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Phenotypic Subgrouping and Multi-Omics Analyses Reveal RESEARCH ARTICLE Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment Chatravee Pichitpunpong1, Surangrat Thongkorn2, Songphon Kanlayaprasit2, a1111111111 Wasana Yuwattana3, Waluga Plaingam4, Siriporn Sangsuthum5, Wan Mohd Aizat6, Syarul 6 7 8 7 a1111111111 Nataqain Baharum , Tewin Tencomnao , Valerie Wailin Hu , Tewarit SarachanaID * a1111111111 a1111111111 1 M.Sc. Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand, 2 PhD Program in Clinical a1111111111 Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand, 3 B.Sc. Program in Medical Technology, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand, 4 College of Oriental Medicine, Rangsit University, Pathum Thani, Thailand, 5 Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand, 6 Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia, 7 Age-related Inflammation and Degeneration Research OPEN ACCESS Unit, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Citation: Pichitpunpong C, Thongkorn S, Thailand, 8 Department of Biochemistry and Molecular Medicine, George Washington University School of Kanlayaprasit S, Yuwattana W, Plaingam W, Medicine and Health Sciences, Washington, DC, United States of America Sangsuthum S, et al. (2019) Phenotypic [email protected] subgrouping and multi-omics analyses reveal * reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment. PLoS ONE 14(3): e0214198. Abstract https://doi.org/10.1371/journal.pone.0214198 Editor: Jon M. Jacobs, Pacific Northwest National Laboratory, UNITED STATES Background Received: April 17, 2018 The mechanisms underlying autism spectrum disorder (ASD) remain unclear, and clinical Accepted: March 8, 2019 biomarkers are not yet available for ASD. Differences in dysregulated proteins in ASD have Published: March 28, 2019 shown little reproducibility, which is partly due to ASD heterogeneity. Recent studies have demonstrated that subgrouping ASD cases based on clinical phenotypes is useful for identi- Copyright: © 2019 Pichitpunpong et al. This is an open access article distributed under the terms of fying candidate genes that are dysregulated in ASD subgroups. However, this strategy has the Creative Commons Attribution License, which not been employed in proteome profiling analyses to identify ASD biomarker proteins for permits unrestricted use, distribution, and specific subgroups. reproduction in any medium, provided the original author and source are credited. Methods Data Availability Statement: All transcriptome profile data are available in the NCBI GEO DataSets We therefore conducted a cluster analysis of the Autism Diagnostic Interview-Revised (ADI- database under GSE numbers GSE15402, R) scores from 85 individuals with ASD to predict subgroups and subsequently identified GSE18123, GSE25507, GSE42133, and GSE6575. dysregulated genes by reanalyzing the transcriptome profiles of individuals with ASD and All ADI-R scores and demographic information were obtained from previous research available in unaffected individuals. Proteome profiling of lymphoblastoid cell lines from these individuals NCBI PubMed article number PMID 19455643. was performed via 2D-gel electrophoresis, and then mass spectrometry. Disrupted proteins Funding: This research was supported by the were identified and compared to the dysregulated transcripts and reported dysregulated Ratchadaphiseksomphot Endowment Fund of proteins from previous proteome studies. Biological functions were predicted using the PLOS ONE | https://doi.org/10.1371/journal.pone.0214198 March 28, 2019 1 / 30 Phenotypic subgrouping and multi-omics analyses of ASD with severe language impairment Chulalongkorn University (CU59-004-HR) granted Ingenuity Pathway Analysis (IPA) program. Selected proteins were also analyzed by West- to TS. CP, ST, and SK are graduate students in the ern blotting. M.Sc. and Ph.D. Programs in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Results Chulalongkorn University. CP and ST were The cluster analysis of ADI-R data revealed four ASD subgroups, including ASD with severe financially supported by the 90th Anniversary Chulalongkorn University Fund language impairment, and transcriptome profiling identified dysregulated genes in each sub- (Ratchadaphiseksomphot Endowment Fund: CP group. Screening via proteome analysis revealed 82 altered proteins in the ASD subgroup GCUGR1125601057M 53-3; ST with severe language impairment. Eighteen of these proteins were further identified by GCUGR1125601056M 53-2) and received nano-LC-MS/MS. Among these proteins, fourteen were predicted by IPA to be associated additional financial support from a ªScholarship from the Graduate School Chulalongkorn with neurological functions and inflammation. Among these proteins, diazepam-binding University to commemorate the 72nd anniversary inhibitor (DBI) protein was confirmed by Western blot analysis to be expressed at signifi- of His Majesty King Bhumibala Aduladejaº. CP was cantly decreased levels in the ASD subgroup with severe language impairment, and the DBI also financially supported by the expression levels were correlated with the scores of several ADI-R items. Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University (CU59-004-HR) as a research assistant. ST received ªThe Overseas Conclusions Research Experience Scholarship for Graduate By subgrouping individuals with ASD based on clinical phenotypes, and then performing an Studentsº from the Graduate School and Faculty of Allied Health Sciences, Chulalongkorn University, integrated transcriptome-proteome analysis, we identified DBI as a novel candidate protein and also received the Royal Golden Jubilee Ph.D. for ASD with severe language impairment. The mechanisms of this protein and its potential Programme Scholarship (Grant No. PHD/0029/ use as an ASD biomarker warrant further study. 2561) by Thailand Research Fund. SK was supported by The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship. VWH is supported by NIEHS grants R21 ES023061 and R21 ES028124. Competing interests: The authors declare that they Introduction have no competing interests. Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders thought to Abbreviations: 2D-PAGE, Two-Dimensional result from both genetic and environmental factors [1±4]. The Centers for Disease Control Polyacrylamide Gel Electrophoresis; ACBP, Acyl- and Prevention (CDC) has reported that the prevalence of ASD is approximately 1 in 59 CoA-Binding Protein; aCGH, Array Comparative children in the United States [5]. According to the Diagnostic and Statistical Manual of Genomic Hybridization; ACN, Acetonitrile; ADI-R, Mental DisordersÐFifth Edition (DSM-5), ASD is characterized by (i) impairments in Autism Diagnostic Interview, Revised; AGRE, Autism Genetic Resource Exchange; AHSG, Alpha- social interaction and communication and (ii) repetitive behaviors and restricted interests, 2-HS-glycoprotein; ANXA5, Annexin A5; ASD, without further subclassification into specific ASD subtypes [6]. However, there is a high Autism Spectrum Disorder; ApoA1, Apolipoprotein degree of variation in ASD symptoms and severity. Some individuals with ASD exhibit A1; ApoA4, Apolipoprotein A4; ATP, Adenosine severe language deficits, including a lack of verbal communication, whereas many others Triphosphate; BCKDH, Branched Chain 2-Oxoacid show little impairment in language and are capable of communication with others [7]. Such Dehydrogenase; BSA, Bovine Serum Albumin; BZD, Benzodiazepine; CALM1, Calmodulin-1; CCT5, heterogeneity in the autism spectrum is thought to result from the combination of multiple T-Complex Protein 1 Subunit Epsilon; CEP290, molecular mechanisms and environmental factors, leading to the pathobiology specific to Centrosomal Protein 290; CDC, Centers for Disease the clinical phenotypes of each ASD subpopulation. There is evidence that dividing individ- Control and Prevention; CHAPS, 3-[(3- uals with ASD into subgroups is meaningful for identifying gene/protein candidates and Cholamidopropyl)Dimethylammonio]-1- molecular mechanisms that are uniquely associated with each ASD subpopulation and Propanesulfonate; CLTA, Clathrin light chain A; shared among subpopulations, which may be useful for ASD diagnosis and/or treatment in CNS, Central Nervous System; CNV, Copy Number Variation; COX5A, Cytochrome c oxidase subunit the future [8±17]. However, clinical biomarkers are not available for ASD diagnosis or the 5A; DBI, Diazepam-Binding Inhibitor; DEGs, specific differentiation of severity. Accurate predictive clinical biomarkers for the diagnosis Differentially Expressed Genes; DLD, Dihydrolipoyl and confirmation of the disorder, as well as for ASD subtyping, are still needed. Dehydrogenase; DSM-5, The Diagnostic and Several studies involving transcriptome profiling analysis of lymphoblastoid cell lines Statistical Manual of Mental Disorders, Fifth (LCLs) derived from patients with ASD and unaffected controls
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