Imprinted DLK1-DIO3 region of 14q32 defines a schizophrenia-associated miRNA signature in peripheral blood mononuclear cells

Abstract

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level and are important for coordinating nervous system development and neuronal function in the mature brain. We have recently identified schizophrenia-associated alteration of cortical miRNA biogenesis and expression in post-mortem brain tissue with implications for the dysregulation of schizophrenia candidate genes. Although these changes were observed in the central nervous system, it is plausible that schizophrenia-associated miRNA expression signatures may also be detected in non-neural tissue. To explore this possibility, we investigated the miRNA expression profile of peripheral blood mononuclear cells (PBMCs) from 112 patients with schizophrenia and 76 non-psychiatric controls. miRNA expression analysis of total RNA conducted using commercial miRNA arrays revealed that 33 miRNAs were significantly downregulated after correction for multiple testing with a false discovery rate (FDR) of 0%, which increased to 83 when we considered miRNA with an FDR<5%. Seven miRNAs altered in microarray analysis of schizophrenia were also confirmed to be downregulated by quantitative real-time reverse transcription-polymerase chain reaction. A large subgroup consisting of 17 downregulated miRNAs is transcribed from a single imprinted locus at the maternally expressed DLK1-DIO3 region on chromosome 14q32. This pattern of differentially expressed miRNA in PBMCs may be indicative of significant underlying genetic or epigenetic alteration associated with schizophrenia.

Figure 1

Schizophrenia associated microRNA (miRNA) expression in peripheral blood mononuclear cells (PBMCs). (a) Significance analysis of microarray (SAM) plot of differentially expressed miRNA in PBMCs from participants with schizophrenia (n=112) compared with non-psychiatric controls (n=76). The central solid blue line indicates equal expression and the upper and lower dashed black lines indicate levels for significantly altered expression (false discovery rate (FDR) of 0%). (b) Quantitative real-time reverse transcription polymerase chain reaction (Q-PCR) validation of miRNA significantly downregulated by miRNA array analysis. Bars indicate mean fold change±s.e.m. of 91 samples (57 schizophrenia cases and 34 non-psychiatric controls). The control cohort is set at 1. ^*P<0.05; ^**P<0.01; ^***P<0.001 by Mann–Whitney U-test.

Figure 2

Heatmap showing hierarchical clustering of significantly downregulated microRNA with diagnosis. Samples are color coded according to diagnosis (blue=control; yellow=schizophrenia). Green indicates low expression and red indicates high expression (Java Treeview).

Figure 3

Genomic organization of 14q32 microRNA (miRNA) clusters neighboring the DLK1-DIO3 imprinted domain. DLK1, RTL1 and DIO3 (blue rectangles) are paternally expressed genes. The maternally expressed non-coding RNA genes (pink) include maternally expressed gene 3 (MEG3), anti-RTL1 (encodes the smaller miRNA cluster A), maternally expressed gene 8 (MEG8) (encodes small nucleolar RNA (snoRNA) cluster) and the larger miRNA cluster B. The intergenic germline derived differentially methylated region (IG-DMR) is methylated on the paternal chromosome denoted by the encircled letter m. The 14q32 miRNA clusters are arranged in two segments: each separated by a C/D snoRNA cluster. miRNA in dark green italic font were significantly downregulated in schizophrenia and miRNA in blue exhibited a trend for downregulation in schizophrenia. miRNA in black were expressed but not differentially expressed, and in gray were not expressed. This figure was adapted from Royo and Cavaille and Hagan et al.