Stephen P Plassmeyer, Colin P Florian, Rebecca Chase, Michael J Kasper, Shayna Mueller et al.
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.
Stephen P Plassmeyer, Colin P Florian, Rebecca Chase, Michael J Kasper, Shayna Mueller, Yating Liu, Kelli McFarland White, Llaelyn Sierra-Cortez, Anthony D Fischer, Courtney F Jungers, Slavica Pavlovic Djuranovic, Sergej Djuranovic, Joseph D Dougherty. Approaches for identification of 5′ UTR mutations impacting translation and protein production from neurodevelopmental disorder genes. 2025-12-03. https://doi.org/10.1016/j.crmeth.2025.101247
An episomal plasmid library containing 1,507 unique 5′ UTR sequence-context reporters representing 997 neurodevelopmental-disorder-family mutations was transiently transfected into human HEK293 cells. Each reference and alternate allele was represented by ten barcodes, and DNA, total RNA, 40S, 80S, polysome, TRAP, and Maxi-library counts were collected across six biological replicates.
Three AAV9 sublibraries, each containing approximately 500 reference/alternate 5′ UTR allele pairs, were delivered transcranially to neonatal mouse cortex and assayed at postnatal day 21. A Cre-dependent reverse-transcription switch separated Vglut2-positive glutamatergic-neuron (CreON) measurements from non-Cre-expressing transduced cells (CreOFF), with DNA, input RNA, monosome, and polysome readouts across six animals per sublibrary.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 11 files (ZIP)GSE246381_hek_combined_umi_counts.csv.gzGSE246381_series.geo.txtGSE246381_vglut_combined_umi_counts.csv.gzHEK_count_QC_summary.csvHEK_sample_index_labels.csvREADME.txtTable_S1_variant_annotation_library_design.xlsxTable_S2_HEK_MPRA_allelic_effects.xlsxTable_S4_LCL_followup.xlsxTable_S5_in_vivo_MPRA_allelic_and_Cre_effects.xlsxVglut_count_QC_summary.csv