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).

Graphical Abstract

中文翻译：

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破坏受体剪接位点的 WFS1 变体揭示了选择性剪接对 Wolfram 综合征患者 β 细胞凋亡的影响

 目标/假设

Wolfram 综合征 1 （WS1） 是一种遗传性疾病，主要表现为儿童期发病的糖尿病和进行性视神经萎缩。致病基因 WFS1 编码 wolframin，这是多种细胞反应的主要调节因子，该基因的突变与临床变异性有关。事实上，无义/移码变体比错义/帧内变体与更严重的症状相关。由于实现基因型-表型相关性对于处理疾病结果至关重要，因此需要研究此类突变引起的转录和翻译景观的影响的工作。因此，我们试图阐明携带 WFS1 复合杂合突变的 WS1 患者病理生理改变背后的分子决定因素：c.316-1G>A，影响外显子 4 上游的受体剪接位点 （ASS）;和 c.757A>T，在外显子 7 中引入过早终止密码子 （PTC）。

 方法

进行生物信息学分析以推断 ASS 破坏后发生的可变剪接事件，然后进行 RNA-seq 和 PCR 以验证转录景观。患者来源的诱导多能干细胞 （iPSC） 用作 WS1 的体外模型，并研究胰腺 β 细胞中的 WFS1 选择性剪接亚型。采用 CRISPR/Cas9 技术纠正 ASS 突变并为内质网应激诱导和免疫毒性测定产生同基因控制。

 结果

我们发现，患者来源的 iPSC 保留了分化为胰腺 β 细胞的能力。我们证明，携带 ASS 突变 c.316-1G>A 的等位基因源自两个含有 PTC 的选择性剪接转录本（c.316del 和 c.316-460del）和两个开放阅读框保守 mRNA（c.271-513del 和 c.316-456del）导致 N 末端截短的多肽。通过保留 C 末端结构域，这些亚型维持了 β 细胞中的内质网应激反应。否则，在基础条件下，携带 PTC 的转录物受无义介导的衰变 （NMD） 的调节。暴露于细胞应激诱导剂和促炎细胞因子影响了 NMD 相关基因 SMG7 的表达水平 （>降低两倍;p<0.001），而不会在 WFS1 β 细胞中引发强烈的未折叠蛋白反应。这导致含 PTC 的亚型 c.316del 的急剧积累（>100 倍于基础增加;p<0.001）和 c.316–460del（比基础增加 >20 倍;p<0.001），使受影响的 β 细胞易发生凋亡。Cas9 介导的 ASS 恢复检索了经典转录景观，挽救了患者来源的 β 细胞中的正常表型。

结论/解释

这项研究代表了一种研究 wolframin 的新模型，强调了 WFS1 基因的每个单个突变如何决定截然不同的功能结果。我们的数据表明 WFS1 β 细胞对压力和炎症的脆弱性增加，我们假设这是由逃逸 NMD 和突变转录本和截短蛋白的积累触发的。这些发现为进一步研究 WS1 中基因型-表型关系的分子基础铺平了道路，以揭示可能旨在改善受这种罕见疾病影响的患者临床结果的关键决定因素。

 数据可用性

将 WT wolframin 的计算机预测 N 端结构域结构文件与程序、ramachandran 图、残基间距离偏差和 IDDT 评分以及 Gromacs 配置文件 （doi/10.5452/马-cg3qd） 一起存入 ModelArchive 中。用于生成 WFS1 的 AS 亚型共有序列的深度测序数据作为 fastq 文件可在 SRA 数据库 （BioProject PRJNA1109747） 中获得。

 图形摘要