破坏接受剪接位点的 WFS1 变体揭示了替代剪接对沃尔夫拉姆综合征患者β细胞凋亡的影响

IF 8.4 1区 医学 Q1 ENDOCRINOLOGY & METABOLISM
Raniero Chimienti, Silvia Torchio, Gabriel Siracusano, Valentina Zamarian, Laura Monaco, Marta Tiffany Lombardo, Silvia Pellegrini, Fabio Manenti, Federica Cuozzo, Greta Rossi, Paola Carrera, Valeria Sordi, Vania Broccoli, Riccardo Bonfanti, Giorgio Casari, Giulio Frontino, Lorenzo Piemonti
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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 <i>WFS1</i>: c.316-1G&gt;A, affecting the acceptor splice site (ASS) upstream of exon 4; and c.757A&gt;T, introducing a premature termination codon (PTC) in exon 7.</p><h3 data-test=\"abstract-sub-heading\">Methods</h3><p>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 <i>WFS1</i> alternative splicing isoforms in pancreatic beta cells. 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引用次数: 0

摘要

目的/假说沃尔夫拉姆综合征 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,它是多种细胞反应的主调控因子,该基因的突变与临床变异有关。事实上,与错义/框架变异相比,无义/框架变异与更严重的症状相关。由于实现基因型与表型的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产生的转录和翻译景观的影响。因此,我们试图阐明一名携带 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,影响第 4 号外显子上游的受体剪接位点(ASS);c.757 A>T,引入第 4 号外显子上游的受体剪接位点(ASS)。方法通过生物信息学分析推断 ASS 被破坏后发生的替代剪接事件,然后通过 RNA-seq 和 PCR 验证转录图谱。患者衍生的诱导多能干细胞(iPSC)被用作WS1的体外模型,并用于研究胰腺β细胞中的WFS1替代剪接异构体。我们利用 CRISPR/Cas9 技术纠正了 ASS 突变,并为内质网应激诱导和免疫毒性试验生成了一个合成对照。我们证明,携带 ASS 突变 c.316-1G>A 的等位基因会产生两个含 PTC 的替代剪接转录本(c.316del 和 c.316-460del),以及两个开放阅读框保留的 mRNA(c.271-513del 和 c.316-456del),导致 N 端截短的多肽。通过保留 C 端结构域,这些异构体可维持β细胞的内质网应激反应。否则,携带 PTC 的转录本在基础条件下会受到无义介导衰变(NMD)的调控。暴露于细胞应激诱导剂和促炎细胞因子会影响 NMD 相关基因 SMG7 的表达水平(>下降两倍; p<0.001),但不会在 WFS1 β 细胞中引起强烈的未折叠蛋白反应。这导致含有 PTC 的同工酶 c.316del(>比基础值增加 100 倍;p<0.001)和 c.316-460del(>比基础值增加 20 倍;p<0.001)的急剧积累,使受影响的 beta 细胞容易发生凋亡。Cas9介导的ASS恢复了典型的转录格局,挽救了患者衍生β细胞的正常表型。我们的数据表明,WFS1 β 细胞更容易受到应激和炎症的影响,我们推测这是由于 NMD 的逃避以及突变转录本和截短蛋白的积累引发的。这些发现为进一步研究 WS1 基因型与表型关系的分子基础铺平了道路,从而揭示出可能成为改善这种罕见疾病患者临床结局目标的关键决定因素。数据可用性WT wolframin的硅学预测N-端结构域文件连同程序、拉马钱德拉图、残基间距离偏差和IDDT评分以及Gromacs配置文件(doi/10.5452/ma-cg3qd)已存入ModelArchive。用于生成 WFS1 的 AS 异构体共识序列的 fastq 文件形式的深度测序数据可在 SRA 数据库(BioProject PRJNA1109747)中获取。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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

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

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来源期刊
Diabetologia
Diabetologia 医学-内分泌学与代谢
CiteScore
18.10
自引率
2.40%
发文量
193
审稿时长
1 months
期刊介绍: Diabetologia, the authoritative journal dedicated to diabetes research, holds high visibility through society membership, libraries, and social media. As the official journal of the European Association for the Study of Diabetes, it is ranked in the top quartile of the 2019 JCR Impact Factors in the Endocrinology & Metabolism category. The journal boasts dedicated and expert editorial teams committed to supporting authors throughout the peer review process.
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