Abstract
An Italian multigenerational family with four members affected by an axonal Charcot-Marie-Tooth type 2D (CMT-2D) or distal spinal muscular atrophy (dSMA) phenotype with upper limb predominance, variable age at onset, degree of disability, and autosomal dominant inheritance is reported. A novel heterozygous missense GARS gene mutation (D500N) was identified.
The axonal forms of Charcot-Marie-Tooth disease (CMT) (CMT2A–G and L according to the locus identified) are characterized by distal predominant lower limb wasting and weakness, sensory loss, and hyporeflexia.1,2 By definition, the median motor conduction velocity is >38 m/sec. Distal spinal muscular atrophy (dSMA) represents a genetically heterogeneous group in which the progressive lower motor neuron degeneration causes limb muscle weakness and atrophy without sensory involvement. The overlap between these two different clinical entities is evident for the CMT2D and dSMA-V phenotypes because they are both associated with a more severe involvement of the upper extremities. Recently, the identification of four missense mutations in the glycyl-tRNA synthetase (GARS) gene, in families affected by features of both CMT2D and dSMA-V has given a genetic explanation for this phenotype overlapping.3–8
In this study, we describe the clinical, electrophysiologic, and neuropathologic features of an Italian family affected by both CMT-2D and dSMA-V phenotypes associated with a novel GARS mutation.
Results.
Clinical findings.
The proband, a 15-year-old boy (III-3, figure 1A), presented with a 5-year history of slowly progressive weakness affecting intrinsic hand and toe dorsiflexors muscles. He occasionally had muscle cramps at the sural triceps, fingers, and toes and of distal pinprick paraesthesias with a glove-stock distribution.
Figure 1. (A) Genealogic tree of the Italian family and segregation of the D500N mutation with CMT2D/dSMA-V phenotype; arrow indicates the proband and shaded square symbol indicates that the subject III-4 may be presymptomatic. (B) Representative section of sequence chromatograms of genomic DNA fragments encompassing the identified D500N mutation in the proband compared with an unaffected subject (GenBank, NM_002047 mRNA).
Neurologic examination showed hands with evident wasting and weakness (2/5 according to the Medical Research Council [MRS] scale) of interosseus and thenar muscles and distal lower limbs hypotrophy with associated mild weakness (MRC scale: 3/5). He was unable to walk on his heels. Deep tendon reflexes were absent distally at the upper limbs and decreased distally at the lower limbs. Sensory examination was normal. Extensive laboratory investigations highlighted an inconsistent mild increase in serum CK. Electrophysiologic assessment (table) revealed a slight reduction of amplitude and increased latency of the compound motor action potential (CMAP) in the right median and common peroneal nerves. Motor nerve conduction velocity (MNCV) was normal in the upper limbs and decreased in the lower limbs, whereas the amplitude of the right median and sural sensory nerve action potential (SNAP) was reduced. Needle EMG showed neurogenic potentials without active denervation in the upper and lower limb muscles.
Table Electrophysiologic studies of four affected members
Patient II-2, the proband’s 47-year-old father, developed bilateral hypotrophy and a mild weakness (MRC scale: 4/5) of the hand interossei and left thenar muscles at the age of 20. On examination, there were also distal leg and foot muscle weakness (MRC scale: 4/5) and sensory loss in stocking-glove distribution. Conduction studies showed a decreased MNCV in the peroneal and ulnar nerves and a reduction in ulnar and sural SNAPs (table). Neurogenic signs were detectable in the opponens pollicis, abductor of the fifth finger, and tibialis anterior muscles, with increased amplitude of muscle unit potentials.
Patient II-5, the 53-year-old paternal uncle of the proband, did not complain of any symptom, but neurologic examination revealed a slight wasting of the distal upper limbs and a reduction in deep tendon reflexes. Electrophysiologic testing evidenced a chronic neurogenic pattern in the distal upper and lower limb muscles, with increased distal latencies of the ulnar, common peroneal, and tibial nerves (table).
Patient I-7, a 70-year-old woman, presented at the age of 35 with a slowly progressive distal and symmetric upper limb weakness with a marked hypotrophy of the forearm and thenar and intrinsic hand muscles without sensory involvement. The EMG showed distal chronic neurogenic potentials in intrinsic hand and tibialis anterior muscles while SNAPs were preserved in the upper and lower limbs (table).
Subject III-4, the proband’s 6-year-old brother, often complains of cramps in the lower limb muscles without a strict correlation with exercise. He has the phenotype of a long-limbed boy and his neurologic examination was normal. EMG studies were not performed.
Subjects II-1, II-6, II-8, III-2, and III-5 had a normal neurologic history and examination. Subject I-2 could not be examined.
Genetic analysis.
For Methods, see appendix E-1 on the Neurology Web site at www.neurology.org. Sequence analysis of the GARS exons3 from the proband’s DNA revealed a heterozygous guanine-to-adenine transition at nucleotide 2016 within exon 13, resulting in an Asp-to-Asn substitution at amino acid residue 500 (D500N, figure 1B). The D500N was absent in 100 unrelated Italian healthy subjects. Analysis of available individuals within the family revealed that the mutation consistently segregates with the disease, following an autosomal dominant mode of transmission. In fact, it was also present in Subjects I-2, I-7, II-2, II-5, and III-4 and absent in the other family members.
SMN1 and SMN2 genes were not deleted; tandem duplication or deletion in the 17p11.2 chromosomal region was excluded.
Histopathology.
Sural nerve biopsy of Patient III-3 showed clear signs of axonal pathology with a substantial decrease in myelinated fibers with a preservation of larger size ones and a focal loss distribution (figure 2A). There was no evidence of active degeneration, and a variable number, two to four, of small regenerative clusters was present. Quantitative analysis performed on semithin sections revealed a fiber density of 3,044 myelinated fibers/mm2 (normal age-matched values: 6,100 myelinated fibers/mm2, SD = 1,100).
Figure 2. Proband’s nerve biopsy. (A) Semithin section stained (×400). (B, C) Electron micrograph ultrathin sections (×20,000). (A) Semithin section, toluidine blue. Loss of all diameter myelinated fibers is observed. (B) Electron-dense and amorphous inclusions (stars) are evident in Schwann cells. (C) Abnormal accumulations of glycogen-like material (stars) are present in Schwann cell cytoplasm.
Ultrastructural examination (figure 2, B and C) disclosed the presence of electron dense and amorphous inclusions in several Schwann cells. Moreover, an accumulation of glycogen-like particles was observed in the cytoplasm of some denervated Schwann cells. Periodic acid–Schiff stain, with and without diastase digestion, was negative.
Discussion.
We describe an Italian family showing autosomal dominant transmission of a distal motor or sensorimotor axonal neuropathy with a novel missense mutation of GARS gene. The findings demonstrate a broad phenotypic variability, although the main clinical aspect is always involvement of the hands, in particular wasting and weakness of the thenar and dorsal interosseus muscles. In fact, our proband (III-3) is affected by a slowly progressive axonal distal polyneuropathy, with disease onset at 10 years of age. The proband’s father and uncle have electrophysiologic and clinical abnormalities suggestive of a chronic progressive distal axonal motor and sensory neuropathy, with minimal disability. Patient I-7, on the other hand, is affected by an adult-onset, slowly progressive distal motor neuropathy mainly involving intrinsic hand muscles, with features very similar to those of dSMA. Finally, subject III-4 may be presymptomatic, being younger than the average age at disease onset.
The sural nerve biopsy of the proband confirmed the sensory axonal loss with preservation of the myelin structure. Our findings are similar to those of a recent report.4 The ultrastructural findings are different: we observed electron-dense material and accumulation of glycogen-like particles within the Schwann cell cytoplasm, whereas they observed filamentous accumulations. Further studies are needed to establish whether these findings are the expression of degradation product accumulation or are related to the altered function of glycyl-tRNA synthetase. This enzyme belongs to the class II aminoacyl-tRNA synthetase family and performs an essential function in protein synthesis by catalyzing the esterification of an amino acid to its cognate tRNA. The human GARS protein is ubiquitously expressed, including the brain and spinal cord. The D500N mutation is located upstream of the motif 3, a very highly conserved sequence (peculiar of class II tRNA synthetases), suggested to be critical for glycyl-tRNA synthetase activity. All the known six mutations (E71G, L129P, G240R, H418R, G526R, and that reported here, D500N)3,4 are randomly located in different portions of the gene spanning diverse functional domains of the protein: the genotype-phenotype correlations, as well as the basis for intrafamilial clinical variability, must still be clarified.
Further studies are needed to better distinguish the clinical continuum associated with GARS mutations and to unravel the relationship between neuropathy and neuronopathy.
Acknowledgment
The authors thank Dr. Avril Mack for language revision.
Footnotes
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Additional material related to this article can be found on the Neurology Web site. Go to www.neurology.org and scroll down the Table of Contents for the March 14 issue to find the title link for this article.
Supported by the Associazione Amici del Centro Dino Ferrari, University of Milan, the Telethon project GTF02008, the Eurobiobank project QLTR-2001-02769, and R.F. 2002 Criobanca Automatizzata di Materiale Biologico.
Disclosure: The authors report no conflicts of interest.
Received May 16, 2005. Accepted in final form November 29, 2005.
References
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