR1, FXN, HTT, and TBP) using samples obtained within the National Health Service in England from patients who were suspected of having neurological disorders; previous PCR test results were used as the reference standard. The clinical accuracy of whole genome sequencing to detect repeat expansions was prospectively examined in previously genetically tested and undiagnosed patients recruited in 2013-17 to the 100 000 Genomes Project in the UK, who were suspected of having a genetic neurological disorder (familial or early-onset forms of ataxia, neuropathy, spastic paraplegia, dementia, motor neuron disease, parkinsonian movement disorders, intellectual disability, or neuromuscular disorders). If a repeat expansion call was made using whole genome sequencing, PCR was used to confirm the result.FINDINGS: The diagnostic accuracy of whole genome sequencing to detect repeat expansions was evaluated against 793 PCR tests previously performed within the NHS from 404 patients. Whole genome sequencing correctly classified 215 of 221 expanded alleles and 1316 of 1321 non-expanded alleles, showing 97·3% sensitivity (95% CI 94·2-99·0) and 99·6% specificity (99·1-99·9) across the 13 disease-associated loci when compared with PCR test results. In samples from 11 631 patients in the 100 000 Genomes Project, whole genome sequencing identified 81 repeat expansions, which were also tested by PCR: 68 were confirmed as repeat expansions in the full pathogenic range, 11 were non-pathogenic intermediate expansions or premutations, and two were non-expanded repeats (16% false discovery rate).INTERPRETATION: In our study, whole genome sequencing for the detection of repeat expansions showed high sensitivity and specificity, and it led to identification of neurological repeat expansion disorders in previously undiagnosed patients. These findings support implementation of whole genome sequencing in clinical laboratories for diagnosis of patients who have a neurological presentation consistent with a repeat expansion disorder.FUNDING: Medical Research Council, Department of Health and Social Care, National Health Service England, National Institute for Health Research, and Illumina.

PurposeGenome sequencing (GS) for diagnosis of rare genetic disease is being introduced into the clinic, but the complexity of the data poses challenges for developing pipelines with high diagnostic sensitivity. We evaluated the performance of the Genomics England 100,000 Genomes Project (100kGP) panel-based pipelines, using craniosynostosis as a test disease.MethodsGS data from 114 probands with craniosynostosis and their relatives (314 samples), negative on routine genetic testing, were scrutinized by a specialized research team, and diagnoses compared with those made by 100kGP.ResultsSixteen likely pathogenic/pathogenic variants were identified by 100kGP. Eighteen additional likely pathogenic/pathogenic variants were identified by the research team, indicating that for craniosynostosis, 100kGP panels had a diagnostic sensitivity of only 47%. Measures that could have augmented diagnoses were improved calling of existing panel genes (+18% sensitivity), review of updated panels (+12%), comprehensive analysis of de novo small variants (+29%), and copy-number/structural variants (+9%). Recent NHS England recommendations that partially incorporate these measures should achieve 85% overall sensitivity (+38%).ConclusionGS identified likely pathogenic/pathogenic variants in 29.8% of previously undiagnosed patients with craniosynostosis. This demonstrates the value of research analysis and the importance of continually improving algorithms to maximize the potential of clinical GS.

Urinary albumin excretion (UAE) is a marker of cardiovascular risk and renal damage in hypertension. MicroRNAs (miRNAs) packaged into exosomes function as paracrine effectors in cell communication and the kidney is not exempt. This study aimed to state an exosomal miRNA profile/signature associated to hypertension with increased UAE and the impact of profibrotic TGF-β1 (transforming growth factor β1) on exosomes miRNA release. Therefore, exosomes samples from patients with hypertension with/without UAE were isolated and characterized. Three individual and unique small RNA libraries from each subject were prepared (total plasma, urinary, and plasma-derived exosomes) for next-generation sequencing profiling. Differentially expressed miRNAs were over-represented in Kyoto Encyclopedia of Genes and Genomes pathways, and selected miRNAs were validated by real-time quantitative polymerase chain reaction in a confirmation cohort. Thus, a signature of 29 dysregulated circulating miRNAs was identified in UAE hypertensive subjects, regulating 21 pathways. Moreover, changes in the levels of 4 exosomes-miRNAs were validated in a confirmation cohort and found associated with albuminuria. In particular miR-26a, major regulator of TGF-β signaling, was found downregulated in both type of exosomes when compared with healthy controls and to hypertension normoalbuminurics ( P

Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Background Biological databases and repositories are incrementing in diversity and complexity over the years. This rapid expansion of current and new sources of biological knowledge raises serious problems of data accessibility and integration. To handle the growing necessity of unification, CellBase was created as an integrative solution. CellBase provides a centralized NoSQL database containing biological information from different and heterogeneous sources. Access to this information is done through a RESTful web service API, which provides an efficient interface to the data. Results In this work we present PyCellBase, a Python package that provides programmatic access to the rich RESTful web service API offered by CellBase. This package offers a fast and user-friendly access to biological information without the need of installing any local database. In addition, a series of command-line tools are provided to perform common bioinformatic tasks, such as variant annotation. CellBase data is always available by a high-availability cluster and queries have been tuned to ensure a real-time performance. Conclusion PyCellBase is an open-source Python package that provides an efficient access to heterogeneous biological information. It allows to perform tasks that require a comprehensive set of knowledge resources, as for example variant annotation. Queries can be easily fine-tuned to retrieve the desired information of particular biological features. PyCellBase offers the convenience of an object-oriented scripting language and provides the ability to integrate the obtained results into other Python applications and pipelines. Electronic supplementary material The online version of this article (10.1186/s12859-019-2726-4) contains supplementary material, which is available to authorized users.

Babelomics has been running for more than one decade offering a user-friendly interface for the functional analysis of gene expression and genomic data. Here we present its fifth release, which includes support for Next Generation Sequencing data including gene expression (RNA-seq), exome or genome re-sequencing. Babelomics has simplified its interface, being now more intuitive. Improved visualization options, such as a genome viewer as well as an interactive network viewer, have been implemented. New technical enhancements at both, client and server sides, makes the user experience faster and more dynamic. Babelomics offers user-friendly access to a full range of methods that cover: (i) primary data analysis, (ii) a variety of tests for different experimental designs and (iii) different enrichment and network analysis algorithms for the interpretation of the results of such tests in the proper functional context. In addition to the public server, local copies of Babelomics can be downloaded and installed. Babelomics is freely available at: http://www.babelomics.org.

Background The 100,000 Genomes Project aims to improve cancer care for NHS patients in the UK through personalised medicine. Our target is to return WGS results to clinicians in a clinically meaningful timescale to facilitate diagnosis and treatment choices for patients, and in parallel to provide a research platform of genomic data linked to longitudinal clinical data. Methods We present here an overview of clinical utility for reported outcomes. To date, bioinformatics reports for WGS, with links to potentially relevant therapies and UK clinical trials, have been produced for more than 14,000 cancer patients in the UK Currently our bioinformatics analysis of WGS includes clinical interpretation of somatic small variants, somatic structural and copy number variants (SV/CNV), germline pertinent findings, mutational burden and signatures. To iteratively develop a high-quality bioinformatics pipeline and to monitor clinical utility of returned results, we are collecting feedback via the 100,000 Genomes Project Interpretation Portal from NHS Molecular Tumour Boards. These data suggest that WGS has the potential to affect patient management. Future applications will include the utilisation of pangenomic markers to better stratify patients within the context of a clinical study. Results WGS has the ability to replace multiple standard of care tests as it has the potential to detect all types of variants (SV/CNV/SNV/indels) as well as emerging pangenome biomarkers in a single test. In this study we use samples submitted as part of the 100,000 Genomes Project to investigate the feasibility of WGS as an alternative to conventional testing. Overall comparison of WGS with the results of NGS panels (96 patients, 156 clinically-relevant SNVs), high-depth exome sequencing (10 patients, 3150 SNVs, 140 indels), cytogenetic FISH tests (70 patients, 259 SVs, 100 CNVs), immunohistochemistry tests for Mismatch Repair Deficiency (265 patients) and HER2 status (154 patients) demonstrated Positive Percentage Agreement > 90% and False Positive Rate Conclusions Further work is required to validate fully all aspects of the WGS analysis pipeline but these results indicate that WGS can reliably detect clinically relevant biomarkers in the genomes of cancer patients. Legal entity responsible for the study Genomics England. Funding Genomics England. Disclosure All authors have declared no conflicts of interest.

The 100,000 Genomes Project aims to improve cancer care for patients in the UK's National Health Service by applying personalized medicine paradigms based on whole-genome sequencing (WGS). The program has been designed to enable return of WGS-based results to clinicians in a clinically meaningful timeframe in order to enable diagnosis and treatment decisions for patient. Validation of WGS analysis results against standard-of-care tests: WGS analysis of tumors has a potential for replacing multiple standard-of-care tests, such as gene panels, genotyping arrays, FISH and karyotyping. For structural variants, WGS also provides much higher genomic resolution in comparison to FISH, the current standard of care. An important challenge when validating the analytical performance of WGS against existing technologies is to be able to systematically compare across a wide range of structural variant classes, low resolution events with WGS ascertained variants, which have often base pair resolution. We present here the results of successful validation for WGS analysis against targeted sequencing, genotyping array and FISH for a cohort of patients with chronic lymphocytic leukaemia (CLL). Quality control of sequencing data: To ensure high sensitivity and specificity of reported variants we developed a protocol for DNA quality control based on PCA analysis of six metrics derived from sequencing data: AT and CG dropout, unevenness of local genome coverage, percentage of mapped reads, percentage of chimeric DNA fragments and average fragment size. For each new sample, these six metrics are projected into the transformed space and outliers are determined by comparing with a cohort of 672 fresh frozen samples. Samples having a p below 10-4 (p: probability density after multivariate normal fitting) are manually reviewed. This procedure allowed us to quickly spot the sets of samples that were extracted with the violation of DNA-extraction protocol and to prevent polluting results with high rates of false positive variant calls. Reporting outcomes: Currently our bioinformatics analysis of WGS includes somatic small variants and somatic structural variants, germline pertinent findings, mutational signatures and mutational burden. Somatic small variants are linked to approved therapies, UK clinical trials and prognosis guidelines. We are working on implementing recently derived genomic signatures for predicting therapeutic response for specific tumor types. We are presenting here an overview of clinical utility for our reported outcomes. Bioinformatics reports for WGS with links to potentially relevant therapies and clinical trials have been already delivered for hundreds of cancer patients in the UK. We are collecting feedback from clinicians and working on improving our analyses to provide better benefits for cancer patients. Citation Format: Alona Sosinsky, Pavlos Antoniou, John Ambrose, Martina Mijuskovic, Antonio Rueda-Martin, Daniel Perez-Gil1, Enric Serra, Shirley Henderson, Angela Hamblin, Anna Schuh, Nirupa Murugaesu, Clare Turnbull, Augusto Rendon, Mark Caulfield. 100,000 Genomes Project: Cancer program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 434.