Front. Neurol. Frontiers in Neurology Front. Neurol. 1664-2295 Frontiers Media S.A. 10.3389/fneur.2022.790082 Neurology Original Research Phenotype of VCP Mutations in Chinese Amyotrophic Lateral Sclerosis Patients Feng Shu-Yan 1 2 † Lin Han 3 † Che Chun-Hui 3 Huang Hua-Pin 3 4 Liu Chang-Yun 3 4 Zou Zhang-Yu 3 4 * 1Department of Neurophysiology, Henan Provincial People's Hospital, Zhengzhou, China 2Zhengzhou University People's Hospital, Zhengzhou, China 3Department of Neurology, Fujian Medical University Union Hospital, Fuzhou, China 4Institute of Clinical Neurology, Fujian Medical University, Fuzhou, China

Edited by: Kewei Chen, Banner Alzheimer's Institute, United States

Reviewed by: Nicola Ticozzi, University of Milan, Italy; Caroline Vance, King's College London, United Kingdom

 *Correspondence: Zhang-Yu Zou fmuzzy@fjmu.edu.cn

This article was submitted to Dementia and Neurodegenerative Diseases, a section of the journal Frontiers in Neurology

†These authors have contributed equally to this work and share first authorship

 07 02 2022 2022 13 790082 06 10 2021 06 01 2022 Copyright © 2022 Feng, Lin, Che, Huang, Liu and Zou. 2022 Feng, Lin, Che, Huang, Liu and Zou

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Mutations in the valosin-containing protein (VCP) gene have been linked to amyotrophic lateral sclerosis (ALS) in the Caucasian populations. However, the phenotype of VCP mutations in Chinese patients with (ALS) remains unclear. Targeted next-generation sequencing covered 28 ALS-related genes including the VCP gene was undertaken to screen in a Chinese cohort of 275 sporadic ALS cases and 15 familial ALS pedigrees. An extensive literature review was performed to identify all patients with ALS carrying VCP mutations previously reported. The clinical characteristics and genetic features of ALS patients with VCP mutations were reviewed. One known p.R155C mutation in the VCP gene was detected in two siblings from a familial ALS pedigree and two sporadic individuals. In addition, the same VCP p.R155C mutation was detected in an additional patient with ALS referred in 2021. Three patients with VCP p.R155C mutation presented with muscular weakness starting from proximal extremities to distal extremities. The other patient developed a phenotype of Paget's disease of bone in addition to the progressive muscular atrophy. We reported the first VCP mutation carrier manifesting ALS with Paget's disease of bone in the Chinese population. Our findings expand the phenotypic spectrum of the VCP mutations in Chinese patients with ALS and suggest that ALS patients with VCP p.R155C mutations tend to present with relatively young onset, symmetrical involvement of proximal muscles weakness of arms or legs, and then progressed to distal muscles of limbs.

 amyotrophic lateral sclerosis Paget's disease of bone valosin-containing protein R155C phenotype 81671271 81974199 National Natural Science Foundation of China10.13039/501100001809 Introduction

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by motor dysfunction in limbs (muscle weakness, atrophy, and spasticity) and bulbar palsy, such as dysarthria and dysphagia. Cognitive decline or behavioral impairment occurs in some cases. Approximate two-thirds of patients with ALS had a limb onset. The typical disease course of ALS cases is aggressive, ending in death due to respiratory failure within 3–5 years (1).

Approximately 5–10% of ALS cases present in a familial pattern, while the others have no family history. The genetic background of ALS is complicated, correlated with a growing spectrum of genes, such as C9orf72, SOD1, FUS, and TDP-43 (1). Valosin-containing protein (VCP) gene codes a highly conserved triple A-adenosine triphosphatase (triple A-ATPase), which operates as a regulatory factor in the procedure of endoplasmic reticulum-associated degradation (2). When the triple A-ATPase has functional deficits, the ubiquitin-dependent recycling or degradation by the proteasome will be disrupted. Additionally, the process of membrane fusion, transcriptional activation, and apoptosis heavily relies on the VCP-coded ATPase (3). Mutations in the VCP gene have been determined as a causative gene of the syndrome, inclusion body myopathy (IBM) with Paget's disease of bone (PDB), and frontotemporal dementia (FTD) (IBMPFD) since 2001 (4). The clinical spectrum of VCP gene-related diseases was expanded to ALS when a four-generation Italian ALS pedigree with VCP mutation was detected by whole-exome sequencing in 2010 (5). Subsequent studies in Caucasian populations detected VCP mutations in both familial and sporadic patients with ALS (5, 6). Mutations in the VCP gene in patients with ALS of Chinese origin have been rarely reported (7). Here, we reported the phenotype of four patients with ALS carrying VCP mutation of Chinese origin.

 Materials and Methods Subjects

In total, a cohort of 275 sporadic ALS cases and 15 familial ALS pedigrees was recruited at Fujian Medical University Union Hospital and Henan Provincial People's Hospital between January 2017 and December 2018. Another sporadic ALS case referred to Fujian Medical University Union Hospital in 2021 was included. Diagnosis of ALS was made according to the revised El Escorial criteria (8). Familial ALS was diagnosed if one or more first- or second-degree relatives developed ALS. The study was approved by the Ethics Committee of Fujian Medical University Union Hospital and Henan Provincial People's Hospital. All subjects involved in this research have offered written consent.

 Genetic Studies

Genomic DNA extracted from venous peripheral blood lymphocytes of both sporadic cases and the proband of the family were subjected to the targeted next-generation sequencing on Illumina Hiseq sequencer (Illumina Inc., San Diego, CA, USA). An ALS-specific gene panel which included 28 genes (SOD1, FUS, TARDBP, VCP, VAPB, SPG11, OPTN, PFN1, ANG, ALS2, DAO, UBQLN2, SIGMAR1, SETX, FIG4, DCTN1, TUBA4A, TBK1, SQSTM1, CHCHD10, MATR3, HNRNPA1, HNRNPA2B1, KIF5A, ANXA11, TIA1, CCNF, and NEK1) was designed. The targeted regions were designed to include all exons and flanking regions of the 29 genes which contained the VCP gene (NM_007126.5). The GGGGCC expansions in C9orf72 were screened as previously described (9).

As a result of sequencing, the mean on-target coverage was 880 × with an average percentage of targets covered greater or equal to 100 × of 100%. Variant filtering process has been described (10). The identified variants were subsequently validated by Sanger sequencing. Bioinformatic analysis of the variants was performed as previously described (10).

 Literature Review

We conducted a literature search in Medline to identify previous studies that screened for VCP mutations in patients with ALS. The following keywords were used: “Valosin-containing protein” OR “VCP”, in combination with “amyotrophic lateral sclerosis” OR “ALS” OR “motor neuron(e) disease” OR “MND”. Only English language literature was included in the review. For each eligible publication, the following information was extracted: name of the first author, publication year, population, the sample size of familial ALS (FALS), and/or sporadic ALS (SALS), numbers of VCP mutation carriers in FALS and/or SALS. For patients with ALS carrying VCP mutations, the following information was extracted from the relevant papers: first author, year of study, population, sex, age, family history, clinical features, and genetic characteristics.

 Statistical Analysis

In each study, the mutation frequencies of the VCP gene were reported as the number of the mutation carriers among all cases of FALS or SALS screened. Single case reports, such as the additional VCP p.R155C mutated ALS referred to our center in 2021 were not included in the meta-analysis. The mutation frequencies in different populations were combined using a fixed-effects model. A statistical analysis was carried out using the Meta function of R (R version 3.64) (https://www.r-project.org/).

 Results Clinical Features of the ALS Cohort

Between January 2017 and December 2018, 275 sporadic cases and 15 familial pedigrees meeting the diagnostic criteria of ALS were enrolled in our study. There were 173 men and 117 women with mean onset age of 55.3 years (SD, 11.6). Of the total cases, 19.3% of patients reported a bulbar onset, 79.0% had a limb onset, and 1.7% had a respiratory onset.

 Genetic Analysis

One known heterozygous missense mutation in the VCP gene, c.463C>T (p.R155C) (Figure 1A) was identified in one familial ALS proband (III-6) and his affected sister (III-5) (Figure 1B), as well as another sporadic patient in the cohort of 275 sporadic ALS cases and 15 familial ALS pedigrees. The same VCP p.R155C mutation was detected in the additional patient referred to Fujian Medical University Union Hospital in 2021. No parental DNA samples of patient 3 and patient 4 were available for sequencing. No variants in other ALS-related genes were identified in these patients.

Genetic information of the valosin-containing protein (VCP) mutation in our study and overview of the VCP mutations associated with amyotrophic lateral sclerosis (ALS). (A) Sequencing chromatograms of the VCP p.R155C mutation. (B) The pedigree of the familial ALS with VCP p.R155C mutation. Arrow indicates the index patient. (C) Schematic graph of the VCP protein and overview of the VCP mutations linked to ALS. The locations of mutations are depicted in the mRNA structure where exons are numbered 1–17.

 Clinical Features of Patients With VCP p.R155C Mutation

Clinical features of the four patients carrying VCP p.R155C mutations in this study are summarized in Table 1.

Clinical features of the amyotrophic lateral sclerosis (ALS) patients with valosin-containing protein (VCP) p.R155C mutation in this study.

Case Gender Age of Site at Bulbar Phenotype Additional Disease Disease
onset (years) onset symptom symptoms progression (ΔALSFRS-R/m) duration (month)
Patient 1 III-6 Male 41 Lower limbs Yes Classic ALS No 0.2 >116 (alive)
Patient 2 III-5 Female 51 Lower limbs No Classic ALS No 0.2 >42 (alive)
Patient 3 Male 48 Lower limbs No PMA No 0.2 >102 (alive)
Patient 4 Male 44 Lower limbs No PMA PDB 0.2 >45 (alive)

ALSFRS-R, revised ALS functional rating scale; PDB, Paget's disease of bone; PMA, progressive muscular atrophy.

The familial ALS proband (III-6, Patient 1) was a 51-year-old male with progressive weakness in four limbs. At the age of 42 years, he complained of weakness in the lower limbs. He felt clumsy when climbing upstairs and unusual fatigue when walking a long distance. He began to have difficulties in uplifting his arms 4 years later. In the following years, he developed weakness of hands. No cognitive deficits or behavioral changes were noted. The neurologic examination at 7 years after onset revealed obvious weakness and atrophy of four limbs (MRC 4/5), more severe in the proximal muscles of limbs, with diffused reduction of deep tendon reflexes. Babinski's sign was elicited bilaterally. The sensory system and cognition were not affected. Serum creatine kinase (CK) level was normal. Electromyography (EMG) demonstrated fibrillations and positive sharp waves in muscles of four limbs and left thoracic paravertebral muscles, with muscle unit action potentials of increased amplitude, prolonged duration, and reduced recruitment in muscles of limbs. The ALS Functional Rating Scale-Revised (ALSFRS-R) score was 39/48. The score of Montreal Cognitive Assessment (MoCA) testing was 27/30. He developed exertional dyspnea 9 years after onset and began to use the non-invasive positive-pressure ventilation. At the last follow-up at 116 months after onset, he could still walk slowly with help and do daily life activities without assistance. He had neither dysarthria nor dysphagia. A repeated ALSFRS-R score was 30 with an estimated progression rate of 0.2 score/month since symptom onset. The sister of proband (III-5, Patient 2) had paraparesis in the legs since she was 51 years. She only had some trouble running and climbing upstairs. Neurological examination at 2 years after onset revealed mild atrophy and weakness of lower limbs (MRC 4/5). The deep tendon reflexes were brisk in four limbs. Hoffmann's sign and Babinski's sign were elicited bilaterally, with no sensory and bulbar involvement. Serum CK level was normal. EMG revealed acute and chronic neurogenic changes in all limbs and thoracic muscles. The ALSFRS-R score was 46/48. The score of MoCA testing was 28/30. At the last follow-up at 42 months after onset, she could still walk and go upstairs slowly with a cane, without the involvement of upper limbs. The ALSFRS-R score was 41 with an estimated progression rate of 0.2 score/month since symptom onset. Their father (II-2) died of acute myocardial infarction at the age of 72 years without evident symptoms of limb weakness, and their unaffected mother was still alive. The grandfather (I-1) of the proband exhibited signs of weakness in lower limbs and stayed bedridden for over 10 years before he died in his 70s. However, he was not formally diagnosed with ALS.

Patient 3 was a 57-year-old male who noticed weakness in the legs at the age of 48 years. Initially, he only had some difficulties in climbing stairs and standing up from a squatting position. In the following years, the weakness progressed distally to the feet and he began to have troubles in running and long-distance walking. Four years later, the patient was referred to physicians for his difficulty in lifting his arms and aggravated trouble of walking. There were no behavioral symptoms. Neurological examination 5 years after onset revealed obvious atrophy of muscles of arms and lower limbs with fasciculations. Deep tendon reflexes were decreased in all limbs. Palm-chin reflexes and Babinski's sign was not elicited. EMG indicated the pattern of neurogenic changes in muscles of four limbs, such as abnormal spontaneous potentials, motor unit potentials of prolonged duration, increased amplitude, and reduced recruitment. Serum CK level was 298 IU/L (normal value 22–270 IU/L). The biopsy sample of the biceps muscle demonstrated grouped atrophic fibers with both type I and type II fibers. He scored 42/48 on ALSFRS-R and 28/30 on MoCA testing. He began to use wheelchair 7 years after onset. At the last follow-up visit at 102 months after onset, he was bedridden, but still independent for daily life activities, such as writing, dressing, and eating meals, without bulbar involvement. The ALSFRS-R score was 31/48 with an estimated progression rate of 0.2 score/month since symptom onset. His parents were in their 80s and healthy.

Patient 4 presented with weakness of the left leg at the age of 44 years and felt clumsy when climbing upstairs. After 2 years, he developed weakness of the right leg. Muscle atrophy with fasciculation in lower limbs was noticed. Serum CK level was normal. Serum alkaline phosphatase level was 1,532 U/L (normal value 50–135 IU/L). Shoulder, pelvic, and lumbar spine radiographs showed osteolysis, osteosclerosis, and cortical thickening. An isotope bone scan showed increased tracer uptake in the affected bones. He did not suffer from bone pain. He was diagnosed with PDB and treated with zoledronic acid injection, calcitriol, and calcium carbonate. The serum alkaline phosphatase level decreased obviously to about 200 U/L. He gradually developed weakness of both arms and had difficulties lifting arms. Neurological examination at 33 months after onset revealed obvious muscle atrophy of arms and legs with fasciculations. Muscle strength was decreased in the upper limbs (MRC 4/5 in distal limbs and MRC 3/5 in proximal limbs) and the lower limbs (MRC 3/5 in distal limbs and MRC 4/5 in proximal limbs). Deep tendon reflexes were decreased in all limbs. Babinski and Hoffman's signs were not elicited. He was cognitively normal, with a score of 28/30 on MoCA testing. He scored 42/48 on ALSFRS-R. EMG demonstrated chronic and active denervation of the upper and lower limbs, rectus abdominis, and sternocleidomastoid muscles. At the last phone follow-up at 45 months after onset, he could still walk independently for 1 km and do daily life activities, such as writing, dressing, eating meals, and driving, without bulbar involvement. The ALSFRS-R score was 39/48 with an estimated progression rate of 0.2 score/month since symptom onset. Both parents of patient 4 were healthy in their 70s.

 Prevalence of VCP Mutations in Patients With ALS in Different Populations

We identified 44 studies screened VCP mutations in patients with FALS and/or SALS (Table 2). The frequency of VCP mutations is 0.08% (95% CI 0.00–1.26%) in patients with FALS and 0.02% (95% CI 0.00–0.15%) in patients with SALS in Chinese. The frequency of VCP mutations is 0.37% (95% CI 0.00–1.43%) in patients with FALS and 0.09% (95% CI 0.00–0.38%) in patients with SALS in Japanese. The frequency of VCP mutations is 0.28% (95% CI 0.12–0.52%) in patients with FALS and 0.08% (95% CI 0.03–0.15%) in patients with SALS in Caucasian populations. In pooled analysis, the frequency of VCP mutations is 0.28% (95% CI 0.12–0.50%) in patients with FALS and 0.06% (95% CI 0.02–0.12%) in patients with SALS (Table 2).

Summary of studies screened VCP variants in patients with ALS.

Race (origin) FALS FALS with VCP variants SALS SALS with VCP variants
Zou et al. (63) Chinese 20 0 324 0
Liu et al. (11) Chinese 20 0 234 0
Pang et al. (12) Chinese 4 0 46 1
Tsai et al. (13) Chinese 39 0 216 0
Zhang et al. (14) Chinese - - 311 0
Liu et al. (15) Chinese 24 0 - -
Chen et al. (16) Chinese 15 0 253 0
This study Chinese 15 1 275 1
Hirano et al. (17) Japanese - - 75 1
Nakamura et al. (18) Japanese 39 1 469 0
Nishiyama et al. (19) Japanese 111 0 - -
Naruse et al. (20) Japanese 89 1 410 1
Narain et al. (21) Indian 5 0 149 0
Johnson et al. (5) Caucasian 215 5 73 0
Williams et al. (22) Caucasian 131 0 48 0
Abramzon et al. (23) Caucasian - - 701 3
González-Pérez et al. (6) Israeli-Arab 274 5 178 0
Koppers et al. (24) Dutch 80 1 1,076 1
Miller et al. (25) British 75 0 101 0
Tiloca et al. (26) Italian 166 0 14 0
Kenna et al. (27) Irish 50 0 389 1
Le Ber et al. (28) French - - 26 0
Couthouis et al. (29) American - - 242 0
McCluskey et al. (30) American 20 1 - -
Cady et al. (31) American 42 1 349 0
Kwok et al. (32) British 102 2 90 0
Krüger et al. (33) German 6 0 74 1
Cooper-Knock et al. (34) British 42 0 - -
Gibson et al. (35) American - - 87 0
McCann et al. (36) Australian 212 0 - -
Morgan et al. (37) British 131 0 995 1
Türk et al. (38) German - - 43 0
Dols-Icardo et al. (39) Spainish 10 0 44 1
Lamp et al. (40) Italian 58 0 210 0
Müller et al. (41) German 301 1 - -
Mehta et al. (42) British 100 0 841 2
Tripolszki et al. (43) Hungarian 3 0 104 0
Kotan et al. (44) Turkish 10 1 45 0
Pensato et al. (45) Italian 34 1 179 3
Ungaro et al. (46) Italian 66 0 931 0
Yilmaz et al. (47) German, Swedish 418 0 - -
McCann et al. (48) Australian - - 616 0
Nunes Gonçalves et al. (49) Brazilian 93 1 - -
Shepheard et al. (50) British 7 0 93 0

ALS, amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; SALS, sporadic amyotrophic lateral sclerosis.

 Literature Review of the Phenotype of ALS Patients With VCP Mutations

In addition, 46 VCP mutated ALS patients with detailed clinical features in previous research were identified. The clinical characteristics of these patients are summarized in Table 3. The mean age at onset was 50.29 ± 10.55 years, ranging from 24 to 68 years. Most of the VCP-related patients with ALS were Caucasian (76.1%, 35/46), only 9 cases were of Asian origin (19.6%, 9/46). Thirty-seven patients (80.4%) had a family history of ALS, FTD, dementia, parkinsonism, or PDB, including 31 patients (67.4%) who had a family history of ALS. Thirty-two (69.6%) patients claimed that the weakness started from limbs, and only six 13.0% patients developed a bulbar-onset. Co-occurrence of FTD, PDB, parkinsonism, myopathy, or psychiatric diseases was reported in 18 (39.1%, 18/46) ALS cases carrying VCP mutations.

Summary of previously published VCP-causing ALS cases with detailed records.

Base Amino Exon Domain Race Family Gender Age of Site of Phenotype Additional Disease Reference
change acid change (origin) history onset (years) onset of ALS symptoms duration
c.179A>G p.K60R 3 CDC48 Italian No F <69 NA Classic ALS Cognitive impairment NA (45)
c.266G>A p.R89Q 3 CDC48 Chinese No M 24 Limbs Classic ALS No 5 months (7)
c.271A>T p.N91Y 3 CDC48 Brazilian Myopathy, FTD M 36 Limbs PMA No >4 years (alive) (51)
c.277C>T p.R93C 3 CDC48 Italian ALS, PBD, AD M 47 Lower limbs Classic ALS No >13 years (alive) (45)
c.293A>T p.D98V 3 CDC48 Japanese ALS M 58 Limbs (proximal right leg, distal right arm) Classic ALS FTD >10 years (alive) (52)
c.340A>G p.I114V 4 CDC48 Dutch Dementia F 52 Lower limbs (bilateral) Classic ALS No >119 months (alive) (24)
c.340A>G p.I114V 4 CDC48 Caucasian ALS NA 45 Upper limbs (distal bilateral) NA No 27 months (6)
NA p.G128A 4 CDC48 Mixed Caucasian ALS, PDB, PD, myopathy M 48 NA NA No NA (53)
c.451A>G p.I151V 5 CDC48 African- American No F 68 Lower limbs Classic ALS No 30 months (19 months to AV) (54)
c.463C>T p.R155C 5 CDC48 Italian No M 42 Limbs Classic ALS Myopathy NA (45)
c.463C>T p.R155C 5 CDC48 Italian ALS F 29 Upper limb (left hand) PMA No >11 years (alive) (55)
c.463C>T p.R155C 5 CDC48 Japanese ALS, FTD F 35 Upper limb (proximal right) Classic ALS No 5 months to AV, alive (52)
c.463C>T p.R155C 5 CDC48 American ALS, FTD, myopathy, PBD F 45 Lower limbs (proximal bilateral) Classic ALS Myopathy 3 years (56)
c.464G>A p.R155H 5 CDC48 Caucasian ALS, PBD, IBM, parkinsonism, dementia NA 53 Upper limb (distal left) NA No 39 months (5)
c.464G>A p.R155H 5 CDC48 Caucasian FTD, IBM, PBD, PD, psychiatric symptoms NA 63 Limbs Classic ALS No 21 years (6)
c.466G>T p.G156C 5 CDC48 Japanese ALS, psychiatric symptoms M 51 Lower limbs Classic ALS No >4 years (alive) (57)
c.466G>T p.G156C 5 CDC48 Japanese ALS F 34 Upper limbs Classic ALS Psychiatric symptoms 34 months (8 months to AV) (57)
c.472A>G p.M158V 5 CDC48 Japanese No M 36 Limbs (right) NA PDB 5 years (2 years to AV) (58)
c.475C>G p.R159G 5 CDC48 American ALS, PDB, dementia NA 53 Lower limbs Classic ALS Cognitive impairment 2 years to AV, alive (5)
c.475C>G p.R159G 5 CDC48 American ALS, dementia NA 46 Lower limbs NA PDB 5 years (5)
c.475C>T p.R159C 5 CDC48 American No F 68 Lower limbs Classic ALS No >5 years (alive) (23)
c.475C>T p.R159C 5 CDC48 Caucasian ALS, PDB NA 62 NA Classic ALS No 24 years (6)
c.475C>T p.R159C 5 CDC48 Caucasian ALS NA 57 Limbs Classic ALS PDB NA (6)
c.475C>T p.R159C 5 CDC48 Caucasian ALS, PDB NA 53 NA Classic ALS No 16 years (6)
c.475C>T p.R159C 5 CDC48 Caucasian ALS, PDB NA 53 NA Classic ALS No NA (6)
c.476G>A p.R159H 5 CDC48 Dutch FTD, MS F 59 Upper limbs (distal bilateral) Classic ALS No 23 months (24)
c.476G>A p.R159H 5 CDC48 Geek FTD, dementia, myopathy M 40s NA Classic ALS No >3 years (alive) (59)
c.571C>G p.R191G 5 Linker 1 Israeli-Arab ALS, myopathy, parkinsonism NA 50 Bulbar Classic ALS No 11 years (6)
c.571C>G p.R191G 5 Linker 1 Israeli-Arab ALS, myopathy, parkinsonism NA 42 Bulbar Classic ALS Myopathy 9 years to AV, alive (6)
c.571C>G p.R191G 5 Linker 1 Israeli-Arab ALS, myopathy, parkinsonism NA <45 Bulbar Classic ALS Myopathy, parkinsonism 9 years (6)
c.571C>G p.R191G 5 Linker 1 Israeli-Arab ALS, myopathy, parkinsonism NA <45 Bulbar Classic ALS Myopathy NA (6)
c.571C>G p.R191G 5 Linker 1 Israeli-Arab ALS, myopathy, parkinsonism NA <45 Bulbar Classic ALS Myopathy, parkinsonism 7 years to AV, alive (6)
c.572G>A p.R191Q 5 Linker 1 Italian ALS, FTD/dementia, parkinsonism, PDB NA 51 Upper limb (proximal right) Classic ALS No 29 months (11 months to AV) (5)
c.572G>A p.R191Q 5 Linker 1 Italian ALS, FTD/dementia, parkinsonism, PDB NA 53 Limbs (left) Classic ALS No 2 years to AV, alive (5)
c.572G>A p.R191Q 5 Linker 1 Italian ALS, FTD/dementia, parkinsonism, PDB NA 50 Right lower limb Classic ALS Cognitive impairment >54 months (alive) (5)
c.572G>A p.R191Q 5 Linker 1 Italian ALS, FTD, dementia, parkinsonism, PDB NA 37 Lower limbs (distal bilateral) Classic ALS No >4 years (alive) (5)
c.572G>A p.R191Q 5 Linker 1 Caucasian ALS M 42 Lower limb Classic ALS No >12 years (alive) (5)
c.572G>A p.R191Q 5 Linker 1 Japanese Demyelinating polyneuropathy, IBM M 53 Lower limbs (distal bilateral) Classic ALS No >1 years (alive) (52)
c.572G>C p.R191P 5 Linker 1 Turkish ALS, FTD F 60 Lower limb Classic ALS FTD NA (44)
c.572G>C p.R191P 5 Linker 1 Turkish ALS, FTD F 48 NA NA No NA (44)
c.572G>C p.R191P 5 Linker 1 Turkish ALS, FTD F 60 NA Classic ALS FTD NA (44)
c.1160A>C p.N387T 10 D1 Caucasian No M 57 Lower limb Classic ALS No >5 years (alive) (23)
c.1460G>A p.R487H 12 D2 Japanese FTD, PD M 61 Upper limbs (proximal bilateral) PMA Dementia 5 years to AV, alive (17)
c.1460G>A p.R487H 12 D2 Japanese ALS M 62 Left lower limb Pyramidal ALS FTD 78 months (60)
c.1774G>A p.D592N 14 D2 Caucasian ALS NA 52 Bulbar Classic ALS No <1 year (5)
c.1984C>T p.R662C 14 D2 Caucasian No M 67 Lower limb Classic ALS No >2 years (alive) (23)

ALS, amyotrophic lateral sclerosis; AV, assisted ventilation; FTD, frontotemporal dementia; MS, multiple sclerosis; NA, not available; PD, Parkinson disease; PDB, Paget's disease of bone; IBM, inclusion body myopathy; IMV, invasive mechanical ventilation.

 Discussion

In the present study, we reported the phenotype of four patients carrying the known VCP p.R155C mutation, such as two siblings from a familial ALS pedigree and a sporadic individual from a Chinese ALS cohort of 275 sporadic and 15 familial ALS pedigrees.

Mutations in the VCP gene have previously been identified in Caucasian patients with ALS (5, 6, 24, 27, 45, 61, 62). Our meta-analysis showed that the presence of VCP mutations was 0.28% (95% CI 0.12–0.52%) in FALS and 0.08% (95% CI 0.03–0.15%) in patients with SALS in the Caucasian populations. The prevalence of VCP mutations in the Asian populations has not been well determined since the published ALS-VCP cases were mostly of Japanese origin (17, 52, 57, 58, 60). A VCP p.R487H mutation was identified in one out of 75 Japanese patients with SALS (1.3%) (17), and a p.R155C mutation was detected in one out of 39 Japanese FALS pedigrees (2.6%) but no VCP mutations were found in 469 SALS individuals (18). Our meta-analysis showed that the presence of VCP mutations was 0.37% (95% CI 0.00–1.43%) in FALS and 0.09% (95% CI 0.00–0.38%) in patients with SALS in Japanese (17–20). No VCP mutations have been discovered among Chinese patients with ALS (11, 13–16, 63) except Pang SY et al. reported a p.G157R mutation in one out of 46 SALS (12) and a recent study identified a p.R89Q mutation in one SALS case in a cohort of 27 unrelated young-onset patients with ALS (7). The higher frequency of VCP mutations in FALS (6.7%, 1/15) and SALS (0.4%, 1/275) in our study may be due to the small sample. Our meta-analysis showed that the frequency of VCP mutations is 0.08% (95% CI 0.00–1.26%) in FALS and 0.02% (95% CI 0.00–0.15%) in SALS in Chinese, which is lower than Japanese and Caucasian populations. The overall pooled mutation frequency of VCP mutations is 0.28% (95% CI 0.12–0.50%) in patients with FALS and 0.06% (95% CI 0.02–0.12%) in patients with SALS.

Valosin-containing protein p.R155C mutation was first associated with ALS in 2012 (6), before which it was found in patients with IBMPFD (64). The mutation was subsequently observed in two ALS cases without FTD or PBD from a large cohort study of 190 individuals carrying VCP variants (61) and another survey on 36 families with diverse VCP mutants (62). The onset age of the VCP p.R155C mutated patients in our study ranged from 42 to 51 years, consistent with the onset age of four VCP p.R155C-mutated ALS cases reported previously (29, 35, 42, 45, respectively) (Table 3). It seems that patients carrying VCP p.R155C mutations tend to have a young onset. The three patients carrying VCP p.R155C mutation in our study all had a limb-onset. Our system review revealed that 69.6% of VCP-related ALS had limb-onset ALS, and only patients carrying VCP p.R191G and p.D592N mutation had bulbar onset (Table 3). Patient 1 and 2 in our study demonstrated a phenotype of ALS while patient 3 had a phenotype of progressive muscular atrophy (PMA). They all presented with rather symmetrical proximal muscle weakness in the lower limbs at onset and subsequently progressed to distal lower limbs and upper limbs. Patient 4 manifested weakness in the left leg and developed a phenotype of PMA. Interestingly, two VCP p.R155C mutated patients with ALS reported previously also presented with symmetrical involvement of proximal muscles weakness of arms or legs (52, 56). The system review of previous studies showed that patients carrying VCP p.R487H and p.R159G mutation demonstrated the same phenotype (5, 17). The four p.R155C mutation carriers in our study demonstrated a slow progression (ΔALSFRS-R/month = 0.2 score/month) and two had a long survival duration of more than 102 months and 116 months, respectively. The grandfather (I-1) of the proband also exhibited signs of weakness in lower limbs and stayed bedridden for over 10 years before he died in his 70s. An Italian ALS patient with VCP p.R155C mutation reported by Battistini et al. also had a survival of more than 11 years (55). However, a 35-year-old female with p.R155C mutation progressed rapidly and received tracheotomy positive-pressure ventilation within 5 months after onset (52). It is interesting that in our study the father of proband who was supposed to carry the VCP p.R155C mutation had no symptom of muscle weakness and atrophy before he died of acute myocardial infarction at the age of 72 years, indicated incomplete penetrance of VCP p.R155C mutation. Phenotypic variability in VCP p.R155C mutated ALS pedigree has been reported. In an Italian family, the proband with harboring VCP p.R155C mutation developed young-onset ALS with diffuse severe weakness and wasting in the limbs and rimmed vacuoles in muscle biopsy, while his mother and maternal aunt suffered from mild symptoms limited to lower limbs (45). Our system review showed that ALS patients with some VCP mutations (p.R93C, p.D98V, p.I114V, p.R155C, p.R155H, p.R159C, p.R191G, and p.R191Q) had a relatively slow progression and survival of more than 10 years (Table 3), which is consistent with the phenotype of our patients. However, some ALS patients with VCP mutations (p.R89Q, p.I114V, p.I151V, p.R155C, p.G156C, p.R159H, p.R191Q, and p.D592N) developed a rapid progression and had a survival of fewer than 3 years (Table 3). Phenotype variability associated with different or the same VCP mutation suggests the possible role of modifying genes and/or environmental factors. Extensive genetic studies in different populations to identify more ALS patients with VCP mutations may provide more insight into the genotype-phenotype correlations and the diversity of clinical phenotypes of VCP mutations.

Patient 4 carrying VCP p.R155C mutation developed a phenotype of PDB in addition to PMA. A VCP p.G97E mutation was reported in a Chinese family with IBMPFD without ALS (65). Patient 4 in our study was the first VCP mutation carrier manifesting PDB in addition to ALS in the Chinese population. The co-occurrence of FTD, PDB, parkinsonism, myopathy, or psychiatric diseases was commonly seen in the ALS cases carrying VCP mutations (Table 3). The co-existence of FTD or cognitive impairment was found in patients with seven VCP mutations (p.K60R, p.D98V, p.R155C, p.R159G, p.R191Q, p.R191P, and p.R487H) (5, 17, 44, 45, 52, 56, 60), while PDB was diagnosed in ALS patients with six VCP mutations (p.R93C, p.G128A, p.R155C, p.M158V, p.R159G, and p.R159C) (5, 6, 45, 53, 56, 58). Co-occurrence of myopathy was reported in ALS patients with VCP p.R155C and p.R191G mutations (6, 45, 56), and psychiatric disorders were found in patients with p.G156C and p.R159G mutations (5, 57). Parkinsonism is presented in familial ALS cases with VCP p.R191G mutation (6). Increasing evidence has shown that VCP-related disease may be a multisystem proteinopathy that has a wide clinical spectrum of IBM, FTD, PDB, and parkinsonism apart from ALS (66–68). The phenotypic diversity of the same VCP mutation may indicate the possible role of modifying genes and/or environmental factors.

Except for the hexanucleotide expansions and single base-pair substitutions in the 5' UTR or 3' UTR region of VCP predicted as pathogenic without definite experimental evidence (32), 26 mutations in 17 exons of the VCP gene have been identified in patients with ALS (Figure 1C). All mutations are heterozygous missense mutations. Residues of R155, R159, and R191 are three hot spots of VCP mutations of patients with ALS. VCP is divided into 4 domains, such as one N-terminal CDC48 region, two AAA ATPase domains (D1 and D2), and one C-terminal domain. VCP mutations are predominantly located within the N-terminal ubiquitin-binding domain (69.2%, 18/26), indicating that the malfunction of poly-ubiquitinated protein degradation may be the major pathogenesis of ALS (2). Within the ring-shaped VCP hexamer, the N-terminal domain serves as an indispensable binding region for interaction with target co-factor molecules (2). When pathogenic mutations occur in the N-domain of VCP protein, such as p.R155C, p.R159H, p.R95G, p.G97E, and p.A232E, the stress response is impaired resulting in the incorrect translocation of this hexameric ATPase and assembly disorder (69). Besides the aberrant aggregation of transactive response DNA-binding protein of 43 kDa (TDP-43) (5, 70), the nuclear-to-cytoplasmic mislocalization of fused in sarcoma (FUS) protein in motor neurons has been identified as another pathogenic feature of VCP-mutated ALS, which was ascribed to the increased intron retention (IR) in splicing factor proline and glutamine rich (SFPQ) transcripts (71, 72). Using ALS patient-specific induced pluripotent stem cell (iPSC) models, Patani et al. further unveiled that the four abnormal accumulated sequence-specific intron retention transcripts (IRTs) in VCP mutations included SFPQ, OGT, TUSC3, and DDX39, and their binding affinity to RNA binding proteins (RBPs) could be the key attributes for RBP localization (73, 74). In future, further experiments on pathomechanism of VCP-mutated ALS should be conducted on transgenic animal models as well as patient-specific iPSC models.

In summary, we reported the first VCP mutation carrier manifesting ALS with PDB of ALS in the Chinese population. Our findings expand the phenotypic spectrum of VCP mutations in Chinese patients with ALS and suggest that ALS patients with VCP p.R155C mutation tend to present with relatively young onset, symmetrical involvement of proximal muscles weakness of arms or legs, and then progressed to distal muscles of limbs.

 Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/, sra/PRJNA791140.

 Ethics Statement

The studies involving human participants were reviewed and approved by Ethics Committee of Fujian Medical Union Hospital and Henan Provincial People's Hospital. The patients/participants provided their written informed consent to participate in this study.

 Author Contributions

Z-YZ and S-YF designed and conceived the study. Z-YZ, S-YF, and HL performed the analysis of mutations in all the patients. HL wrote the manuscript. Z-YZ critically revised the manuscript. All remaining authors participated in the analysis of data, discussion of the final manuscript.

 Funding

This study was supported by grants from the National Natural Science Foundation of China (81671271 and 81974199) and Joint Funds for the Innovation of Science and Technology, Fujian province (2017Y9002).

 Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

We thank the patients and their families for their cooperation. Dr. Ton-Hai Dou from Shanghai Amplicongene Bioscience Co., Ltd., Shanghai, China, for kind help in the analysis of the sequencing data.

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