Charcot-Marie-Tooth disease type 2D (CMT2D) is an autosomal-dominant axonal 
peripheral neuropathy characterized by impaired motor and sensory function in 
the distal extremities. Mutations in the glycyl-tRNA synthetase (GARS) gene 
cause CMT2D. GARS is a member of the ubiquitously expressed aminoacyl-tRNA 
synthetase (ARS) family and is responsible for charging tRNA with glycine. To 
date, 13 GARS mutations have been identified in patients with CMT disease. While 
functional studies have revealed loss-of-function characteristics, only four 
GARS mutations have been rigorously studied. Here, we report the functional 
evaluation of nine CMT-associated GARS mutations in tRNA charging, yeast 
complementation, and subcellular localization assays. Our results demonstrate 
that impaired function is a common characteristic of CMT-associated GARS 
mutations. Additionally, one mutation previously associated with CMT disease 
(p.Ser581Leu) does not demonstrate impaired function, was identified in the 
general population, and failed to segregate with disease in two newly identified 
families with CMT disease. Thus, we propose that this variant is not a 
disease-causing mutation. Together, our data indicate that impaired function is 
a key component of GARS-mediated CMT disease and emphasize the need for careful 
genetic and functional evaluation before implicating a variant in disease onset.