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Investigation of a Fused in Sarcoma Splicing Mutation in a Chinese Amyotrophic Lateral Sclerosis Patient

Published online by Cambridge University Press:  12 December 2022

Wanli Yang ,
Xi Chen ,
Yu Zhou ,
Xuelin Tang ,
Yimin Sun ,
Yi Dong ,
Hong Yang ,
Yan Chen  and
Ming Zhang Open the ORCID record for Ming Zhang [Opens in a new window]
Wanli Yang
Affiliation:
The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, China
Xi Chen
Affiliation:
Department of Neurology and National Center for Neurological Disorders, Huashan Hospital, Fudan University, Shanghai, China
Yu Zhou
Affiliation:
The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, China
Xuelin Tang
Affiliation:
The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, China
Yimin Sun
Affiliation:
Department of Neurology and National Center for Neurological Disorders, Huashan Hospital, Fudan University, Shanghai, China
Yi Dong
Affiliation:
Department of Neurology and National Center for Neurological Disorders, Huashan Hospital, Fudan University, Shanghai, China
Hong Yang
Affiliation:
The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, China
Yan Chen*
Affiliation:
Department of Neurology and National Center for Neurological Disorders, Huashan Hospital, Fudan University, Shanghai, China
Ming Zhang*
Affiliation:
The First Rehabilitation Hospital of Shanghai, Department of Medical Genetics, School of Medicine, Tongji University, Shanghai, China Clinical Center for Brain and Spinal Cord Research, Tongji University, Shanghai, China Institute for Advanced Study, Tongji University, Shanghai, China
*
Corresponding authors: Yan Chen. E-mail: chenyanhs@fudan.edu.cn; Ming Zhang. E-mail: mingzhang@tongji.edu.cn
Corresponding authors: Yan Chen. E-mail: chenyanhs@fudan.edu.cn; Ming Zhang. E-mail: mingzhang@tongji.edu.cn
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Abstract:

Objective:

Genetic mutations of fused in sarcoma (FUS) causing amyotrophic lateral sclerosis (ALS) may disrupt mRNA splicing events. For example, the FUS c.1394-2delA variant was reported in two western ALS patients, but its molecular mechanism is unclear. In this study, we aim to investigate FUS splice site mutations in Chinese ALS patients.

Methods:

Sanger sequencing was used to identify FUS splicing mutations in Chinese ALS patients. We combined a deep learning tool (SpliceAI), RNA sequencing, and RT-PCR/RT-qPCR to analyze the effect of FUS c.1394-2delA mutation on RNA splicing and expression. AlphaFold was used to predict the protein structure of mutant FUS. In transfected cell lines, we used immunofluorescence to assess cytoplasmic mislocalization of mutant FUS protein.

Results:

We identified a de novo FUS splice acceptor site mutation (c.1394-2delA, p. Gly466Valfs*14) in one Chinese sporadic ALS patient, which is linked to exon 14 skipping, and upregulated total FUS mRNA expression. The FUS splice site mutation was predicted to be translated into a truncated protein product at C-terminal. In vitro studies revealed that the FUS mutation increased cytoplasmic mislocalization in both HEK293T and SH-SY5Y cells.

Conclusions:

We identified a de novo FUS splicing mutation (c.1394-2delA, p. Gly466Valfs*14) in 1 out of 233 Chinese ALS patients. It caused abnormal RNA splicing, upregulated gene expression, truncated FUS translation, and cytosolic mislocalization. Our findings suggested that FUS splice site mutation is rare in Chinese ALS patients and extended our knowledge of molecular mechanisms of the FUS c.1394-2delA mutation.

Résumé :

RÉSUMÉ :

Étude d’une mutation affectant des sites d’épissage dans le cas de protéines de fusion d’un sarcome chez un patient chinois atteint de sclérose latérale amyotrophique.

Objectif :

Les mutations génétiques des protéines de fusion d’un sarcome (PFS), lesquelles sont à l’origine de la sclérose latérale amyotrophique (SLA), peuvent perturber les sites d’épissage de l’ARN messager. À titre d’exemple, la variante c.1394-2delA des PFS a été signalée chez deux patients occidentaux atteints de SLA mais son mécanisme moléculaire demeure nébuleux. Dans la présente étude, nous entendons étudier les mutations des sites d’épissage des PFS chez des patients chinois atteints de SLA.

Méthodes :

Le séquençage génétique de Sanger a été utilisé pour identifier les mutations des sites d’épissage des PFS chez des patients chinois atteints de SLA. Nous avons aussi combiné un outil d’apprentissage profond (SpliceAI), le séquençage de l’ARN messager et la méthode dite « RT-PCR/RT-qPCR » pour analyser l’effet de la mutation des PFS (variante c.1394-2delA) sur l’épissage et l’expression de l’ARN messager. Le programme AlphaFold a été par ailleurs utilisé pour prédire la structure protéique des PFS mutantes. Dans les lignées cellulaires transfectées, nous avons utilisé l’immunofluorescence pour évaluer la mauvaise localisation cytoplasmique des PFS mutantes.

Résultats :

Nous avons identifié une mutation de novo du site accepteur d’épissage des PFS (c.1394-2delA, p.Gly466Valfs*14) chez un patient chinois atteint de SLA sporadique, laquelle est liée à un saut de l’exon 14, ce qui a augmenté l’expression totale de l’ARN messager des PFS. La mutation des sites d’épissage des PFS est supposée se traduire par un produit protéique tronqué à l'extrémité C-terminale. Des études in vitro ont aussi révélé que les mutations des PFS augmentait la mauvaise localisation cytoplasmique dans les cellules HEK293T et SH-SY5Y.

Conclusions :

Nous avons identifié une mutation de novo de l’épissage des PFS (c.1394-2delA, p.Gly466Valfs*14) chez un des 233 patients chinois atteints de SLA. Cette mutation a provoqué un épissage anormal de l’ARN messager, une expression génétique régulée, une traduction tronquée des PFS ainsi qu’une mauvaise localisation cytoplasmique. En somme, nos résultats suggèrent que les mutations des sites d’épissage des PFS sont rares chez les patients chinois atteints de SLA. Ils permettent également d’élargir nos connaissances quant aux mécanismes moléculaires de la mutation c.1394-2delA des PFS.

Keywords

ALSFUSRNA splicingCytoplasmic mislocalization
Type
Original Article
Information
Canadian Journal of Neurological Sciences , Volume 50 , Issue 6 , November 2023 , pp. 891 - 896
Copyright
© The Author(s), 2022. Published by Cambridge University Press on behalf of Canadian Neurological Sciences Federation

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