Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V) are axonal peripheral neuropathies inherited in an autosomal dominant fashion. Our previous genetic and physical mapping efforts localized the responsible gene(s) to a well-defined region on human chromosome 7p. Here, we report the identification of four disease-associated missense mutations in the glycyl tRNA synthetase gene in families with CMT2D and dSMA-V. This is the first example of an aminoacyl tRNA synthetase being implicated in a human genetic disease, which makes genes that encode these enzymes relevant candidates for other inherited neuropathies and motor neuron diseases. Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V) are axonal peripheral neuropathies inherited in an autosomal dominant fashion. Our previous genetic and physical mapping efforts localized the responsible gene(s) to a well-defined region on human chromosome 7p. Here, we report the identification of four disease-associated missense mutations in the glycyl tRNA synthetase gene in families with CMT2D and dSMA-V. This is the first example of an aminoacyl tRNA synthetase being implicated in a human genetic disease, which makes genes that encode these enzymes relevant candidates for other inherited neuropathies and motor neuron diseases. Charcot-Marie-Tooth (CMT) disease constitutes a heterogeneous group of peripheral neuropathies estimated to affect 1 in 2,500 individuals (Skre Skre, 1974Skre H Genetic and clinical aspects of Charcot-Marie-Tooth’s disease.Clin Genet. 1974; 6: 98-118Crossref PubMed Scopus (663) Google Scholar). The clinical features of CMT include muscular weakness and atrophy in the distal extremities, steppage gait, pes cavus, absent or diminished deep-tendon reflexes, and impaired sensation (Murakami et al. Murakami et al., 1996Murakami T Garcia CA Reiter LT Lupski JR Charcot-Marie-Tooth disease and related neuropathies.Medicine. 1996; 75: 233-250Crossref PubMed Scopus (68) Google Scholar). Through the measurement of motor nerve conductance velocities (MNCVs), CMT can be subdivided into two classes (Dyck and Lambert Dyck and Lambert, 1968Dyck PJ Lambert EH Lower motor and primary sensory neuron diseases with peroneal muscular atrophy.Arch Neurol. 1968; 18: 619-625Crossref PubMed Scopus (348) Google Scholar). In CMT1, patients exhibit decreased MNCVs with demyelinating axons. In CMT2, patients exhibit normal MNCVs and no demyelination but have decreased amplitudes of evoked motor and sensory nerve responses. To date, six subtypes of CMT2 have been reported (CMT2A–F), with the genes responsible for three of these now identified: (1) CMT2A: kinesin superfamily gene (KIF1B) (Zhao et al. Zhao et al., 2001Zhao C Takita J Tanaka Y Setou M Nakagawa T Takeda S Yang HW Terada S Nakata T Takei Y Saito M Tsuji S Hayashi Y Hirokawa N Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bβ.Cell. 2001; 105: 587-597Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar); (2) CMT2B: RAS-related GTP-binding protein 7 gene (RAB7) (Verhoeven et al. Verhoeven et al., 2003Verhoeven K De Jonghe P Coen K Verpoorten N Auer-Grumbach M Kwon JM FitzPatrick D Schmedding E De Vriendt E Jacobs A Van Gerwen V Wagner K Hartung H-P Timmerman V Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy.Am J Hum Genet. 2003; 72: 722-727Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar); and (3) CMT2E: neurofilament light chain gene (NEFL) (Mersiyanova et al. Mersiyanova et al., 2000Mersiyanova IV Perepelov AV Polyakov AV Sitnikov VF Dadali EL Oparin RB Petrin AN Evgrafov OV A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene.Am J Hum Genet. 2000; 67: 37-46Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). CMT2D (MIM 601472) was first mapped to chromosome 7p in a large North American family exhibiting a peripheral neuropathy that was more pronounced in the upper extremities (family 1 in table 1) (Ionasescu et al. Ionasescu et al., 1996Ionasescu V Searby C Sheffield VC Roklina T Nishimura D Ionasescu R Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D).Hum Mol Genet. 1996; 5: 1373-1375Crossref PubMed Scopus (119) Google Scholar). Subsequently, an additional North American family was identified with a CMT2 phenotype that mapped to 7p (family 2 in table 1) (Pericak-Vance et al. Pericak-Vance et al., 1997Pericak-Vance MA Speer MC Lennon F West SG Menold MM Stajich JM Wolpert CM Slotterbeck BD Saito M Tim RW Rozear MP Middleton LT Tsuji S Vance JM Confirmation of a second locus for CMT2 and evidence for additional genetic heterogeneity.Neurogenetics. 1997; 1: 89-93Crossref PubMed Scopus (25) Google Scholar). The CMT2D region overlaps an interval shown elsewhere to contain a locus for distal spinal muscular atrophy type V (dSMA-V) mapped in a Bulgarian kindred (family 3 in table 1) (Christodoulou et al. Christodoulou et al., 1995Christodoulou K Kyriakides T Hristova AH Georgiou DM Kalaydjieva L Yshpekova B Ivanova T Weber JL Middleton LT Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p.Hum Mol Genet. 1995; 4: 1629-1632Crossref PubMed Scopus (100) Google Scholar). dSMA-V is a neuromuscular disorder with a phenotype similar to CMT2D; both diseases are associated with a more severe phenotype in the upper extremities that generally affects the thenar eminence and first dorsal interosseous muscle groups. The main characteristic that distinguishes these disorders is a distal sensory loss in patients with CMT2D. Genetic mapping of a Mongolian kindred (family 4 in table 1), with phenotypic features of both CMT2D and dSMA-V, implicated the same region on chromosome 7p, thereby raising the likely possibility that the two diseases are allelic (Sambuughin et al. Sambuughin et al., 1998Sambuughin N Sivakumar K Selenge B Lee HS Friedlich D Baasanjav D Dalakas MC Goldfarb LG Autosomal dominant distal spinal muscular atrophy type V (dSMA-V) and Charcot-Marie-Tooth disease type 2D (CMT2D) segregate within a single large kindred and map to a refined region on chromosome 7p15.J Neurol Sci. 1998; 161: 23-28Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). In addition, we recently identified an Algerian Sephardic Jewish family presenting with a dSMA phenotype (family 5 in table 1). Affected members are present in multiple generations, and the pattern of inheritance is consistent with an autosomal dominant trait. A 53-year-old man and his 27-year-old daughter presented with symptoms of bilateral hand amyotrophy. Hand weakness began at ages 13 and 26 years, respectively, and physical examination revealed decreased muscle strength limited to thenar and dorsal interosseous muscles of both hands. Strength was deemed normal in all other muscles in both patients. Tendon reflexes were normal, and physical exam revealed no sensory impairment. Electromyography showed a pure motor neuropathy limited to the hands, with preserved conduction velocities; sensory conduction studies were normal. On the basis of these studies, we classified this family as having dSMA type V.Table 1Families with CMT2D and dSMA-V Analyzed in This StudyFamilyClassificationAge at Onset (years)Symptoms More Severe in Upper ExtremitiesSymptoms Prominent in Thenar Eminence and First Dorsal InterosseousReferences1CMT216–30aRange in age at onset.+NDIonasescu et al. Ionasescu et al., 1996Ionasescu V Searby C Sheffield VC Roklina T Nishimura D Ionasescu R Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D).Hum Mol Genet. 1996; 5: 1373-1375Crossref PubMed Scopus (119) Google Scholar2CMT2NDNDNDPericak-Vance et al. Pericak-Vance et al., 1997Pericak-Vance MA Speer MC Lennon F West SG Menold MM Stajich JM Wolpert CM Slotterbeck BD Saito M Tim RW Rozear MP Middleton LT Tsuji S Vance JM Confirmation of a second locus for CMT2 and evidence for additional genetic heterogeneity.Neurogenetics. 1997; 1: 89-93Crossref PubMed Scopus (25) Google Scholar3dSMA17bMedian age at onset.++Christodoulou et al. Christodoulou et al., 1995Christodoulou K Kyriakides T Hristova AH Georgiou DM Kalaydjieva L Yshpekova B Ivanova T Weber JL Middleton LT Mapping of a distal form of spinal muscular atrophy with upper limb predominance to chromosome 7p.Hum Mol Genet. 1995; 4: 1629-1632Crossref PubMed Scopus (100) Google Scholar4CMT2/dSMA18bMedian age at onset.++Sambuughin et al. Sambuughin et al., 1998Sambuughin N Sivakumar K Selenge B Lee HS Friedlich D Baasanjav D Dalakas MC Goldfarb LG Autosomal dominant distal spinal muscular atrophy type V (dSMA-V) and Charcot-Marie-Tooth disease type 2D (CMT2D) segregate within a single large kindred and map to a refined region on chromosome 7p15.J Neurol Sci. 1998; 161: 23-28Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar5dSMAND++The present study (see fig. 3D)Note.—+ = finding present; ND = not determined.a Range in age at onset.b Median age at onset. Open table in a new tab Note.— + = finding present; ND = not determined. Our previous studies indicated that the CMT2D/dSMA-V critical region encompasses an ∼1.25-Mb region on chromosome 7p14 (Ellsworth et al. Ellsworth et al., 1999Ellsworth RE Ionasescu V Searby C Sheffield VC Braden VV Kucaba TA McPherson JD Marra MA Green ED The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.Genome Res. 1999; 9: 568-574PubMed Google Scholar). To refine this interval, additional genotyping was performed on families 1, 2, and 4. We identified a single recombination event at marker 7p-3180 (a novel dinucleotide repeat at chr7:29490559–29490683 of the November 2002 build on the UCSC Genome Browser) in the unaffected offspring of an affected individual from family 4 (data not shown). This finding narrows the CMT2D/dSMA-V critical region to ∼980 kb between markers 7p-3180 (telomeric) and D7S632 (centromeric) (fig. 1A). Of note, families 1 and 2 bear the same haplotype across this region, raising the possibility that they carry the same CMT2D allele (data not shown). Analysis of the CMT2D/dSMA-V critical region reveals the presence of 11 known genes (fig. 1A). These genes were analyzed for mutations by sequencing PCR-amplified exons generated from representative members of each family. Screening of the glycyl tRNA synthetase gene (GARS [MIM 600287]) revealed mutations in all five families (fig. 2A). Specifically, the following four heterozygous missense mutations were detected: (1) in families 1 and 2, a 1236g→c variant that results in a predicted G240R amino acid change (note that this is consistent with the above haplotype data for these two families); (2) in family 3, a 904c→t variant that results in a predicted L129P amino acid change; (3) in family 4, a 730a→g variant that results in a predicted E71G amino acid change; and (4) in family 5, a 2094g→c nucleotide change that results in a predicted G526R amino acid change. Analysis of available individuals from each pedigree revealed that the mutations consistently segregate with the disease, with representative examples shown in figure 3A,3C, and 3D. We also tested human-rodent hybrid cell lines derived from family 1, one carrying the “affected” (i.e., CMT2D-associated) chromosome 7 haplotype and one carrying an “unaffected” chromosome 7 haplotype. Sequence analysis revealed that the G240R mutation resides only on the copy of chromosome 7 bearing the affected haplotype (fig. 3B). To rule out the possibility that the above sequence variants represent rare polymorphisms, we genotyped appropriate control populations by PCR amplification and DNA sequencing or RFLP analysis. None of these mutations were encountered in the GARS gene of any control sample. For all mutations, screening was performed with a mixed-ethnic population collected in North America (described in Struewing et al. [Struewing et al., 1995Struewing JP Abeliovich D Peretz T Avishai N Kaback MM Collins FS Brody LC The carrier frequency of the BRCA1 185delAG mutation is approximately 1 percent in Ashkenazi Jewish individuals.Nat Genet. 1995; 11: 198-200Crossref PubMed Scopus (578) Google Scholar]); for the mutations found in the Mongolian, Bulgarian, and Sephardic Jewish families, additional screening was performed with suitably matched controls. Our results revealed that: (1) E71G was absent in 398 unrelated chromosomes (130 from a Mongolian population and 268 from the mixed-ethnic population); (2) L129P was absent in 376 unrelated chromosomes (200 from an Eastern European population and 176 from the mixed-ethnic population); (3) G240R was absent in 368 unrelated chromosomes from the mixed-ethnic population; and (4) G526R was absent in 360 unrelated chromosomes (160 from a Sephardic Jewish population and 200 from the mixed-ethnic population). The human GARS protein is encoded by a 17-exon gene that spans ∼40 kb on chromosome 7p14 (fig. 1B) and that is expressed in a ubiquitous fashion (fig. 1C), including the brain and spinal cord (tissues relevant for neurodegenerative diseases). Examination of the four CMT2D/dSMA-V–associated mutations reveals striking conservation of the altered amino acids. Specifically, in all but one case, the variant amino acid is identical in organisms distributed across wide taxonomic spans, from primates to yeast (fig. 2C; see the “Electronic-Database Information” section for GenBank accession numbers for multispecies amino acid sequences). The one exception is the human residue G240 (a glycine). In roundworm, there is an alanine at this position (albeit these two amino acids are quite similar in terms of side chain size and charge). The above genetic data implicate mutations in the GARS gene as the cause of the neurodegenerative disorders, CMT2D and dSMA-V. GARS is a member of the family of aminoacyl tRNA synthetases responsible for charging tRNAs with their cognate amino acids. The functional holoenzyme exists as a homodimer (reviewed in Freist et al. [Freist et al., 1996Freist W Logan DT Gauss DH Glycyl-tRNA synthetase.Biol Chem Hoppe Seyler. 1996; 377: 343-356PubMed Google Scholar]) and contains three major functional domains (fig. 2B): (1) the WHEP-TRS domain (residues 13–63; pfam00458 in the NCBI Conserved Domain Database) for conjugation with other aminoacyl tRNA synthetases in enzyme complexes; (2) the core catalytic domain (residues 92–168 and 241–324; pfam00587) for ligation; and (3) the anticodon-binding domain (residues 557–655; pfam03129) for recognition of glycine-specific tRNAs. One of the identified mutations (L129P) falls within the catalytic core (fig. 2B), whereas another (G240R) lies one residue upstream of the catalytic core (fig. 2B) and two residues upstream of a highly conserved LRPETAQ sequence. This stretch of amino acids is fully conserved between human and Thermus thermophilus; in the latter species, it composes a loop and helix structure essential for forming the glycine-binding pocket (reviewed in Freist et al. [Freist et al., 1996Freist W Logan DT Gauss DH Glycyl-tRNA synthetase.Biol Chem Hoppe Seyler. 1996; 377: 343-356PubMed Google Scholar]). The positions of these two mutations in conjunction with the presence of mutations across the GARS protein suggest a corresponding loss or decrease in enzyme activity. It is possible that these missense mutations act in a dominant negative fashion, such that the presence of a mutant subunit within dimers (i.e., mutant/wild type, mutant/mutant) greatly reduces overall GARS activity. To investigate further the possible functional impact of these mutations, we attempted to model the affected amino acids on three-dimensional structures available for the T. thermophilus GARS protein. Human GARS exhibits high overall similarity to these structures; however, the absolute identity between the human and T. thermophilus GARS is only 27%–29% (A. Baxevanis, personal communication). Although some of the identified mutations fall within structured regions, standard threading techniques (e.g., Bryant and Lawrence Bryant and Lawrence, 1993Bryant SH Lawrence CE An empirical energy function for threading protein sequence through the folding motif.Proteins. 1993; 16: 92-112Crossref PubMed Scopus (350) Google Scholar) to examine the effect of these mutations on the thermodynamic stability of GARS cannot be applied. As a rule, a minimum of 40% sequence identity is required for the results to be biologically meaningful (A. Baxevanis, personal communication). The mechanism by which mutations in such a ubiquitously expressed gene lead to such a highly specific phenotype (i.e., peripheral neuropathy/neuronopathy) requires additional investigation. There are two possibilities, in the likely event that the molecular pathology is due to decreased enzyme activity. First, the phenotype may arise owing to a general defect in translation efficiency, since every protein-bearing glycine would be affected. In the environment of a typical cell, this defect may not give rise to a detectable phenotype. However, cells bearing long axons may be more prone to ensuing pathology owing to a reduction of protein products reaching axon termini. Second, there may be a specific need for more glycine-rich proteins in neurons affected in CMT2D and dSMA-V. Also intriguing is the fact that the pathologic mechanism must account for the more prominent phenotype in specific muscle groups of the hand. In addition to studies designed to resolve these issues, it will be of interest to perform GARS mutation screening in: (1) a broader group of patients with CMT2 and dSMA to assess the frequency of mutations in these disorders; and (2) patients with neuropathology affecting the thenar eminence in a predominant fashion, for example, as seen in carpal tunnel syndrome (Sternbach Sternbach, 1999Sternbach G The carpal tunnel syndrome.J Emerg Med. 1999; 17: 519-523Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). These findings represent the first example of a defect in an aminoacyl tRNA synthetase being directly associated with a human genetic disease. It is interesting that two mutant forms of the superoxide dismutase 1 gene (SOD1 [MIM 147450]), the gene implicated in the neurodegenerative disease amyotrophic lateral sclerosis 1 (ALS1 [MIM 105400]), have been shown to interact directly with lysyl tRNA synthetase (KARS) (Kunst et al. Kunst et al., 1997Kunst CB Mezey E Brownstein MJ Patterson D Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions.Nat Genet. 1997; 15: 91-94Crossref PubMed Scopus (106) Google Scholar). Since these interactions are not observed with wild-type SOD1, this raises the possibility that KARS inhibition may be involved in the pathology of ALS1. Thus, the genes for all aminoacyl tRNA synthetases should be considered relevant candidates for inherited neuropathies and motor neuron diseases. We thank the members of the families for their participation in this study. We also thank: Larry Brody for providing screening populations, the Washington University Genome Sequencing Center for sequencing, Don Hadley for sample collection, Veneta Georgieva for family 3 pedigree data, Andy Baxevanis for protein modeling, and Robert Nussbaum and Francis Collins for critical review of the manuscript. This work was supported in part by grant NS 26630 (to J.M.V.). All studies performed herein were approved by the National Institute of Neurological Disorders and Stroke Institutional Review Board, with informed consent obtained from all subjects.