Yoshikawa et al. Translational Psychiatry (2021) 11:331 https://doi.org/10.1038/s41398-021-01460-1 Translational Psychiatry ARTICLE Open Access Dysregulation of post-transcriptional modification by copy number variable microRNAs in schizophrenia with enhanced glycation stress Akane Yoshikawa1,2,ItaruKushima 3,4, Mitsuhiro Miyashita 1,5,6,KazuyaToriumi 1, Kazuhiro Suzuki 1,5, Yasue Horiuchi 1, Hideya Kawaji 7, Shunya Takizawa8,NorioOzaki 3, Masanari Itokawa1 and Makoto Arai 1 Abstract Previously, we identified a subpopulation of schizophrenia (SCZ) showing increased levels of plasma pentosidine, a marker of glycation and oxidative stress. However, its causative genetic factors remain largely unknown. Recently, it has been suggested that dysregulated posttranslational modification by copy number variable microRNAs (CNV-miRNAs) may contribute to the etiology of SCZ. Here, an integrative genome-wide CNV-miRNA analysis was performed to investigate the etiology of SCZ with accumulated plasma pentosidine (PEN-SCZ). The number of CNV-miRNAs and the gene ontology (GO) in the context of miRNAs within CNVs were compared between PEN-SCZ and non-PEN-SCZ groups. Gene set enrichment analysis of miRNA target genes was further performed to evaluate the pathways affected in PEN-SCZ. We show that miRNAs were significantly enriched within CNVs in the PEN-SCZ versus non-PEN-SCZ groups (p = 0.032). Of note, as per GO analysis, the dysregulated neurodevelopmental events in the two groups may have different origins. Additionally, gene set enrichment analysis of miRNA target genes revealed that miRNAs involved in glycation/oxidative stress and synaptic neurotransmission, especially glutamate/GABA receptor signaling, fi 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; were possibly affected in PEN-SCZ. To the best of our knowledge, this is the rst genome-wide CNV-miRNA study suggesting the role of CNV-miRNAs in the etiology of PEN-SCZ, through effects on genes related to glycation/oxidative stress and synaptic function. Our findings provide supportive evidence that glycation/oxidative stress possibly caused by genetic defects related to the posttranscriptional modification may lead to synaptic dysfunction. Therefore, targeting miRNAs may be one of the promising approaches for the treatment of PEN-SCZ. Introduction educational achievement, leading to lifelong social and/or Schizophrenia (SCZ) is a neurodevelopmental disorder occupational dysfunction. Accumulating evidence sug- with a worldwide prevalence of around 1.0%, leading to gests that both genetic and epigenetic factors are involved – abnormalities in function and connectivity of key brain in the etiology of SCZ2 7. An integrative genetic and regions1. The most common symptoms are auditory epigenetic analysis would accelerate the elucidation of the hallucinations, delusions, withdrawal, flat affect, lack of etiology. motivation, and disorganized thinking. The onset, during Recent advances in genomic technologies have facili- early adolescence, strongly impacts school life and tated large-scale genomic studies. Previous research has successfully uncovered the polygenic nature of multiple common risk variants4,5,8 as well as the involvement of Correspondence: Makoto Arai ([email protected]) 9,10 1Schizophrenia Research Project, Department of Psychiatry and Behavioral rare but highly penetrant variants , and structural Sciences, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, genetic variants called copy number variations (CNVs) – Setagaya-ku 156-8506 Tokyo, Japan in SCZ11 15. In the largest-scale CNV study to date, 2Department of Adolescent Psychiatry, Nihonbashi Sun Clinic, 2-1-21, Nihonbashi, Chuou-ku, Tokyo 103-0027, Japan using 41,321 subjects, deletions in 1q21.1, 2p16.3, 3q29, Full list of author information is available at the end of the article © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Yoshikawa et al. Translational Psychiatry (2021) 11:331 Page 2 of 11 and 22q11.21 were confirmed as risk factors for SCZ Science Institute near Tokyo, for both genetic and bio- with high odds ratios (ORs) ranging from 3.8–67.714. chemical analyses. All subjects were ethnically Japanese. The Psychiatric Genomics Consortium (PGC)-CNV The patients were diagnosed according to the DSM-5 analysis group has demonstrated that genes disrupted by criteria (American Psychiatric Association) for SCZ or CNVs in SCZ are enriched within the synaptic net- schizoaffective disorders as per the consensus of at least works14. Kushima et al. performed the largest genome- two experienced psychiatrists. Patients with a history of wide CNV analysis in the Japanese population so far and drug addiction or alcohol abuse/dependence were confirmed an increased burden of rare exonic CNVs in excluded. Patients with accompanying diabetes mellitus 2458 SCZ and 1108 autism spectrum disorder (ASD) and chronic renal disease were also excluded because cases16. these diseases may affect the plasma pentosidine levels. MicroRNAs (miRNAs) are a family of short (~22 nt), Medical records were obtained with the approval of single-stranded, non-coding RNAs primarily involved in patients. The study was performed in accordance with the posttranscriptional downregulation of gene expres- the Declaration of Helsinki. This study was approved by sion via binding to the 3′UTR of target genes17.CNV- the research ethics committee of each participating miRNA genes generate the complexity of miRNA reg- institute and written informed consent was obtained ulation and function17,18. Genomic CNVs can induce the from all subjects. The subjects’ demographics are pre- aberrant expression of integral miRNAs and their target sented in Table 1. Of note, to define the PEN-SCZ and genes18, resulting in the dysregulation of posttransla- non-PEN-SCZ cases, plasma concentration of pentosi- tional modification, crucial for the adaptation to envir- dine was measured via high-performance liquid chro- onmental factors. Recently, it has been shown that the matography as described previously25. genetically dysregulated posttranslational modification caused by CNV-miRNAs contribute to the etiology of Identification of CNV-miRNAs neurodevelopmental disorders including SCZ, ASD, and Genomic DNA was extracted from blood samples. intellectual disability (ID)19. For example, in the context Array comparative genomic hybridization (array CGH) of SCZ, miRNA contents within CNV regions were sig- was performed to identify rare (<1%) CNVs in PEN-SCZ nificantly enriched compared to those in control sub- and non-PEN-SCZ patients; NimbleGen 720 K Whole- jects19.Additionally,inASD,miRNAspresentinCNV Genome Tiling Arrays (Roche NimbleGen, Madison, loci were shown to regulate synaptic transmission and WI, USA) were used for the CNV analysis. CNV calls may, thus, contribute to the disorder phenotype20. were conducted using the Nexus Copy Number software Genetic heterogeneity is a hallmark of SCZ; the genetic v9.0 (Bio Discovery, El Segundo, CA, USA) with the Fast architecture of this common disorder is largely com- Adaptive States Segmentation Technique 2 algorithm. plex21,22. To overcome the heterogeneity, the identifica- Quality control (QC) was performed as described pre- tion of SCZ subpopulations presenting with disturbances viously16,26.Briefly, QC scores for each sample based on of specific metabolic pathways is a promising strategy. We the statistical variance of the probe-to-probe log ratios previously reported an SCZ subtype with enhanced gly- were calculated and samples with QC >0.15 were cation/oxidative stress that showed accumulated plasma removed. As sample QC, subjects with excessive num- pentosidine (PEN-SCZ)23. Although around 20% of SCZ bers of autosomal CNVs were also removed from the patients were reported to be PEN-SCZ24, the genetic and analysis. After filtering out common CNVs (≥1%), rare epigenetic factors that account for the accumulation of CNVs (<1%) in 185 patients were used for further ana- pentosidine and their contribution to this phenotype have lyses. All genomic locations are given in GRCh38 not yet been fully elucidated. coordinates, and gene annotation was based on GEN- Here, we performed an integrative, genome-wide, CODE version 27. Of note, previously, we confirmed CNV-miRNA analysis to investigate the genetic factors that CNV calls from NimbleGen arrays are highly associated with SCZ with enhanced glycation/oxidative accurate with a validation rate >99%27.ThemiRNAs stress and to provide novel insights into the etiology of present in CNVs were then identified via the analysis of PEN-SCZ, focusing on the disturbance of post- the chromosomal coordinates as per the UCSC Genome transcriptional modification caused by CNV-miRNAs. Browser