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Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K
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Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K Jun Fu, Shixu Dai, Yuanyuan Lu, Rui Wu, Zhaoxia Wang, Yun Yuan, He Lv * Department of Neurology, First Hospital, Peking University, Beijing 100034, China Received 29 October 2016; received in revised form 24 March 2017; accepted 4 April 2017
Abstract Mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) cause rare subtypes of Charcot–Marie–Tooth disease (CMT2K and CMT4A). CMT2K is an axonal neuropathy while CMT4A is a demyelinating type. In a series of 169 Chinese CMT patients (79 CMT1, 52 CMT2 and 38 unclassified), four unrelated patients (2.37%) were identified with GDAP1 mutations, including two with autosomal recessive CMT2K (AR-CMT2K) and two dominant CMT2K (AD-CMT2K). All patients had disease onset before 5 years of age, and presented with muscle weakness, atrophy, and mild sensory disturbance in distal limbs. Motor nerve conduction velocities of the median nerve were within normal ranges, and compound muscle action potential ranged from 1.5 to 3.8 mV. Sural nerve biopsy revealed loss of large myelinated fibers with regeneration clusters and a few onion bulbs. Electron microscopy showed mitochondrial aggregation in both axons and Schwann cells, and neurofilament accumulation in giant unmyelinated fibers. The p.H256R mutation was found in all patients with GDAP1 compound heterozygous mutations, suggesting that it might be a common mutation in Chinese patients. This study observed no difference in the disease onset, phenotype severity, electrophysiological findings, or pathological changes between AR-CMT2K and AD-CMT2K patients. © 2017 Published by Elsevier B.V. Keywords: Charcot–Marie–Tooth disease; GDAP1; Chinese population; Axonal CMT
1. Introduction Ganglioside-induced differentiation-associated protein 1 (GDAP1) localizes to the outer membrane of the mitochondrion and plays an important role in the maintenance of mitochondrial morphology and function [1,2]. Mutations in GDAP1 (OMIM 606598) are responsible for the autosomal recessive demyelinating, axonal, and intermediate forms of Charcot– Marie–Tooth disease (CMT), known as CMT4A [3] (OMIM 214400), AR-CMT2K [4] (OMIM 607831), and RI-CMTA [5,6] (OMIM 608340), respectively. Besides numerous recessive GDAP1 mutations (http://gdap1.mitodyn.org), dominant GDAP1 mutations have also been reported in a few cases of autosomal dominant (AD)-CMT2K [7–11] (OMIM 607831). AR-CMT2K patients reported in the literature have an earlier onset of disease and a more severe phenotype. Proximal muscles become affected later and often lead to an inability to walk in the second or third decade of life [12]. By contrast,
* Corresponding author. Department of Neurology, Peking University First Hospital, 8 Xishiku St, Xicheng District, Beijing 100034, China. Fax: +86 10 66176450. E-mail address: [email protected] (H. Lv).
AD-CMT2K is much milder with a later onset and slow progression [7]. Recently, many cases of CMT caused by GDAP1 mutations have been described worldwide. There is a broad variability in GDAP1 mutation frequency reported in different regions [7,10,11,13,14]. However, genetically confirmed GDAP1related CMT has rarely been documented in the Asian population [15–18]. The frequency of GDAP1-related CMT in the Chinese population, and their main clinical features and pathological changes remain unclear. Herein we report the GDAP1 mutation frequency in a cohort of Chinese CMTs and compare the clinical, pathological, and genetic features between Chinese patients with AR-CMT2K and AD-CMT2K. 2. Patients and methods 2.1. Patients We performed a genetic examination of 169 patients with suspected CMT who attended to our clinic from January 2007 to December 2015. Of the 131 patients with sufficient nerve conduction data, 79 cases were classified as demyelinating CMT1 and 52 as axonal CMT2. The mode of inheritance was autosomal or X-linked dominant in 20 familial cases, autosomal recessive in 2 cases, and apparently isolated in 147 with no
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2.2. Pathological examination Sural nerve biopsies were performed in four index patients. Part of each sample was fixed in 4% formaldehyde, paraffinembedded, then 5 µm sections were stained with hematoxylin and eosin, Luxol fast blue, and Congo red. The rest of each specimen was fixed in 2.5% glutaraldehyde followed by 1% buffered osmium tetroxide, then dehydrated in ascending grades of ethanol, and embedded in Epon. Semi-thin sections for light microscopy were stained with toluidine blue. Ultrathin sections were double-stained with uranyl acetate and lead citrate, and then examined under an electron microscope. We randomly selected three high-resolution micrographic images to perform the morphometrical analysis of myelinated fibers (MFs), including MF density and fiber diameter. 2.3. Mutation analysis
Fig. 1. (A) The pedigrees and genotypes of four families. (B) Location of mutations in the GDAP1 protein in Chinese patients. #, novel mutation; *, mutations occurred more than one time in Chinese patients. The numbers below the GDAP1 protein indicate the amino acid positions of the structural domains. Patients with heterozygous mutations are in red and compound heterozygous mutations in black.
evidence of family history of peripheral neuropathy. Among the 169 patients, 96 patients had a genetic diagnosis, including 34 cases with CMT1A (caused by PMP22 duplication), five with hereditary neuropathy with liability to pressure palsies (HNPP) (caused by PMP22 deletion), two with CMT1E (caused by PMP22 mutations), one with CMT1B (caused by MPZ mutation), one with CMT1D (caused by EGR2 mutation), one with CMT4F (caused by PRX mutation), 20 with CMT2A (caused by MFN2 mutations), four with CMT2K (caused by GDAP1 mutations), one with CMT2E (caused by NEFL mutation), one with CMT2O (caused by DYNC1H1 mutation), 24 with CMTX1 (caused by GJB1 mutations), and two with CMTDIE (caused by INF2 mutations). Five patients from the four families (Fig. 1A) with GDAP1related CMT underwent detailed clinical evaluation to determine the family medical history, age of disease onset, gait disturbance, distribution of muscle weakness and atrophy, reflex response, level of foot deformity, sensory disturbance, and nerve conduction velocity (NCV). The CMT Neuropathy Score (CMTNS) was applied by two different neurologists to evaluate disease severity as mild (CMTNS ≤10), moderate (CMTNS 11–20), and severe (CMTNS 21–36). Detailed clinical and electrophysiological characteristics of all patients were summarized. Ethical approval for this study was obtained from the health authority ethical committee of Peking University First Hospital. After written informed consent was provided, GDAP1 mutation screening and sural nerve biopsies were performed in all index patients.
Genomic DNA was extracted from peripheral blood samples from all index patients following standard procedures. GDAP1 mutations were analyzed by Sanger sequencing before 2011 and by next generation sequencing (NGS) after 2011. NGS was conducted using a NGS panel covering all exons and their flanking sequences of genes known to be associated with hereditary neuropathies (Supplementary Table S1). Mutations were described according to HGVS nomenclature using nucleotide and amino acid numbering based on published mRNA (NM_018972) and protein (NP_061845) sequences of GDAP1. Sanger sequencing with specific primers was conducted to confirm the mutations in index patients. Segregation analysis of the mutations was performed in available family members. For the novel mutations, 100 healthy control participants of Chinese origin were screened, and we also checked for allele frequencies in ExAC, 1000 genome project database, and Exome Variant Server database (http://evs.gs.washington.edu/EVS/). We used the HomoloGene Web server (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=homologene) to compare GDAP1 sequences among different species. In silico predictions of the functional effect of mutations were performed with Mutation Taster (mutationtaster.org), PolyPhen 2 (genetics.bwh.harvard.edu/ pph2/), and SIFT (sift.jcvi.org/) prediction software. 3. Results 3.1. Clinical data Three index patients were isolated and one index patient (index 4) had a positive family history for disease and was confirmed to have an autosomal dominant transmission pattern. The onset of disease in all patients ranged from infancy to 5 years of age (Table 1). Initial symptoms were muscle weakness and atrophy, predominately involving the distal part of the lower limbs. After 2–4 years, four patients presented with weakness in the hand (patients 2, 3, 4, and 5). Proximal muscle weakness appeared in two cases around 4 years after the onset of disease (patients 4 and 5). No patients complained of any sensory disturbance, while pinprick and vibration sense in patient 2 and vibration sense in patient 3 were mildly decreased in distal lower limbs on physical examination. Foot deformities were
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Table 1 Clinical and genetic data of the present patients with GDAP1 mutations. Patients
Patient 1 (index 1)
Patient 2 (index 2)
Patient 3 (index 3)
Patient 4 (index 4)
Patient 5 (Father of index 4)
Onset/Exam age Initial symptoms Muscle weakness Muscle atrophy Tendon reflexes Sensory loss Pinprick Vibration Deformity
5/6 Unsteady gait DLL Yes Decrease
4/9 Unsteady gait DLL > DUL Yes Absent
1/5 Delayed milestone DLL > DUL Yes Decrease
3/7 Unsteady gait DLL > DUL > PLL > PUL Yes Absent
5/32 Unsteady gait DLL > DUL > PLL > PUL Yes Absent
No No Pes equinusvarus, tendon contracture Orthopedic shoes 45.2/1.7 12 c.845G>A+c.767A>G p.R282H+ p.H256R GST-C+GST-C Father + de novo AR
Mildly Mildly Tendon contracture
No Mildly No
No No Pes equinusvarus
No No Tendon contracture
Orthopedic shoes 48.1/3.8 14 c.767A>G+c.466G>A p.H256R+ p.A156T GST-C+α4α5 loop NA Likely AR
Ankle foot orthosis 55/1.5 13 c.719G>A p.C240Y GST-C de novo AD
Ankle foot orthosis 57.3/2.7 12 c.358C>T p.R120W None Father AD
Wheelchair at 30y – – c.358C>T p.R120W
Early onset Moderate CMT2 [15]
Early onset Moderate CMT2 [15–17]
Early onset Moderate CMT2 [9]
Early onset Moderate CMT2 [10]
Motor ability* Median MCV/CMAP CMTNS Nucleotides Amino acids Domains Parental origin Mode of inheritance Phenotype Onset age Severity Subtype Reference
DLL, distal lower limbs; DUL, distal upper limbs; PLL, proximal lower limbs; PUL, proximal upper limbs; MCV, motor nerve conduction velocity; CMAP, compound muscle action potential; GST-C, glutathione-S-transferase C-terminal; NA, parents not available for genetic testing; AD, autosomal dominant; AR, autosomal recessive. * Motor ability in patients 1 and 2 indicated independent ambulation with orthopedic shoes, and in patients 3 and 4 indicated independent ambulation with ankle foot orthoses.
seen in four patients (patients 1, 2, 4, and 5), including ankle contracture and pes equinus-varus. Four indexes were able to walk with assistance at the time of consultation; however, patient 5 (the father of index 4) was wheelchair-bound before 30 years old. All patients denied visual impairment and mental retardation, and no patient had evidence of dysmorphic features, hoarseness, or diaphragmatic paralysis. All index patients showed moderate CMTNS scores (CMTNS 12–14). Nerve conduction velocity studies in four indexes revealed axonal neuropathy changes, with both motor and sensory nerves affected (Supplementary Table S2). Motor nerve conduction velocities (MNCVs) in median nerves were between 45.2 and 57.3 m/s. Compound muscle action potential (CMAP) amplitudes of median nerves were reduced and ranged from 1.5 to 3.8 mV. CMAP, MNCV, and sensory nerve action potential (SNAP) amplitudes in the lower extremities were severely reduced or absent. 3.2. Nerve pathology All index patients showed normal to mildly decreased myelinated fiber densities that ranged from 6894 to 9026/mm2 (Supplementary Table S3). A unimodal distribution of myelinated fibers was observed (Fig. 2A), with a marked reduction of large diameter fibers (fibers > 6 µm ranging from 1.88% to 5.32% in four patients). Regenerating clusters of small myelinated fibers were visible in all cases under both light and electron microscopy (Fig. 2B and C). Additionally, occasional onion bulb formations (Fig. 2D), thinly myelinated fibers and focal
folded myelin were observed. Electron microscopy of longitudinal sections revealed mitochondrial aggregation within myelinated and unmyelinated axons and Schwann cells in all cases (Fig. 2E). In all nerve biopsies, a minor proportion of unmyelinated fibers (2%–5%) appeared as giant axons that were filled with accumulated neurofilaments (Fig. 2F and G). No neurofilament accumulation was found in the axons of myelinated fibers in either longitudinal or cross sections. 3.3. GDAP1 mutation analysis We identified two compound heterozygous and two heterozygous GDAP1 mutations in four index patients (4/169, 2.37%) (Table 1 and Fig. 1A). Index 1 had p.R282H and p.H256R mutations, which were previously reported in a Taiwanese patient [15]. Index 2 harbored mutations p.H256R and p.A156T. The p.H256R has also been previously reported as one of the mutations detected in two Chinese compound heterozygotes [15,16] and in a Korean homozygote [17]. The p.A156T (c.466G>A) mutation has not been reported previously. No specimens from the family of index 2 were available, so segregation analysis could not be performed. However, p.A156T was not detected in 100 healthy controls, and was not found in ExAC, 1000 Genomes or EVS databases. The amino acid residue A156 is highly evolutionarily conserved among different species. The p.A156T was predicted to be disease causing by Mutation Taster, possibly damaging by PolyPhen 2 (score of 0.879), and tolerated by SIFT, with a score of 0.06. Mutation p.A156G at the same amino acid residue was
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Fig. 2. Neuropathological findings. (A) Histograms of myelinated fibers in four patients show unimodal distribution with marked reduction of large myelinated fibers, compared with the control. (B) Loss of large myelinated fibers with regenerating cluster (black arrow) and axon with dark staining which suggested abnormal structure of the axon (white arrow) from index 4 (semithin section, touluding blue staining). (C) A regenerating cluster of myelinated fibers under EM from index 3. (D) An onion bulb from index 4. (E) Mitochondrial aggregation in axons in longitudinal section from index 1. (F) A giant unmyelinated fiber (black arrow) near two normal unmyelinated fibers (white arrow) and a degeneration fiber (arrowhead) from index 4. (G) Neurofilament was relatively aggregated in the giant axon (black arrow) compared with the normal axon (white arrow). Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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previously reported to be pathogenic [11]. Index 3 had a heterozygous mutation p.C240Y, and index 4 had another heterozygous mutation p.R120W. Both of the two mutations have been previously reported [9,10]. 4. Discussion In this study, we identified GDAP1-related CMT as a rare subtype of CMT with mutation frequency of 2.37% in a cohort containing both demyelinating and axonal CMT patients. This was the second most common type of CMT2 in our cohort, and much less frequent than CMT2A caused by MFN2 mutations (20/169, 11.8%). GDAP1 mutations are rare in Asian populations, with a reported frequency of 0.6% in Japanese CMT patients [18,19], and detection in only three Korean patients [8,17]. A low frequency of GDAP1 mutations (0.8%) has also been reported in a CMT series from the United States [20]. Conversely, high GDAP1 mutation frequencies are observed in European CMT patients, with reported frequencies of 9.6% in Spanish CMT patients [14], 6.9% in Italian CMT2 patients [7], and 14% in Finnish CMT2 patients [13] (Supplementary Table S4). Axonal CMT2K may be a major type of GDAP1-related CMT in Chinese patients. All patients in our series presented with axonal neuropathy as identified by a nerve conduction study. Additionally, the previously reported Taiwanese patient carrying GDAP1 mutations also had CMT2K [15]. In Japan, GDAP1 mutations are rare, and AR-CMT4A was only reported in one of 103 demyelinating CMT patients [19]. CMT2K has also been reported in just one patient among 127 axonal Japanese CMT patients [18]. Similar to our own series, CMT2K is also more common than CMT4A in American [20], Spanish [14] and Italian [7,21] patients. Clinically, we were able to confirm that all our AR-CMT2K patients had an early onset of disease. However, they all showed a moderate phenotype, as reported previously in a Taiwanese patient [15], which differs from the severe phenotype seen in Caucasian AR-CMT2K patients [7,22–24]. Our patients showed no vocal cord paresis reported in Italian patients [22], nor diaphragmatic palsy reported in Spanish patients [24]. Besides, AD-CMT2K patients in the present series also showed symptoms in the early childhood with moderate phenotype, which is different from other reports that dominant GDAP1 mutations cause a late onset and mild phenotype [8,11,13,23]. Our study findings therefore broaden the clinical picture of AD-CMT2K with great phenotypic variability, especially in the age of disease onset ranging from early childhood to adulthood and severity from mild to moderate [10,25]. Pathologically, AR-CMT2K and AD-CMT2K are not pure axonal neuropathies. In both our AR-CMT2K and AD-CMT2K patients, sural nerve biopsy revealed a predominant axonal neuropathy with mild demyelinating change, as previously reported in Caucasian patients [22,24]. The density of myelinated fibers was not largely decreased because of the increase of small myelinated fibers; however, there was a marked loss of large myelinated fibers. Demyelinating features were visible as atypical onion bulb formations and thinly myelinated fibers. Histopathological findings of previous Korean patients also
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showed mixed features of demyelinating and axonal neuropathies [8,17]. The pathological involvement of both axons and myelin might be associated with the mechanism that GDAP1 is expressed in both neurons and Schwann cells [26]. Although the histological findings might also be largely compatible with intermediate CMT, the patients should be classified into CMT2 as the median motor NCVs are above 45 m/s. We also observed mitochondrial aggregation in both axons and Schwann cells, as previously reported [10,27]. It is noteworthy that giant unmyelinated axons were visible in all patients of the present series. The giant unmyelinated axons were filled with accumulated neurofilament. Giant axons of unmyelinated fibers are also documented in CMT2A caused by MFN2 mutations [28]. We suggest that neurofilament aggregation in giant unmyelinated axon might be a common ultrastructural feature of mitochondrial neuropathies. Mutations of GDAP1 have been identified in a total of six Chinese index patients, including four in our present series and two AR-CMT patients reported previously [15,16] (Fig. 1B). Both mutations causing AR-CMT2K and AD-CMT2K are located in or near the glutathione-S-transferase (GST) domain, which is commonly targeted in Caucasian AD-CMT2K patients [1,12,29], or the α4–α5 loop of GDAP1 protein (Fig. 1B). We observed that the p.H256R is the most frequent mutation in the present series, and is present in all patients with compound heterozygous mutations. This same mutation has also been reported in a Korean patient with AR-CMT2K [17], but not in other populations. We therefore suggest that p.H256R might be a common mutation in Chinese CMT patients, despite the overall rarity of GDAP1 mutations in such patients. Case 1 inherited the p.R282H variant from his father while the p.H256R variant was apparently de novo (neither found in the father nor in the mother). Based on family history, clinical presentation and autosomal recessive inheritance of the p.H256R variant in other CMT families, it seems plausible that both variants are compound heterozygous; however, we cannot confirm this experimentally. In conclusion, both AR-CMT2K and AD-CMT2K patients in the current series presented with similar symptoms including an early onset and a moderate to severe phenotype, and similar pathological features including axonal neuropathy with mild demyelination and mitochondrial aggregation in axons and Schwann cells. This study also highlights the presence of giant axons of unmyelinated fibers filled with accumulated neurofilament in GDAP1-related CMT patients. Acknowledgements We thank the patients and their parents for cooperation. Ms. Yuehuan Zuo contributed for technical assistance in nerve biopsy preparation. This research was supported by the National Natural Science Foundation of China (No. 81471185) and by the Ministry of Science and Technology of the People’s Republic of China (No. 2011ZX09307-001-07). Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.nmd.2017.04.001.
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K Jun Fu, Shixu Dai, Yuanyuan Lu, Rui Wu, Zhaoxia Wang, Yun Yuan, He Lv * Department of Neurology, First Hospital, Peking University, Beijing 100034, China Received 29 October 2016; received in revised form 24 March 2017; accepted 4 April 2017
Abstract Mutations in the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) cause rare subtypes of Charcot–Marie–Tooth disease (CMT2K and CMT4A). CMT2K is an axonal neuropathy while CMT4A is a demyelinating type. In a series of 169 Chinese CMT patients (79 CMT1, 52 CMT2 and 38 unclassified), four unrelated patients (2.37%) were identified with GDAP1 mutations, including two with autosomal recessive CMT2K (AR-CMT2K) and two dominant CMT2K (AD-CMT2K). All patients had disease onset before 5 years of age, and presented with muscle weakness, atrophy, and mild sensory disturbance in distal limbs. Motor nerve conduction velocities of the median nerve were within normal ranges, and compound muscle action potential ranged from 1.5 to 3.8 mV. Sural nerve biopsy revealed loss of large myelinated fibers with regeneration clusters and a few onion bulbs. Electron microscopy showed mitochondrial aggregation in both axons and Schwann cells, and neurofilament accumulation in giant unmyelinated fibers. The p.H256R mutation was found in all patients with GDAP1 compound heterozygous mutations, suggesting that it might be a common mutation in Chinese patients. This study observed no difference in the disease onset, phenotype severity, electrophysiological findings, or pathological changes between AR-CMT2K and AD-CMT2K patients. © 2017 Published by Elsevier B.V. Keywords: Charcot–Marie–Tooth disease; GDAP1; Chinese population; Axonal CMT
1. Introduction Ganglioside-induced differentiation-associated protein 1 (GDAP1) localizes to the outer membrane of the mitochondrion and plays an important role in the maintenance of mitochondrial morphology and function [1,2]. Mutations in GDAP1 (OMIM 606598) are responsible for the autosomal recessive demyelinating, axonal, and intermediate forms of Charcot– Marie–Tooth disease (CMT), known as CMT4A [3] (OMIM 214400), AR-CMT2K [4] (OMIM 607831), and RI-CMTA [5,6] (OMIM 608340), respectively. Besides numerous recessive GDAP1 mutations (http://gdap1.mitodyn.org), dominant GDAP1 mutations have also been reported in a few cases of autosomal dominant (AD)-CMT2K [7–11] (OMIM 607831). AR-CMT2K patients reported in the literature have an earlier onset of disease and a more severe phenotype. Proximal muscles become affected later and often lead to an inability to walk in the second or third decade of life [12]. By contrast,
* Corresponding author. Department of Neurology, Peking University First Hospital, 8 Xishiku St, Xicheng District, Beijing 100034, China. Fax: +86 10 66176450. E-mail address: [email protected] (H. Lv).
AD-CMT2K is much milder with a later onset and slow progression [7]. Recently, many cases of CMT caused by GDAP1 mutations have been described worldwide. There is a broad variability in GDAP1 mutation frequency reported in different regions [7,10,11,13,14]. However, genetically confirmed GDAP1related CMT has rarely been documented in the Asian population [15–18]. The frequency of GDAP1-related CMT in the Chinese population, and their main clinical features and pathological changes remain unclear. Herein we report the GDAP1 mutation frequency in a cohort of Chinese CMTs and compare the clinical, pathological, and genetic features between Chinese patients with AR-CMT2K and AD-CMT2K. 2. Patients and methods 2.1. Patients We performed a genetic examination of 169 patients with suspected CMT who attended to our clinic from January 2007 to December 2015. Of the 131 patients with sufficient nerve conduction data, 79 cases were classified as demyelinating CMT1 and 52 as axonal CMT2. The mode of inheritance was autosomal or X-linked dominant in 20 familial cases, autosomal recessive in 2 cases, and apparently isolated in 147 with no
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2.2. Pathological examination Sural nerve biopsies were performed in four index patients. Part of each sample was fixed in 4% formaldehyde, paraffinembedded, then 5 µm sections were stained with hematoxylin and eosin, Luxol fast blue, and Congo red. The rest of each specimen was fixed in 2.5% glutaraldehyde followed by 1% buffered osmium tetroxide, then dehydrated in ascending grades of ethanol, and embedded in Epon. Semi-thin sections for light microscopy were stained with toluidine blue. Ultrathin sections were double-stained with uranyl acetate and lead citrate, and then examined under an electron microscope. We randomly selected three high-resolution micrographic images to perform the morphometrical analysis of myelinated fibers (MFs), including MF density and fiber diameter. 2.3. Mutation analysis
Fig. 1. (A) The pedigrees and genotypes of four families. (B) Location of mutations in the GDAP1 protein in Chinese patients. #, novel mutation; *, mutations occurred more than one time in Chinese patients. The numbers below the GDAP1 protein indicate the amino acid positions of the structural domains. Patients with heterozygous mutations are in red and compound heterozygous mutations in black.
evidence of family history of peripheral neuropathy. Among the 169 patients, 96 patients had a genetic diagnosis, including 34 cases with CMT1A (caused by PMP22 duplication), five with hereditary neuropathy with liability to pressure palsies (HNPP) (caused by PMP22 deletion), two with CMT1E (caused by PMP22 mutations), one with CMT1B (caused by MPZ mutation), one with CMT1D (caused by EGR2 mutation), one with CMT4F (caused by PRX mutation), 20 with CMT2A (caused by MFN2 mutations), four with CMT2K (caused by GDAP1 mutations), one with CMT2E (caused by NEFL mutation), one with CMT2O (caused by DYNC1H1 mutation), 24 with CMTX1 (caused by GJB1 mutations), and two with CMTDIE (caused by INF2 mutations). Five patients from the four families (Fig. 1A) with GDAP1related CMT underwent detailed clinical evaluation to determine the family medical history, age of disease onset, gait disturbance, distribution of muscle weakness and atrophy, reflex response, level of foot deformity, sensory disturbance, and nerve conduction velocity (NCV). The CMT Neuropathy Score (CMTNS) was applied by two different neurologists to evaluate disease severity as mild (CMTNS ≤10), moderate (CMTNS 11–20), and severe (CMTNS 21–36). Detailed clinical and electrophysiological characteristics of all patients were summarized. Ethical approval for this study was obtained from the health authority ethical committee of Peking University First Hospital. After written informed consent was provided, GDAP1 mutation screening and sural nerve biopsies were performed in all index patients.
Genomic DNA was extracted from peripheral blood samples from all index patients following standard procedures. GDAP1 mutations were analyzed by Sanger sequencing before 2011 and by next generation sequencing (NGS) after 2011. NGS was conducted using a NGS panel covering all exons and their flanking sequences of genes known to be associated with hereditary neuropathies (Supplementary Table S1). Mutations were described according to HGVS nomenclature using nucleotide and amino acid numbering based on published mRNA (NM_018972) and protein (NP_061845) sequences of GDAP1. Sanger sequencing with specific primers was conducted to confirm the mutations in index patients. Segregation analysis of the mutations was performed in available family members. For the novel mutations, 100 healthy control participants of Chinese origin were screened, and we also checked for allele frequencies in ExAC, 1000 genome project database, and Exome Variant Server database (http://evs.gs.washington.edu/EVS/). We used the HomoloGene Web server (http://www.ncbi.nlm.nih.gov/ sites/entrez?db=homologene) to compare GDAP1 sequences among different species. In silico predictions of the functional effect of mutations were performed with Mutation Taster (mutationtaster.org), PolyPhen 2 (genetics.bwh.harvard.edu/ pph2/), and SIFT (sift.jcvi.org/) prediction software. 3. Results 3.1. Clinical data Three index patients were isolated and one index patient (index 4) had a positive family history for disease and was confirmed to have an autosomal dominant transmission pattern. The onset of disease in all patients ranged from infancy to 5 years of age (Table 1). Initial symptoms were muscle weakness and atrophy, predominately involving the distal part of the lower limbs. After 2–4 years, four patients presented with weakness in the hand (patients 2, 3, 4, and 5). Proximal muscle weakness appeared in two cases around 4 years after the onset of disease (patients 4 and 5). No patients complained of any sensory disturbance, while pinprick and vibration sense in patient 2 and vibration sense in patient 3 were mildly decreased in distal lower limbs on physical examination. Foot deformities were
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Table 1 Clinical and genetic data of the present patients with GDAP1 mutations. Patients
Patient 1 (index 1)
Patient 2 (index 2)
Patient 3 (index 3)
Patient 4 (index 4)
Patient 5 (Father of index 4)
Onset/Exam age Initial symptoms Muscle weakness Muscle atrophy Tendon reflexes Sensory loss Pinprick Vibration Deformity
5/6 Unsteady gait DLL Yes Decrease
4/9 Unsteady gait DLL > DUL Yes Absent
1/5 Delayed milestone DLL > DUL Yes Decrease
3/7 Unsteady gait DLL > DUL > PLL > PUL Yes Absent
5/32 Unsteady gait DLL > DUL > PLL > PUL Yes Absent
No No Pes equinusvarus, tendon contracture Orthopedic shoes 45.2/1.7 12 c.845G>A+c.767A>G p.R282H+ p.H256R GST-C+GST-C Father + de novo AR
Mildly Mildly Tendon contracture
No Mildly No
No No Pes equinusvarus
No No Tendon contracture
Orthopedic shoes 48.1/3.8 14 c.767A>G+c.466G>A p.H256R+ p.A156T GST-C+α4α5 loop NA Likely AR
Ankle foot orthosis 55/1.5 13 c.719G>A p.C240Y GST-C de novo AD
Ankle foot orthosis 57.3/2.7 12 c.358C>T p.R120W None Father AD
Wheelchair at 30y – – c.358C>T p.R120W
Early onset Moderate CMT2 [15]
Early onset Moderate CMT2 [15–17]
Early onset Moderate CMT2 [9]
Early onset Moderate CMT2 [10]
Motor ability* Median MCV/CMAP CMTNS Nucleotides Amino acids Domains Parental origin Mode of inheritance Phenotype Onset age Severity Subtype Reference
DLL, distal lower limbs; DUL, distal upper limbs; PLL, proximal lower limbs; PUL, proximal upper limbs; MCV, motor nerve conduction velocity; CMAP, compound muscle action potential; GST-C, glutathione-S-transferase C-terminal; NA, parents not available for genetic testing; AD, autosomal dominant; AR, autosomal recessive. * Motor ability in patients 1 and 2 indicated independent ambulation with orthopedic shoes, and in patients 3 and 4 indicated independent ambulation with ankle foot orthoses.
seen in four patients (patients 1, 2, 4, and 5), including ankle contracture and pes equinus-varus. Four indexes were able to walk with assistance at the time of consultation; however, patient 5 (the father of index 4) was wheelchair-bound before 30 years old. All patients denied visual impairment and mental retardation, and no patient had evidence of dysmorphic features, hoarseness, or diaphragmatic paralysis. All index patients showed moderate CMTNS scores (CMTNS 12–14). Nerve conduction velocity studies in four indexes revealed axonal neuropathy changes, with both motor and sensory nerves affected (Supplementary Table S2). Motor nerve conduction velocities (MNCVs) in median nerves were between 45.2 and 57.3 m/s. Compound muscle action potential (CMAP) amplitudes of median nerves were reduced and ranged from 1.5 to 3.8 mV. CMAP, MNCV, and sensory nerve action potential (SNAP) amplitudes in the lower extremities were severely reduced or absent. 3.2. Nerve pathology All index patients showed normal to mildly decreased myelinated fiber densities that ranged from 6894 to 9026/mm2 (Supplementary Table S3). A unimodal distribution of myelinated fibers was observed (Fig. 2A), with a marked reduction of large diameter fibers (fibers > 6 µm ranging from 1.88% to 5.32% in four patients). Regenerating clusters of small myelinated fibers were visible in all cases under both light and electron microscopy (Fig. 2B and C). Additionally, occasional onion bulb formations (Fig. 2D), thinly myelinated fibers and focal
folded myelin were observed. Electron microscopy of longitudinal sections revealed mitochondrial aggregation within myelinated and unmyelinated axons and Schwann cells in all cases (Fig. 2E). In all nerve biopsies, a minor proportion of unmyelinated fibers (2%–5%) appeared as giant axons that were filled with accumulated neurofilaments (Fig. 2F and G). No neurofilament accumulation was found in the axons of myelinated fibers in either longitudinal or cross sections. 3.3. GDAP1 mutation analysis We identified two compound heterozygous and two heterozygous GDAP1 mutations in four index patients (4/169, 2.37%) (Table 1 and Fig. 1A). Index 1 had p.R282H and p.H256R mutations, which were previously reported in a Taiwanese patient [15]. Index 2 harbored mutations p.H256R and p.A156T. The p.H256R has also been previously reported as one of the mutations detected in two Chinese compound heterozygotes [15,16] and in a Korean homozygote [17]. The p.A156T (c.466G>A) mutation has not been reported previously. No specimens from the family of index 2 were available, so segregation analysis could not be performed. However, p.A156T was not detected in 100 healthy controls, and was not found in ExAC, 1000 Genomes or EVS databases. The amino acid residue A156 is highly evolutionarily conserved among different species. The p.A156T was predicted to be disease causing by Mutation Taster, possibly damaging by PolyPhen 2 (score of 0.879), and tolerated by SIFT, with a score of 0.06. Mutation p.A156G at the same amino acid residue was
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Fig. 2. Neuropathological findings. (A) Histograms of myelinated fibers in four patients show unimodal distribution with marked reduction of large myelinated fibers, compared with the control. (B) Loss of large myelinated fibers with regenerating cluster (black arrow) and axon with dark staining which suggested abnormal structure of the axon (white arrow) from index 4 (semithin section, touluding blue staining). (C) A regenerating cluster of myelinated fibers under EM from index 3. (D) An onion bulb from index 4. (E) Mitochondrial aggregation in axons in longitudinal section from index 1. (F) A giant unmyelinated fiber (black arrow) near two normal unmyelinated fibers (white arrow) and a degeneration fiber (arrowhead) from index 4. (G) Neurofilament was relatively aggregated in the giant axon (black arrow) compared with the normal axon (white arrow). Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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previously reported to be pathogenic [11]. Index 3 had a heterozygous mutation p.C240Y, and index 4 had another heterozygous mutation p.R120W. Both of the two mutations have been previously reported [9,10]. 4. Discussion In this study, we identified GDAP1-related CMT as a rare subtype of CMT with mutation frequency of 2.37% in a cohort containing both demyelinating and axonal CMT patients. This was the second most common type of CMT2 in our cohort, and much less frequent than CMT2A caused by MFN2 mutations (20/169, 11.8%). GDAP1 mutations are rare in Asian populations, with a reported frequency of 0.6% in Japanese CMT patients [18,19], and detection in only three Korean patients [8,17]. A low frequency of GDAP1 mutations (0.8%) has also been reported in a CMT series from the United States [20]. Conversely, high GDAP1 mutation frequencies are observed in European CMT patients, with reported frequencies of 9.6% in Spanish CMT patients [14], 6.9% in Italian CMT2 patients [7], and 14% in Finnish CMT2 patients [13] (Supplementary Table S4). Axonal CMT2K may be a major type of GDAP1-related CMT in Chinese patients. All patients in our series presented with axonal neuropathy as identified by a nerve conduction study. Additionally, the previously reported Taiwanese patient carrying GDAP1 mutations also had CMT2K [15]. In Japan, GDAP1 mutations are rare, and AR-CMT4A was only reported in one of 103 demyelinating CMT patients [19]. CMT2K has also been reported in just one patient among 127 axonal Japanese CMT patients [18]. Similar to our own series, CMT2K is also more common than CMT4A in American [20], Spanish [14] and Italian [7,21] patients. Clinically, we were able to confirm that all our AR-CMT2K patients had an early onset of disease. However, they all showed a moderate phenotype, as reported previously in a Taiwanese patient [15], which differs from the severe phenotype seen in Caucasian AR-CMT2K patients [7,22–24]. Our patients showed no vocal cord paresis reported in Italian patients [22], nor diaphragmatic palsy reported in Spanish patients [24]. Besides, AD-CMT2K patients in the present series also showed symptoms in the early childhood with moderate phenotype, which is different from other reports that dominant GDAP1 mutations cause a late onset and mild phenotype [8,11,13,23]. Our study findings therefore broaden the clinical picture of AD-CMT2K with great phenotypic variability, especially in the age of disease onset ranging from early childhood to adulthood and severity from mild to moderate [10,25]. Pathologically, AR-CMT2K and AD-CMT2K are not pure axonal neuropathies. In both our AR-CMT2K and AD-CMT2K patients, sural nerve biopsy revealed a predominant axonal neuropathy with mild demyelinating change, as previously reported in Caucasian patients [22,24]. The density of myelinated fibers was not largely decreased because of the increase of small myelinated fibers; however, there was a marked loss of large myelinated fibers. Demyelinating features were visible as atypical onion bulb formations and thinly myelinated fibers. Histopathological findings of previous Korean patients also
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showed mixed features of demyelinating and axonal neuropathies [8,17]. The pathological involvement of both axons and myelin might be associated with the mechanism that GDAP1 is expressed in both neurons and Schwann cells [26]. Although the histological findings might also be largely compatible with intermediate CMT, the patients should be classified into CMT2 as the median motor NCVs are above 45 m/s. We also observed mitochondrial aggregation in both axons and Schwann cells, as previously reported [10,27]. It is noteworthy that giant unmyelinated axons were visible in all patients of the present series. The giant unmyelinated axons were filled with accumulated neurofilament. Giant axons of unmyelinated fibers are also documented in CMT2A caused by MFN2 mutations [28]. We suggest that neurofilament aggregation in giant unmyelinated axon might be a common ultrastructural feature of mitochondrial neuropathies. Mutations of GDAP1 have been identified in a total of six Chinese index patients, including four in our present series and two AR-CMT patients reported previously [15,16] (Fig. 1B). Both mutations causing AR-CMT2K and AD-CMT2K are located in or near the glutathione-S-transferase (GST) domain, which is commonly targeted in Caucasian AD-CMT2K patients [1,12,29], or the α4–α5 loop of GDAP1 protein (Fig. 1B). We observed that the p.H256R is the most frequent mutation in the present series, and is present in all patients with compound heterozygous mutations. This same mutation has also been reported in a Korean patient with AR-CMT2K [17], but not in other populations. We therefore suggest that p.H256R might be a common mutation in Chinese CMT patients, despite the overall rarity of GDAP1 mutations in such patients. Case 1 inherited the p.R282H variant from his father while the p.H256R variant was apparently de novo (neither found in the father nor in the mother). Based on family history, clinical presentation and autosomal recessive inheritance of the p.H256R variant in other CMT families, it seems plausible that both variants are compound heterozygous; however, we cannot confirm this experimentally. In conclusion, both AR-CMT2K and AD-CMT2K patients in the current series presented with similar symptoms including an early onset and a moderate to severe phenotype, and similar pathological features including axonal neuropathy with mild demyelination and mitochondrial aggregation in axons and Schwann cells. This study also highlights the presence of giant axons of unmyelinated fibers filled with accumulated neurofilament in GDAP1-related CMT patients. Acknowledgements We thank the patients and their parents for cooperation. Ms. Yuehuan Zuo contributed for technical assistance in nerve biopsy preparation. This research was supported by the National Natural Science Foundation of China (No. 81471185) and by the Ministry of Science and Technology of the People’s Republic of China (No. 2011ZX09307-001-07). Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.nmd.2017.04.001.
Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001
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Please cite this article in press as: Jun Fu, et al., Similar clinical, pathological, and genetic features in Chinese patients with autosomal recessive and dominant Charcot–Marie–Tooth disease type 2K, Neuromuscular6 Disorders (2017), doi: 10.1016/j.nmd.2017.04.001