First-Trimester Cell-Free DNA Fetal Fraction and Birth Weight in Twin Pregnancies.

BACKGROUND: The relationship between fetal fraction and birth weight in twin gestations is poorly understood. OBJECTIVE: To investigate the relationship between first trimester cfDNA fetal fraction and birth weight < 10th percentile in twin gestations. STUDY DESIGN: This is a planned secondary analysis of the Twin cfDNA Study, a 17-center retrospective cohort of twin pregnancies screened for aneuploidy using cfDNA in the first trimester from 12/2011 - 2/2022, excluding those with positive screen results for chromosomal aneuploidy. CfDNA testing was performed by a single lab using massively parallel sequencing (MPSS). Baseline characteristics and birth weight of pregnancies with normal fetal fraction were compared to those with low (<5%) and high (>95%) fetal fraction using univariable analyses and multivariable regression. RESULTS: A total of 1041 twin pregnancies were included. Chronic hypertension, elevated BMI, and self-identified Black race were associated with fetal fraction <5th percentile. There was no difference in median fetal fraction between those with birth weight <10th percentile in at least one twin (median [IQR] fetal fraction 12.2% [9.8, 14.8] versus those with normal birth weight (10th percentile) in both twins (median [IQR] fetal fraction 12.3% [9.7, 15.2] for normal birth weight, p = 0.49). There was no association between high or low fetal fraction and birth weight <10th percentile for one (p=0.45) or both (p=0.81) twins, and there was no association between high or low fetal fraction and birth weight <5th percentile for one (p=0.44) or both (p=0.74) twins. The results were unchanged after adjustment for potential confounders. CONCLUSION: In this large cohort, there was no association between the extremes of cfDNA fetal fraction and birthweight < 10th percentile, suggesting that first trimester fetal fraction may not predict impaired fetal growth in twin gestations.

Navigation of Prenatal Care With Sex Discordance Between Cell-free DNA and Ultrasound Findings.

The utilization of cell-free DNA (cfDNA) screening has expanded rapidly across the age spectrum of pregnant persons. With cfDNA's widespread adoption, genetic fetal sex is now often known before a phenotypic assessment on anatomic survey. CfDNA detects sex discordance in 1/1500 to 2000 pregnancies. Upon detection of sex discordance, lab error or other factors should first be assessed. Once other causes have been ruled out, this may indicate an underlying disorder/difference in sex development. A multidisciplinary team should coordinate diagnosis, treatment, and support for the family. This review discusses the diagnostic workup, emphasizing the multidisciplinary counseling and management of disorder/differences in sex development.

A pilot randomized control trial of topical capsaicin as adjunctive therapy for nausea and vomiting of pregnancy.

BACKGROUND: Nausea and vomiting is one of the most common complications of pregnancy, affecting 50% to 80% of pregnant persons. Moreover, despite its prevalence, it remains a challenging condition to treat. Treatment often involves oral and intravenous medications with potential side effects, particularly when taken in combination. Capsaicin cream is proven to decrease nausea and vomiting in cyclic vomiting syndrome; however, its use has not been well studied among pregnant patients. OBJECTIVE: This study aimed to test the feasibility of the off-label use of capsaicin for the treatment of nausea and vomiting in pregnancy. STUDY DESIGN: This was a double-blinded randomized controlled trial of pregnant individuals in their first trimester of pregnancy seeking care at a tertiary care hospital for nausea and vomiting. Consenting participants were randomized to abdominal application of topical capsaicin vs placebo. All participants received intravenous hydration and metoclopramide. The primary outcome, total treatment time, was recorded for all participants. In addition, symptom severity was assessed every 30 minutes using a visual analog scale. Data were analyzed using the Wilcoxon rank-sum test for continuous variables and the Fisher exact test for binary variables. RESULTS: Of the 38 eligible individuals approached, 30 were randomized. There was a trend toward decreased mean treatment time in the capsaicin group compared with the placebo group (79.9 vs 97.3 minutes; P=.1). There was no significant difference in visual analog scale scores at any time point between groups. Furthermore, capsaicin was well tolerated, with only 1 individual requesting the medication be removed. CONCLUSION: This study demonstrated that capsaicin is an acceptable treatment of nausea and vomiting in pregnancy and additional explorations of its use as treatment are feasible. A larger randomized controlled trial is needed to determine the efficacy of capsaicin in this population.

Lactation Consultation by an International Board Certified Lactation Consultant Improves Breastfeeding Rates for Mothers With Gestational Diabetes Mellitus.

BACKGROUND: In patients with gestational diabetes, breastfeeding decreases the lifetime risk of Type 2 diabetes by half. Lactation consultation has been shown to increase breastfeeding rates in the general population but has not been assessed in a gestational diabetes population. RESEARCH AIMS: To determine if (1) a postpartum International Board Certified Lactation Consultant (IBCLC) consultation during delivery hospitalization improved inclusive (any) or exclusive breastfeeding rates at hospital discharge and 3 months postpartum in participants with GDM; and if (2) obstetrical providers' acknowledgement of maternal feeding preference affected the rates of IBCLC consultation for patients. METHODS: This was a retrospective, comparative, secondary analysis of a prospective cohort (N = 517) study of women gestational diabetes. Participants who received a IBCLC consultation (n = 386; 74.5%) were compared to those who did not (n = 131; 25.5%). Baseline demographics, antepartum characteristics, neonatal information, mode of infant feeding at hospital discharge and 3 months postpartum, and IBCLC consultation during postpartum hospitalization were measured. RESULTS: After adjusting for baseline differences, participants who received an IBCLC consultation were more likely to report any breastfeeding at postpartum discharge (aOR 4.87; 95% CI [2.67, 8.86]) and at 3 months postpartum (aOR 5.39; 95% CI [2.61, 11.16]) compared to participants who did not. However, there was no difference in exclusive breastfeeding rates between those who did and did not receive IBCLC consultation. CONCLUSION: Inpatient IBCLC consultation during the immediate postpartum period was associated with improved rates of any breastfeeding in participants with GDM.

Breastfeeding Success Among Women with Gestational Diabetes Managed by Diet Only Compared with Those Requiring Medications.

Objective: Breastfeeding is known to have a positive impact on maternal and neonatal health. Some have suggested that gestational diabetes mellitus (GDM) is associated with lower breastfeeding rates, but it is not known whether rates are further impacted by glucose control in pregnancy. Thus, we examined whether patients with GDM requiring medication (A2 GDM) were more likely to not initiate or discontinue breastfeeding compared with patients with GDM well controlled by diet (A1 GDM). Research Design and Methods: This is a secondary analysis of a prospective cohort study of 600 patients with GDM. Eligible patients were enrolled during their delivery hospitalization and followed prospectively postpartum. The primary outcome was exclusive breastfeeding at hospital discharge and secondary outcomes included breastfeeding rates at 3 months postpartum. Patients classified as A2 GDM were compared with those classified as A1 GDM. Results: Of the 600 patients enrolled, 301 had A2 GDM and 299 had A1 GDM. Patients who needed medication were observed to be older and more likely to be parous and obese. There were no significant differences in labor outcomes or neonatal complications. After adjusting for baseline differences between the two groups, adjusted odds ratios (aORs) for exclusive breastfeeding rates were similar in mothers with A2 GDM compared with those with A1 GDM at hospital discharge (aOR 0.83 [0.54-1.28]) and 3 months postpartum (aOR 0.58 [0.34-1.01]). Additionally, any breastfeeding rates were similar in mothers with A2 GDM compared with those with A1 GDM, both at hospital discharge (aOR 0.72 [0.44-1.16]) and 3 months postpartum (aOR 0.63 [0.34-1.17]). Conclusions: After adjusting for baseline differences, there was no difference in any or exclusive breastfeeding rates at hospital discharge or 3 months postpartum among patients with A2 GDM compared with those with A1 GDM.

Effect of Novel Breastfeeding Smartphone Applications on Breastfeeding Rates.

Background: Low-income women are less likely to breastfeed than high-income women. Technology-based interventions demonstrate promise in decreasing health disparities. We assessed whether increased use of breastfeeding smartphone applications (apps) impacts breastfeeding rates for low-income women. Materials and Methods: This is a secondary analysis of a randomized control trial (RCT), including nulliparous, low-income women. Women were randomized to one of two novel apps: control app containing digital breastfeeding handouts and BreastFeeding Friend (BFF), an interactive app containing on-demand breastfeeding educational and video content. App usage was securely tracked. The highest quartile of BFF and control app users were combined and compared to the lowest quartile of app users. The primary outcome was breastfeeding initiation. Secondary outcomes included breastfeeding outcomes and resource preferences through 6 months. Results: In the RCT, BFF and control app median uses were 15 (interquartile range [IQR] 4-24) and 9 (IQR 5-19) (p = 0.1), respectively. Breastfeeding initiation did not differ with app usage (84.1% in highest quartile versus 78.2% for lowest quartile; p = 0.5). Rates of sustained and exclusive breastfeeding through 6 months were similar between groups. Among both groups, smartphone apps were the most preferred breastfeeding resource at 6 weeks. Low quartile users also preferred alternative online breastfeeding resources: >50% of all users preferred technology-based breastfeeding resources. Conclusions: Increased usage of breastfeeding apps did not improve breastfeeding rates among low-income women. However, technology-based resources were the most preferred breastfeeding resource after hospital discharge, indicating ongoing development of technology-based interventions has potential to increase breastfeeding in this high-needs population. clinicaltrials.gov (NCT03167073).

Allele-specific RNA interference prevents neuropathy in Charcot-Marie-Tooth disease type 2D mouse models.

Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.

Homozygosity for a mutation affecting the catalytic domain of tyrosyl-tRNA synthetase (YARS) causes multisystem disease.

Aminoacyl-tRNA synthetases (ARSs) are critical for protein translation. Pathogenic variants of ARSs have been previously associated with peripheral neuropathy and multisystem disease in heterozygotes and homozygotes, respectively. We report seven related children homozygous for a novel mutation in tyrosyl-tRNA synthetase (YARS, c.499C > A, p.Pro167Thr) identified by whole exome sequencing. This variant lies within a highly conserved interface required for protein homodimerization, an essential step in YARS catalytic function. Affected children expressed a more severe phenotype than previously reported, including poor growth, developmental delay, brain dysmyelination, sensorineural hearing loss, nystagmus, progressive cholestatic liver disease, pancreatic insufficiency, hypoglycemia, anemia, intermittent proteinuria, recurrent bloodstream infections and chronic pulmonary disease. Related adults heterozygous for YARS p.Pro167Thr showed no evidence of peripheral neuropathy on electromyography, in contrast to previous reports for other YARS variants. Analysis of YARS p.Pro167Thr in yeast complementation assays revealed a loss-of-function, hypomorphic allele that significantly impaired growth. Recombinant YARS p.Pro167Thr demonstrated normal subcellular localization, but greatly diminished ability to homodimerize in human embryonic kidney cells. This work adds to a rapidly growing body of research emphasizing the importance of ARSs in multisystem disease and significantly expands the allelic and clinical heterogeneity of YARS-associated human disease. A deeper understanding of the role of YARS in human disease may inspire innovative therapies and improve care of affected patients.

Compound heterozygosity for loss-of-function FARSB variants in a patient with classic features of recessive aminoacyl-tRNA synthetase-related disease.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in phenotypically diverse dominant and recessive human diseases. The charging of tRNAPHE with phenylalanine is performed by a tetrameric enzyme that contains two alpha (FARSA) and two beta (FARSB) subunits. To date, mutations in the genes encoding these subunits (FARSA and FARSB) have not been implicated in any human disease. Here, we describe a patient with a severe, lethal, multisystem, developmental phenotype who was compound heterozygous for FARSB variants: p.Thr256Met and p.His496Lysfs*14. Expression studies using fibroblasts isolated from the proband revealed a severe depletion of both FARSB and FARSA protein levels. These data indicate that the FARSB variants destabilize total phenylalanyl-tRNA synthetase levels, thus causing a loss-of-function effect. Importantly, our patient shows strong phenotypic overlap with patients that have recessive diseases associated with other ARS loci; these observations strongly support the pathogenicity of the identified FARSB variants and are consistent with the essential function of phenylalanyl-tRNA synthetase in human cells. In sum, our clinical, genetic, and functional analyses revealed the first FARSB variants associated with a human disease phenotype and expand the locus heterogeneity of ARS-related human disease.

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

Predicting the pathogenicity of aminoacyl-tRNA synthetase mutations.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes responsible for charging tRNA with cognate amino acids-the first step in protein synthesis. ARSs are required for protein translation in the cytoplasm and mitochondria of all cells. Surprisingly, mutations in 28 of the 37 nuclear-encoded human ARS genes have been linked to a variety of recessive and dominant tissue-specific disorders. Current data indicate that impaired enzyme function is a robust predictor of the pathogenicity of ARS mutations. However, experimental model systems that distinguish between pathogenic and non-pathogenic ARS variants are required for implicating newly identified ARS mutations in disease. Here, we outline strategies to assist in predicting the pathogenicity of ARS variants and urge cautious evaluation of genetic and functional data prior to linking an ARS mutation to a human disease phenotype.

A novel FGD1 mutation in a family with Aarskog-Scott syndrome and predominant features of congenital joint contractures.

Mutations in FGD1 cause Aarskog-Scott syndrome (AAS), an X-linked condition characterized by abnormal facial, skeletal, and genital development due to abnormal embryonic morphogenesis and skeletal formation. Here we report a novel FGD1 mutation in a family with atypical features of AAS, specifically bilateral upper and lower limb congenital joint contractures and cardiac abnormalities. The male proband and his affected maternal uncle are hemizygous for the novel FGD1 mutation p.Arg921X. This variant is the most carboxy-terminal FGD1 mutation identified in a family with AAS and is predicted to truncate the FGD1 protein at the second to last amino acid of the carboxy-terminal pleckstrin homology (PH) domain. Our study emphasizes the importance of the 3' peptide sequence in the structure and/or function of the FGD1 protein and further demonstrates the need to screen patients with X-linked congenital joint contractures for FGD1 mutations.

Dimerization is required for GARS-mediated neurotoxicity in dominant CMT disease.

Charcot-Marie-Tooth (CMT) disease is a genetically heterogeneous group of peripheral neuropathies. Mutations in several aminoacyl-tRNA synthetase (ARS) genes have been implicated in inherited CMT disease. There are 12 reported CMT-causing mutations dispersed throughout the primary sequence of the human glycyl-tRNA synthetase (GARS). While there is strong genetic evidence linking GARS mutations to CMT disease, the molecular pathology underlying the neuromuscular and sensory phenotypes is still not fully understood. In particular, it is unclear whether the mutations result in a toxic gain of function, a partial loss of activity related to translation, or a combination of these mechanisms. We identified a zebrafish allele of gars (gars(s266)). Homozygous mutant embryos carry a C->A transversion, that changes a threonine to a lysine, in a residue next to a CMT-associated human mutation. We show that the neuromuscular phenotype observed in animals homozygous for T209K Gars (T130K in GARS) is due to a loss of dimerization of the mutated protein. Furthermore, we show that the loss of function, dimer-deficient and human disease-associated G319R Gars (G240R in GARS) mutant protein is unable to rescue the above phenotype. Finally, we demonstrate that another human disease-associated mutant G605R Gars (G526 in GARS) dimerizes with the remaining wild-type protein in animals heterozygous for the T209K Gars and reduces the function enough to elicit a neuromuscular phenotype. Our data indicate that dimerization is required for the dominant neurotoxicity of disease-associated GARS mutations and provide a rapid, tractable model for studying newly identified GARS variants for a role in human disease.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy.

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder.

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

Loss of function mutations in HARS cause a spectrum of inherited peripheral neuropathies.

Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.

A novel AARS mutation in a family with dominant myeloneuropathy.

OBJECTIVE: To determine the genetic cause of neurodegeneration in a family with myeloneuropathy. METHODS: We studied 5 siblings in a family with a mild, dominantly inherited neuropathy by clinical examination and electrophysiology. One patient had a sural nerve biopsy. After ruling out common genetic causes of axonal Charcot-Marie-Tooth disease, we sequenced 3 tRNA synthetase genes associated with neuropathy. RESULTS: All affected family members had a mild axonal neuropathy, and 3 of 4 had lower extremity hyperreflexia, evidence of a superimposed myelopathy. A nerve biopsy showed evidence of chronic axonal loss. All affected family members had a heterozygous missense mutation c.304G>C (p.Gly102Arg) in the alanyl-tRNA synthetase (AARS) gene; this allele was not identified in unaffected individuals or control samples. The equivalent change in the yeast ortholog failed to complement a strain of yeast lacking AARS function, suggesting that the mutation is damaging. CONCLUSION: A novel mutation in AARS causes a mild myeloneuropathy, a novel phenotype for patients with mutations in one of the tRNA synthetase genes.

Loss-of-function alanyl-tRNA synthetase mutations cause an autosomal-recessive early-onset epileptic encephalopathy with persistent myelination defect.

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.

Impaired function is a common feature of neuropathy-associated glycyl-tRNA synthetase mutations.

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.