A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on beta cell apoptosis in a patient with Wolfram syndrome

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 wolframin, a master regulator of several cellular responses, and the gene's mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame variants. As achieving a genotype-phenotype correlation is crucial for dealing 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: c.316-1G>A, affecting the acceptor splice site (ASS) upstream of 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 RNA-seq 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 in pancreatic beta cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the endoplasmic reticulum stress induction and immunotoxicity assays.

Results: We showed that patient-derived iPSCs retained the ability to differentiate into pancreatic beta 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 open reading frame-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 stress response in beta cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense-mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and proinflammatory cytokines affected expression levels of the NMD-related gene SMG7 (>twofold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 beta cells. This resulted 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), predisposing affected beta cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived beta cells.

Conclusions/interpretation: This study represents a new model to study wolframin, highlighting how each single mutation of the WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 beta 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.

Data availability: The in silico predicted N-terminal domain structure file of WT wolframin was deposited in the ModelArchive, together with procedures, ramachandran plots, inter-residue distance deviation and IDDT scores, and Gromacs configuration files (doi/10.5452/ma-cg3qd). The deep-sequencing data as fastq files used to generate consensus sequences of AS isoforms of WFS1 are available in the SRA database (BioProject PRJNA1109747).

Keywords: Cell stress; IPSCs; Inflammation; Nonsense-mediated decay; Wolfram syndrome; Wolframin.

Fig. 1

(a, b) Sanger sequencing chromatograms showing the heterozygous c.316-1G>A transition and the c.757A>T transversion in the WFS1 gene of the patient. The modified nucleotides are highlighted in blue and red, respectively, as well as the resulting amino-acid substitution in the protein sequence, if known. (c) Genetic pedigree of the patient’s family. The first line below each symbol represents generation and identification number. The heterozygous mutations found in the patient’s father and mother are reported; the patient’s brother carries no mutated allele. (d) Schematic representation of WFS1 gene and wolframin protein. The exons 1 (non-coding, grey boxes), 2–7 (encoding the N-terminal domain of wolframin, green boxes) and 8 (encoding the transmembrane and C-terminal domains of wolframin, orange box) are reported. The genomic position of the patient’s WFS1 mutations are highlighted and colour-coded as in (a, b). The previously reported ASS variants are reported as well. The N-terminal domain (green) and transmembrane/C-terminal domains (orange) are shown along with the c.757A>T transversion introducing a premature stop codon

Fig. 2

(a) Genomic positions of the natural and cryptic donor (green) and acceptor (red) splice sites along WFS1 exon 4 (145 bp) and its flanking regions (150 bp/each) as predicted by Human Splice Finder. Only the splice sites with an HSF score >75 and a MaxEnt score >4 are plotted. (b) ESR calculated as a ratio between auxiliary splicing signals (ESE and ESS) along the same genomic region. The binding sites for the splicing factors SC35 (dark blue), SRp40 (purple) and 9G8 (light blue) are noted for the region of interest surrounding the cryptic ASSs inside exon 4. (c) Position of the natural and alternative branch points (light blue) along the analysed genomic region. Only the branch points with a BP score >65 are plotted. The coordinates indicate the base number counting from the p-arm telomere of chromosome 4 (according to the UCSC Genome Browser on Human GRCh38/hg38 Assembly). (d) Results of maximum entropy modelling in the presence of the WT ASS and after the c.316-1G>A transition. The two most likely alternatives upon the canonical splice site disruption are shown. ASSs scored by the model are underlined and highlighted in yellow. Exonic bases are in upper case letters; intronic bases in lower case letters. The grey area represents the spliced-out sequence. Coordinates refer to the nucleotide position in the coding sequence

Fig. 3

(a) Schematic representation of PCR strategy for the amplification of the WFS1 cDNA among exons 3, 4 and 5. Coloured arrows show the mapping of the four primers along the WFS1 gene sequence. The red cross indicates the mutated canonical ASS. The theoretical length of PCR products obtained by performing amplification with different combinations of the indicated primers is shown. (b) Representative PCR results for the indicated primer pairs, performed on WT and five patient-derived clones. The yellow rectangle highlights the ~180 bp-long amplicon obtained with the Ex3fw-Ex5rev primer pair. (c) Electropherograms from Sanger sequencing of WFS1-derived PCR products. (d) Agarose gel electrophoresis of amplification with Ex3fw-Ex5rev from WT donor- and WFS1 patient-derived PBMCs. The additional amplicons, including the 180 bp-long amplicon observed in WFS1 iPSCs, are indicated by red triangles. (e) High-throughput evaluation of RNA-seq data quality and goodness of alignment: the sequence quality graph reports the mean quality value (Phred score) across each base position in the R1 (green line) and R2 (orange line) reads. (f) STAR alignment score, reporting the number of mapped/unmapped reads per sample. (g) Per sequence quality score graph reports the number of reads with average quality scores (red, yellow and green backgrounds highlight the Phred score ranges of bad, poor and good quality, respectively). (h) Percentage of reads per sample after adaptor removal by Trimmomatic. (i) Percentage of NGS reads aligned to the WT or the four alternative splice isoform consensus sequences in WT and WFS1 iPSCs. Alignment data for WFS1 are reported as mean of the two analysed clones

Fig. 4

(a) Schematic representation of the WFS1 AS variants found by NGS, showing the conserved exonic sequence (green) and spliced-out sequence (grey). Sequences at the bottom of each structure zoom at the donor/acceptor AS sites. (b) RT-qPCR of total WFS1 mRNA, in WT and WFS1 iPSCs. Data are plotted as mean ± SD. n=3. *p<0.05 (by Student’s unpaired one-tailed t test). (c) RT-qPCR of the four WFS1 isoforms in WFS1 iPSCs after treatment with NMDI-14 or vehicle (untreated). Data are plotted as mean ± SD. n=3 independent experiments. *p<0.05, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction)

Fig. 5

(a, b) Immunoblot analysis of wolframin in WT and WFS1 iPSC clones recognised by polyclonal antibodies targeting the C-terminal (a) and N-terminal (b) domains. GAPDH was used as housekeeping gene; Fibro denotes fibroblasts with the indicated genotype. The densitometric analysis of the wolframin/GAPDH ratio is shown; n=3. ***p<0.001 (by Student’s unpaired two-tailed t test). (c, d) Three-dimensional models of WT (residues 1–402), and mutated p.Val106-Arg152del (residues 1–355) (c), and p.Val91–Asp171del (residues 1–322) (d) N-terminal domains, including the first two transmembrane regions. Proteins are represented as ribbons. Region lost in mutated isoforms (green), the mutation-induced conformational changes (blue) and the unaltered portion of the domain (red) are highlighted. In the images displaying the mutated isoforms, magnifications show the superimposed view of transmembrane region α helices in WT (light blue) and p.Val106–Arg152del (red) (c), and the superimposed view of wolframin-CaM binding domain (yellow) loss in p.Val91–Asp171del (d). In p.Val106–Arg152del, the partially conserved wolframin-CaM binding interface is shown as Connolly surface (c). (e, f) Results of molecular dynamics simulation, reporting RMSD of protein backbone atoms and radius of gyration (Rg) of the WT (grey) and mutated (orange/purple) N-terminal domains. Flattening of the RMSD and Rg plots of protein was observed around 10 ns at 300 K

Fig. 6

(a) Schematic representation of the c.316-1G>A point mutation correction strategy based on the Cas9/gRNA-mediated targeting of WFS1 exon 4. Mapping of the ssODN (blue) and the fw/rev primer pair (orange) used for screening of the homology-directed repair is shown. Magnification highlights the comparison between the pathogenic variant containing the c.316-1G>A mutation with the ssODN sequence re-establishing the natural ASS upstream the exon 4. Protospacer adjacent motif (PAM) sequence and gRNA recognition site are emphasised in dark green. Silent mutations and SacI cut site in ssODN sequence are shown in light blue and framed by a pink rectangle, respectively. (b) Screening by SacI restriction enzyme digestion of GFP-positive iPSC clones and immunoblotting of wolframin in WT and WFS1 fibroblasts (Fibro; as positive and negative controls, respectively), and WT, WFS1 and WFS1^wt/757A>T iPSC clones recognised by the polyclonal antibody targeting the N-terminal domain. GAPDH was used as housekeeping gene. (c) Immunoblot analysis of wolframin expression in WT, WFS1^wt/757A>T and WFS1 iPSCs. The relative quantification of wolframin/GAPDH ratio is reported and expressed as mean ± SD. n=3 different iPSC clones. ***p<0.001 (by Student’s unpaired two-tailed t test). (d, e) RT-qPCR of total WFS1 mRNA (d) and of AS isoforms (e) in WFS1 and WFS1^wt/757A>T iPSCs expressed as mean ± SD; n=3. *p<0.05, **p<0.01, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction)

Fig. 7

(a) Representative immunoblot showing wolframin protein recognised by the anti-wolframin C-terminal antibody before (day 0) and after (day 24) iPSC differentiation into beta cells. The monomeric form (100 kDa) and homotetrameric form (>250 kDa) are indicated by red and violet triangles, respectively. The signal was acquired at different exposure times to detect homotetramers. (b) Relative quantification of wolframin in WFS1 and WFS1^wt/757A>T iPSCs (day 0) and iBeta (day 24), expressed as mean ± SD. n=3 independent in vitro differentiation experiments. *p<0.05, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction). (c) RT-qPCR analysis of total WFS1 mRNA in WFS1 and WFS1^wt/757A>T iPSCs (day 0) vs iBeta (day 24), presented as mean ± SD. n=3–6 independent in vitro differentiation experiments. ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction). (d) Quantification of the AS isoforms in iPSCs and iBeta expressed as mean ± SD. n=3. ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction)

Fig. 8

(a, d, g) Relative expression of ATF4, HSPA5 and DDIT3 in WFS1 and WFS1^wt/757A>T beta cells at 8 h or 16 h post-TG exposure. Data are plotted as mean ± SEM. n=4–9 independent replicates. *p<0.05, **p<0.01, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction). (b, c, e, f, h, i) Estimation plot including the mean of difference in ATF4, HSPA5 and DDIT3 mRNAs calculated at 48 h after cytokine treatment (50 U/ml IL-1β, 1000 U/ml IFN-γ and 10 ng/ml TNF-α) in WFS1 (red) and WFS1^wt/757A>T (green) beta cells. n=6 for WFS1, n=5 for WFS1^wt/757A>T iBeta. *p<0.05 (by paired Student’s t test)

Fig. 9

(a–d) Early and late apoptosis were measured as percentage (%) of Annexin-V and PI-positive cells, respectively, in WFS1 and WFS1wt/757A>T iBeta after 50 nmol/l TG or inflammatory cytokine exposure. Data are plotted as mean ± SD. n=3–5. **p<0.01, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction). (e, f) Gene expression of the splicing factors UPF1 and SMG7 in WFS1 and WFS1wt/757A>T iBeta upon TG or inflammatory cytokine treatment at the indicated times. Data are expressed as fold change over the untreated control. Data are expressed as mean ± SD; n=3. **p<0.01, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction). (g, h) Relative quantification of AS isoforms in WFS1 iBeta upon treatment for 8 h and 16 h with TG (g) or treatment for 48 h with inflammatory cytokines (h). Data are plotted as mean ± SD. n=3 independent experiments. **p<0.01, ***p<0.001 (by two-way ANOVA with Dunn–Šídák correction)