HETEROGENEITY OF AXONAL PATHOLOGY IN CHINESE PATIENTS WITH GIANT AXONAL NEUROPATHY LU WANG, PhD, DANHUA ZHAO, MD, ZHAOXIA WANG, MD, WEI ZHANG, MD, HE LV, MD, XIAO LIU, PhD, LINGCHAO MENG, MD, and YUN YUAN, MD, PhD Department of Neurology, Peking University First Hospital, 8 Xishiku Street, Xicheng District, Beijing 100034, China Accepted 19 November 2013 ABSTRACT: Introduction: Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder caused by mutations in the GAN gene. Herein we report ultrastructural changes in Chinese patients with GAN. Methods: General clinical assessment, sural nerve biopsy, and genetic analysis were performed. Results: Sural biopsy revealed giant axons in 3 patients, 2 with a mild phenotype and 1 with a classical phenotype. Ultrastructurally, all patients had giant axons filled with closely packed neurofilaments. In addition, the classical patient had some axons containing irregular tubular-like structures. GAN mutation analysis revealed novel compound heterozygous c.98A>C and c.158C>T mutations in the BTB domain in 1 mild patient, a novel homozygous c.371T>G mutation in the BACK domain in another mild patient, and a novel c.1342G>T homozygous mutation in the Kelch domain in the classical patient. Conclusion: Closely packed neurofilaments in giant axons are common pathological changes in Chinese patients with GAN, whereas irregular tubular-like structures appear in the classical type of this neuropathy. Muscle Nerve 50: 200–205, 2014
Giant axonal neuropathy (GAN; MIM256850) is a rare autosomal recessive neurodegenerative disorder characterized by early-onset, severe peripheral neuropathy, varying central nervous system (CNS) involvement, and strikingly curled hair.1 Since the first report in 1972, an increasing number of GAN patients of various ethnic origins have been diagnosed through molecular genetic approaches, and mutations in the GAN gene are responsible for the disease.2–5 The patients can be divided into 2 clinical groups based on phenotype, either classical or mild.6,7 The classical clinical phenotype is characterized by severe peripheral neuropathy with kinky hair, early onset of cerebellar and pyramidal signs, and mental deterioration.7–9 In contrast, the mild clinical phenotype of GAN usually has a relatively later onset, less severe clinical features, and a more protracted course.7,10,11 The presence of giant axons filled with closely packed neurofilaments on Abbreviations: CMAP, compound muscle action potential; CNS, central nervous system; DL, distal latency; EMG, electromyography; GAN, Giant axonal neuropathy; LFB, Luxol Fast Blue; MT, microtubule; MAP1B, microtubule-associated protein 1B; MNCV, motor nerve conduction velocity; NF, neurofilament; PCR, polymerase chain reaction; SNAP, sensory nerve action potential Key words: gene mutation; giant axonal neuropathy; gigaxonin protein; neurofilament protein; tubular-like structure This research was supported by a grant from the Twelfth Five-Year Plan of China (2011ZX09307-001-07). Correspondence to: Y. Yuan; e-mail: [email protected] C 2013 Wiley Periodicals, Inc. V
Published online 24 November 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24130
200
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nerve biopsy is a distinctive pathological feature of GAN.12,13 In this study, we report 3 Chinese patients with 4 novel mutations in the GAN gene, and we describe a new type of abnormal axons containing irregular tubular-like structures in a patient with classical phenotype. METHODS Patients. Patient 1 was the 18-year-old son of unrelated parents (Table 1). There was no history of neuromuscular disorders in his family. At age 6 years, he developed weakness and wasting of distal muscles in the lower limbs and bilateral pes cavus. He had progressive walking problems. At age 12, he had weakness and wasting of hand muscles accompanied by urinary incontinence. Physical examination at the time of diagnosis revealed no kinky hair. He had normal intelligence. Cranial nerves were intact. Muscle strength was 3/5 in distal muscles and normal in proximal muscles in the upper limbs and 1/5 in distal muscles and 4/5 in proximal muscles in the lower limbs (grades 0–5 on the Medical Research Council scale). He had atrophy of bilateral calf and intrinsic hand muscles and bilateral pes cavus. Finger-to-nose and heel-to-knee tests were normal. He had sensory loss in the upper and lower limbs, distal more than proximal. Abdominal and cremasteric reflexes were not elicited. Biceps brachii, triceps, Achilles, and patellar reflexes were absent. Babinski signs were not found. The main conduction findings included absent compound muscle action potentials (CMAPs) in the common fibular nerve; motor nerve conduction velocity (MNCV) 37.9 m/s, amplitude 2.1 mV, and distal latency (DL) 7.7 ms in the left median nerve; MNCV 38.6 m/s, amplitude 4.6 mV, and DL of 11.4 ms in the right ulnar nerve; and MNCV 30.4 m/s, amplitude 1.6 mV, and DL 19.9 ms in the right tibial nerve. Sensory nerve action potentials (SNAPs) were unobtainable in the left ulnar, median, and sural nerves. Needle electromyography (EMG) showed a reduced recruitment pattern in the tibialis anterior and quadriceps femoris muscles, with polyphasic motor unit potentials of increased amplitude and duration but no abnormal spontaneous activity. Cranial magnetic resonance imaging showed no abnormal signal. Visual evoked potentials showed prolonged latencies of bilateral P100 with normal amplitude. MUSCLE & NERVE
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Table 1. Summary of clinical data in the 3 patients with GAN.
Gender/age (years) Onset age (years) Initial symptoms Motor milestones Kinky hair Walking difficulty Cranial nerve lesions Sensory loss Weakness Pyramidal signs Cerebellar signs Dementia Scoliosis Other symptoms MRI abnormality Domain affected Clinical type
Patient 1
Patient 2
Patient 3
Male/18 6 Waddling gait Normal No Without aid No
Female/19 6 Waddling gait Normal No With aid No
Female/25 4 Waddling gait Delayed No Wheelchair Yes
Yes Yes No No No Slight Incontinence Slight BTB Mild
Yes Yes No Yes No Slight Incontinence NA BACK Mild
Yes Yes No Yes Yes Severe Seizures Severe Kelch Classical
NA, not available.
Patient 2 was a 19-year-old girl, the only child of consanguineous parents. Developmental milestones were normal. She presented at 6 years with weakness of lower limbs, ankle contractures, and walking difficulty. She was unable to jump and climb stairs. At age 9, she had difficulty squatting, with bilateral pes cavus. The muscle weakness developed slowly, and she needed orthopedic shoes to walk. Physical examination showed bilateral pes cavus and contracture of Achilles tendons, but no kinky hair. Neurological examination at the time of diagnosis showed normal intelligence. She had horizontal nystagmus. Muscle strength was almost normal in the upper limbs, 2/5 in distal muscles, and 4/5 in proximal muscles in the lower limbs with decreased muscle tone. She had a positive Romberg sign. Loss of cutaneous and vibration sensation was present in the distal lower limbs. Abdominal reflexes could not be elicited. The biceps brachii, triceps, patellar, and Achilles reflexes were absent. She did not have Babinksi signs. Creatine kinase was 270 IU/L (normal 25–175 IU/L). Nerve conduction studies of the lower limbs disclosed no distal CMAPs from bilateral fibular nerves, MNCV was 34.8 m/s, amplitude was 0.7 mV, and DL was 12.3 ms in the left tibial nerve. MNCV was 33.4 m/s, amplitude was 0.6 mV, and DL was 13.1 ms in the right tibial nerve. MNCVs were within normal limits in the upper limbs. SNAPs were absent in the left median, ulnar, and right sural nerves. Needle EMG of the left quadriceps femoris and tibialis anterior muscles showed marked high-amplitude, long-duration motor unit potentials (MUPs) with reduced recruitment. Fibrillation potentials were observed in the left tibialis anterior muscle. Axonal Pathology in GAN
Patient 3, a 25-year-old woman, was the only child of consanguineous parents. She had delayed motor milestones. Gait disturbance with pes cavus was observed at 4 years of age. After orthopedic correction of her ankle, she developed scoliosis, weakness, and wasting, as well as sensory disturbance in the distal lower limbs. At around age 16 she could not stand, even with support, and used a wheelchair. At the same time, she developed upper limb muscle wasting and weakness, dysphagia, dysarthria, and cognitive decline. At age 24 she had progressive hearing loss and seizures. Physical examination at age 25 revealed severe scoliosis and arthrogryposis in the hands and feet. She had normal hair. Neurological examination showed dementia, nystagmus, mild facial weakness, and severe hearing loss. Marked atrophy was observed in all limbs, especially in distal muscles. Muscle strength was 3/5 in the upper limbs and 0/5 in the lower limbs. Hypotonia, areflexia, and impairment of cutaneous and vibration sensation distally in the lower extremities were found. Electrophysiological studies showed MNCV was 21.7 m/s, amplitude was 0.2 mV, and DL was 14.2 ms in the left median nerve. MNCV was 32.5 m/s, amplitude was 1.1 mV, and DL was 11.5 ms in the right median nerve. No distal CMAPs from tibial or fibular nerves and SNAPs from median, ulnar, and sural nerves were recorded. Fibrillation potentials and positive sharp waves were detected on needle EMG in both abductor pollicis brevis, tibialis anterior, and quadriceps femoris muscles. Cranial magnetic resonance imaging (MRI) revealed general brain atrophy with diffuse T2 hyperintensity in bilateral subcortical and periventricular white matter, internal and external capsules, posteromedial thalamus, brainstem, superior and middle cerebellar peduncles, and cerebellar white matter (Fig. 1A and B). METHODS
The ethics committee of Peking University First Hospital, China, approved this study. Written informed consent was signed by each participant before initiation of the investigation in compliance with the Declaration of Helsinki. Sural nerve biopsy samples were obtained from the 3 patients. One piece of the nerve was fixed in 4% formaldehyde, paraffin-embedded, cut into 8lm sections, and stained with hematoxylin and eosin, Luxol Fast Blue (LFB), and Congo Red. The rest of the specimen was fixed in 3% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated through serial alcohol baths, and embedded in Epon 812. Semithin sections for light microscopy were stained with toluidine blue. Ultrathin sections were contrasted with uranyl acetate and lead citrate, then examined under electron microscopy. MUSCLE & NERVE
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FIGURE 1. Cranial MRI of patient 3 showed diffuse white matter changes with involvement of the posteromedial thalamus and internal and external capsules (A). T2 hyperintensity was present in the brainstem, superior and middle cerebellar peduncles, and cerebellar white matter (B).
Gene sequencing of GAN was analyzed in DNA from the 3 patients, the parents of patient 1, the mother of patient 3, and 100 healthy Chinese controls. We took 5 ml of peripheral blood and used the salt precipitation method to extract genomic DNA. Polymerase chain reaction (PCR) primers for exons 1–11 of GAN were designed using Primer 3 software. PCR was performed using standard protocols. PCR products were purified and then sequenced on an automatic sequencing machine (ABI 3730XL). RESULTS Nerve Biopsy.
Sural nerve biopsies from the 3 patients showed moderate to severe loss of myelinated fibers. The total myelinated fiber density was 3200/mm2 (range 2880–3648/mm2) in patient 1, 2240/mm2 (range 1920–2688/mm2) in patient 2, and 1280/mm2 (range 1024–1728/mm2) in patient 3 (Fig. 2A). The most striking feature in the nerve
biopsies was abnormally enlarged axons (diameter 15–25 lm) with a homogeneous and mildly eosinophilic axoplasm wrapped with thin or absent myelin sheaths. Semithin sections revealed giant axons with homogeneous material in all patients (Fig. 2B). Occasionally, atypical onion bulbs and a few regenerating clusters were observed. Ultrastructural examination disclosed 2 types of abnormal axons. The typical type was a giant axon filled with closely packed, irregularly oriented neurofilaments in all 3 patients (Fig. 3A and B). Atypical abnormal axons, which contain irregular tubular-like structures (diameters 37–52 nm), were found only in patient 3 (Fig. 4A). These materials were distributed in the whole axon, and small bundles of fine filaments were scattered between them (Fig. 4B). In some areas, microtubules could be observed beneath the axolemmal membrane (Fig. 4A). All affected axons exhibited thin or absent myelin sheaths. There were no irregular
FIGURE 2. In patient 3, LFB staining showed reduction of myelinated nerve fibers (A). Semithin sections of the sural nerve from patient 2 revealed giant axons (arrow) and few regenerating clusters (B). 202
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FIGURE 3. Electron microscopic examination of the sural nerve from patient 2 showed a giant axon (A) filled with closely packed neurofilaments (B).
tubular-like structures or neurofilament accumulation in Schwann cells, vascular endothelial cells, perineurial cells, or endoneurial fibroblasts. Four novel missense mutations were identified in the GAN gene (Fig. 5). Patient 1 had compound heterozygous c.98A>C and c.158C>T mutations in exon 1, resulting in amino acid substitutions of histidine to proline (p.H33P) and proline to leucine (p.P53L) in the BTB domain (Fig. 5A and B). The mother carried the c.98A>C mutation, whereas the father had the c.158C>T mutation. Patient 2 had a homozygous c.371T>G mutation in exon 3, leading to an amino acid substitution of phenylalanine to cysteine (p.F124C) in the BACK domain (Fig. 5C). Patient 3 had a homozygous c.1342G>T mutation in exon 8, resulting in a missense mutation of tryptophan to leucine (p.W448L) in the Kelch domain (Fig. 5D). Her mother was a heterozygous carrier. These mutations were not present in 100 healthy Chinese controls or listed in the Human Gene Mutation Database (www.hgmd.cf.ac.uk).
Genetic Analysis.
DISCUSSION
We describe 3 Chinese patients with GAN. They all presented clinically with progressive neu-
ropathy. Although they did not have thick curly hair, they had the typical giant axons in peripheral nerves. The diagnosis of GAN was confirmed genetically. Patients 1 and 2 had mild phenotypes. In addition to severe motor and sensory neuropathy with an onset in early childhood, patient 1 had no central nervous system (CNS) signs, and patient 2 had only minor cerebellar signs. Patient 3 had the classical phenotype with severe CNS signs. The brain MRI showed no abnormal signal in the mild case (patient 1), whereas diffuse hyperintensity in the white matter and in the posteromedial thalamus was obvious in the classical patient (patient 3). Involvement of the posteromedial thalamus has been described previously in other reported cases of classical GAN.14–16 To our knowledge, high signal in peripheral areas of the midbrain and pons has not been described previously. Nerve biopsy demonstrated 2 types of abnormal axons. The typical giant axons filled with closely packed neurofilaments were found in all 3 patients. However, there were atypical axons containing irregular tubular-like structures in patient 3 with the classical phenotype. Meanwhile, microtubules and fine filaments were also observed in these abnormal axons. The irregular, tubular-like structures could be distinguished from neurofilaments morphologically
FIGURE 4. Electron microscopic examination of the sural nerve from patient 3 showed axons filled with irregular tubular-like structures (arrows). Microtubules appear beneath the axolemmal membrane (A), and the tubular-like masses are distributed peripherally to the fine filaments in small patches (B). MT, microtubule; NF, neurofilaments. Axonal Pathology in GAN
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FIGURE 5. Compound heterozygous mutations c.98A>C and c.158C>T in the GAN gene were identified in patient 1 (A, B). Homozygous mutations c.371T>G and c.1342G>T were found in patients 2 and 3, respectively (C, D). P, sequence from the proband; M, sequence from the proband’s mother; F, sequence from the proband’s father; C, sequence from a healthy control (arrows indicate mutation sites).
by their higher density. Compared with the diameter of neurofilaments, the minimal diameter of the tubular-like structures was larger. Donaghy et al. described 2 types of abnormal material within GAN axons: osmiophilic amorphous matter and paracrystalline arrays, and it was impossible to state that the major component of the amorphous matter was of neurofilamentous origin.17 Ding et al. proposed that toxic accumulation of microtubule-associated protein 8 was related to a disturbed microtubule network in GAN-null mice.18 Based on these observations, we hypothesize that the irregular, tubular-like structures consist of abnormal microtubule-associated proteins. The GAN gene encodes gigaxonin, which contains an N-terminal BTB domain, with the Cterminal Kelch domain and the BACK domain between them.19 Four novel compound heterozygous or homozygous mutations in the GAN gene in our patients were located in the BTB, BACK, and Kelch domains, respectively. The mutation in the patient with irregular tubular-like structures in axons was located in the C-terminal Kelch domain. The Kelch domain of gigaxonin interacts with microtubule-associated protein 1B (MAP1B), microtubule-associated protein 8, and tubulinfolding cofactor B. Gigaxonin can also bind to the ubiquitin-activating enzyme E1 through its amino-terminal BTB domain.18 Allen et al. concluded that ablation of gigaxonin results in sub204
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stantial accumulation of MAP1B light chains, and aberrant increases in MAP1B levels affected transport processes indirectly by altering microtubule dynamics.20 A previous proteomics study suggested that disturbed cytoskeletal regulation was responsible for aggregation of intermediate filaments.21 Thus, we propose that the tubular-like structures may result from disruption of cytoskeletal regulation. Since the discovery of gigaxonin, more than 50 mutated alleles of GAN, including our patients, have been reported, and most of them are unique. Houlden et al. reported that mutations in the BTB domain seem to cause an earlier onset, with loss of ambulation at an earlier age compared with mutations in the Kelch repeats or other sites.8 However, our patients showed contrary findings, including a mild phenotype with mutations in the BTB or BACK domain and a classical phenotype with mutation in the Kelch domain. Our study further supports the opinion that there is no consistent relationship between genotype and phenotype among GAN patients.6,22 The mutation in the patient with irregular, tubular-like structures in axons was located in the C-terminal Kelch domain. Further studies are needed to elucidate the relationship between the genotype and different types of abnormal axons. In conclusion, these findings highlight the pathologic, genetic, and phenotypic heterogeneity in Chinese patients with GAN. Irregular, tubularMUSCLE & NERVE
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like structures appear in the classical Chinese patient, and closely packed neurofilaments are observed in all patients. The authors gratefully acknowledge the participation of all patients and their families. We also thank Yuehuan Zuo and Qiurong Zhang for skillful technical assistance. REFERENCES 1. Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M, et al. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/Kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet 2000;26:370–374. 2. Asbury AK, Gale MK, Cox SC, Baringer JR, Berg BO. Giant axonal neuropathy: a unique case with segmental neurofilamentous masses. Acta Neuropathol 1972;20:237–247. 3. Tazir M, Vallat JM, Bomont P, Zemmouri R, Sindou P, Assami S, et al. Genetic heterogeneity in giant axonal neuropathy: an Algerian family not linked to chromosome 16q24.1. Neuromuscul Disord 2002;12:849–852. 4. Bruno C, Bertini E, Federico A, Tonoli E, Lispi ML, Cassandrini D, et al. Clinical and molecular findings in patients with giant axonal neuropathy (GAN). Neurology 2004;62:13–16. 5. Bomont P, Ioos C, Yalcinkaya C, Korinthenberg R, Vallat JM, Assami S, et al. Identification of seven novel mutations in the GAN gene. Hum Mutat 2003;21:446. 6. Akagi M, Mohri I, Iwatani Y, Kagitani-Shimono K, Okinaga T, Sakai N, et al. Clinicogenetical features of a Japanese patient with giant axonal neuropathy. Brain Devel 2012;34:156–162. 7. Tazir M, Nouioua S, Magy L, Huehne K, Assami S, Urtizberea A, et al. Phenotypic variability in giant axonal neuropathy. Neuromuscul Disord 2009;19:270–274. 8. Houlden H, Groves M, Miedzybrodzka Z, Roper H, Willis T, Winer J, et al. New mutations, genotype phenotype studies and manifesting carriers in giant axonal neuropathy. J Neurol Neurosurg Psychiatry 2007;78:1267–1270. 9. Zhang LP, Zou LP. Clinical and genetic studies in a Chinese family with giant axonal neuropathy. J Child Neurol 2009;24:1552–1556.
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10. Kuhlenb€aumer G, Young P, Oberwittler C, H€ unermund G, Schirmacher A, Domschke K, et al. Giant axonal neuropathy (GAN): case report and two novel mutations in the gigaxonin gene. Neurology 2002;58:1273–1276. 11. Zemmouri R, Azzedine H, Assami S, Kitouni N, Vallat JM, Maisonobe T, et al. Charcot–Marie–Tooth 2 like presentation of an Algerian family with giant axonal neuropathy. Neuromuscul Disord 2000;10: 592–598. 12. Carpenter S, Karpati G, Andermann F, Gold R. Giant axonal neuropathy. A clinically and morphologically distinct neurological disease. Arch Neurol 1974;31:312–316. 13. Yuan Y, Bergmann M, Gerfelmeyer G, Kuchelmeister K. Giant axonal neuropathy. A case report. Pathologe 1996;17:213–218. 14. Alkan A, Kutlu R, Sigirci A, Baysal T, Altinok T, Yakinci C. Giant axonal neuropathy: MRS findings. J Neuroimaging 2003;13:371–375. 15. Demir E, Bomont P, Erdem S, Cavalier L, Demirci M, Kose G, et al. Giant axonalneuropathy: clinical and genetic study in six cases. J Neurol Neurosurg Psychiatry 2005;76:825–832. 16. Ravishankar S, Goel G, Rautenstrauss CP, Nalini A. Spectrum of magnetic resonance imaging findings in a family with giant axonal neuropathy confirmed by genetic studies. Neurol India 2009;57:181–184. 17. Donaghy M, King RH, Thomas PK, Workman JM. Abnormalities of the axonal cytoskeleton in giant axonal neuropathy. J Neurocytol 1988;17:197–208. 18. Ding J, Allen E, Wang W, Valle A, Wu C, Nardine T, et al. Gene targeting of GAN in mouse causes a toxic accumulation of microtubule associated protein 8 and impaired retrograde axonal transport. Hum Mol Genet 2006;15:1451–1463. 19. Stogios PJ, Downs GS, Jauhal JJ, Nandra SK, Priv e GG. Sequence and structural analysis of BTB domain proteins. Genome Biol 2005;6:R82. 20. Allen E, Ding J, Wang W, Pramanik S, Chou J, Yau V, et al. Gigaxonin-controlled degradation of MAP1B light chain is critical to neuronal survival. Nature 2005;438:224–228. 21. Mussche S, De Paepe B, Smet J, Devreese K, Lissens W, Rasic VM, et al. Proteomic analysis in giant axonal neuropathy: new insights into disease mechanisms. Muscle Nerve 2012;46:246–256. 22. Koop O, Schirmacher A, Nelis E, Timmerman V, de Jonghe P, Ringelstein B, et al. Genotype–phenotype analysis in patients with giant axonal neuropathy (GAN). Neuromuscul Disord 2007;17: 624–630.
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Giant axonal neuropathy (GAN; MIM256850) is a rare autosomal recessive neurodegenerative disorder characterized by early-onset, severe peripheral neuropathy, varying central nervous system (CNS) involvement, and strikingly curled hair.1 Since the first report in 1972, an increasing number of GAN patients of various ethnic origins have been diagnosed through molecular genetic approaches, and mutations in the GAN gene are responsible for the disease.2–5 The patients can be divided into 2 clinical groups based on phenotype, either classical or mild.6,7 The classical clinical phenotype is characterized by severe peripheral neuropathy with kinky hair, early onset of cerebellar and pyramidal signs, and mental deterioration.7–9 In contrast, the mild clinical phenotype of GAN usually has a relatively later onset, less severe clinical features, and a more protracted course.7,10,11 The presence of giant axons filled with closely packed neurofilaments on Abbreviations: CMAP, compound muscle action potential; CNS, central nervous system; DL, distal latency; EMG, electromyography; GAN, Giant axonal neuropathy; LFB, Luxol Fast Blue; MT, microtubule; MAP1B, microtubule-associated protein 1B; MNCV, motor nerve conduction velocity; NF, neurofilament; PCR, polymerase chain reaction; SNAP, sensory nerve action potential Key words: gene mutation; giant axonal neuropathy; gigaxonin protein; neurofilament protein; tubular-like structure This research was supported by a grant from the Twelfth Five-Year Plan of China (2011ZX09307-001-07). Correspondence to: Y. Yuan; e-mail: [email protected] C 2013 Wiley Periodicals, Inc. V
Published online 24 November 2013 in Wiley Online Library (wileyonlinelibrary. com). DOI 10.1002/mus.24130
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nerve biopsy is a distinctive pathological feature of GAN.12,13 In this study, we report 3 Chinese patients with 4 novel mutations in the GAN gene, and we describe a new type of abnormal axons containing irregular tubular-like structures in a patient with classical phenotype. METHODS Patients. Patient 1 was the 18-year-old son of unrelated parents (Table 1). There was no history of neuromuscular disorders in his family. At age 6 years, he developed weakness and wasting of distal muscles in the lower limbs and bilateral pes cavus. He had progressive walking problems. At age 12, he had weakness and wasting of hand muscles accompanied by urinary incontinence. Physical examination at the time of diagnosis revealed no kinky hair. He had normal intelligence. Cranial nerves were intact. Muscle strength was 3/5 in distal muscles and normal in proximal muscles in the upper limbs and 1/5 in distal muscles and 4/5 in proximal muscles in the lower limbs (grades 0–5 on the Medical Research Council scale). He had atrophy of bilateral calf and intrinsic hand muscles and bilateral pes cavus. Finger-to-nose and heel-to-knee tests were normal. He had sensory loss in the upper and lower limbs, distal more than proximal. Abdominal and cremasteric reflexes were not elicited. Biceps brachii, triceps, Achilles, and patellar reflexes were absent. Babinski signs were not found. The main conduction findings included absent compound muscle action potentials (CMAPs) in the common fibular nerve; motor nerve conduction velocity (MNCV) 37.9 m/s, amplitude 2.1 mV, and distal latency (DL) 7.7 ms in the left median nerve; MNCV 38.6 m/s, amplitude 4.6 mV, and DL of 11.4 ms in the right ulnar nerve; and MNCV 30.4 m/s, amplitude 1.6 mV, and DL 19.9 ms in the right tibial nerve. Sensory nerve action potentials (SNAPs) were unobtainable in the left ulnar, median, and sural nerves. Needle electromyography (EMG) showed a reduced recruitment pattern in the tibialis anterior and quadriceps femoris muscles, with polyphasic motor unit potentials of increased amplitude and duration but no abnormal spontaneous activity. Cranial magnetic resonance imaging showed no abnormal signal. Visual evoked potentials showed prolonged latencies of bilateral P100 with normal amplitude. MUSCLE & NERVE
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Table 1. Summary of clinical data in the 3 patients with GAN.
Gender/age (years) Onset age (years) Initial symptoms Motor milestones Kinky hair Walking difficulty Cranial nerve lesions Sensory loss Weakness Pyramidal signs Cerebellar signs Dementia Scoliosis Other symptoms MRI abnormality Domain affected Clinical type
Patient 1
Patient 2
Patient 3
Male/18 6 Waddling gait Normal No Without aid No
Female/19 6 Waddling gait Normal No With aid No
Female/25 4 Waddling gait Delayed No Wheelchair Yes
Yes Yes No No No Slight Incontinence Slight BTB Mild
Yes Yes No Yes No Slight Incontinence NA BACK Mild
Yes Yes No Yes Yes Severe Seizures Severe Kelch Classical
NA, not available.
Patient 2 was a 19-year-old girl, the only child of consanguineous parents. Developmental milestones were normal. She presented at 6 years with weakness of lower limbs, ankle contractures, and walking difficulty. She was unable to jump and climb stairs. At age 9, she had difficulty squatting, with bilateral pes cavus. The muscle weakness developed slowly, and she needed orthopedic shoes to walk. Physical examination showed bilateral pes cavus and contracture of Achilles tendons, but no kinky hair. Neurological examination at the time of diagnosis showed normal intelligence. She had horizontal nystagmus. Muscle strength was almost normal in the upper limbs, 2/5 in distal muscles, and 4/5 in proximal muscles in the lower limbs with decreased muscle tone. She had a positive Romberg sign. Loss of cutaneous and vibration sensation was present in the distal lower limbs. Abdominal reflexes could not be elicited. The biceps brachii, triceps, patellar, and Achilles reflexes were absent. She did not have Babinksi signs. Creatine kinase was 270 IU/L (normal 25–175 IU/L). Nerve conduction studies of the lower limbs disclosed no distal CMAPs from bilateral fibular nerves, MNCV was 34.8 m/s, amplitude was 0.7 mV, and DL was 12.3 ms in the left tibial nerve. MNCV was 33.4 m/s, amplitude was 0.6 mV, and DL was 13.1 ms in the right tibial nerve. MNCVs were within normal limits in the upper limbs. SNAPs were absent in the left median, ulnar, and right sural nerves. Needle EMG of the left quadriceps femoris and tibialis anterior muscles showed marked high-amplitude, long-duration motor unit potentials (MUPs) with reduced recruitment. Fibrillation potentials were observed in the left tibialis anterior muscle. Axonal Pathology in GAN
Patient 3, a 25-year-old woman, was the only child of consanguineous parents. She had delayed motor milestones. Gait disturbance with pes cavus was observed at 4 years of age. After orthopedic correction of her ankle, she developed scoliosis, weakness, and wasting, as well as sensory disturbance in the distal lower limbs. At around age 16 she could not stand, even with support, and used a wheelchair. At the same time, she developed upper limb muscle wasting and weakness, dysphagia, dysarthria, and cognitive decline. At age 24 she had progressive hearing loss and seizures. Physical examination at age 25 revealed severe scoliosis and arthrogryposis in the hands and feet. She had normal hair. Neurological examination showed dementia, nystagmus, mild facial weakness, and severe hearing loss. Marked atrophy was observed in all limbs, especially in distal muscles. Muscle strength was 3/5 in the upper limbs and 0/5 in the lower limbs. Hypotonia, areflexia, and impairment of cutaneous and vibration sensation distally in the lower extremities were found. Electrophysiological studies showed MNCV was 21.7 m/s, amplitude was 0.2 mV, and DL was 14.2 ms in the left median nerve. MNCV was 32.5 m/s, amplitude was 1.1 mV, and DL was 11.5 ms in the right median nerve. No distal CMAPs from tibial or fibular nerves and SNAPs from median, ulnar, and sural nerves were recorded. Fibrillation potentials and positive sharp waves were detected on needle EMG in both abductor pollicis brevis, tibialis anterior, and quadriceps femoris muscles. Cranial magnetic resonance imaging (MRI) revealed general brain atrophy with diffuse T2 hyperintensity in bilateral subcortical and periventricular white matter, internal and external capsules, posteromedial thalamus, brainstem, superior and middle cerebellar peduncles, and cerebellar white matter (Fig. 1A and B). METHODS
The ethics committee of Peking University First Hospital, China, approved this study. Written informed consent was signed by each participant before initiation of the investigation in compliance with the Declaration of Helsinki. Sural nerve biopsy samples were obtained from the 3 patients. One piece of the nerve was fixed in 4% formaldehyde, paraffin-embedded, cut into 8lm sections, and stained with hematoxylin and eosin, Luxol Fast Blue (LFB), and Congo Red. The rest of the specimen was fixed in 3% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated through serial alcohol baths, and embedded in Epon 812. Semithin sections for light microscopy were stained with toluidine blue. Ultrathin sections were contrasted with uranyl acetate and lead citrate, then examined under electron microscopy. MUSCLE & NERVE
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FIGURE 1. Cranial MRI of patient 3 showed diffuse white matter changes with involvement of the posteromedial thalamus and internal and external capsules (A). T2 hyperintensity was present in the brainstem, superior and middle cerebellar peduncles, and cerebellar white matter (B).
Gene sequencing of GAN was analyzed in DNA from the 3 patients, the parents of patient 1, the mother of patient 3, and 100 healthy Chinese controls. We took 5 ml of peripheral blood and used the salt precipitation method to extract genomic DNA. Polymerase chain reaction (PCR) primers for exons 1–11 of GAN were designed using Primer 3 software. PCR was performed using standard protocols. PCR products were purified and then sequenced on an automatic sequencing machine (ABI 3730XL). RESULTS Nerve Biopsy.
Sural nerve biopsies from the 3 patients showed moderate to severe loss of myelinated fibers. The total myelinated fiber density was 3200/mm2 (range 2880–3648/mm2) in patient 1, 2240/mm2 (range 1920–2688/mm2) in patient 2, and 1280/mm2 (range 1024–1728/mm2) in patient 3 (Fig. 2A). The most striking feature in the nerve
biopsies was abnormally enlarged axons (diameter 15–25 lm) with a homogeneous and mildly eosinophilic axoplasm wrapped with thin or absent myelin sheaths. Semithin sections revealed giant axons with homogeneous material in all patients (Fig. 2B). Occasionally, atypical onion bulbs and a few regenerating clusters were observed. Ultrastructural examination disclosed 2 types of abnormal axons. The typical type was a giant axon filled with closely packed, irregularly oriented neurofilaments in all 3 patients (Fig. 3A and B). Atypical abnormal axons, which contain irregular tubular-like structures (diameters 37–52 nm), were found only in patient 3 (Fig. 4A). These materials were distributed in the whole axon, and small bundles of fine filaments were scattered between them (Fig. 4B). In some areas, microtubules could be observed beneath the axolemmal membrane (Fig. 4A). All affected axons exhibited thin or absent myelin sheaths. There were no irregular
FIGURE 2. In patient 3, LFB staining showed reduction of myelinated nerve fibers (A). Semithin sections of the sural nerve from patient 2 revealed giant axons (arrow) and few regenerating clusters (B). 202
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FIGURE 3. Electron microscopic examination of the sural nerve from patient 2 showed a giant axon (A) filled with closely packed neurofilaments (B).
tubular-like structures or neurofilament accumulation in Schwann cells, vascular endothelial cells, perineurial cells, or endoneurial fibroblasts. Four novel missense mutations were identified in the GAN gene (Fig. 5). Patient 1 had compound heterozygous c.98A>C and c.158C>T mutations in exon 1, resulting in amino acid substitutions of histidine to proline (p.H33P) and proline to leucine (p.P53L) in the BTB domain (Fig. 5A and B). The mother carried the c.98A>C mutation, whereas the father had the c.158C>T mutation. Patient 2 had a homozygous c.371T>G mutation in exon 3, leading to an amino acid substitution of phenylalanine to cysteine (p.F124C) in the BACK domain (Fig. 5C). Patient 3 had a homozygous c.1342G>T mutation in exon 8, resulting in a missense mutation of tryptophan to leucine (p.W448L) in the Kelch domain (Fig. 5D). Her mother was a heterozygous carrier. These mutations were not present in 100 healthy Chinese controls or listed in the Human Gene Mutation Database (www.hgmd.cf.ac.uk).
Genetic Analysis.
DISCUSSION
We describe 3 Chinese patients with GAN. They all presented clinically with progressive neu-
ropathy. Although they did not have thick curly hair, they had the typical giant axons in peripheral nerves. The diagnosis of GAN was confirmed genetically. Patients 1 and 2 had mild phenotypes. In addition to severe motor and sensory neuropathy with an onset in early childhood, patient 1 had no central nervous system (CNS) signs, and patient 2 had only minor cerebellar signs. Patient 3 had the classical phenotype with severe CNS signs. The brain MRI showed no abnormal signal in the mild case (patient 1), whereas diffuse hyperintensity in the white matter and in the posteromedial thalamus was obvious in the classical patient (patient 3). Involvement of the posteromedial thalamus has been described previously in other reported cases of classical GAN.14–16 To our knowledge, high signal in peripheral areas of the midbrain and pons has not been described previously. Nerve biopsy demonstrated 2 types of abnormal axons. The typical giant axons filled with closely packed neurofilaments were found in all 3 patients. However, there were atypical axons containing irregular tubular-like structures in patient 3 with the classical phenotype. Meanwhile, microtubules and fine filaments were also observed in these abnormal axons. The irregular, tubular-like structures could be distinguished from neurofilaments morphologically
FIGURE 4. Electron microscopic examination of the sural nerve from patient 3 showed axons filled with irregular tubular-like structures (arrows). Microtubules appear beneath the axolemmal membrane (A), and the tubular-like masses are distributed peripherally to the fine filaments in small patches (B). MT, microtubule; NF, neurofilaments. Axonal Pathology in GAN
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FIGURE 5. Compound heterozygous mutations c.98A>C and c.158C>T in the GAN gene were identified in patient 1 (A, B). Homozygous mutations c.371T>G and c.1342G>T were found in patients 2 and 3, respectively (C, D). P, sequence from the proband; M, sequence from the proband’s mother; F, sequence from the proband’s father; C, sequence from a healthy control (arrows indicate mutation sites).
by their higher density. Compared with the diameter of neurofilaments, the minimal diameter of the tubular-like structures was larger. Donaghy et al. described 2 types of abnormal material within GAN axons: osmiophilic amorphous matter and paracrystalline arrays, and it was impossible to state that the major component of the amorphous matter was of neurofilamentous origin.17 Ding et al. proposed that toxic accumulation of microtubule-associated protein 8 was related to a disturbed microtubule network in GAN-null mice.18 Based on these observations, we hypothesize that the irregular, tubular-like structures consist of abnormal microtubule-associated proteins. The GAN gene encodes gigaxonin, which contains an N-terminal BTB domain, with the Cterminal Kelch domain and the BACK domain between them.19 Four novel compound heterozygous or homozygous mutations in the GAN gene in our patients were located in the BTB, BACK, and Kelch domains, respectively. The mutation in the patient with irregular tubular-like structures in axons was located in the C-terminal Kelch domain. The Kelch domain of gigaxonin interacts with microtubule-associated protein 1B (MAP1B), microtubule-associated protein 8, and tubulinfolding cofactor B. Gigaxonin can also bind to the ubiquitin-activating enzyme E1 through its amino-terminal BTB domain.18 Allen et al. concluded that ablation of gigaxonin results in sub204
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stantial accumulation of MAP1B light chains, and aberrant increases in MAP1B levels affected transport processes indirectly by altering microtubule dynamics.20 A previous proteomics study suggested that disturbed cytoskeletal regulation was responsible for aggregation of intermediate filaments.21 Thus, we propose that the tubular-like structures may result from disruption of cytoskeletal regulation. Since the discovery of gigaxonin, more than 50 mutated alleles of GAN, including our patients, have been reported, and most of them are unique. Houlden et al. reported that mutations in the BTB domain seem to cause an earlier onset, with loss of ambulation at an earlier age compared with mutations in the Kelch repeats or other sites.8 However, our patients showed contrary findings, including a mild phenotype with mutations in the BTB or BACK domain and a classical phenotype with mutation in the Kelch domain. Our study further supports the opinion that there is no consistent relationship between genotype and phenotype among GAN patients.6,22 The mutation in the patient with irregular, tubular-like structures in axons was located in the C-terminal Kelch domain. Further studies are needed to elucidate the relationship between the genotype and different types of abnormal axons. In conclusion, these findings highlight the pathologic, genetic, and phenotypic heterogeneity in Chinese patients with GAN. Irregular, tubularMUSCLE & NERVE
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like structures appear in the classical Chinese patient, and closely packed neurofilaments are observed in all patients. The authors gratefully acknowledge the participation of all patients and their families. We also thank Yuehuan Zuo and Qiurong Zhang for skillful technical assistance. REFERENCES 1. Bomont P, Cavalier L, Blondeau F, Ben Hamida C, Belal S, Tazir M, et al. The gene encoding gigaxonin, a new member of the cytoskeletal BTB/Kelch repeat family, is mutated in giant axonal neuropathy. Nat Genet 2000;26:370–374. 2. Asbury AK, Gale MK, Cox SC, Baringer JR, Berg BO. Giant axonal neuropathy: a unique case with segmental neurofilamentous masses. Acta Neuropathol 1972;20:237–247. 3. Tazir M, Vallat JM, Bomont P, Zemmouri R, Sindou P, Assami S, et al. Genetic heterogeneity in giant axonal neuropathy: an Algerian family not linked to chromosome 16q24.1. Neuromuscul Disord 2002;12:849–852. 4. Bruno C, Bertini E, Federico A, Tonoli E, Lispi ML, Cassandrini D, et al. Clinical and molecular findings in patients with giant axonal neuropathy (GAN). Neurology 2004;62:13–16. 5. Bomont P, Ioos C, Yalcinkaya C, Korinthenberg R, Vallat JM, Assami S, et al. Identification of seven novel mutations in the GAN gene. Hum Mutat 2003;21:446. 6. Akagi M, Mohri I, Iwatani Y, Kagitani-Shimono K, Okinaga T, Sakai N, et al. Clinicogenetical features of a Japanese patient with giant axonal neuropathy. Brain Devel 2012;34:156–162. 7. Tazir M, Nouioua S, Magy L, Huehne K, Assami S, Urtizberea A, et al. Phenotypic variability in giant axonal neuropathy. Neuromuscul Disord 2009;19:270–274. 8. Houlden H, Groves M, Miedzybrodzka Z, Roper H, Willis T, Winer J, et al. New mutations, genotype phenotype studies and manifesting carriers in giant axonal neuropathy. J Neurol Neurosurg Psychiatry 2007;78:1267–1270. 9. Zhang LP, Zou LP. Clinical and genetic studies in a Chinese family with giant axonal neuropathy. J Child Neurol 2009;24:1552–1556.
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