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Full Disclosures ARTICLE OPEN ACCESS Critical exon indexing improves clinical interpretation of copy number variants in neurodevelopmental disorders E. Robert Wassman, MD,* Karen S. Ho, PhD,* Diana Bertrand, MS, Kyle W. Davis, ScM, Megan M. Martin, MS, Correspondence Stephanie Page, BS, Andreas Peiffer, MD, PhD, Aparna Prasad, PhD, Moises A. Serrano, PhD, Hope Twede, BS, Dr. Wassman [email protected] Rena Vanzo, MS, Stephen W. Scherer, PhD, Mohammed Uddin, PhD, and Charles H. Hensel, PhD* Neurol Genet 2019;5:e378. doi:10.1212/NXG.0000000000000378 Abstract Objective To evaluate a new tool to aid interpretation of copy number variants (CNVs) in individuals with neurodevelopmental disabilities. Methods Critical exon indexing (CEI) was used to identify genes with critical exons (CEGs) from clinically reported CNVs, which may contribute to neurodevelopmental disorders (NDDs). The 742 pathogenic CNVs and 1,363 variants of unknown significance (VUS) identified by chromosomal microarray analysis in 5,487 individuals with NDDs were subjected to CEI to identify CEGs. CEGs identified in a subsequent random series of VUS were evaluated for relevance to CNV interpretation. Results CEI identified a total of 2,492 unique CEGs in pathogenic CNVs and 953 in VUS compared with 259 CEGs in 6,965 CNVs from 873 controls. These differences are highly significant (p < 0.00001) whether compared as frequency, average, or normalized by CNV size. Twenty-one percent of VUS CEGs were not represented in Online Mendelian Inheritance in Man, high- lighting limitations of existing resources for identifying potentially impactful genes within CNVs. CEGs were highly correlated with other indices and known pathways of relevance. Separately, 136 random VUS reports were reevaluated, and 76% of CEGs had not been commented on. In multiple cases, further investigation yielded additional relevant literature aiding interpretation. As one specific example, we discuss GTF2I as a CEG, which likely alters interpretation of several reported duplication VUS in the Williams-Beuren region. Conclusions Application of CEI to CNVs in individuals with NDDs can identify genes of potential clinical relevance, aid laboratories in effectively searching the clinical literature, and support the clinical reporting of poorly annotated VUS. *These authors contributed equally to this work. From Lineagen Inc. (E.R.W., K.S.H., D.B., K.W.D., M.M.M., S.P., A. Peiffer, A. Prasad, M.A.S., H.T., R.V., C.H.H.); Life Designs Ventures (E.R.W.), Park City, UT; Department of Pediatrics (K.S.H., A. Peiffer), University of Utah; The Centre for Applied Genomics (S.W.S., M.U.), The Hospital for Sick Children, Toronto, Ontario, Canada; Program in Genetics and Genome Biology (S.W.S), The Hospital for Sick Children; McLaughlin Centre (S.W.S), University of Toronto, Toronto, Ontario, Canada; and Department of Molecular Genetics (S.W.S), University of Toronto, Toronto, Ontario, Canada. Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by Lineagen, Inc. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ASD = autism spectrum disorder; CEG = critical exon gene; CEI = critical exon indexing; CMA = chromosomal microarray analysis; CNV = copy number variant; DD = developmental delay; FMRP = fragile X mental retardation protein; ID = intellectual disability; IPA = ingenuity pathway analysis; Mbp = megabase pairs; MCA = multiple congenital anomaly; NDD = neurodevelopmental disorder; OMIM = Online Mendelian Inheritance in Man; pLI = probability of loss of function index; VUS = variants of unknown significance; WBS = Williams-Beuren syndrome. Neurodevelopmental disorders (NDDs) including de- Standard protocol approvals, registrations, velopmental delay (DD), intellectual disability (ID), and au- and patient consents tism spectrum disorder (ASD) are often caused by copy Written informed consent was obtained for all participants’ – number variants (CNVs) in the genome.1 6 Chromosomal data in this study. Deidentified data were collected under microarray analysis (CMA) for the detection of CNVs is the Western Institutional Review Board Protocol #20162032. – first-tier clinical diagnostic test for the evaluation of NDDs.2 6 fi fi CMA frequently identi es variants of uncertain signi cance Chromosomal microarray and reporting (VUS), which limit clinical utility in such cases. Interpretation Between September 2012 and December 2015, 5,487 ge- of VUS is relevant to clinical care of these children and im- 7,8 nomic DNAs were extracted with either a buccal swab or portant to their families. blood Gentra Puregene kit (Qiagen, Redwood City, CA), fragmented, labeled, and hybridized to a previously de- Clinical application of an ultra-high-resolution whole-genome scribed microarray optimized for NDDs9 (FSDX; Lineagen, chromosomal microarray optimized for the detection of genetic Salt Lake City, UT) as specified by the manufacturer changes associated with a variety of NDDs provides an in- (Affymetrix).15 Washed arrays were scanned and raw data creased diagnostic yield, particularly due to careful attention to files analyzed as CYCHP files using Chromosome Analysis VUS of potential clinical relevance, constituting up to 67% of Suite version 2.0.1 (Affymetrix, Santa Clara, CA) with ref- reported CNVs.9 These VUS could not be definitively linked to ’ erence genome from the Genome Reference Consortium: patients neurodevelopmental traits despite extensive analysis of human build 37/human genome 19 (GRCh37/hg19). the extant literature using standard approaches, which include 10 American Board of Medical Genetics and Genomics- analysis of relevant gene content. certified cytogeneticists used established interpretation 10 fi 11–14 fi “ ” standards. In brief, CNVs were classi ed pathogenic if The recently described identi cation of critical exons multiple peer-reviewed publications, representing at least 2 with high levels of brain expression and low mutation burden 14 families indicate clinical features result from sensitivity of the recognizes genes (e.g., OTUD7A) that might be NDD can- region or gene(s) to dosage.10 If there was preliminary but didate genes. This was adapted to create a categorical classifi- ffi fi fi insu cient evidence for pathogenicity, CNVs were classi ed cation tool that focuses analysis on speci c genes among many as variants of unknown significance (VUS). CNVs are clas- contained in CNVs, which are likely to contribute to in- sified as benign and not reported if the region had been well terpretation. This tool retrospectively identified many genes fi characterized as a common variant in the general population that have relevant clinical literature, but had not been speci cally by multiple peer-reviewed publications or by curated data- detailed in the clinical reports previously issued. Prospective bases (e.g., the Database of Genomic Variants16). application of this tool may aid in the classification of VUS and affect diagnosis and management of a wide range of NDDs. Critical exon indexing Methods Identification of CEGs by CEI was adapted from previously described methods11,12 The mutation burden of rare (<0.05)11 Patient data missense and loss-of-function mutations for each exon CMA results were reviewed from 1,602 cases with clinically was calculated from the 1000 Genomes Project (inter- relevant findings from a previously described retrospective se- nationalgenome.org). The BrainSpan Atlas provided spatio- ries of 5,487 consecutive patients with indications for testing for temporal exon level RNA-seq expression from 388 tissue broader NDDs that includes ASD, DD, ID, multiple congenital samples from 16 brain regions of prenatal, early childhood, anomaly (MCA), and seizures or combinations of these.9 and adult brains from 32 postmortem donors.17 Our cutoffs, Medical records and indications for testing provided by re- which were modified for high stringency clinical translation, ferring providers were used to collect phenotype information. considered an exon critical if expressed at >90th percentile Subsequent to this series’ analysis for critical exon genes throughout all 3 developmental stages, and has a mutation (CEGs) prevalence, a separate group of 136 randomly selected burden <10th percentile (normalized by exon size). “Critical reports of VUS was assessed to determine the impact critical exon” enrichment between CNVs classified as pathogenic, exon indexing (CEI) might have on searching for the relevant VUS, or normal was tested with the Fisher exact test, Welch t literature and revised clinical reporting. test, or χ2 analysis depending on the nature of the variables. 2 Neurology: Genetics | Volume 5, Number 6 | December 2019 Neurology.org/NG Online Mendelian Inheritance in Man (OMIM, omim.org) found in both pathogenic CNVs and VUS alike. The presence and PubMed (ncbi.nlm.nih.gov/pubmed/) were used to search of at least 1 CEG in a CNV was significantly higher in path- for published data and disorder associations for each CEG. ogenic CNVs (69.9%) compared with either VUS (39.6%) (p < 0.00001) or a control population13 (11.4%) (p < 0.00001). Ingenuity Pathway Analysis
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