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CHCHD10 mutations in patients with amyotrophic lateral sclerosis in Mainland China
Accepted Manuscript CHCHD10 mutations in patients with amyotrophic lateral sclerosis in mainland China Shen Shen, Ji He, Lu Tang, Nan Zhang, Dongsheng Fan PII:
S0197-4580(17)30055-6
DOI:
10.1016/j.neurobiolaging.2017.02.011
Reference:
NBA 9850
To appear in:
Neurobiology of Aging
Received Date: 20 October 2016 Revised Date:
11 February 2017
Accepted Date: 16 February 2017
Please cite this article as: Shen, S., He, J., Tang, L., Zhang, N., Fan, D., CHCHD10 mutations in patients with amyotrophic lateral sclerosis in mainland China, Neurobiology of Aging (2017), doi: 10.1016/j.neurobiolaging.2017.02.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CHCHD10 mutations in patients with amyotrophic lateralMANUSCRIPT sclerosis in mainland China ACCEPTED
Shen Shen1 Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
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Ji He1 Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China Lu Tang
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Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
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Nan Zhang
Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China Dongsheng Fan*
1
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Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
ShenShen and Ji He contributed equally
*Corresponding Author:
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Dongsheng Fan (e-mail: [email protected]; Tel: +86 10 82266699; Fax: +86 10 62017700)
ACKNOWLEDGMENTS This work was supported by the National Natural Sciences Foundations of China [81030019, 81601105].
CONFLICT OF INTEREST STATEMENT None declared
ACCEPTED MANUSCRIPT
Abstract
Many genes have been found to be pathogenic for amyotrophic lateral sclerosis (ALS). Among them, the coiled-coil-helixcoiled-coil-helix domain containing 10 (CHCHD10) has been reported to play a controversial role in ALS. We examined the coding region of this gene in 424 unrelated Chinese sporadic ALS (SALS) subjects, 73 familial ALS (FALS) subjects, and
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204 healthy controls using a PCR-direct sequencing strategy. Two types of variants were identified, and of these, one variant (g.877C>T, p. P23L) was identified to be damaging, and the other one was (g.648G>A) in intron. The mutation (g.877C>T, p. P23L) has been previously reported in a Chinese frontotemporal dementia (FTD) patient. Our study is the first to report
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the clinical heterogeneity of specific mutations in CHCHD10 in ALS in an Asian population and to report the possible new
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mutation hotspot. Our findings support the major role of CHCHD10 in the FTD-ALS disease spectrum and stress the importance of mitochondrial abnormalities in the pathogenesis of diseases in Asian cohorts.
INTRODUCTION
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Keywords: amyotrophic lateral sclerosis, genetics, CHCHD10 mutation, clinical heterogeneity
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that selectively affects upper and lower motor
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neurons. The disease typically has a late onset, and the average survival expectancy is 3-5 years. The worldwide incidence
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of ALS is 0.3 to7.0 new cases per 100,000 individuals each year (Cronin et al., 2007). Nearly 10% of ALS cases are classified as familial ALS (FALS), in which a family history of the disease is identified. The remaining 90% of ALS cases are termed sporadic ALS (SALS). While approximately 60% of the familial cases can be explained by specific genes, the genetic aetiology of SALS cases remains uncertain. Although the exact pathogenesis and natural course of ALS are still unclear, mitochondrial dysfunction plays an important role in the pathogenesis of this disease (He et al., 2014). The CHCHD10 gene is located on chromosome 22q.11.23 and encodes a protein that is enriched at cristae junctions in mitochondria. This gene may play a role in mitochondrial cristae morphology, maintenance, or oxidative phosphorylation. Mutations of CHCHD10 have been reported to cause a large variety of neurological disorders, including those in the
frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy ACCEPTED MANUSCRIPT (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2).
In 2014, mutations of CHCHD10 were described to be the cause of a late-onset phenotype including motor neuron disease, cognitive decline resembling FTD, cerebellar ataxia, and myopathy in a large family (Bannwarthet al., 2014).
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Further analysis indicated that mutations were present in familial cases of FTD-ALS (Johnson et al., 2014). To date, approximately 20 different mutations of CHCHD10 have been shown to be associated with SALS and FALS in Asian and European patients. Mutation frequencies vary substantially among populations. The prevalence of CHCHD10 mutations has
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been reported to be 2.3% in German, 2.6% in French, 0.68% in Spanish, and 1% in Italian populations (Chaussenotet al.,
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2014; Chiò et al., 2015; Dols-Icardoet al., 2015; Mulleret al., 2014; Wonget al., 2015). However, in recent studies in China (including in the area of Taiwan), which is where most Asian studies have been conducted, the prevalence has been reported to be between 0% and 0.2% (Li et al., 2016; Tsai et al., 2016; Zhou et al., 2016). However, these studies were limited by
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small sample sizes, especially for the familial cases, in which only approximately 40 probands were included.
To assess the frequency of mutations of CHCHD10 in Chinese ALS patients, especially for familial cases, and to enhance the accuracy of our findings, we screened the clinical characteristics of patients with CHCHD10 variants in what is
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currently the largest cohort of Chinese ALS patients and control subjects of Chinese Han descent.
MATERIALS AND METHODS
Participants
This study included 424 SALS patients, 73 FALS patients, and 204 healthy controls. The patients were diagnosed with definite, probable, or possible ALS according to the E1 criteria (Brooks, 1994) at the ALS Clinic of Peking University Third Hospital from 2013 to 2014.All participants resided in mainland China. Detailed clinical features of the patients, including the age of onset, initial symptoms, and family history, were analysed. DNA samples were extracted from peripheral blood that was obtained from participants with their written informed consent. All patients and controls provided written informed
consent for the clinical and genetic studies during their visit to the neurologist. The Peking University Third Hospital ethics ACCEPTED MANUSCRIPT committee approved the collection of DNA samples from case and control subjects Genetic analyses To detect mutations of CHCHD10, all 4 coding exons (NM_213720) were sequenced. Primers were designed using
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Primer3Plus (http://www.primer3plus.com) and are listed in Supplementary Table 1. The assays were performed with an I-5 2X High-Fidelity Master Mix and JinpaiGreen (Tsingke, Beijing, China) according to the recommended protocol, and the PCR amplicons were sequenced using a BigDye Terminator v3.1 cycle sequencing kit on a 3730 ABI Prism Genetic
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Analyzer. A search was conducted for variants in the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000
http://exac.broadinstitute.org/).
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genomes project database (http://www.1000genomes.org/) and Exome Aggregation Consortium (ExAC,
The primer sequences and PCR conditions are shown in Supplementary Table 1. All patients who had mutations of the CHCHD10 gene were screened for mutations of the common ALS-related genes, including the C9orf72, SOD1, TARDBP,
Bioinformatics prediction
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FUS, DCTN1, TBK1, and SQSTM1 genes.
Mutationtaster (http://www.mutationtaster.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT
RESULTS
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(http://sift.jcvi.org/) were used to evaluate the potential effect of the identified missense mutations on protein structures.
In the presented cohorts, the mean ages of symptom onset inpatients with SALS and FALS were 51.29±11.87 and 43.72±12.10 years, respectively. Of the patients included in the study, 90(21.03%) patients with SALS and 12(16.44%) with FALS had bulbar site onsets. The ratios of male to female patients were 275:153 and 44:29 in the SALS and FALS groups, respectively. Additional demographic and clinical characteristics of the patients are presented in Table 1. Genetic findings Two variants were identified among the SALS cohort. They were predicted to be possibly damaging by several bio-
functional software programs and negatively screened in genetic datasets (including 1000genomes, ExAC and dbSNP) and ACCEPTED MANUSCRIPT the control group. One missense mutation (ID: 13095, g.877C>T, p. P23L) was present in a heterozygous state in a SALS patient. The P23L variant was predicted by the SIFT bioinformatics program to affect protein function, while it was predicted to be benign by PolyPhen-2. The other nucleotide change (g.648G>A) was identified in a heterozygous state in
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one SALS patient (ID:13308). The g.648G>A variant was found in an intron between exon 1 and 2. Though the variant of g.648G>A is not adjoined the exons (13 base pair away), the software (http://www.mutationtaster.org) predicted a possible damaging affection to the splicing function. No mutations were found in the common ALS-related genes, including the
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C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1 genes, in the patients with CHCHD10 variants. The remaining three
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variants detected were all included in the SNP database. We did not find significant differences in the minor allele frequencies (MAF) of the three SNPs between the patient and control groups (Table 2) or in other databases referenced. Clinical information
Patient 13095, who was revealed to have the missense mutation p.P23L, was a 36-year-old female who developed
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weakness in her left hand at 32 years of age. A neurological examination that was performed 13 months after the onset of symptoms revealed marked atrophy and weakness of the small muscles of the left hand, generalized hyperreflexia, and positive Babinski and Hoffman signs. A neurophysiological examination showed signs of active and chronic denervation in
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the cervical and lumbar regions. The patient gradually developed weakness in her three other limbs, dysarthria, and
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dysphagia during her disease duration of 4.3 years and was still alive with no manifestations of FTD at her last follow-up. Her family history was negative.
The g.648G>A variant was found in Patient 13308, a female who presented with upper limb weakness at the age of 52 years old and developed dysarthria and manifestations of progressive non fluent aphasia after the onset of symptoms. A neurological examination revealed tongue atrophy and fasciculation and weakness and hypotrophy of her upper limb muscles. Neurophysiological testing showed signs of active and chronic denervation in the cervical and bulbar regions. A detailed cognitive examination was performed to investigate dementia and verbal fluency, and progressive non fluent aphasia was identified in the patient. The patient died of the disease 4.5 years after onset. Her family history was also
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negative.
DISCUSSION
CHCHD10 encodes a mitochondrial protein that is enriched at cristae junctions in the intermembrane space.
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Alternative splicing of this gene leads to multiple transcript variants. The prevalence of CHCHD10 mutations in ALS patients in Chinese populations is generally lower than in European groups. Controversial results have been reported in Asian ALS cohorts; therefore, the role of CHCHD10 needs to be elucidated.
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The current study confirmed the significant role of CHCHD10 in the spectrum of FTD-ALS diseases, especially in
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Chinese cohorts. This study is also the first to report the clinical heterogeneity of specific mutations of CHCHD10 in an Asian population with ALS. One mutation (g.877C>T, p. P23L) was identified in our Chinese patient with ALS who had classic pyramidal damage, while the same mutation was independently identified in another aged Chinese patient with pure semantic dementia (Table 3). Combined with previous findings (Table 3), the relatively high prevalence of CHCHD10
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mutations at p. P23 (g.877C) might indicate that this is a new mutation hotspot. Findings across the FTD-ALS disease spectrum in the current study indicate the core role of CHCHD10 in the pathogenic mechanisms underlying ALS. Further
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biological studies investigating this hotspot are required.
As a limited numbers of carriers were identified, no significant and distinguishing clinical phenotypes of CHCHD10
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variants were reported. However, most CHCHD10 variant carriers were reported to have a longer survival time of more than 3–5 years. Patient 13095, with the (g.877C>T, p. P23L) mutation, had a very young onset age of 31 years old, which was significantly different than other patients (Table 3). The other patient (13308 with variant g.648G>A) with had classic motor symptoms combined with progressive non fluent aphasia, which is the first FTD-ALS case reported in an Asian patient with a CHCHD10 variant in intron. Although the prevalence of Chinese patients with FTD is higher, the low prevalence of CHCHD10 mutations was previously suggested to indicate that it was not associated with ALS in Chinese populations (Jiao et al., 2015; Li et al., 2016). The incidence of CHCHD10 in pure ALS is always low in Asian cohorts (ranging from 0.20 to 0.47%). The
prevalence of CHCHD10 mutations in SALS in the current study was approximately 0.24%(1/424), which is generally ACCEPTED MANUSCRIPT consistent with previous studies. However, when considering the disease spectrum of FTD-ALS, the incidence of mutations in CHCHD10 would obviously increase when combined with previous studies, especially because the incidence of FTD in Chinese cohorts is approximately 8% (Jiao et al., 2015). The same mutation (p. P23L) was identified across the spectrum of
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FTD-ALS diseases and confirms the important role of CHCHD10 in the FTD-ALS spectrum in Chinese populations. The current study failed to identify any disease-relevant variants in FALS, suggesting that mutations in CHCHD10 are not a common cause of FALS. However, the familial cases and probands recruited in this study mostly had pure ALS that
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would be useful for elucidation of the role of CHCHD10 in ALS.
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was not combined with cognitive disorders. Further examinations of FALS cases combined with cognitive dysfunction
Our genetic findings of rare variants did not replicate previous findings, especially for the mutations (including R15L, P34S, S59L, and G66V) primarily present in European patients. However, the mutations identified were all located in exon 2, which is consistent with previous findings. The difference in mutations might be caused by genetic differences in patients
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from different population of origin. In addition, the relatively small sample size limits the power for genetic exploration. To summarize, this is the first study involving CHCHD10 mutation screening in a large cohort of patients with ALS in Mainland China. Our findings support the major role of CHCHD10 in the disease spectrum of FTD-ALS, with particular
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emphasis on the importance of mitochondrial abnormalities in the pathogenesis in Asian cohorts. Combined with recent
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experimental evidence, the clinical heterogeneity in our findings across ALS and FTD suggests that pathogenic mechanisms involving CHCHD10 are complex in Chinese cohorts. Further identification of the role of CHCHD10 and mitochondria dysfunction in this disease could lead to novel preventive or therapeutic avenues of disease management of ALS in Asian populations.
ACCEPTED MANUSCRIPT References Bannwarth S, Ait-El-Mkadem S, Chaussenot A, Genin EC, Lacas-Gervais S, Fragaki K, Berg-Alonso L, Kageyama Y, Serre V, Moore DG, Verschueren A, Rouzier C, Le Ber I, Auge G, Cochaud C, Lespinasse F, N'Guyen K, de Septenville A, Brice A, Yu-Wai-Man P, Sesaki H, Pouget J, Paquis-Flucklinger V. A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement. Brain 2014;137:2329–45.
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Brooks BR. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. J NeurolSci 1994;124:96–107.
Chaussenot A, Ber I, Ait-El-Mkadem S, Camuzat A, Septenville A, Bannwarth S, Genin EC, Serre V, Augé G, Brice A, Pouget J, Paquis-Flucklinger V. Screening of CHCHD10 in a French cohort confirms the involvement of this gene
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in frontotemporal dementia with amyotrophic lateral sclerosis patients. Neurobiol Aging 2014;35:2884.e1-e4. Chiò A, Mora G, Sabatelli M, Caponnetto C, Traynor BJ, Johnson JO, Nalls MA, Calvo A, Moglia C, Borghero G, Monsurrò MR, Bella V, Volanti P, Simone I, Salvi F, Logullo FO, Nilo R, Battistini S, Mandrioli J, Tanel R, Murru MR,
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Mandich P, Zollino M, Conforti FL, Brunetti M, Barberis M, Restagno G, Penco S, Lunetta C. CHCH10 mutations in an Italian cohort of familial and sporadic amyotrophic lateral sclerosis patients. Neurobiol Aging 2015;36:1767.e3-e6.
Cronin S, Hardiman O, Traynor BJ. 2007. Ethnic variation in the incidence of ALS: a systematic review. Neurology 68(13):1002–7.
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Dols-Icardo O, Nebot I, Gorostidi A, Ortega-Cubero S, Hernández I, Rojas-García R, García-Redondo A, Povedano M, Lladó A, Álvarez V, Sánchez-Juan P, Pardo J, Jericó I, Vázquez-Costa J, Sevilla T, Cardona F, Indakoechea B, Moreno F, Fernández-Torrón R, Muñoz-Llahuna L, Moreno-Grau S, Rosende-Roca M, Vela Á, Muñoz-Blanco JL, Combarros O, Coto E, Alcolea D, Fortea J, Lleó A, Sánchez-Valle R, Esteban-Pérez J, Ruiz A, Pastor P, Munain A,
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Pérez-Tur J, Clarimón J. Analysis of theCHCHD10gene in patients with frontotemporal dementia and amyotrophic lateral sclerosis from Spain. Brain 2015;138:e400. He J, Mangelsdorf M, Fan D, Bartlett P, Brown MA. Amyotrophic lateral sclerosis genetic studies: from genome-wide
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association mapping to genome sequencing. Neuroscientist 2014;21:599–615. Jiao B, Xiao T, Hou L, Gu X, Zhou Y, Zhou L, Tang B, Xu J, Shen L. High prevalence ofCHCHD10mutation in patients with frontotemporal dementia from China. Brain 2015;139:e21. Johnson JO, Glynn SM, Gibbs JR, Nalls MA, Sabatelli M, Restagno G, Drory VE, Chiò A, Rogaeva E, Traynor BJ. Mutations in the CHCHD10 gene are a common cause of familial amyotrophic lateral sclerosis. Brain 2014; 137:e311 Li XL, Shu S, Li XG, Liu Q, Liu F, Cui B, Liu MS, Peng B, Cui LY, Zhang X. CHCHD10 is not a frequent causative gene in Chinese ALS patients. Amyotroph Lateral SclerFrontotemporalDegener 2016;17:458–60. Muller K, Andersen PM, Hubers A, Marroquin N, Volk AE, Danzer KM, Meitinger T, Ludolph AC, Strom TM, Weishaupt JH. Two novel mutations in conserved codons indicate that CHCHD10 is a gene associated with motor neuron disease. Brain 2014;137:e309.
Tsai P-C, Liu Y-C, Lin K-P, Liu Y-T, Liao Y-C, Hsiao C-T, Soong B-W, Yip P-K, Lee Y-C. Mutational analysis of TBK1 in
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Taiwanese patients with amyotrophic lateral sclerosis. Neurobiol Aging 2016;40:191.e11-e16. Wong CH, Topp S, Gkazi AS, Troakes C, Miller JW, Majo M, Kirby J, Shaw PJ, Morrison KE, Belleroche J, Vance CA, AlChalabi A, Al-Sarraj S, Shaw CE, Smith BN. The CHCHD10 P34S variant is not associated with ALS in a UK cohort of familial and sporadic patients. Neurobiol Aging 2015;36:2908.e17-e18. Zhou Q, Chen Y, Wei Q, Cao B, Wu Y, Zhao B, Ou R, Yang J, Chen X, Hadano S, Shang H-F. Mutation screening of the CHCHD10 gene in Chinese patients with Amyotrophic Lateral Sclerosis. MolNeurobiol 2016.doi:10.1007/s12035-
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ACCEPTED MANUSCRIPT Tables
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Table 1. Demographic and clinical characteristics of disease cases and controls. Variable FALS SALS CON Case, N 73 424 204 Sex, F (%) 29 (39.73) 153 (36.08) 98 (48.04) Mean Onset Age (years) 43.72±12.10 51.29±11.87 Site of Onset (bulbar, %) 12 (16.44) 90 (21.22) SALS, sporadic amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; CON, control subjects
ACCEPTED MANUSCRIPT Table 2. Single nucleotide polymorphisms identified in Chinese patients with ALS. Position
Base change (Alt/Ref)
Annotation
MAF in SALS
MAF in FALS
MAF in Con
rs9153
Exon 3
T/C
Synonymous
0.32
0.30
0.28
rs179468
Exon 2
C/A
Synonymous
0.13
0.17
0.10
rs183564385
Intron
A/G
Synonymous
0.012
0.011
0.09
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SNP ID
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MAF, minor allele frequency; SNP, single nucleotide polymorphism; Alt, alteration allele; Ref, reference allele; Con, controls; SALS, sporadic ALS; FALS, familial ALS
ACCEPTED MANUSCRIPT Table 3.Clinical phenotypes of p.P23 mutations of CHCHD10 in the FTD-ALS disease spectrum. Patient count
Amino acid change
Clinical phenotype
Familial history
Age of onset
Inherited model
Jiao et al. (2016)
1
p.P23S
FTD
Negative
66
Homozygous
Jiao et al. (2016)
1
p.P23L
FTD (semantic dementia)
Negative
52
Heterozygous
This study
1
p.P23L
ALS (no cognitive disorders)
Negative
31
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FTD: frontotemporal dementia; ALS: amyotrophic lateral sclerosis.
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Reference
Heterozygous
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Figure captions
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Fig. 1. Results of genetic analyses of patients.
A. Forward sequence chromatograms of portions of polymerase chain reaction (PCR) products show the heterozygousg.877C>T, p. P23L mutation in patient 13095 but not in the healthy control. B. Forward sequence chromatograms of portions of PCR products show a heterozygousg.648G>A mutation in patient 13308 but not in the
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healthy control.
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Highlights We examined the coding region of CHCHD10 gene in 424 unrelated Chinese sporadic
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ALS (SALS) subjects, 73 familial ALS (FALS) subjects, and 204 healthy controls. We found two types of variants, one was a missense mutation, and one might be a splicing point mutation. Our study is the first to report the clinical heterogeneity of specific mutations in CHCHD10 in ALS in an Asian population and to report the possible novel mutation hotspot.
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We declare no conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could
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inappropriately influence our work. Our institutions have not any contract relating to this research through which it or any other organization may stand to gain financially now or in the future. No
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other agreements of the authors or our institutions that could be
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seen as involving a financial interest in this work.
The data are not published or submitted elsewhere and we will not be submitted elsewhere while under consideration at
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Neurobiology of Aging.
All authors have reviewed the contents of the manuscript being
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the data.
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submitted, approved of its contents and validated the accuracy of
S0197-4580(17)30055-6
DOI:
10.1016/j.neurobiolaging.2017.02.011
Reference:
NBA 9850
To appear in:
Neurobiology of Aging
Received Date: 20 October 2016 Revised Date:
11 February 2017
Accepted Date: 16 February 2017
Please cite this article as: Shen, S., He, J., Tang, L., Zhang, N., Fan, D., CHCHD10 mutations in patients with amyotrophic lateral sclerosis in mainland China, Neurobiology of Aging (2017), doi: 10.1016/j.neurobiolaging.2017.02.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
CHCHD10 mutations in patients with amyotrophic lateralMANUSCRIPT sclerosis in mainland China ACCEPTED
Shen Shen1 Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
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Ji He1 Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China Lu Tang
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Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
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Nan Zhang
Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China Dongsheng Fan*
1
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Department of Neurology, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, China
ShenShen and Ji He contributed equally
*Corresponding Author:
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Dongsheng Fan (e-mail: [email protected]; Tel: +86 10 82266699; Fax: +86 10 62017700)
ACKNOWLEDGMENTS This work was supported by the National Natural Sciences Foundations of China [81030019, 81601105].
CONFLICT OF INTEREST STATEMENT None declared
ACCEPTED MANUSCRIPT
Abstract
Many genes have been found to be pathogenic for amyotrophic lateral sclerosis (ALS). Among them, the coiled-coil-helixcoiled-coil-helix domain containing 10 (CHCHD10) has been reported to play a controversial role in ALS. We examined the coding region of this gene in 424 unrelated Chinese sporadic ALS (SALS) subjects, 73 familial ALS (FALS) subjects, and
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204 healthy controls using a PCR-direct sequencing strategy. Two types of variants were identified, and of these, one variant (g.877C>T, p. P23L) was identified to be damaging, and the other one was (g.648G>A) in intron. The mutation (g.877C>T, p. P23L) has been previously reported in a Chinese frontotemporal dementia (FTD) patient. Our study is the first to report
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the clinical heterogeneity of specific mutations in CHCHD10 in ALS in an Asian population and to report the possible new
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mutation hotspot. Our findings support the major role of CHCHD10 in the FTD-ALS disease spectrum and stress the importance of mitochondrial abnormalities in the pathogenesis of diseases in Asian cohorts.
INTRODUCTION
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Keywords: amyotrophic lateral sclerosis, genetics, CHCHD10 mutation, clinical heterogeneity
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that selectively affects upper and lower motor
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neurons. The disease typically has a late onset, and the average survival expectancy is 3-5 years. The worldwide incidence
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of ALS is 0.3 to7.0 new cases per 100,000 individuals each year (Cronin et al., 2007). Nearly 10% of ALS cases are classified as familial ALS (FALS), in which a family history of the disease is identified. The remaining 90% of ALS cases are termed sporadic ALS (SALS). While approximately 60% of the familial cases can be explained by specific genes, the genetic aetiology of SALS cases remains uncertain. Although the exact pathogenesis and natural course of ALS are still unclear, mitochondrial dysfunction plays an important role in the pathogenesis of this disease (He et al., 2014). The CHCHD10 gene is located on chromosome 22q.11.23 and encodes a protein that is enriched at cristae junctions in mitochondria. This gene may play a role in mitochondrial cristae morphology, maintenance, or oxidative phosphorylation. Mutations of CHCHD10 have been reported to cause a large variety of neurological disorders, including those in the
frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy ACCEPTED MANUSCRIPT (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2).
In 2014, mutations of CHCHD10 were described to be the cause of a late-onset phenotype including motor neuron disease, cognitive decline resembling FTD, cerebellar ataxia, and myopathy in a large family (Bannwarthet al., 2014).
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Further analysis indicated that mutations were present in familial cases of FTD-ALS (Johnson et al., 2014). To date, approximately 20 different mutations of CHCHD10 have been shown to be associated with SALS and FALS in Asian and European patients. Mutation frequencies vary substantially among populations. The prevalence of CHCHD10 mutations has
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been reported to be 2.3% in German, 2.6% in French, 0.68% in Spanish, and 1% in Italian populations (Chaussenotet al.,
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2014; Chiò et al., 2015; Dols-Icardoet al., 2015; Mulleret al., 2014; Wonget al., 2015). However, in recent studies in China (including in the area of Taiwan), which is where most Asian studies have been conducted, the prevalence has been reported to be between 0% and 0.2% (Li et al., 2016; Tsai et al., 2016; Zhou et al., 2016). However, these studies were limited by
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small sample sizes, especially for the familial cases, in which only approximately 40 probands were included.
To assess the frequency of mutations of CHCHD10 in Chinese ALS patients, especially for familial cases, and to enhance the accuracy of our findings, we screened the clinical characteristics of patients with CHCHD10 variants in what is
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currently the largest cohort of Chinese ALS patients and control subjects of Chinese Han descent.
MATERIALS AND METHODS
Participants
This study included 424 SALS patients, 73 FALS patients, and 204 healthy controls. The patients were diagnosed with definite, probable, or possible ALS according to the E1 criteria (Brooks, 1994) at the ALS Clinic of Peking University Third Hospital from 2013 to 2014.All participants resided in mainland China. Detailed clinical features of the patients, including the age of onset, initial symptoms, and family history, were analysed. DNA samples were extracted from peripheral blood that was obtained from participants with their written informed consent. All patients and controls provided written informed
consent for the clinical and genetic studies during their visit to the neurologist. The Peking University Third Hospital ethics ACCEPTED MANUSCRIPT committee approved the collection of DNA samples from case and control subjects Genetic analyses To detect mutations of CHCHD10, all 4 coding exons (NM_213720) were sequenced. Primers were designed using
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Primer3Plus (http://www.primer3plus.com) and are listed in Supplementary Table 1. The assays were performed with an I-5 2X High-Fidelity Master Mix and JinpaiGreen (Tsingke, Beijing, China) according to the recommended protocol, and the PCR amplicons were sequenced using a BigDye Terminator v3.1 cycle sequencing kit on a 3730 ABI Prism Genetic
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Analyzer. A search was conducted for variants in the SNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000
http://exac.broadinstitute.org/).
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genomes project database (http://www.1000genomes.org/) and Exome Aggregation Consortium (ExAC,
The primer sequences and PCR conditions are shown in Supplementary Table 1. All patients who had mutations of the CHCHD10 gene were screened for mutations of the common ALS-related genes, including the C9orf72, SOD1, TARDBP,
Bioinformatics prediction
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FUS, DCTN1, TBK1, and SQSTM1 genes.
Mutationtaster (http://www.mutationtaster.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and SIFT
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(http://sift.jcvi.org/) were used to evaluate the potential effect of the identified missense mutations on protein structures.
In the presented cohorts, the mean ages of symptom onset inpatients with SALS and FALS were 51.29±11.87 and 43.72±12.10 years, respectively. Of the patients included in the study, 90(21.03%) patients with SALS and 12(16.44%) with FALS had bulbar site onsets. The ratios of male to female patients were 275:153 and 44:29 in the SALS and FALS groups, respectively. Additional demographic and clinical characteristics of the patients are presented in Table 1. Genetic findings Two variants were identified among the SALS cohort. They were predicted to be possibly damaging by several bio-
functional software programs and negatively screened in genetic datasets (including 1000genomes, ExAC and dbSNP) and ACCEPTED MANUSCRIPT the control group. One missense mutation (ID: 13095, g.877C>T, p. P23L) was present in a heterozygous state in a SALS patient. The P23L variant was predicted by the SIFT bioinformatics program to affect protein function, while it was predicted to be benign by PolyPhen-2. The other nucleotide change (g.648G>A) was identified in a heterozygous state in
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one SALS patient (ID:13308). The g.648G>A variant was found in an intron between exon 1 and 2. Though the variant of g.648G>A is not adjoined the exons (13 base pair away), the software (http://www.mutationtaster.org) predicted a possible damaging affection to the splicing function. No mutations were found in the common ALS-related genes, including the
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C9orf72, SOD1, TARDBP, FUS, PFN1, and SQSTM1 genes, in the patients with CHCHD10 variants. The remaining three
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variants detected were all included in the SNP database. We did not find significant differences in the minor allele frequencies (MAF) of the three SNPs between the patient and control groups (Table 2) or in other databases referenced. Clinical information
Patient 13095, who was revealed to have the missense mutation p.P23L, was a 36-year-old female who developed
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weakness in her left hand at 32 years of age. A neurological examination that was performed 13 months after the onset of symptoms revealed marked atrophy and weakness of the small muscles of the left hand, generalized hyperreflexia, and positive Babinski and Hoffman signs. A neurophysiological examination showed signs of active and chronic denervation in
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the cervical and lumbar regions. The patient gradually developed weakness in her three other limbs, dysarthria, and
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dysphagia during her disease duration of 4.3 years and was still alive with no manifestations of FTD at her last follow-up. Her family history was negative.
The g.648G>A variant was found in Patient 13308, a female who presented with upper limb weakness at the age of 52 years old and developed dysarthria and manifestations of progressive non fluent aphasia after the onset of symptoms. A neurological examination revealed tongue atrophy and fasciculation and weakness and hypotrophy of her upper limb muscles. Neurophysiological testing showed signs of active and chronic denervation in the cervical and bulbar regions. A detailed cognitive examination was performed to investigate dementia and verbal fluency, and progressive non fluent aphasia was identified in the patient. The patient died of the disease 4.5 years after onset. Her family history was also
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negative.
DISCUSSION
CHCHD10 encodes a mitochondrial protein that is enriched at cristae junctions in the intermembrane space.
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Alternative splicing of this gene leads to multiple transcript variants. The prevalence of CHCHD10 mutations in ALS patients in Chinese populations is generally lower than in European groups. Controversial results have been reported in Asian ALS cohorts; therefore, the role of CHCHD10 needs to be elucidated.
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The current study confirmed the significant role of CHCHD10 in the spectrum of FTD-ALS diseases, especially in
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Chinese cohorts. This study is also the first to report the clinical heterogeneity of specific mutations of CHCHD10 in an Asian population with ALS. One mutation (g.877C>T, p. P23L) was identified in our Chinese patient with ALS who had classic pyramidal damage, while the same mutation was independently identified in another aged Chinese patient with pure semantic dementia (Table 3). Combined with previous findings (Table 3), the relatively high prevalence of CHCHD10
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mutations at p. P23 (g.877C) might indicate that this is a new mutation hotspot. Findings across the FTD-ALS disease spectrum in the current study indicate the core role of CHCHD10 in the pathogenic mechanisms underlying ALS. Further
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biological studies investigating this hotspot are required.
As a limited numbers of carriers were identified, no significant and distinguishing clinical phenotypes of CHCHD10
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variants were reported. However, most CHCHD10 variant carriers were reported to have a longer survival time of more than 3–5 years. Patient 13095, with the (g.877C>T, p. P23L) mutation, had a very young onset age of 31 years old, which was significantly different than other patients (Table 3). The other patient (13308 with variant g.648G>A) with had classic motor symptoms combined with progressive non fluent aphasia, which is the first FTD-ALS case reported in an Asian patient with a CHCHD10 variant in intron. Although the prevalence of Chinese patients with FTD is higher, the low prevalence of CHCHD10 mutations was previously suggested to indicate that it was not associated with ALS in Chinese populations (Jiao et al., 2015; Li et al., 2016). The incidence of CHCHD10 in pure ALS is always low in Asian cohorts (ranging from 0.20 to 0.47%). The
prevalence of CHCHD10 mutations in SALS in the current study was approximately 0.24%(1/424), which is generally ACCEPTED MANUSCRIPT consistent with previous studies. However, when considering the disease spectrum of FTD-ALS, the incidence of mutations in CHCHD10 would obviously increase when combined with previous studies, especially because the incidence of FTD in Chinese cohorts is approximately 8% (Jiao et al., 2015). The same mutation (p. P23L) was identified across the spectrum of
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FTD-ALS diseases and confirms the important role of CHCHD10 in the FTD-ALS spectrum in Chinese populations. The current study failed to identify any disease-relevant variants in FALS, suggesting that mutations in CHCHD10 are not a common cause of FALS. However, the familial cases and probands recruited in this study mostly had pure ALS that
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would be useful for elucidation of the role of CHCHD10 in ALS.
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was not combined with cognitive disorders. Further examinations of FALS cases combined with cognitive dysfunction
Our genetic findings of rare variants did not replicate previous findings, especially for the mutations (including R15L, P34S, S59L, and G66V) primarily present in European patients. However, the mutations identified were all located in exon 2, which is consistent with previous findings. The difference in mutations might be caused by genetic differences in patients
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from different population of origin. In addition, the relatively small sample size limits the power for genetic exploration. To summarize, this is the first study involving CHCHD10 mutation screening in a large cohort of patients with ALS in Mainland China. Our findings support the major role of CHCHD10 in the disease spectrum of FTD-ALS, with particular
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emphasis on the importance of mitochondrial abnormalities in the pathogenesis in Asian cohorts. Combined with recent
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experimental evidence, the clinical heterogeneity in our findings across ALS and FTD suggests that pathogenic mechanisms involving CHCHD10 are complex in Chinese cohorts. Further identification of the role of CHCHD10 and mitochondria dysfunction in this disease could lead to novel preventive or therapeutic avenues of disease management of ALS in Asian populations.
ACCEPTED MANUSCRIPT References Bannwarth S, Ait-El-Mkadem S, Chaussenot A, Genin EC, Lacas-Gervais S, Fragaki K, Berg-Alonso L, Kageyama Y, Serre V, Moore DG, Verschueren A, Rouzier C, Le Ber I, Auge G, Cochaud C, Lespinasse F, N'Guyen K, de Septenville A, Brice A, Yu-Wai-Man P, Sesaki H, Pouget J, Paquis-Flucklinger V. A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement. Brain 2014;137:2329–45.
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Brooks BR. El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. J NeurolSci 1994;124:96–107.
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in frontotemporal dementia with amyotrophic lateral sclerosis patients. Neurobiol Aging 2014;35:2884.e1-e4. Chiò A, Mora G, Sabatelli M, Caponnetto C, Traynor BJ, Johnson JO, Nalls MA, Calvo A, Moglia C, Borghero G, Monsurrò MR, Bella V, Volanti P, Simone I, Salvi F, Logullo FO, Nilo R, Battistini S, Mandrioli J, Tanel R, Murru MR,
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Mandich P, Zollino M, Conforti FL, Brunetti M, Barberis M, Restagno G, Penco S, Lunetta C. CHCH10 mutations in an Italian cohort of familial and sporadic amyotrophic lateral sclerosis patients. Neurobiol Aging 2015;36:1767.e3-e6.
Cronin S, Hardiman O, Traynor BJ. 2007. Ethnic variation in the incidence of ALS: a systematic review. Neurology 68(13):1002–7.
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Dols-Icardo O, Nebot I, Gorostidi A, Ortega-Cubero S, Hernández I, Rojas-García R, García-Redondo A, Povedano M, Lladó A, Álvarez V, Sánchez-Juan P, Pardo J, Jericó I, Vázquez-Costa J, Sevilla T, Cardona F, Indakoechea B, Moreno F, Fernández-Torrón R, Muñoz-Llahuna L, Moreno-Grau S, Rosende-Roca M, Vela Á, Muñoz-Blanco JL, Combarros O, Coto E, Alcolea D, Fortea J, Lleó A, Sánchez-Valle R, Esteban-Pérez J, Ruiz A, Pastor P, Munain A,
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Pérez-Tur J, Clarimón J. Analysis of theCHCHD10gene in patients with frontotemporal dementia and amyotrophic lateral sclerosis from Spain. Brain 2015;138:e400. He J, Mangelsdorf M, Fan D, Bartlett P, Brown MA. Amyotrophic lateral sclerosis genetic studies: from genome-wide
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association mapping to genome sequencing. Neuroscientist 2014;21:599–615. Jiao B, Xiao T, Hou L, Gu X, Zhou Y, Zhou L, Tang B, Xu J, Shen L. High prevalence ofCHCHD10mutation in patients with frontotemporal dementia from China. Brain 2015;139:e21. Johnson JO, Glynn SM, Gibbs JR, Nalls MA, Sabatelli M, Restagno G, Drory VE, Chiò A, Rogaeva E, Traynor BJ. Mutations in the CHCHD10 gene are a common cause of familial amyotrophic lateral sclerosis. Brain 2014; 137:e311 Li XL, Shu S, Li XG, Liu Q, Liu F, Cui B, Liu MS, Peng B, Cui LY, Zhang X. CHCHD10 is not a frequent causative gene in Chinese ALS patients. Amyotroph Lateral SclerFrontotemporalDegener 2016;17:458–60. Muller K, Andersen PM, Hubers A, Marroquin N, Volk AE, Danzer KM, Meitinger T, Ludolph AC, Strom TM, Weishaupt JH. Two novel mutations in conserved codons indicate that CHCHD10 is a gene associated with motor neuron disease. Brain 2014;137:e309.
Tsai P-C, Liu Y-C, Lin K-P, Liu Y-T, Liao Y-C, Hsiao C-T, Soong B-W, Yip P-K, Lee Y-C. Mutational analysis of TBK1 in
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Taiwanese patients with amyotrophic lateral sclerosis. Neurobiol Aging 2016;40:191.e11-e16. Wong CH, Topp S, Gkazi AS, Troakes C, Miller JW, Majo M, Kirby J, Shaw PJ, Morrison KE, Belleroche J, Vance CA, AlChalabi A, Al-Sarraj S, Shaw CE, Smith BN. The CHCHD10 P34S variant is not associated with ALS in a UK cohort of familial and sporadic patients. Neurobiol Aging 2015;36:2908.e17-e18. Zhou Q, Chen Y, Wei Q, Cao B, Wu Y, Zhao B, Ou R, Yang J, Chen X, Hadano S, Shang H-F. Mutation screening of the CHCHD10 gene in Chinese patients with Amyotrophic Lateral Sclerosis. MolNeurobiol 2016.doi:10.1007/s12035-
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Table 1. Demographic and clinical characteristics of disease cases and controls. Variable FALS SALS CON Case, N 73 424 204 Sex, F (%) 29 (39.73) 153 (36.08) 98 (48.04) Mean Onset Age (years) 43.72±12.10 51.29±11.87 Site of Onset (bulbar, %) 12 (16.44) 90 (21.22) SALS, sporadic amyotrophic lateral sclerosis; FALS, familial amyotrophic lateral sclerosis; CON, control subjects
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Base change (Alt/Ref)
Annotation
MAF in SALS
MAF in FALS
MAF in Con
rs9153
Exon 3
T/C
Synonymous
0.32
0.30
0.28
rs179468
Exon 2
C/A
Synonymous
0.13
0.17
0.10
rs183564385
Intron
A/G
Synonymous
0.012
0.011
0.09
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SNP ID
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MAF, minor allele frequency; SNP, single nucleotide polymorphism; Alt, alteration allele; Ref, reference allele; Con, controls; SALS, sporadic ALS; FALS, familial ALS
ACCEPTED MANUSCRIPT Table 3.Clinical phenotypes of p.P23 mutations of CHCHD10 in the FTD-ALS disease spectrum. Patient count
Amino acid change
Clinical phenotype
Familial history
Age of onset
Inherited model
Jiao et al. (2016)
1
p.P23S
FTD
Negative
66
Homozygous
Jiao et al. (2016)
1
p.P23L
FTD (semantic dementia)
Negative
52
Heterozygous
This study
1
p.P23L
ALS (no cognitive disorders)
Negative
31
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FTD: frontotemporal dementia; ALS: amyotrophic lateral sclerosis.
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Reference
Heterozygous
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Figure captions
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Fig. 1. Results of genetic analyses of patients.
A. Forward sequence chromatograms of portions of polymerase chain reaction (PCR) products show the heterozygousg.877C>T, p. P23L mutation in patient 13095 but not in the healthy control. B. Forward sequence chromatograms of portions of PCR products show a heterozygousg.648G>A mutation in patient 13308 but not in the
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healthy control.
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Highlights We examined the coding region of CHCHD10 gene in 424 unrelated Chinese sporadic
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ALS (SALS) subjects, 73 familial ALS (FALS) subjects, and 204 healthy controls. We found two types of variants, one was a missense mutation, and one might be a splicing point mutation. Our study is the first to report the clinical heterogeneity of specific mutations in CHCHD10 in ALS in an Asian population and to report the possible novel mutation hotspot.
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We declare no conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work submitted that could
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inappropriately influence our work. Our institutions have not any contract relating to this research through which it or any other organization may stand to gain financially now or in the future. No
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other agreements of the authors or our institutions that could be
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seen as involving a financial interest in this work.
The data are not published or submitted elsewhere and we will not be submitted elsewhere while under consideration at
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Neurobiology of Aging.
All authors have reviewed the contents of the manuscript being
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the data.
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submitted, approved of its contents and validated the accuracy of