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June 16, 2015; 84 (24) Article

A SIGMAR1 splice-site mutation causes distal hereditary motor neuropathy

Xiaobo Li, Zhengmao Hu, Lei Liu, Yongzhi Xie, Yajing Zhan, Xiaohong Zi, Junling Wang, Lixiang Wu, Kun Xia, Beisha Tang, Ruxu Zhang
First published May 15, 2015, DOI: https://doi.org/10.1212/WNL.0000000000001680
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Abstract

Objective: To identify the underlying genetic cause in a consanguineous Chinese family segregating distal hereditary motor neuropathy (dHMN) in an autosomal recessive pattern.

Methods: We used whole-exome sequencing and homozygosity mapping to detect the genetic variant in 2 affected individuals of the consanguineous Chinese family with dHMN. RNA analysis of peripheral blood leukocytes and immunofluorescence and immunoblotting of stable cell lines were performed to support the pathogenicity of the identified mutation.

Results: We identified 3 shared novel homozygous variants in 3 shared homozygous regions of the affected individuals. Sequencing of these 3 variants in family members revealed the c.151+1G>T mutation in SIGMAR1 gene, which located in homozygous region spanning approximately 5.3 Mb at chromosome 9p13.1-p13.3, segregated with the dHMN phenotype. The mutation causes an alternative splicing event and generates a transcript variant with an in-frame deletion of 60 base pairs in exon 1 (c.92_151del), and results in an internally shortened protein σ1R31_50del. The proteasomal inhibitor treatment increased the intracellular amount of σ1R31_50del and led to the formation of nuclear aggregates. Stable expressing σ1R31_50del induced endoplasmic reticulum stress and enhanced apoptosis.

Conclusion: The homozygous c.151+1G>T mutation in SIGMAR1 caused a novel form of autosomal recessive dHMN in a Chinese consanguineous family. Endoplasmic reticulum stress may have a role in the pathogenesis of dHMN.

GLOSSARY

ALS=
amyotrophic lateral sclerosis;
cDNA=
complementary DNA;
CMT=
Charcot-Marie-Tooth;
dHMN=
distal hereditary motor neuropathy;
ER=
endoplasmic reticulum;
ERAD=
endoplasmic reticulum–associated degradation;
GFP=
green fluorescent protein;
HEK293=
human embryonic kidney 293;
MN=
motor neuron;
SNP=
single-nucleotide polymorphism;
TDP-43=
TAR DNA-binding protein 43;
WES=
whole-exome sequencing

Distal hereditary motor neuropathies (dHMN), also called spinal Charcot-Marie-Tooth (CMT) diseases, form a clinically and genetically heterogeneous group of inherited diseases that share the common feature of progressive distal muscle weakness and wasting without sensory abnormalities.1 To date, at least 19 causative genes and 4 loci have been identified with autosomal dominant, recessive, or X-linked inheritance (http://neuromuscular.wustl.edu/). Autosomal recessive dHMN can be due to mutations in IGHMBP2, PLEKHG5, HINT1, HSPB1, and HSJ1 genes, and has also been linked to 9p21.1-p12 (HMN-Jerash) and 11q13.3 (normal IGHMBP2 gene).2,–,6 However, it is estimated that more than 80% of patients with dHMN have mutations in undiscovered genes.

SIGMAR1, also termed σ1R, is a nonopioid endoplasmic reticulum (ER) protein with a molecular mass of 25 kDa and is involved in a large diversity of cell functions including the ER stress response, ion channel regulation, synaptogenesis, and neuronal plasticity.7,8 SIGMAR1 is expressed ubiquitously in both central and peripheral nervous systems, but is enriched in α motor neurons (MNs) of the brainstem and spinal cord. At the subcellular level, it is predominantly localized in cholinergic postsynaptic densities of MNs termed C-terminals.9 The homozygous p.E102Q mutation in SIGMAR1 has been associated with juvenile amyotrophic lateral sclerosis 16 (ALS16), and the 3′UTR heterozygous point mutations in this gene might be involved in frontotemporal lobar degeneration–MN disease.10,11 However, no reported study linked SIGMAR1 mutations to the dHMN phenotype.

In the present study, we report a consanguineous Chinese family with dHMN caused by a novel splice-site mutation in the SIGMAR1 gene, and propose that the ER stress caused by the mutant σ1R31_50del may have a role in the pathogenesis of dHMN.

METHODS

Standard protocol approvals, registrations, and patient consents.

All individuals involved in the study signed informed written consent before enrollment. This study was approved by the ethics committee of the Third Xiangya Hospital of Central South University.

Family material and clinical evaluation.

A consanguineous Chinese family with 3 affected siblings and 500 Chinese healthy controls participated in our study. The representative pedigree of the family is shown in figure 1. Twelve family members including the affected individuals were comprehensively checked by 2 experienced neurologists. The nerve electrophysiologic examination, EMG examination, and sural nerve biopsy followed by semithin staining were performed on the proband. A brain and whole-spine MRI, and routine hematologic and biochemical examinations including creatine kinase level were also performed on the proband. Ten-milliliter venous blood was obtained from all participants for genetic analysis.

Figure 1
Figure 1 Pedigree of the Chinese family with distal hereditary motor neuropathy

Black boxes indicate the patients affected. The arrow indicates the proband (patient IV:5). Patients IV:1 and IV:5 underwent whole-exome sequencing.

Whole-exome sequencing and homozygosity mapping.

We performed whole-exome sequencing (WES) on 2 affected individuals (IV:1 and IV:5) using the Agilent SureSelect Human All Exon 50-Mb kit (Agilent, Santa Clara, CA). The exome libraries were prepared with an Illumina Paired-End DNA Sample Preparation kit. A total of 90–base pair paired-end reads were obtained by sequencing on a HiSeq2000 (Illumina, San Diego, CA). The coverage of the targeted regions was more than 99%, and the mean depth of the targeted regions was more than 47-fold. All sequenced reads were mapped to the human genome reference (UCSC hg19) with the SOAPaligner.12 Single-nucleotide polymorphisms (SNPs) and insertions-deletions were detected by using SOAPsnp software and the BWA (http://bio-bwa.sourceforge.net/), respectively.13 Nonsynonymous variants, splice acceptor and donor site variants, and insertions-deletions were filtered against the dbSNP129, HapMap 8, and 1000 Genome Project. Synonymous changes were identified and removed from the variant list using SIFT software. The selection of homozygous regions met the following parameters: 500 of marker-windows' size (allowing 2 heterozygous markers per window), 1 Mb of the minimal length of homozygous regions, and 500 kb of the maximal distance of adjacent homozygous regions. Candidate variants were confirmed with Sanger sequencing.

Transcript study.

Total RNA was extracted from peripheral blood lymphocytes of 2 affected individuals (IV:1 and IV:5), 1 carrier subject (III:4), and 1 healthy family member (IV:4), and from 1 healthy frontal brain tissue supplied by the Chinese Brain Bank Center using TRIZOL (Ambion/Life Technologies). The complementary DNAs (cDNAs) were obtained using standard protocol. A region between exon 1 and exon 4 of the cDNA of SIGMAR1 was amplified by PCR using primers SIGMAR1-1F (5′-GTGCTGACCCAGGTCGTC-3′) and SIGMAR1-4R (5′-CCAAAGAGGTAGGTGGTGAGC-3′). The PCR products were analyzed by agarose gel electrophoresis and Sanger sequencing.

Plasmid construction, cell culture, transfection, and stable cell line generation.

The full length and c.92_151del shortened SIGMAR1 sequence were amplified by PCR of the cDNAs of IV:4 and IV:5 using primers SIGMAR1-F (5′-CCGGAATTCATGCAGTGGGCCGTGGG-3′) and SIGMAR1-R (5′-CGCGGATCCACAGGGTCCTGGCCAAAGAGGT-3′) and transferred into pEGFP-N1 vectors in the sense orientation. Identity with the reference sequence (NM 005866.3) and the c.92_151del were confirmed by Sanger sequencing. Human embryonic kidney 293 (HEK293) cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HEK293 cells were grown in Dulbecco minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum in a 5% CO2 incubator at 37°C. Cells grown on 10-cm culture plates were transfected with expression vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Twenty-four hours after transfection, the medium was exchanged for medium containing 800 µg/mL G418. Ten days after growing under selective pressure, cells were serially diluted in 96-well culture plates and continued to grow under selection. Single colonies were then isolated and expanded. Positive colonies were confirmed by immunofluorescence and immunoblotting.

Immunofluorescence.

Cells were cultured on glass coverslips for 24 hours, then treated with MG132 (Sigma, St. Louis, MO) or dimethyl sulfoxide for 6 hours at a final concentration of 5 µmol/L. For immunostaining, the cells were fixed with 4% paraformaldehyde, stained with rabbit anti-calnexin polyclonal antibody (1:100, Sigma), and detected with Cy3-conjugated secondary antibody. Cell nuclei were stained with DAPI (Invitrogen). Images were taken under a Leica confocal microscope with appropriate excitation and emission filter pairs.

Immunoblotting.

Protein was isolated from stable cell lines using standard protocols. After separating on a sodium dodecyl sulfate–polyacrylamide gel, samples were transferred to polyvinylidene fluoride membrane. The membranes were incubated for 1 hour in blocking solution (5% nonfat dry milk in 0.1% Triton X-100/phosphate-buffered saline buffer), followed by incubation with the appropriate primary antibodies in blocking solution overnight. After washing in 0.1% Triton X-100/phosphate-buffered saline buffer, the membrane was incubated with the appropriate secondary antibodies for 1 hour and visualized via an enhanced chemiluminescence kit (GE Healthcare, Waukesha, WI) according to the manufacturer's instruction. Rabbit anti-σ1R (1:125; Abcam, Cambridge, MA), which recognizes the C-terminal of σ1R, anti-Bip (1:1,000; Cell Signaling Technology, Danvers, MA), anti-β-actin (1:1,000; Cell Signaling Technology), and mouse anti-GFP (1:4,000; Clontech, Mountain View, CA) were used as primary antibodies. Horseradish peroxidase–conjugated anti-mouse or anti-rabbit immunoglobulin G (1:10,000; Jackson ImmunoResearch, West Grove, PA) was used as secondary antibody. The difference in protein levels between samples was normalized using β-actin levels. Data were analyzed using Image J software (NIH, Bethesda, MD) and Prism 5.0 (GraphPad Software Inc., San Diego, CA).

Flow cytometry assay.

HEK293 cells stably expressing σ1R or σ1R31_50del were stained with an Annexin V Apoptosis Detection Kit APC (eBioscience, San Diego, CA) according to the manufacturer's instruction and detected using a BD FACSCanto II kit (BD Biosciences, San Jose, CA) within 1 hour. Experiments were performed in triplicate.

Statistical analysis.

Values are represented as means ± SEM. The data were analyzed by 2-sample t test. A p value <0.05 was considered statistically significant.

RESULTS

Clinical assessment.

The proband (IV:5) had insidious wasting and weakness of distal lower limbs involving the ankle dorsiflexors more than the plantar flexors and the evertors more than the invertors, and developed foot drop and pes varus at the age of 10 years. Progressive wasting of distal upper limbs and disability to straighten his fingers became noted at 13 years. His condition stopped deterioration after the age of 20. Although with difficulty, he functioned independently when examined at the age of 30. Physical examination revealed symmetrically severe to moderate muscle wasting and weakness in distal lower and upper extremities, normal muscle tension, absence of fasciculation, brisk knee reflexes, absence of ankle reflexes, positive Babinski signs, normal sensory examination including pain sensation, touch sensation, temperature sensation, position sensation, and vibration sensation, claw hands, hammer toes, and pes varus. Representative pictures of the distal limbs are shown in figure 2. The motor nerve conduction velocities moderately reduced and the amplitudes of compound muscle action potentials severely decreased. The sensory nerve conduction velocities and the sensory nerve action potentials were in the normal range. Detailed nerve electrophysiologic data are listed in table 1. Needle EMG revealed distal denervation featured by large motor unit potentials and reduced recruitment on voluntary contraction recorded in distal limbs. The sural nerve biopsy was within normal limits (figure e-1 on the Neurology® Web site at Neurology.org). The brain and whole spinal MRI scanning was normal. The serum creatine kinase level was also at normal level.

Figure 2
Figure 2 Photographs of the Chinese proband with dHMN (IV:5)

Photographs of the proband with dHMN demonstrating severe muscle wasting in the legs and moderate muscle wasting in the hands. dHMN = distal hereditary motor neuropathy.

View this table:
Table 1

Clinical features of the affected individuals: IV:1, IV:3, and IV:5

Patient IV:1 presented with progressive distal wasting and weakness of the lower limbs and developed foot drop and pes varus at the age of 12 years. Involvement of the upper limbs became noted in 3 years. He had accepted right and left ankle arthrodesis at the age of 17 and 18, respectively. His condition stopped deterioration after the orthopedic procedures. He functioned independently when examined at the age of 42. Physical examination revealed symmetrically moderate to mild muscle wasting and weakness in distal lower and upper extremities, normal muscle tension, absence of fasciculation, brisk knee reflexes, absence of ankle reflexes, positive Babinski signs, normal sensation of all modalities, and hammer toes.

Patient IV:3 presented with progressive distal wasting and weakness of the lower limbs and developed foot drop and pes varus at the age of 9 years. Involvement of the distal upper limbs became noted in 2 years. She had accepted right and left ankle arthrodesis at the age of 15 and 16, respectively. Her condition stopped deterioration after the age of 20. She functioned independently when examined at the age of 37. Physical examination revealed symmetrically moderate to mild muscle wasting and weakness in distal lower and upper extremities, normal muscle tension, absence of fasciculation, brisk knee reflexes, absence of ankle reflexes, normal sensation of all modalities, and hammer toes.

Affected individuals showed no delay in developmental milestone achievements. Their clinical manifestations are summarized in table 1.

Genetic analysis.

Neither a homozygous nor heterozygous nucleotide variant was detected in the exons of known causative genes of dHMN. The statistic data of WES and homozygosity mapping are shown in table e-1. After filtering all of the conditions, we identified 3 shared homozygous variants in 3 shared homozygous regions located at chromosome 9: c.151+1G>T in SIGMAR1 in position 33797681-39103742 (length: 5.3 Mb), c.2036G>A in DAPK1 in position 89560863-92221114 (length: 2.7 Mb), and c.935G>A in ROR2 in position 93375457-94841979 (length: 1.5 Mb). Using Sanger sequencing, we detected the heterozygous c.2036G>A variation in DAPK1 and normal c.935G>A in ROR2 in patient IV:3, which indicated these 2 variants did not segregate with the dHMN phenotype. However, c.151+1G>T in SIGMAR1 segregated perfectly with the phenotype in the family members. All affected individuals (IV:1, IV:3, and IV:5) harbored the homozygous c.151+1G>T variant, while their parents and children were heterozygous carriers with normal phenotype. The variant was absent in 500 Chinese healthy controls and thus considered to be the pathogenic mutation. The genomic DNA sequence chromatograms of SIGMAR1 are shown in figure 3A. To assess the impact of the mutation at transcriptional level, the reverse transcription–PCR products were analyzed by agarose gel electrophoresis and sequencing. We found that the c.151+1G>T mutation resulted in a transcript variant with an in-frame deletion of 60 base pairs in exon 1 (c.92_151del), which predicted an internally shortened SIGMAR1 protein with deletion of 20 amino acids (p.31_50del) (see figure 3, B–E).

Figure 3
Figure 3 The c.151G>T mutation of SIGMAR1

(A) Chromatograms of the SIGMAR1 gene. The carrier and the patient harbored the heterozygous and homozygous c.151+1G>T mutation, respectively (arrows). (B) The agarose gel electrophoresis of reverse transcription–PCR products. A 599–base pair (bp) band corresponding to wild-type SIGMAR1 transcript was obtained in a healthy control (IV:4 and brain). A 539-bp band corresponding to the shortened SIGMAR1 transcript with an in-frame deletion of 60 bp in exon 1 was obtained in affected individuals (IV:1 and IV:5). Both bands were obtained in the heterozygous carrier (III:4). (C) Transcript chromatograms of the complementary DNA of SIGMAR1. The 60-bp deletion (c.92_151del) in the transcript of SIGMAR1 caused by the mutation was confirmed. (D) Schematic representation of the alternative splicing event leading to an in-frame deletion of 60 bp in exon 1 (c.92_151del). (E) Schematic representation of the predicted protein with the deletion of 20 amino acids (σ1R31_50del).

Cellular expression of σ1R31_50del.

To investigate the cellular expression of σ1R31_50del, we stably expressed σ1R-GFP or σ1R31_50del-GFP in HEK293 cells, and several stable clones were isolated and maintained in G418-containing medium. The expression of σ1R-GFP or σ1R31_50del-GFP was verified by immunofluorescence and immunoblotting (figure e-2). By immunofluorescence staining, we found that σ1R31_50del distributed diffusively in the cytoplasm and the nucleus, and showed lower steady-state levels of expression when compared with σ1R. Both cytoplasmic σ1R and σ1R31_50del were detected predominantly in the ER colocalized with calnexin, a Ca2+-binding ER chaperone. To investigate whether σ1R31_50del is degraded through the ER-associated degradation (ERAD) pathway, we examined protein expression in the presence or absence of the proteasomal inhibitor MG132. The MG132 treatment led to an elevated intracellular amount of σ1R31_50del, which further proved by immunoblotting, increased relocation of σ1R31_50del to nuclear envelope and the formation of nuclear aggregates. However, the expression of σ1R was not affected by MG132 treatment (figure 4, A and B). These findings suggested that σ1R31_50del is proteasomally degraded through the ERAD pathway, and prone to form nuclear aggregates upon ubiquitin proteasome system inhibition.

Figure 4
Figure 4 Localization of σ1R or σ1R31_50del conjugated with GFP and the effect of σ1R31_50del on the ERAD pathway, ER stress, and apoptosis in HEK293 cells

(A) Representative confocal images of HEK293 stably expressing the σ1R-GFP or σ1R31_50del-GFP (green) in the presence or absence of MG132 (a proteasomal inhibitor, 5 μM for 6 hours). The ER was stained with the ER marker calnexin (red). Nuclei were stained with DAPI (blue). Note the increased intracellular expression and nuclear aggregate formation in σ1R31_50del-GFP–expressing cells after the MG132 treatment. Scale bar = 25 μm. (B.a) Immunoblotting analysis of σ1R and σ1R31_50del in stable cell lines with or without MG132 for 6 hours, using β-actin as a loading control. (B.b) Densitometry quantification showed elevated σ1R31_50del expression after MG132 treatment. (C.a) Immunoblotting analysis of Bip in the σ1R-GFP– or σ1R31_50del-GFP–expressing cells, using β-actin as a loading control. (C.b) Densitometry quantification showed elevated Bip expression in σ1R31_50del-GFP–expressing cells. (D) Flow cytometry assay showed higher percentage of apoptotic cells in σ1R31_50del–expressing cells compared with σ1R-expressing cells. ***p value <0.001; **p value <0.01. DAPI = 4',6-diamidino-2-phenylindole; DMSO = dimethyl sulfoxide; ER = endoplasmic reticulum; ERAD = endoplasmic reticulum–associated degradation; GFP = green fluorescent protein; HEK293 = human embryonic kidney 293.

σ1R31_50del induces ER stress and enhances apoptosis.

σ1R31_50del was proved to undergo ERAD, which causes the unfolded protein response and apoptosis consequently. We therefore performed immunoblotting of the stable cell lines with an anti-GRP78/Bip antibody to observe the ER stress induction, and flow cytometry assay to examine apoptosis. The σ1R31_50del cell line showed an elevated expression level of Bip in contrast to the σ1R control, which indicated that the stable expression of σ1R31_50del induced ER stress (figure 4C). Statistically significant higher percentage of apoptosis cells was observed in the σ1R31_50del cell line compared with the σ1R cell line under normal conditions (figure 4D).

DISCUSSION

The clinical features of 3 affected siblings consist of progressive muscle wasting and weakness in distal limbs, normal sensory function, and pyramidal tract signs in the lower limbs. A relatively benign disease course was concluded because the conditions stopped deterioration after the age of 20 years, and all patients functioned independently at the time of examination. There were pyramidal tract signs in lower limbs including positive Babinski signs in 2 affected individuals and brisk knee reflexes in 3 affected individuals. However, the length-dependent involvement of distal limbs, no limb spasticity, and no muscle fasciculation indicate the axons of lower MNs are predominantly affected. The lack of sensory involvement and the distal predominance of muscle weakness and wasting define a dHMN, also called spinal CMT or distal spinal muscular atrophy.

The autosomal recessive manner of transmission in this family with dHMN allowed us to use WES and homozygosity mapping to identify the genetic variant. The absence of nucleotide variant detected in all known dHMN genes indicated a novel form of dHMN. The cosegregation of a novel homozygous c.151+1G>T variant in SIGMAR1 with the dHMN phenotype in the family members and the absence of this variant in normal controls suggested that SIGMAR1 is the causative gene underlying this novel form of autosomal recessive dHMN. It is noteworthy that HMN-Jerash, a form of autosomal recessive dHMN with pyramidal tract signs identified in a Jerash population of Jordan, was mapped to 9p21.1-p12 encompassing SIGMAR1 but the causative gene remained unpublished.5 These 2 forms of dHMN shared similar phenotype regarding hereditary trait, age at onset, distribution of muscle involvement, presence of pyramidal signs, and disease progression. For these reasons, we suggest that SIGMAR1 gene mutation should be tested in the HMN-Jerash family, although it is also possible that a distinct gene accounts for HMN-Jerash due to the genetic heterogeneity of this region.

It is increasingly recognized that some forms of dHMN, ALS, and CMT have significant clinical overlap and share the same causative gene.14,–,18 SETX and DCTN1 are reported to be associated with both dHMN and familial ALS with autosomal dominant inheritance.14,17,18 The aforementioned ALS16 caused by p.E102Q mutation in SIGMAR1 differed from the Chinese dHMN in clinical features, including early age at onset, equally significant involvement of both upper and lower MNs, and severe disability.10 Therefore, we propose that SIGMAR1 can be added to the list of genes that cause both lower and upper MN syndromes to a varying degree termed dHMN or familial ALS clinically.

The SIGMAR1 gene contains 4 exons and spans approximately 7 kb on chromosome 9p13.3.19 Seven distinct transcript variants generated from alternative splicing have been reported online (http://genome.ucsc.edu/). The canonical transcript containing all 4 exons and the encoded σ1R structurally possesses an intracellular N-terminal end, 2 transmembrane domains (amino acids 10–30 and 80–100, respectively), an extracellular loop, and an intracellular C-terminal end.20 σ1R31_50del encoded by the c.92_151del transcript is predicted to lack the initial 20 amino acids of the putative extracellular loop with an estimated molecular mass of 23 kDa. This shortened transcript (NM 001282207.1) was once indentified in a human breast cancer biopsy sample. However, we could not detect this form in lymphocytes and brain tissue of a healthy control in this study. Since it was found once only in a tumor biopsy and absent in healthy tissue, we inferred that it is not a functional variant present under physiologic conditions.

Accumulating evidence suggested that σ1R is associated with the pathogenesis of different neurodegenerative diseases. The 3′UTR alterations in SIGMAR1 detected in frontotemporal lobar degeneration–MN disease families were hypothesized to alter the expression level of σ1R and have pathogenic effects.11 The p.E102Q mutation in SIGMAR1 was demonstrated to alter membrane distribution of σ1R and reduce cell viability.10 Cells overexpressing σ1RE102Q showed cytoplasmic aggregation, aberrant extranuclear localization of the TAR DNA-binding protein 43 (TDP-43), impaired mitochondrial adenosine triphosphate production, and compromised proteasome activity.21 Altered localization and dysfunction of σ1R were observed in both sporadic ALS and familial ALS.22 Accumulation of σ1R was detected in neuronal nuclear inclusions of TDP-43 proteinopathy and several forms of polyglutamine disease, and overexpression of σ1R reduced the accumulation of nuclear inclusions containing mutant huntingtin.23,24 In the present study, we showed that the σ1R31_50del is cleared largely by ERAD, and expression of σ1R31_50del induces ER stress and enhances apoptosis in vitro. Although the alteration of this mutation to the normal σ1R function such as ion channel regulation and chaperone activity remains unknown, our study indicates that ER stress has a role in the pathogenesis of dHMN.

AUTHOR CONTRIBUTIONS

X. Li: provision of patient information, ascertainment of the family with dHMN, acquisition of clinical data, and cellular studies. Dr. Hu: genetic studies, interpretation of genetic data. L. Liu: genetic studies and cellular studies. Y. Xie: genetic studies and cellular studies. Y. Zhan: genetic studies. Dr. Zi: acquisition of clinical data. Dr. Wang: interpretation of genetic data. Dr. Wu: revision of the manuscript. Dr. Xia: interpretation of genetic data, revision of the manuscript. Dr. Tang: study design, revision of the manuscript. Dr. Zhang: study design, data analysis and interpretation, writing of the manuscript.

STUDY FUNDING

Supported by grants from the National Natural Science Foundation of China (81071001) and Hunan Provincial Natural Science Foundation of China (13JJ2014).

DISCLOSURE

The authors report no disclosures relevant to the manuscript. Go to Neurology.org for full disclosures.

ACKNOWLEDGMENT

The authors thank the patients and their family members for generous participation in this study. The authors greatly thank BGI Tech Solutions Co., Ltd., for technical support in genetic analysis and Dr. J. Hu (Departments of Neuromuscular Disease, Third Hospital of Hebei Medical University, China) for pathologic support. The authors also thank all colleagues for their valuable advice in experimental procedures.

Footnotes

  • Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

  • Supplemental data at Neurology.org

  • Received September 22, 2014.
  • Accepted in final form March 6, 2015.

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