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 1974). 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. 1996). Through the measurement of motor nerve conductance velocities (MNCVs), CMT can be subdivided into two classes (Dyck and Lambert 1968). 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

Charcot-Marie-Tooth disease type 2D (CMT2D) and distal spinal muscular atrophy type V (dSMA-V) are axonal neuropathies characterized by a phenotype that is more severe in the upper extremities. We previously implicated mutations in the gene encoding glycyl-tRNA synthetase (GARS) as the cause of CMT2D and dSMA-V. GARS is a member of the family of aminoacyl-tRNA synthetases responsible for charging tRNA with cognate amino acids; GARS ligates glycine to tRNA Gly . Here, we present functional analyses of disease-associated GARS mutations and show that there are not any significant mutation-associated changes in GARS expression levels; that the majority of identified GARS mutations modeled in yeast severely impair viability; and that, in most cases, mutant GARS protein mislocalizes in neuronal cells. Indeed, four of the five mutations studied show loss-of-function features in at least one assay, suggesting that tRNAcharging deficits play a role in disease pathogenesis. Finally, we detected endogenous GARS-associated granules in the neurite projections of cultured neurons and in the peripheral nerve axons of normal human tissue. These data are particularly important in light of the recent identification of CMT-associated mutations in another tRNA synthetase gene [YARS (tyrosyl-tRNA synthetase gene)]. Together, these findings suggest that tRNA-charging enzymes play a key role in maintaining peripheral axons.

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest–derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression.

The transcription factor SOX10 is mutated in the human neurocristopathy Waardenburg-Shah syndrome (WS4), which is characterized by enteric aganglionosis and pigmentation defects. SOX10 directly regulates genes expressed in neural crest lineages, including the enteric ganglia and melanocytes. Although some SOX10 target genes have been reported, the mechanisms by which SOX10 expression is regulated remain elusive. Here, we describe a transgene-insertion mutant mouse line (Hry) that displays partial enteric aganglionosis, a loss of melanocytes, and decreased Sox10 expression in homozygous embryos. Mutation analysis of Sox10 coding sequences was negative, suggesting that non-coding regulatory sequences are disrupted. To isolate the Hry molecular defect, Sox10 genomic sequences were collected from multiple species, comparative sequence analysis was performed and software was designed (ExactPlus) to identify identical sequences shared among species. Mutation analysis of conserved sequences revealed a 15.9 kb deletion located 47.3 kb upstream of Sox10 in Hry mice. ExactPlus revealed three clusters of highly conserved sequences within the deletion, one of which shows strong enhancer potential in cultured melanocytes. These studies: (i) present a novel hypomorphic Sox10 mutation that results in a WS4-like phenotype in mice; (ii) demonstrate that a 15.9 kb deletion underlies the observed phenotype and likely removes sequences essential for Sox10 expression; (iii) combine a novel in silico method for comparative sequence analysis with in vitro functional assays to identify candidate regulatory sequences deleted in this strain. These studies will direct further analyses of Sox10 regulation and provide candidate sequences for mutation detection in WS4 patients lacking a SOX10-coding mutation.