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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.

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.

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.

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.

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.

Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable."We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided.Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P.001).Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.

International audience; 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.

Using linkage analysis and whole-exome sequencing, Safka Brozkova et al. reveal missense mutations in the histidyl-tRNA synthetase gene in 23 patients from four families with axonal and demyelinating neuropathies of varying severity. The mutations cause loss of function in yeast complementation assays and neurotoxicity in a C. elegans model.Using linkage analysis and whole-exome sequencing, Safka Brozkova et al. reveal missense mutations in the histidyl-tRNA synthetase gene in 23 patients from four families with axonal and demyelinating neuropathies of varying severity. The mutations cause loss of function in yeast complementation assays and neurotoxicity in a C. elegans model.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.

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, thirteen 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.

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.

Crossing over during meiotic prophase I is required for sexual reproduction in mice and contributes to genome-wide genetic diversity. Here we report on the characterization of an N-ethyl-N-nitrosourea-induced, recessive allele called mei4, which causes sterility in both sexes owing to meiotic defects. In mutant spermatocytes, chromosomes fail to congress properly at the metaphase plate, leading to arrest and apoptosis before the first meiotic division. Mutant oocytes have a similar chromosomal phenotype but in vitro can undergo meiotic divisions and fertilization before arresting. During late meiotic prophase in mei4 mutant males, absence of cyclin dependent kinase 2 and mismatch repair protein association from chromosome cores is correlated with the premature separation of bivalents at diplonema owing to lack of chiasmata. We have identified the causative mutation, a transversion in the 5′ splice donor site of exon 1 in the mouse ortholog of Human Enhancer of Invasion 10 (Hei10; also known as Gm288 in mouse and CCNB1IP1 in human), a putative B-type cyclin E3 ubiquitin ligase. Importantly, orthologs of Hei10 are found exclusively in deuterostomes and not in more ancestral protostomes such as yeast, worms, or flies. The cloning and characterization of the mei4 allele of Hei10 demonstrates a novel link between cell cycle regulation and mismatch repair during prophase I., Author Summary Human infertility and reproductive complications have devastating social and monetary costs. Errors in meiosis during reproduction may lead to birth defects, spontaneous abortion, or infertility. Many of the genes essential for meiosis function in DNA repair and mutations in several of these genes have been shown to contribute to cancer. The identification of the genes necessary for normal meiosis is an important goal and will potentially influence the fields of reproductive and cancer biology. In this study, genetic screens in mice have generated the mutation mei4. mei4 causes male and female sterility by disrupting meiosis and altering the function of the DNA repair system known as mismatch repair. We have identified the causative mutation behind the mei4 phenotype in a gene called Human Enhancer of Invasion 10 or Hei10. This work demonstrates that Hei10 is essential for the completion of meiosis and that it functions to coordinate the DNA repair system and the progression of the cell cycle during meiosis.