Molecular Psychiatry (2015) 20, 472–481 © 2015 Macmillan Publishers Limited All rights reserved 1359-4184/15 www.nature.com/mp ORIGINAL ARTICLE MIR137 variants identified in psychiatric patients affect synaptogenesis and neuronal transmission gene sets M Strazisar1,2,10, S Cammaerts1,2,10, K van der Ven1,2,11, DA Forero1,2,3, A-S Lenaerts1,2,12, A Nordin4, L Almeida-Souza2,5, G Genovese6,7,8, V Timmerman2,5, A Liekens1,2, P De Rijk1,2, R Adolfsson4, P Callaerts9 and J Del-Favero1,2,11 Sequence analysis of 13 microRNA (miRNA) genes expressed in the human brain and located in genomic regions associated with schizophrenia and/or bipolar disorder, in a northern Swedish patient/control population, resulted in the discovery of two functional variants in the MIR137 gene. On the basis of their location and the allele frequency differences between patients and controls, we explored the hypothesis that the discovered variants impact the expression of the mature miRNA and consequently influence global mRNA expression affecting normal brain functioning. Using neuronal-like SH-SY5Y cells, we demonstrated significantly reduced mature miR-137 levels in the cells expressing the variant miRNA gene. Subsequent transcriptome analysis showed that the reduction in miR-137 expression led to the deregulation of gene sets involved in synaptogenesis and neuronal transmission, all implicated in psychiatric disorders. Our functional findings add to the growing data, which implicate that miR-137 has an important role in the etiology of psychiatric disorders and emphasizes its involvement in nervous system development and proper synaptic function. Molecular Psychiatry (2015) 20, 472–481; doi:10.1038/mp.2014.53; published online 3 June 2014 INTRODUCTION association studies and genome scans provided an indication of Schizophrenia (SZ) and bipolar disorder (BD) are two separate additional miRNAs being relevant for both etiologies on the basis – entities, but the presence of coinciding symptoms, frequent of their genomic location.18 21 co-occurrence of both disorders in affected families, similar Variants in the miRNA seed sequence can alter its target epidemiological risks in relatives and overlapping genetic findings spectrum, but the importance of the correct hairpin structure, suggest that they could have at least a partially uniform genetic thermodynamic influences on strand loading and base-pairing base.1,2 The polygenic nature of both disorders, missing reprodu- requirements outside the seed sequence imply that variants cibility in genetics research and often conflicting results originat- located outside the seed region can also influence miRNA ing from a plentitude of meta-analyses, linkage and association biogenesis and/or targeting and therefore are, as such, important studies shows that the search for complex disorder genes is far factors influencing miRNA function.22–24 miRNA profiling using from trivial. The realization that only a low percent of the genome massively parallel sequencing provided an insight into the hetero- codes for proteins and that nonprotein coding genes have an 3,4 geneity in length and sequence of mature miRNAs originating important but poorly understood role in gene regulation, from the same gene (isomiRs).25–27 The genesis of isomiRs can be initiated the interest in the nonprotein coding DNA. MicroRNA caused by different biological phenomena, including imprecise genes (miRNAs) regulate a variety of processes in eukaryotes by cleavage by Drosha or Dicer, which may be influenced by genetic mediating mRNA cleavage, translational repression and transcrip- 28 tional or translational activation.5–9 Strong evidence for the variants. Whether the presence of isomiRs is a regulated process involvement of miRNAs in SZ and/or BD emerged from several and whether isomiRs have a functional role in the regulation is a fi separate observations. First, the identification of brain-expressed matter of a discussion in the scienti c community, but reports miRNAs revealed specific miRNA subsets, involved in normal brain show that at least some of the isomiRs can affect miRNA stability, function.10–13 Second, the studies exploring the importance of target spectra and efficiency of incorporation into the RISC 28–30 miRNA regulation in neurological processes and studies of miRNA complex, when compared with the canonical miRNA. expression signatures in different neuropsychiatric disorders On the basis of expression, association, linkage and functional discerned possible implication of specific miRNAs in SZ and studies, 13 miRNA genes were selected for variant screening that BD.14–17 Third, the combination of the increasing number of impact mature miRNA levels. With this study we provide com- miRNA genes being identified, and data from previously published pelling evidence that variants in the MIR137 gene, observed in BD 1Applied Molecular Genomics Unit, Department of Molecular Genetics, VIB, Antwerp, Belgium; 2University of Antwerp, Antwerp, Belgium; 3Biomedical Sciences Research Group, School of Medicine, Universidad Antonio Nariño, Bogotá, Colombia; 4Department of Clinical Sciences, Division of Psychiatry, Umeå University, Umeå, Sweden; 5Peripheral Neuropathy Group, Department of Molecular Genetics, VIB, Antwerp, Belgium; 6Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; 7Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; 8Department of Genetics, Harvard Medical School, Boston, MA, USA and 9VIB Laboratory of Behavioral and Developmental Genetics, KU Leuven, Leuven, Belgium. Correspondence: Professor Dr J Del-Favero, Applied Molecular Genomics Unit, Department of Molecular Genetics, VIB, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail: [email protected] 10These authors contributed equally to this work. 11Multiplicom, Niel, Belgium. 12BRC hiPSC Core Facility, University of Cambridge, Cambridge, UK. Received 7 October 2013; revised 24 April 2014; accepted 28 April 2014; published online 3 June 2014 Reduced miR-137 deregulates synaptic gene sets M Strazisar et al 473 and SZ patients, reduce the levels of the mature miR-137 and catalog number CRL-1573) to test the expression differences in cells known affect gene sets involved in synapse formation and function. not to express detectable levels of miR-137. MATERIALS AND METHODS RNA isolation Total RNA from untransduced replicates, stably transduced SH-SY5Y and Subjects HEK replicates; for each of the variants, was extracted with the mirVana The association sample, recruited from the Västerbotten region in Northern miRNA Isolation Kit (Life Technologies) according to the manufacturer’s Sweden, included 345 BD patients, 426 SZ patients and 1376 healthy protocol. Aliquots of total RNA were subsequently used for miRNA individuals, all unrelated. The patients were clinically characterized by expression analysis, transduction efficiency normalization and transcrip- trained research nurses and research psychiatrists using register data, data tome profiling. Additional information on RNA isolation is provided in from psychiatric records and semi-structured interviews. Additional Supplementary File S1. information of the selection is provided in Supplementary File S1. The study was approved by the Medical Ethical Committees of the Expression of miR-137 and EGFP universities of Umeå and Antwerp. MiR-137 and EGFP expression was analyzed on ABI Prism 7900 HT Sequence Detection System with either TaqMan microRNA assays or SYBR Gene selection green (Life Technologies). Thirteen miRNA genes were selected on the basis of two criteria: (1) RT–qPCR (quantitative PCR with reverse transcription) reactions were expression in the human brain and (2) location in genomic regions performed on technical triplicates for each biological replicate (wild-type previously associated or linked with BD and/or SZ. As a result of the and variant transduced cells) and for each assay. Reverse transcription for literature mining MIR34A, MIR99B, MIR103A1, MIR103B1, MIR128-1, MIR132, miRNA expression was performed according to the manufacturer’s MIR135B, MIR137, MIR301A, MIR448, MIR764, MIRLET7E and MIRLET7A2 protocol (Life Technologies) with reverse transcription primers for were selected for Sanger sequencing-based variant discovery. The miR-137 (mmu-miR-137 assay, ID 001129) and two endogenous controls references of the published data used for the selection are provided in miR-16 (hsa-miR-16 assay, ID 00391) and RNU24 (RNU24 assay, ID 001001). Supplementary File S1. To determine transduction efficiency we used relative EGFP quantification as transduction efficiency factor. The reaction was done using SYBR Green I mix (Life Technologies) and appropriate primers in final concentrations of Sanger sequencing-based variant discovery 0.3 μM per reaction. The primers used for relative quantification are listed in The DNA sequences containing 500 bases flanking the 3′ and 5′ regions of Supplementary File S1. selected miRNA stem-loop sequences were downloaded from the UCSC The RT–qPCR data were normalized by geometric averaging of multiple genome browser (February 2009, GRCh37/hg19 assembly). The PCR and internal control genes.36 Processing of raw data and calculation of norma- 31 sequencing primers were designed using Primer3 software. lized relative quantities were done with ddCt-method (ΔΔCt) including the The 13 selected miRNA genes were PCR amplified and sequenced from amplification efficiencies of all primer