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Systematic Phenomics Analysis of Autism-Associated Genes Reveals Parallel Networks Underlying Reversible Impairments in Habituation

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Systematic Phenomics Analysis of Autism-Associated Genes Reveals Parallel Networks Underlying Reversible Impairments in Habituation Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation Troy A. McDiarmida, Manuel Belmadanib,c, Joseph Lianga, Fabian Meilia, Eleanor A. Mathewsd, Gregory P. Mullene, Ardalan Hendif, Wan-Rong Wongg, James B. Randd,h, Kota Mizumotof, Kurt Haasa, Paul Pavlidisa,b,c, and Catharine H. Rankina,i,1 aDjavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, BC V6T 2B5, Canada; bDepartment of Psychiatry, University of British Columbia, Vancouver, BC V6T 2A1, Canada; cMichael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; dGenetic Models of Disease Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; eBiology Program, Oklahoma City University, Oklahoma City, OK 73106; fDepartment of Zoology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; gDivision of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125; hOklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and iDepartment of Psychology, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Edited by Gene E. Robinson, University of Illinois at Urbana–Champaign, Urbana, IL, and approved October 25, 2019 (received for review July 16, 2019) A major challenge facing the genetics of autism spectrum disorders have dramatically increased the pace of gene discovery in ASD (ASDs) is the large and growing number of candidate risk genes and (5–9). There are now >100 diverse genes with established ties gene variants of unknown functional significance. Here, we used to ASD, many of which are being used in diagnosis. Impor- Caenorhabditis elegans to systematically functionally characterize tantly, each gene accounts for <1% of cases and none have ASD-associated genes in vivo. Using our custom machine vision sys- shown complete specificity for ASD, with many implicated in tem, we quantified 26 phenotypes spanning morphology, locomo- multiple neurodevelopmental disorders (3, 4, 8). Some of these tion, tactile sensitivity, and habituation learning in 135 strains each genes have fallen into an encouragingly small set of broadly carrying a mutation in an ortholog of an ASD-associated gene. We defined biological processes such as gene expression regulation identified hundreds of genotype–phenotype relationships ranging (e.g., chromatin modification) and synaptic neuronal commu- from severe developmental delays and uncoordinated movement nication (3, 6–8, 10). Seminal studies using mouse models, NEUROSCIENCE to subtle deficits in sensory and learning behaviors. We clustered genetically stratified populations of individuals with ASD, human genes by similarity in phenomic profiles and used epistasis analysis induced pluripotent stem cells (iPSCs), and, more recently, high- to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 throughput genetic model organisms such as Drosophila and that underlie mechanosensory hyperresponsivity and impaired ha- bituation learning. We then leveraged our data for in vivo func- Significance tional assays to gauge missense variant effect. Expression of wild- type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and Although >100 genes have been implicated in the etiology of learning impairments. Testing the rescuing ability of conserved autism spectrum disorder (ASD), we need to better understand ASD-associated neuroligin variants revealed varied partial loss of their functions to reveal mechanisms underlying ASD and de- function despite proper subcellular localization. Finally, we used velop treatments. We developed a pipeline to discover functions CRISPR-Cas9 auxin-inducible degradation to determine that phe- of ASD-associated genes by inactivating each gene in the model notypic abnormalities caused by developmental loss of NLG-1 can organism Caenorhabditis elegans and observing the phenotypic be reversed by adult expression. This work charts the phenotypic consequences using machine vision. We quantified 26 pheno- landscape of ASD-associated genes, offers in vivo variant func- types spanning morphology, locomotion, tactile sensitivity, and tional assays, and potential therapeutic targets for ASD. learning in >27,000 animals representing 135 genotypes, allow- ing us to identify disruptions in habituation (a neural circuit’s autism spectrum disorder | neurodevelopmental disorders | Caenorhabditis plastic ability to decrease responding to repeated sensory stim- elegans | habituation learning | variants of uncertain significance uli) as a shared impairment. We then demonstrated how this database can facilitate experiments that determine the func- utism spectrum disorders (ASDs) encompass a clinically and tional consequences of missense variants and whether pheno- Agenetically heterogeneous group of neurodevelopmental typic alterations are reversible. disorders characterized by deficits in social communication and interaction, restrictive repetitive behaviors, and profound sensory Author contributions: T.A.M., J.B.R., K.M., K.H., P.P., and C.H.R. designed research; T.A.M., processing abnormalities (1–4). The fifth edition of the Diagnostic J.L., E.A.M., G.P.M., A.H., J.B.R., and P.P. performed research; T.A.M., M.B., J.L., F.M., E.A.M., G.P.M., A.H., W.-R.W., J.B.R., K.M., K.H., P.P., and C.H.R. contributed new and Statistical Manual of Mental disorders combines autistic dis- reagents/analytic tools; T.A.M., M.B., J.L., E.A.M., G.P.M., J.B.R., K.M., K.H., P.P., and C.H.R. order, Asperger disorder, childhood disintegrative disorder, and analyzed data; T.A.M. and C.H.R. wrote the paper; and M.B., J.B.R., K.M., K.H., and P.P. pervasive developmental disorder not otherwise specified into the edited completed manuscript. single grouping of autism spectrum disorder (1). Despite extensive The authors declare no competing interest. study, there is currently no unanimously agreed upon structural or This article is a PNAS Direct Submission. functional neuropathology common to all individuals with ASD, Published under the PNAS license. and there is little understanding of the biological mechanisms that Data deposition: All data generated in this work are available free online in their raw and cause ASD (3). The most promising avenue for research into ASDs processed forms at https://dataverse.scholarsportal.info/citation?persistentId=doi:10. 5683/SP2/FJWIL8. All analysis code is freely available at https://github.com/PavlidisLab/ has stemmed from the observation that they have a strong genetic McDiarmid-etal-2019_Multi-Worm-Tracker-analysis. All strains, sequence confirmation component, with monozygotic concordance estimates of ∼70 to files, and reagents are available from the corresponding author upon request. 90% and several distinct highly penetrant genetic syndromes (3, 4). 1To whom correspondence may be addressed. Email: [email protected]. Rapid advances in copy number variation association, whole- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ exome, and more recently, whole-genome sequencing tech- doi:10.1073/pnas.1912049116/-/DCSupplemental. nology and the establishment of large sequencing consortia, www.pnas.org/cgi/doi/10.1073/pnas.1912049116 PNAS Latest Articles | 1of12 Downloaded by guest on September 26, 2021 zebrafish have investigated the molecular, circuit, and behavioral et al. (8), the largest exome sequencing study of ASD to date phenotypic disruptions that result from mutations in diverse ASD- (35,584 individuals, 11,986 ASD cases). We also identified associated genes. These systems have offered valuable insights into numerous orthologs from SFARI Gene (https://gene.sfari.org/) the biological mechanisms underlying this heterogeneous group of “syndromic” and “high confidence” categories and the top 50 ASD- disorders (11–24). However, thousands of additional mutations in associated genes by variant count from our ongoing meta-analysis these and many other genes have been identified in individuals of rare variants, VariCarta (https://varicarta.msl.ubc.ca/index) (38). with ASD, and their roles as causative agents, or their pathoge- Of note, 72% (18/25) of genes in the SFARI high confidence nicity, remain ambiguous. Thus, there are 2 major challenges category and 78% (80/102) of genes associated with ASD by facing ASD genetics: (1) the large, growing number of candidate Satterstrom et al. (8) have a clear C. elegans ortholog according to risk genes with poorly characterized biological functions and (2) OrthoList2 and the Alliance of Genome Resources, compendiums the inability to predict the functional consequences of the large of human–C. elegans orthology prediction algorithm results. This is number of rare missense variants. Difficulties in rare missense substantially higher than the 53% estimated genome-wide orthol- variant interpretation stem in part from constraints on computa- ogy between humans and C. elegans (10,678/23,010) (32), sug- tional variant effect prediction and a paucity of in vivo experi- gesting an exceptionally high conservation of biological processes mental variant functional assays (25, 26). This lag between gene core to ASD pathology (SI Appendix,Fig.S1B and C). discovery and functional characterization is even more pro- Rapid advances in gene discovery and orthology prediction nounced when assessing the role of putative ASD risk genes and have altered the gene lists used during the course of this
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