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PLoS One. 2019 Mar 28;14(3):e0214198. doi: 10.1371/journal.pone.0214198. eCollection 2019.

Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment.

Author information

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 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 Unit, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
8
Department of Biochemistry and Molecular Medicine, George Washington University School of Medicine and Health Sciences, Washington, DC, United States of America.

Abstract

BACKGROUND:

The mechanisms underlying autism spectrum disorder (ASD) remain unclear, and clinical biomarkers are not yet available for ASD. Differences in dysregulated proteins in ASD have 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 identifying candidate genes that are dysregulated in ASD subgroups. However, this strategy has not been employed in proteome profiling analyses to identify ASD biomarker proteins for specific subgroups.

METHODS:

We therefore conducted a cluster analysis of the Autism Diagnostic Interview-Revised (ADI-R) scores from 85 individuals with ASD to predict subgroups and subsequently identified dysregulated genes by reanalyzing the transcriptome profiles of individuals with ASD and unaffected individuals. Proteome profiling of lymphoblastoid cell lines from these individuals was performed via 2D-gel electrophoresis, and then mass spectrometry. Disrupted proteins were identified and compared to the dysregulated transcripts and reported dysregulated proteins from previous proteome studies. Biological functions were predicted using the Ingenuity Pathway Analysis (IPA) program. Selected proteins were also analyzed by Western blotting.

RESULTS:

The cluster analysis of ADI-R data revealed four ASD subgroups, including ASD with severe language impairment, and transcriptome profiling identified dysregulated genes in each subgroup. Screening via proteome analysis revealed 82 altered proteins in the ASD subgroup with severe language impairment. Eighteen of these proteins were further identified by nano-LC-MS/MS. Among these proteins, fourteen were predicted by IPA to be associated with neurological functions and inflammation. Among these proteins, diazepam-binding inhibitor (DBI) protein was confirmed by Western blot analysis to be expressed at significantly decreased levels in the ASD subgroup with severe language impairment, and the DBI expression levels were correlated with the scores of several ADI-R items.

CONCLUSIONS:

By subgrouping individuals with ASD based on clinical phenotypes, and then performing an integrated transcriptome-proteome analysis, we identified DBI as a novel candidate protein for ASD with severe language impairment. The mechanisms of this protein and its potential use as an ASD biomarker warrant further study.

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