Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes

Coding mutations can cause neurodevelopmental disorders (NDDs), including autism. Yet, predicting which non-coding (e.g., 5' untranslated region [UTR]) mutations are functional is challenging. We tested assays of various throughput for the assessment of 997 mutations from NDD families. A massively parallel reporter assay (MPRA) using polysomes from cell lines identified >100 altering translation, with a subset subsequently altering endogenous protein production in patient lymphoblastoid cell lines. Next, since UTR function varies by cell type, we optimized Cre-dependent MPRAs, enabling assessment in neurons in vivo. We demonstrate that neurons have different principles of regulation by 5' UTRs and discover mutations altering translational activity. Finally, we tested whether polysome-MPRAs predict changes in canonical open reading frame (ORF) protein production. Only for mutations altering UTR structure was there a reasonable correlation. Overall, we benchmarked a variety of approaches for assessing impacts of 5' UTR mutation and identified functional 5' UTR mutations from known NDD genes, including LRRC4 and ZNF644.