Hostname: page-component-6766d58669-tq7bh Total loading time: 0 Render date: 2026-05-19T01:28:03.375Z Has data issue: false hasContentIssue false
  • English
  • Français

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
Rights & Permissions [Opens in a new window]

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

Information

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

Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease, characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Reference Brown and Al-Chalabi1 More than 50 fused in sarcoma (FUS) mutations have been reported, accounting for about 5.8% familial ALS (fALS) and 1.3% sporadic ALS (sALS) cases in China. Reference Zou, Zhou, Che, Liu, He and Huang2 The majority of FUS mutations occur in exons 5–6 and exons 13–15 of different protein domains, suggesting variable pathogenic mechanisms. Reference Chen, Ding, Akram, Xue and Luo3 For example, FUS-Arg244Cys, FUS-Arg514Ser, and FUS-Arg521Cys mutations cause DNA damage by impairing FUS interaction with histone deacetylase 1 (HDAC1) and reducing double-strand breaks repair efficiency, Reference Qiu, Lee and Shang4,Reference Shang and Huang5 FUS-Pro525Leu causes abnormal RNA metabolism by inhibiting splicing of minor introns, Reference Reber, Stettler and Filosa6 and C-terminal mutations usually cause cytoplasmic mislocalization of FUS. Reference Dormann, Rodde and Edbauer7

Pathogenic genetic mutations in the intron/exon flanking regions (e.g. classical splicing sites) may lead to complete exon skipping, intron retention, or the introduction of a new splice site within an exon or intron. Reference Baralle and Baralle8 Splice site mutations are DNA sequence changes that alter or abolish correct mRNA splicing during the process of precursor mRNA maturation. Reference Skjørringe, Tümer and Møller9 It has been estimated that about 15% of human disease-causing mutations may disrupt mRNA splicing. Reference Krawczak, Reiss and Cooper10 For example, a C/T change at position 6 in exon 7 of survival of motor neuron 2 (SMN2) gene causing frequent skipping of exon 7 that produces an inactive and unstable protein lacking the last 16 amino acids, caused spinal muscular atrophy. Reference Cooper, Wan and Dreyfuss11

So far, few splice site mutations in FUS have been reported in the ALSoD database with unclear pathogenicity. The FUS splicing site mutations (IVS13-2A > G, p. Gly466Valfs*14; c.1542-2A > C, p. Gly515Serfs*8; c.1542-1G > T, p. Gly515Serfs*8) at C-terminal were reported to cause exon skipping, truncated protein products, and cytosolic mislocalization. Reference DeJesus-Hernandez, Kocerha and Finch12–Reference Canosa, Lomartire and De Marco14 Previously, two independent studies reported the FUS c.1394-2delA variant in western ALS patients. Briefly, the heterozygous FUS c.1394-2delA variant was found in 1 out of 301 German ALS patients, Reference Müller, Brenner and Weydt15 and 1 out of 391 US ALS patients. Reference Cady, Allred and Bali16 However, the frequency of FUS c.1394-2delA variant in Chinese ALS patients is unclear, and its functional mechanisms are largely unknown.

In this study, we aimed to investigate FUS splice site mutations in Chinese ALS patients and reported one Chinese sALS patient carrying the FUS splice acceptor site mutation (c.1394-2delA, p. Gly466Valfs*14). We also characterized the molecular mechanisms of this splice site FUS mutation at mRNA and protein levels.

Materials and Methods

Participants

The included patients were diagnosed with ALS according to the revised EI Escorial criteria Reference Ludolph, Drory and Hardiman17 in the ALS clinic at Huashan Hospital (Shanghai, China). The parents of the FUS splice mutation carrier were also included. Age of onset was defined as the age of initial appearance of symptoms. The functional impairment of the proband was assessed according to the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R). Reference Cedarbaum, Stambler and Malta18 This study was approved by the ethical review boards of Huashan Hospital (Shanghai, China).

Genetic Analysis

We collected the peripheral blood using the EDTA-containing vacuum blood collection tube (BD Vacutainer, 367863, USA). Genomic DNA was extracted using the QIAGEN DNA extract kit (QIAGEN, 51206, Germany) according to the standard protocols. We amplified all coding regions of Cu/Zn superoxide dismutase (SOD1), exon 6 of TAR DNA binding protein (TARDBP) and selected FUS coding regions (exon 5, exon 6, exons 13–14, and exon 15) for polymerase chain reaction (PCR), followed by sequencing with the ABI-3730XL genetic analyzer (Applied Biosystems, USA). We used CodonCode Aligner software (V.10.0.2) to analyze the amplicon sequences.

The minor allele frequency was obtained from the gnomAD database (Version 2.1.1) (http://gnomad-sg.org) (n = 15,708 Genomes data) and the China Metabolic Analytics Project (ChinaMAP) database (http://www.mbiobank.com) (n = 10,588 Genomes data). We used the SpliceAI deep learning algorithm (https://github.com/Illumina/SpliceAI) Reference Jaganathan, Kyriazopoulou Panagiotopoulou and McRae19 to predict the effect of mutation on splicing. The software sets a cutoff of 0.8 to indicate a high-precision prediction result.

Prediction of Protein Structure

AlphaFold deep learning algorithm was used to predict protein structures of FUS carrying wild type or mutant variant (https://github.com/deepmind/alphafold#running-alphafold). Reference Jumper, Evans and Pritzel20 We used the ranked_0.pdb file for further analysis, which contained the prediction result with the highest confidence. The results were visualized by PyMol (Version 2.5.2) software.

RNA Sequencing and RT-PCR

Lymphocytes were isolated from 3 ml EDTA-containing venous blood using LymphoprepTM (Stem Cell, Germany) and density-gradient centrifugation. Total RNA extraction from lymphocytes was performed using the total RNA extraction kit (Aidlab, China). 1 μg RNA was used for RNA sequencing at BerryGenomics Co., Ltd. In brief, the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, 20037135, USA) was used to remove the ribosomal RNA and TruSeq Stranded mRNA kit (Illumina, RS-122-2001, USA). Strand-specific sequencing libraries were prepared and sequenced by the Illumina Novaseq 6000 platform (Illumina, San Diego, USA). The alternative splicing was analyzed by LeafCutter using the RNA sequencing data with the default setting, which is available on https://github.com/davidaknowles/leafcutter/. Reference Li, Knowles and Humphrey21 In short, the RNA sequencing reads were aligned by STAR (Version 2.7.10a). Reference Dobin, Davis and Schlesinger22 Reads that span exon–exon junctions and map with a minimum of 6 nt into each exon were extracted from the bam files with filter_cs.py script. Intron clustering was performed using the leafcutter_cluster.py script. We used the leafcutter_ds.R script to calculate the differential excision of FUS (wild type vs mutant).

To validate abnormal RNA splicing event, we used the PrimeScriptTM RT reagent Kit (Takara Bio, Japan) to convert RNA to cDNA. FUS-specific primers targeting the exon 14 were used for RT-PCR (Supplementary Figure 1). The PCR products were separated by 2% agarose gel electrophoresis and visualized using iBrightCL1000 Imager (Thermo Fisher Scientific, USA). We then cut the target gel band, extracted the cDNA by the gel extraction kit (TianGen, China) and sequenced it with the ABI-3730XL genetic analyzer (Applied Biosystems, USA).

Preparation of Plasmids and Cell Culture

The human full-length coding sequence of wild-type FUS (NM_004960.3) was amplified from pEGFP-N1-FUS/TLS-FLAGC (addgene:60362) and was cloned into pEGFP-C1 vector at the XhoI/BamHI restriction site. The cDNA of the patient was used as a template to generate the truncated mutant FUS sequences and cloned into the XhoI/BamHI site of the pEGFP-C1 vector. HEK293T cells (purchased from Cell Bank of Chinese Academic of Science) and SH-SY5Y cells (a gift from Dr. Jian Wang in Fudan University) were cultured in DMEM or DF12 supplemented with 10% fetal bovine serum (FBS, Gibco) and Glutamax (Gibco), MEM non-essential amino acids solution (Gibco), and 1% penicillin/streptomycin (Gibco) respectively.

Immunofluorescence

HEK293T and SH-SY5Y cells were plated on 20 mm Glass Bottom Cell Culture Dishes (NEST, 801001, China) and transfected with wild type or mutant FUS plasmids using Lipofectamine 2000 (Thermo Fisher Scientific, USA). Reference Dalby, Cates and Harris23 After 36 h of plasmids transfection, we washed cells with phosphate-buffered saline and fixed cells with 4% cold paraformaldehyde for 15 min at room temperature. We used the Hoechst 33342 (Thermo Fisher Scientific, H3570, USA) to label the nuclei and Nikon’s Eclipse Ti2 inverted microscope confocal system (Nikon, Japan) to acquire images at 60× magnification with a Z stack interval of 2 μm. ImageJ software was used to quantify the intensity of the GFP fluorescence (NIH, USA, https://imagej.nih.gov/ij/). Briefly, we selected the cells with the nucleus stained by Hoechst and GFP for quantifying the intensities. The cytoplasmic FUS intensities were calculated by the total GFP intensities minus the GFP intensities of the nucleus.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was reverse transcribed to cDNA using the PrimeScriptTM RT reagent Kit (Takara Bio, Japan). RT-qPCR was performed in a 20-μl reaction volume using TB Green® Premix Ex TaqTM II (Tli RNaseH Plus) reagent (Takara Bio, Japan) and specific primers (Supplementary Table 1) in Bio-Rad CFX96 Real-Time PCR Detection System (Bio-rad, USA). The expression level of total FUS mRNA was estimated based on the 2−ΔΔCT method and was normalized to the expression level of GAPDH housekeeping gene to obtain a relative expression level.

Statistics

We used the Wilcoxon rank sum nonparametric test (R Version 4.0.4) to analyze the mean cytoplasmic/total FUS ratio between wild-type and mutant groups. P value < 0.05 was accepted as statistically significant.

Results

Identification of the FUS Splicing Mutation in Chinese ALS Patients

In 233 Chinese ALS patients (Table 1), we found that one sporadic case carried a heterozygous FUS splicing mutation (NM_004960.4, chr16:31202282, c.1394-2delA, p. Gly466Valfs*14, rs1555509569) (Figure 1(A) and (B)). Sanger sequencing excluded other mutations of SOD1 or TARDBP in the proband. The healthy parents were further analyzed and did not carry this variant, suggesting that the FUS c.1394-2delA is a de novo variant (Figure 1(B)). It was absent in the gnomAD and ChinMAP databases. The proband has a younger sister [II-2] (39 years old), who had no complaint of any neurological problems and refused any genetic tests.

Table 1:

Characteristics of 233 Chinese ALS patients

Abbreviations: ALS: amyotrophic lateral sclerosis; fALS: familial ALS; sALS: sporadic ALS; N, number; M: male; SD: standard deviation.

Figure 1:

A novel FUS mutation affected RNA splicing in a sporadic ALS patient. (A) The pedigree of the investigated family. Proband was indicated by the arrowhead, and the age of family members and age at onset (AAO) were indicated. FUS genotype status was indicated as WT (wild type), MT (mutant); (B) Chromatography of Sanger sequencing. It showed that the proband carried FUS c.1394-2delA variant; (C) Visualization of altered RNA splicing of FUS exon13-exon15. The RNA sequencing data of the proband and one healthy control sample was processed by the LeafCutter tool (https://github.com/davidaknowles/leafcutter/). Location of the mutation site was indicated; (D) Agarose gel electrophoresis of FUS cDNA product. The patient II-1 (lane 1) and the health parents (I-1 and I-2, lane 2–3) had different FUS mRNA transcripts; and the patient had two dominant cDNA bands, corresponding to the normal transcript (393 bp) and the abnormal transcript (∼245 bp); (E) The Sanger sequencing chromatogram of the smaller cDNA product (∼245 bp on the gel). It validated the skipping of FUS exon 14 in the proband.

Clinical Presentation

The pedigree of this family is presented in Figure 1(A). The proband (II-1) developed muscle weakness in the right upper limb at the age of 39. The symptoms spread quickly to the left upper limb. Seven months later, he developed dysarthria, dysphagia, and dyspnea, with fasciculation in four limbs and obvious atrophy in both upper limbs. Neurologic examination at 11 months after initial symptoms showed reduced muscle strength of both distal and proximal upper extremities (4/5). Deep tendon reflexes were reduced in upper limbs and brisk in lower limbs. Hoffmann and Babinski’s signs were not present. His ALSFRS-R score was 40/48 with normal cognition and significant weight loss. Electromyography showed acute and chronic denervation in the upper and lower limb, thoracic, and sternocleidomastoid muscles. Blood biochemical tests (routine blood test, thyroid function, retinal and liver function, and immune-related antibodies [anti-ANA, anti-ENA, anti-CCP, anti-ACA, and anti-ANCAs]) and related examinations (cerebral magnetic resonance imaging and cerebrospinal fluid basic analyses) were normal. His blood gas analysis showed the high pressure of carbon dioxide (PaCO2: 46.3 mmHg) which indicated decreased respiratory function. The ALSFRS-R score of the patient was 15/48 at 17 months after symptom onset. The patient underwent tracheostomy 19 months after symptom onset due to respiratory failure.

The FUS Splicing Mutation is Linked to Altered RNA Splicing and Upregulated Gene Expression

Functional mechanisms of the FUS splice site mutation are largely unclear. SpliceAI analysis suggested that the FUS mutation (c.1394-2delA) may alter the normal splicing at chr16:31202284 (hg19) (SpliceAI = T|FUS|0.15|1.00|0.00|0.00|37|3|5|29), suggesting that the variant may cause a loss of splice acceptor. Sequencing of lymphocyte RNA revealed aberrant splicing leading to the skipping of FUS exon 14 (Figure 1(C)). The agarose gel electrophoresis of FUS cDNA products from the proband (II-1) revealed two PCR bands corresponding to a 393 bp (containing exon 14) and a 245 bp band (Figure 1(D)). Sanger sequencing chromatogram of the mutant products showed the skipping of FUS exon 14 (148bp) (Figure 1(E)). In addition, the mutation carrier showed elevated total FUS mRNA expression level (Supplementary Figure 2) when compared to the FUS expression of lymphocytes of the parents and another independent neurologically healthy individuals carrying the wild-type allele. RNA-seq further confirmed the upregulation of FUS expression in the mutation carrier (Supplementary Figure 3).

The FUS Splicing Mutation Induces Prominent Cytosolic Mislocalization

The FUS c.1394-2delA mutation was predicted to cause the frameshift and premature termination in exon 15, leading to a truncated FUS (p. Gly466Valfs*14) (Supplementary Figure 4), which might cause the loss of the nuclear localization signal (NLS) domain (Figure 2(A)).

Figure 2:

Mislocalization of FUS in cell lines. (A) Schematic diagram of the EGFP-FUS fusion protein of different FUS plasmids. The protein domains of FUS are indicated by different boxes. The G in the mutant plasmid represents the truncated last RGG domain; (B) In HEK293T cells, mutant FUS caused mislocalization of FUS into the cytoplasm. GFP: EGFP-FUS fusion protein, Hoechst: nuclear staining, Merge: colocalization of GFP signal with the nuclear Hoechst fluorescence; (C) The histogram of the mean ratio of cytoplasmic/total FUS intensities in HEK293T cells (mean ratio: 0.45 vs 0.03 in MT and WT groups); (D) In SH-SY5Y cells, mutant FUS caused mislocalization of FUS into the cytoplasm. GFP: EGFP-FUS fusion protein, Hoechst: nuclear staining, Merge: colocalization of GFP signal with the nuclear Hoechst fluorescence; (E) The histogram of the mean ratio of cytoplasmic/total FUS in SH-SY5Y cells (mean ratio: 0.50 vs 0.05 in MT and WT groups); Scale bar: 10 μm; p < 10−4.

Since the C-terminus of FUS is a non-classical Proline/Tyrosine-nuclear localization signal (PY-NLS) domain and essential for FUS nuclear import, we tested whether the novel FUS c.1394-2delA mutation may affect the FUS cytosolic/nucleus mislocalization. In both HEK293T kidney and SH-SY5Y neuronal cell lines, we found that cells with the mutant FUS (c.1394-2delA, p. Gly466Valfs*14) showed the mislocalization of nucleus FUS into the cytoplasm (p < 0.0001; mean ratio:0.45 vs 0.03 in HEK293T cells, 0.50 vs 0.05 in SH-SY5Y cells) (Figure 2).

Discussion

Our study investigated FUS splice site mutations in Chinese ALS patients and identified one Chinese sALS patient carrying the rare FUS splice site mutation (c.1394-2delA, p. Gly466Valfs*14), which was previously reported in two western ALS patients, Reference Müller, Brenner and Weydt15,Reference Cady, Allred and Bali16 with unclear functional mechanisms. In lymphocytes of the patient, we found that the splice site mutation caused the splice acceptor loss at intron 13, leading to exon 14 skipping and upregulation of FUS gene expression. The mutant allele was predicted to encode a truncated protein. In both kidney and neuronal cell lines, we demonstrated that the FUS splice site mutation significantly induced cytosolic mislocalization, which is a key feature of FUS NLS domain mutations. Reference Dormann, Rodde and Edbauer7,Reference An, Rabesahala de Meritens, Buchman and Shelkovnikova24 Our findings support the pathogenicity of the FUS c.1394-2delA mutation in ALS.

A previous study reported a different mutation (g.10747A > G; IVS13-2A > G, p. Gly466Valfs*14) at the same FUS genomic locus in an American female sALS patient. Reference DeJesus-Hernandez, Kocerha and Finch12 It also caused abnormal RNA splicing, truncated FUS product, and cytoplasmic mislocalization of FUS in a mouse neuroblastoma cell line (N2A cells). Reference DeJesus-Hernandez, Kocerha and Finch12 In addition, both FUS c.1394-2delA mutation and the previously reported mutation were absent in the large public datasets (the ChinaMAP and the gnomAD databases), supporting that it might be pathogenic.

FUS is a DNA/RNA-binding protein involved in RNA metabolism processes, including gene transcription regulation, RNA splicing, and transport. Reference Niu, Zhang and Gao25 Wild-type FUS predominantly resides in the nucleus and shuttles between the nucleus and cytoplasm. Reference Zinszner, Sok, Immanuel, Yin and Ron26 Our in vitro experiments support that the FUS splicing mutation may cause truncated FUS protein lacking the PY-NLS domain, disrupt the interaction between FUS and karyopherin β2 (Kapβ2), and cytoplasmic mislocalization (Figure 2(B)–(E)). The results are in line with the previous report that FUS C-terminal NLS domain is important for FUS nuclear transportation by binding with Kapβ2 in a Ran-sensitive manner, Reference Lee, Cansizoglu, Süel, Louis, Zhang and Chook27 which would form the large TDP-43 negative inclusions and incorporate into the stress granules. Reference Daigle, Lanson and Smith28 Splicing site mutations may cause complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron, which could affect the balance of isoforms produced by alternatively spliced exons and cause disease. Reference Krawczak, Reiss and Cooper10 ALS has been reported to be caused by other splicing mutations, such as annexin A11 (ANXA11) c.1086+1G>A by promoting cytoplasmic aggregation of the mutant protein, SOD1 c.240-7T>G by reducing its stability and enzymic activity, and TANK-binding kinase 1 (TBK1) c.1522-3T>G, and c.2066+4A>G by reducing gene expression. Reference Sainouchi, Hatano and Tada29–Reference Lu, Chen and Lin31 Our findings support that abnormal splicing is an important molecular mechanism of ALS.

Genetic variants at C-terminal may escape nonsense-mediated RNA decay (NMD), Reference Amrani, Sachs and Jacobson32 but it is unusual to increase the gene expression level. To maintain its gene expression level in vivo, FUS has a unique homeostasis mechanism, in which FUS protein bind to exon 7 and flanking introns of its pre-mRNAs promoting intron retention Reference Zhou, Liu, Liu, Oztürk and Hicks33,Reference Humphrey, Birsa and Milioto34 that is followed by the NMD-mediated RNA degradation. Reference Zhou, Liu, Liu, Oztürk and Hicks33,Reference Humphrey, Birsa and Milioto34 The FUS splicing mutation may disrupt this homeostasis mechanism, because FUS mislocalization reduced the nucleus FUS protein level and may disrupt the intron retention and NMD related degradation, which might be related to upregulated gene expression level observed in this study.

The current study was limited to a moderate sample set of Chinese ALS patients. Future studies should investigate FUS splice mutations in a larger sample set with different ethnical backgrounds to understand the mechanisms of splicing mutations in ALS etiology.

In conclusion, we reported a de novo FUS splice site mutation (c.1394-2delA, p. Gly466Valfs*14) in 1 out of 233 (0.4%) Chinese ALS patients. It caused abnormal RNA splicing, upregulated gene expression, truncated FUS translation, and cytosolic mislocalization. Our findings suggested the pathogenic mechanism of the FUS c.1394-2delA mutation in ALS.

Supplementary Material

To view supplementary material for this article, please visit https://doi.org/10.1017/cjn.2022.336.

Acknowledgements

We would like to thank the patients and their family members for participating in this study.

Funding

This work was supported by Shanghai Municipal Natural Science Foundation General Program (22ZR1466400) (MZ), the National Natural Science Foundation of China (82071430) (MZ), and the Fundamental Research Funds for the Central Universities (MZ).

Conflict of Interest

There is no conflict of interest to disclose.

Statement of Authorship

Conceptualization: MZ and YC; Methodology: WLY, YZ, and XLT; Clinical data collection: XC, YMS, YD, and HY; Writing–original draft preparation: WLY, MZ, and XC; Writing—review and editing: MZ and YC.

References

Brown, RH, Al-Chalabi, A. Amyotrophic lateral sclerosis. N Engl J Med. 2017;377:162–72. DOI 10.1056/NEJMra1603471.CrossRefGoogle ScholarPubMed
Zou, ZY, Zhou, ZR, Che, CH, Liu, CY, He, RL, Huang, HP. Genetic epidemiology of amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry. 2017;88:540–9. DOI 10.1136/jnnp-2016-315018.CrossRefGoogle ScholarPubMed
Chen, C, Ding, X, Akram, N, Xue, S, Luo, SZ. Fused in sarcoma: properties, self-assembly and correlation with neurodegenerative diseases. Molecules. 2019;24:1622. DOI 10.3390/molecules24081622.CrossRefGoogle ScholarPubMed
Qiu, H, Lee, S, Shang, Y, et al. ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects. J Clin Invest. 2021;131:e149564. DOI 10.1172/jci149564.CrossRefGoogle ScholarPubMed
Shang, Y, Huang, EJ. Mechanisms of FUS mutations in familial amyotrophic lateral sclerosis. Brain Res. 2016;1647:65–78. DOI 10.1016/j.brainres.2016.03.036.CrossRefGoogle ScholarPubMed
Reber, S, Stettler, J, Filosa, G, et al. Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants. EMBO J. 2016;35:1504–21. DOI 10.15252/embj.201593791.CrossRefGoogle ScholarPubMed
Dormann, D, Rodde, R, Edbauer, D, et al. ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. EMBO J. 2010;29:2841–57. DOI 10.1038/emboj.2010.143.CrossRefGoogle ScholarPubMed
Baralle, D, Baralle, M. Splicing in action: assessing disease causing sequence changes. J Med Genet. 2005;42:737–48. DOI 10.1136/jmg.2004.029538.CrossRefGoogle ScholarPubMed
Skjørringe, T, Tümer, Z, Møller, LB. Splice site mutations in the ATP7A gene. PLoS One. 2011;6:e18599. DOI 10.1371/journal.pone.0018599.CrossRefGoogle ScholarPubMed
Krawczak, M, Reiss, J, Cooper, DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet. 1992;90:41–54. DOI 10.1007/bf00210743.CrossRefGoogle ScholarPubMed
Cooper, TA, Wan, L, Dreyfuss, G. RNA and disease. Cell. 2009;136:777–93. DOI 10.1016/j.cell.2009.02.011.CrossRefGoogle ScholarPubMed
DeJesus-Hernandez, M, Kocerha, J, Finch, N, et al. De novo truncating FUS gene mutation as a cause of sporadic amyotrophic lateral sclerosis. Hum Mutat. 2010;31:E1377–89. DOI 10.1002/humu.21241.CrossRefGoogle ScholarPubMed
Belzil, VV, St-Onge, J, Daoud, H, et al. Identification of a FUS splicing mutation in a large family with amyotrophic lateral sclerosis. J Hum Genet. 2011;56:247–9. DOI 10.1038/jhg.2010.162.CrossRefGoogle Scholar
Canosa, A, Lomartire, A, De Marco, G, et al. A novel splice site FUS mutation in a familial ALS case: effects on protein expression. Amyotroph Lateral Scler Frontotemporal Degener. 2022;23:128–36. DOI 10.1080/21678421.2021.1909065.CrossRefGoogle Scholar
Müller, K, Brenner, D, Weydt, P, et al. Comprehensive analysis of the mutation spectrum in 301 German ALS families. J Neurol Neurosurg Psychiatry. 2018;89:817–27. DOI 10.1136/jnnp-2017-317611.CrossRefGoogle ScholarPubMed
Cady, J, Allred, P, Bali, T, et al. Amyotrophic lateral sclerosis onset is influenced by the burden of rare variants in known amyotrophic lateral sclerosis genes. Ann Neurol. 2015;77:100–13. DOI 10.1002/ana.24306.CrossRefGoogle ScholarPubMed
Ludolph, A, Drory, V, Hardiman, O, et al. A revision of the El Escorial criteria – 2015. Amyotroph Lateral Scler Frontotemporal Degener. 2015;16:291–2. DOI 10.3109/21678421.2015.1049183.CrossRefGoogle Scholar
Cedarbaum, JM, Stambler, N, Malta, E, et al. The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. BDNF ALS Study Group (Phase III). J Neurol Sci. 1999;169:13–21. DOI 10.1016/s0022-510x(99)00210-5.CrossRefGoogle ScholarPubMed
Jaganathan, K, Kyriazopoulou Panagiotopoulou, S, McRae, JF, et al. Predicting splicing from primary sequence with deep learning. Cell. 2019;176:535.e24–48. DOI 10.1016/j.cell.2018.12.015.CrossRefGoogle ScholarPubMed
Jumper, J, Evans, R, Pritzel, A, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–9. DOI 10.1038/s41586-021-03819-2.CrossRefGoogle ScholarPubMed
Li, YI, Knowles, DA, Humphrey, J, et al. Annotation-free quantification of RNA splicing using LeafCutter. Nat Genet. 2018;50:151–8. DOI 10.1038/s41588-017-0004-9.CrossRefGoogle ScholarPubMed
Dobin, A, Davis, CA, Schlesinger, F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21. DOI 10.1093/bioinformatics/bts635.CrossRefGoogle ScholarPubMed
Dalby, B, Cates, S, Harris, A, et al. Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications. Methods. 2004;33:95–103. DOI 10.1016/j.ymeth.2003.11.023.CrossRefGoogle ScholarPubMed
An, H, Rabesahala de Meritens, C, Buchman, VL, Shelkovnikova, TA. Frameshift peptides alter the properties of truncated FUS proteins in ALS-FUS. Mol Brain. 2020;13:77. DOI 10.1186/s13041-020-00618-0.CrossRefGoogle ScholarPubMed
Niu, C, Zhang, J, Gao, F, et al. FUS-NLS/Transportin 1 complex structure provides insights into the nuclear targeting mechanism of FUS and the implications in ALS. PLoS One. 2012;7:e47056. DOI 10.1371/journal.pone.0047056.CrossRefGoogle ScholarPubMed
Zinszner, H, Sok, J, Immanuel, D, Yin, Y, Ron, D. TLS (FUS) binds RNA in vivo and engages in nucleo-cytoplasmic shuttling. J Cell Sci. 1997;110:1741–50. DOI 10.1242/jcs.110.15.1741.CrossRefGoogle ScholarPubMed
Lee, BJ, Cansizoglu, AE, Süel, KE, Louis, TH, Zhang, Z, Chook, YM. Rules for nuclear localization sequence recognition by karyopherin beta 2. Cell. 2006;126:543–58. DOI 10.1016/j.cell.2006.05.049.CrossRefGoogle ScholarPubMed
Daigle, JG, Lanson, NA Jr, Smith, RB, et al. RNA-binding ability of FUS regulates neurodegeneration, cytoplasmic mislocalization and incorporation into stress granules associated with FUS carrying ALS-linked mutations. Hum Mol Genet. 2013;22:1193–205. DOI 10.1093/hmg/dds526.CrossRefGoogle ScholarPubMed
Sainouchi, M, Hatano, Y, Tada, M, et al. A novel splicing variant of ANXA11 in a patient with amyotrophic lateral sclerosis: histologic and biochemical features. Acta Neuropathol Commun. 2021;9:106. DOI 10.1186/s40478-021-01202-w.CrossRefGoogle Scholar
Birve, A, Neuwirth, C, Weber, M, et al. A novel SOD1 splice site mutation associated with familial ALS revealed by SOD activity analysis. Hum Mol Genet. 2010;19:4201–6. DOI 10.1093/hmg/ddq338.CrossRefGoogle ScholarPubMed
Lu, YQ, Chen, JM, Lin, H, et al. Novel intronic mutations of TBK1 promote aberrant splicing modes in amyotrophic lateral sclerosis. Front Mol Neurosci. 2022;15:691534. DOI 10.3389/fnmol.2022.691534.CrossRefGoogle ScholarPubMed
Amrani, N, Sachs, MS, Jacobson, A. Early nonsense: mRNA decay solves a translational problem. Nat Rev Mol Cell Biol. 2006;7:415–25. DOI 10.1038/nrm1942.CrossRefGoogle Scholar
Zhou, Y, Liu, S, Liu, G, Oztürk, A, Hicks, GG. ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. PLos Genet. 2013;9:e1003895. DOI 10.1371/journal.pgen.1003895.CrossRefGoogle ScholarPubMed
Humphrey, J, Birsa, N, Milioto, C, et al. FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention. Nucleic Acids Res. 2020;48:6889–905. DOI 10.1093/nar/gkaa410.CrossRefGoogle ScholarPubMed
Figure 0

Table 1: Characteristics of 233 Chinese ALS patients

Figure 1

Figure 1: A novel FUS mutation affected RNA splicing in a sporadic ALS patient. (A) The pedigree of the investigated family. Proband was indicated by the arrowhead, and the age of family members and age at onset (AAO) were indicated. FUS genotype status was indicated as WT (wild type), MT (mutant); (B) Chromatography of Sanger sequencing. It showed that the proband carried FUS c.1394-2delA variant; (C) Visualization of altered RNA splicing of FUS exon13-exon15. The RNA sequencing data of the proband and one healthy control sample was processed by the LeafCutter tool (https://github.com/davidaknowles/leafcutter/). Location of the mutation site was indicated; (D) Agarose gel electrophoresis of FUS cDNA product. The patient II-1 (lane 1) and the health parents (I-1 and I-2, lane 2–3) had different FUS mRNA transcripts; and the patient had two dominant cDNA bands, corresponding to the normal transcript (393 bp) and the abnormal transcript (∼245 bp); (E) The Sanger sequencing chromatogram of the smaller cDNA product (∼245 bp on the gel). It validated the skipping of FUS exon 14 in the proband.

Figure 2

Figure 2: Mislocalization of FUS in cell lines. (A) Schematic diagram of the EGFP-FUS fusion protein of different FUS plasmids. The protein domains of FUS are indicated by different boxes. The G in the mutant plasmid represents the truncated last RGG domain; (B) In HEK293T cells, mutant FUS caused mislocalization of FUS into the cytoplasm. GFP: EGFP-FUS fusion protein, Hoechst: nuclear staining, Merge: colocalization of GFP signal with the nuclear Hoechst fluorescence; (C) The histogram of the mean ratio of cytoplasmic/total FUS intensities in HEK293T cells (mean ratio: 0.45 vs 0.03 in MT and WT groups); (D) In SH-SY5Y cells, mutant FUS caused mislocalization of FUS into the cytoplasm. GFP: EGFP-FUS fusion protein, Hoechst: nuclear staining, Merge: colocalization of GFP signal with the nuclear Hoechst fluorescence; (E) The histogram of the mean ratio of cytoplasmic/total FUS in SH-SY5Y cells (mean ratio: 0.50 vs 0.05 in MT and WT groups); Scale bar: 10 μm; p < 10−4.

Supplementary material: File

Yang et al. supplementary material

Yang et al. supplementary material

Download Yang et al. supplementary material(File)
File 480.8 KB