Abstract Aims/hypothesis Wolfram Syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes for Wolframin, a master regulator of several cellular responses, whose mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame ones. As achieving a genotype-phenotype correlation is crucial to deal with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1 gene: c.316-1G>A, affecting the acceptor splice site (ASS) upstream exon 4, and c.757A>T, introducing a premature termination codon (PTC) in exon 7. Methods Bioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNAseq and PCR to validate the transcriptional landscape. Patient-derived induced Pluripotent Stem Cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms into pancreatic β cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the ER-stress induction and immunotoxicity assays. Results We showed that patient-derived iPSCs retained the ability to differentiate into pancreatic β cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two ORF-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum (ER)-stress response in β cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and pro-inflammatory cytokines affected the NMD-related gene SMG7 expression levels (>2 fold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 β cells, thus resulting in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001) and predisposing affected β cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived β cells. Conclusions/interpretation This study represents a new model to study Wolframin, highlighting how each single mutation of WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 β cells to stress and inflammation, and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease.
