Functional Variant Rs2270363 on 16p13.3 Confers Schizophrenia Risk by Regulating NMRAL1

Recent genome-wide association studies have reported multiple schizophrenia risk loci, yet the functional variants and their roles in schizophrenia remain to be characterized. Here we identify a functional single nucleotide polymorphism (rs2270363: G>A) at the schizophrenia risk locus 16p13.3. rs2270363 lies in the E-box element of the promoter of NMRAL1 and disrupts binding of the basic helix-loop-helix leucine zipper family proteins, including USF1, MAX and MXI1. We validated the regulatory effects of rs2270363 using reporter gene assays and electrophoretic mobility shift assay. Besides, expression quantitative trait loci analysis showed that the risk allele (A) of rs2270363 was significantly associated with elevated NMRAL1 expression in the human brain. Transcription factors knockdown and CRISPR-Cas9-mediated editing further confirmed the regulatory effects of the genomic region containing rs2270363 on NMRAL1. Intriguingly, NMRAL1 was significantly downregulated in the brain of schizophrenia patients compared with healthy subjects, and knockdown of Nmral1 expression affected proliferation and differentiation of mouse neural stem cells, as well as genes and pathways associated with brain development and synaptic transmission. Of note, Nmral1 knockdown resulted in significant decrease of dendritic spine density, revealing the potential pathophysiological mechanisms of NMRAL1 in schizophrenia. Finally, we independently confirmed the association between rs2270363 and schizophrenia in the Chinese population and found that the risk allele of rs2270363 was the same in European and Chinese populations. These lines of evidence suggest that rs2270363 may confer schizophrenia risk by regulating NMRAL1, a gene whose expression dysregulation might be involved in the pathogenesis of schizophrenia by affecting neurodevelopment and synaptic plasticity.