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      Diagnostic Yield of Next Generation Sequencing in Genetically Undiagnosed Patients with Primary Immunodeficiencies: a Systematic Review

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          Abstract

          Background

          As the application of next generation sequencing (NGS) is moving to earlier stages in the diagnostic pipeline for primary immunodeficiencies (PIDs), re-evaluation of its effectiveness is required. The aim of this study is to systematically review the diagnostic yield of NGS in PIDs.

          Methods

          PubMed and Embase databases were searched for relevant studies. Studies were eligible when describing the use of NGS in patients that had previously been diagnosed with PID on clinical and/or laboratory findings. Relevant data on study characteristics, technological performance and diagnostic yield were extracted.

          Results

          Fourteen studies were eligible for data extraction. Six studies described patient populations from specific PID subcategories. The remaining studies included patients with unsorted PIDs. The studies were based on populations from Italy, Iran, Turkey, Thailand, the Netherlands, Norway, Saudi Arabia, Sweden, the UK, and the USA. Eight studies used an array-based targeted gene panel, four used WES in combination with a PID filter, and two used both techniques. The mean reported reading depth ranged from 98 to 1337 times. Five studies described the sensitivity of the applied techniques, ranging from 83 to 100%, whereas specificity ranged from 45 to 99.9%. The percentage of patients who were genetically diagnosed ranged from 15 to 79%. Several studies described clinical implications of the genetic findings.

          Discussion

          NGS has the ability to contribute significantly to the identification of molecular mechanisms in PID patients. The diagnostic yield highly depends on population and on the technical circumstances under which NGS is employed. Further research is needed to determine the exact diagnostic yield and clinical implications of NGS in patients with PID.

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          Most cited references25

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          Exome and genome sequencing for inborn errors of immunity.

          The advent of next-generation sequencing (NGS) in 2010 has transformed medicine, particularly the growing field of inborn errors of immunity. NGS has facilitated the discovery of novel disease-causing genes and the genetic diagnosis of patients with monogenic inborn errors of immunity. Whole-exome sequencing (WES) is presently the most cost-effective approach for research and diagnostics, although whole-genome sequencing offers several advantages. The scientific or diagnostic challenge consists in selecting 1 or 2 candidate variants among thousands of NGS calls. Variant- and gene-level computational methods, as well as immunologic hypotheses, can help narrow down this genome-wide search. The key to success is a well-informed genetic hypothesis on 3 key aspects: mode of inheritance, clinical penetrance, and genetic heterogeneity of the condition. This determines the search strategy and selection criteria for candidate alleles. Subsequent functional validation of the disease-causing effect of the candidate variant is critical. Even the most up-to-date dry lab cannot clinch this validation without a seasoned wet lab. The multifariousness of variations entails an experimental rigor even greater than traditional Sanger sequencing-based approaches in order not to assign a condition to an irrelevant variant. Finding the needle in the haystack takes patience, prudence, and discernment.
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            Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies.

            Primary immunodeficiency (PID) disorders are a heterogeneous group of inherited disorders caused by a variety of monogenetic immune defects. Thus far, mutations in more than 170 different genes causing PIDs have been described. A clear genotype-phenotype correlation is often not available, which makes a genetic diagnosis in patients with PIDs complex and laborious. We sought to develop a robust, time-effective, and cost-effective diagnostic method to facilitate a genetic diagnosis in any of 170 known PID-related genes by using next-generation sequencing (NGS). We used both targeted array-based and in-solution enrichment combined with a SOLiD sequencing platform and a bioinformatic pipeline developed in house to analyze genetic changes in the DNA of 41 patients with PIDs with known mutations and 26 patients with undiagnosed PIDs. This novel NGS-based method accurately detected point mutations (sensitivity and specificity >99% in covered regions) and exonic deletions (100% sensitivity and specificity). For the 170 genes of interest, the DNA coverage was greater than 20× in 90% to 95%. Nine PID-related genes proved not eligible for evaluation by using this NGS-based method because of inadequate coverage. The NGS method allowed us to make a genetic diagnosis in 4 of 26 patients who lacked a genetic diagnosis despite routine functional and genetic testing. Three of these patients proved to have an atypical presentation of previously described PIDs. This novel NGS tool facilitates accurate simultaneous detection of mutations in 161 of 170 known PID-related genes. In addition, these analyses will generate more insight into genotype-phenotype correlations for the different PID disorders. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.
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              Application of whole genome and RNA sequencing to investigate the genomic landscape of common variable immunodeficiency disorders

              Common Variable Immunodeficiency Disorders (CVIDs) are the most prevalent cause of primary antibody failure. CVIDs are highly variable and a genetic causes have been identified in <5% of patients. Here, we performed whole genome sequencing (WGS) of 34 CVID patients (94% sporadic) and combined them with transcriptomic profiling (RNA-sequencing of B cells) from three patients and three healthy controls. We identified variants in CVID disease genes TNFRSF13B, TNFRSF13C, LRBA and NLRP12 and enrichment of variants in known and novel disease pathways. The pathways identified include B-cell receptor signalling, non-homologous end-joining, regulation of apoptosis, T cell regulation and ICOS signalling. Our data confirm the polygenic nature of CVID and suggest individual-specific aetiologies in many cases. Together our data show that WGS in combination with RNA-sequencing allows for a better understanding of CVIDs and the identification of novel disease associated pathways.
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                Author and article information

                Contributors
                +31 88 75 55 555 , J.vanMontfrans@umcutrecht.nl
                Journal
                J Clin Immunol
                J. Clin. Immunol
                Journal of Clinical Immunology
                Springer US (New York )
                0271-9142
                1573-2592
                28 June 2019
                28 June 2019
                2019
                : 39
                : 6
                : 577-591
                Affiliations
                [1 ]ISNI 0000000090126352, GRID grid.7692.a, Department of Pediatric Immunology and Infectious Diseases, , University Medical Centre Utrecht, ; Utrecht, The Netherlands
                [2 ]ISNI 0000000084992262, GRID grid.7177.6, Department of Pediatric Hematology, Immunology and Infectious Diseases, Emma Children’s Hospital, Academic Medical Centre, , University of Amsterdam, ; Amsterdam, The Netherlands
                [3 ]ISNI 0000000090126352, GRID grid.7692.a, Department of Medical Genetics, , University Medical Centre Utrecht, ; Utrecht, The Netherlands
                [4 ]ISNI 0000000090126352, GRID grid.7692.a, Julius Center for Health Sciences and Primary Care, , University Medical Centre Utrecht, ; Utrecht, The Netherlands
                Author information
                http://orcid.org/0000-0001-6764-0211
                Article
                656
                10.1007/s10875-019-00656-x
                6697711
                31250335
                702653f1-10d5-4520-b661-42adbf0a7e67
                © The Author(s) 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 25 January 2019
                : 10 June 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001826, ZonMw;
                Award ID: 846002001
                Award Recipient :
                Categories
                Original Article
                Custom metadata
                © Springer Science+Business Media, LLC, part of Springer Nature 2019

                Immunology
                primary immunodeficiency (pid),next generation sequencing (ngs),diagnostic yield,technological performance,clinical performance,whole exome sequencing (wes),whole genome sequencing (wgs)

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