M58 - FUNCTIONAL ANALYSIS OF THE SCHIZOPHRENIA ASSOCIATED GENE AS3MT IN SH-SY5Y NEUROBLASTOMA CELLS

Abstract

Background Schizophrenia is a neuropsychiatric disorder with a prevalence of 1%, characterised by episodes of psychosis and an alteration in cognitive function. The aetiology of Schizophrenia is still largely unknown but evidence suggests an underlying neurodevelopmental aspect despite onset occurring in adulthood as well as a considerable genetic burden. Recently 108 genomic loci have robustly been associated with Schizophrenia. However, of these identified genes, very few have been characterised for their role in brain development. Arsenite Methyltransferase (AS3MT) is located in the10q24.32 GWAS locus, which is the most statistically significant locus outside the Major Histocompatibility Complex (MHC) region associated with schizophrenia. Furthermore, both mQTLs and expression quantitative trait loci (eQTL) map to this gene and a recent publication has identified an alternative splice variant which is increased in schizophrenia brain. AS3MT encodes for a methyltransferase involved in arsenic metabolism, however the role of AS3MT in brain development has not been explored. Methods CRISPR-Cas9 technology was used to create a knockout cell line of AS3MT in SH-SY5Y neuroblastoma cells. Two guide RNAs (gRNA) were designed, one to cut in exon 4 and one in exon 6 of the AS3MT gene, where the methyltransferase domain is located. These guides were cloned into two expression vectors which express the gRNA, Cas9 enzyme and either EGFP or mCherry. These vectors were transfected into P11 SH-SY5Y (neuroblastoma) cell line and screened for double fluorescence and sorted into single cells by Fluorescent Activated Cell Sorting after 24 hours. These cells were allowed to clonally expand for 3 weeks before DNA was extracted for genotyping by PCR. Protein expression of AS3MT was measured by western blotting, gene expression by RT-PCR and DNA methylation by the Ilumina EPIC array. Immunocytochemistry techniques will be used to identify any alterations in cell morphology. Results Of the cells transfected with EGPF and mCherry expressing CRISPR-Cas9 vectors 15.5% of the single sorted cells expressed both EGPF and mCherry and were sorted into 96 well plates. Following a week of clonal expansion approximately 38% of wells contained colonies, where 41% un-transfected control sorted cells had colonies. Following genotyping 27.8% (5/18) were identified as heterozygous mutants, 16.7% (3/18) were homozygous mutants and 55.6% (10/18) were wild type. Discussion We have developed a robust protocol for creating deletion mutants using CRISPR-Cas9 technology and a functional domain of AS3MT has been deleted in the neuroblastoma cell line SH-SY5Y using CRISPR-Cas9 technology, further work is ongoing to characterise the molecular consequences of this mutation.