Functional consequences of ITPase deficiency and the impact of inosine misincorporation into the transcriptome

Project Abstract Bi-allelic loss of function variants in the ITPA gene cause a fatal infantile multisystem developmental disorder characterized by epileptic encephalopathy and dilated cardiomyopathy. ITPA encodes the inosine triphosphate pyrophosphatase (ITPase) enzyme which hydrolyzes inosine triphosphate (ITP) and deoxyITP to prevent their accumulation in cellular nucleotide pools and subsequent misincorporation during RNA and DNA synthesis. Although the biochemical activity of ITPase is well understood, the molecular and cellular mechanisms underlying the pathology resulting from ITPase deficiency are unknown. ITPase-deficient patient cells, mouse embryonic stem cells and tissues contain high levels of misincorporated inosine within bulk RNA but deoxyinosine is undetectable within DNA, due to active DNA repair processes. To investigate the functional consequences of ITPase deficiency and inosine misincorporation into the transcriptome; Aim 1) Illumina RNA sequencing (RNA-seq) and Oxford Nanopore Long-Read sequencing will be performed on CRISPR/Cas9-derived ITPA-null human induced pluripotent stem cells (iPSCs), pre- and post-differentiation into neurons and cardiomyocytes to evaluate transcriptome deregulation (transcript abundance, splicing and polyadenylation) and novel transcript isoforms. In parallel, inosine accumulation as a direct driver of transcriptome dysregulation will be tested by culturing ITPA-null cells with inosine nucleoside in the culture media. The resulting inosine misincorporation into total RNA will be measured using mass spectrometry prior to RNA-seq and long read sequencing. Aim 2) The impact of ITPase deficiency and inosine misincorporation into RNA on translation will be determined. Ribosome profiling (Ribo-Seq) of ITPA-null neurons and cardiomyocytes will be performed under basal and inosine nucleoside treatment conditions to determine translation efficiency and to pinpoint mRNA targets of altered translation. Inosine misincorporation into rRNA, tRNA and mRNA will be quantified using mass spectrometry and potential activation of cellular stress markers will be investigated. The proposed work will identify transcriptome and translation alterations resulting from ITPase deficiency. The findings of this research will aid in elucidating the unknown pathogenic mechanism(s) underlying ITPase-deficiency and may reveal novel therapeutic targets for treating this rare but fatal disorder. The impact of this work extends to other disorders of nucleotide pool maintenance and may shed new light on our understanding of inosine in A-to-I RNA editing and gene expression during development and disease from a unique perspective.