Identification of a Novel Indel Variant in the DARS2 Gene in Russian Patients with Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement. CASE PRESENTATION: Peripheral blood samples were collected from four patients from four unrelated families to extract genomic DNA. All patients underwent partial exon analysis of the DARS2 gene using Sanger sequencing, which detected the c.228-21_228-20delinsC variant in a heterozygous state. Further DNA from three patients was analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies, and one of the patients underwent whole genome sequencing. We identified a novel pathogenic variant c.1675-1256_*115delinsGCAACATTTCGGCAACATTCCAACC in the DARS2 gene. Three patients (patients 1, 2, and 4) had slowly progressive cerebellar ataxia, and two patients (patients 1 and 2) had spasticity. In addition, two patients (patients 2 and 4) showed signs of axonal neuropathy, such as decreased tendon reflexes and loss of distal sensitivity. Three patients (patients 1, 2, and 3) also had learning difficulties. It should be noted the persistent presence of characteristic changes in brain MRI in all patients, which emphasizes its importance as the main diagnostic tool for suspicion and subsequent confirmation of LBSL. Conclusions: We found a novel indel variant in the DARS2 gene in four patients with LBSL and described their clinical and genetic characteristics. These results expand the mutational spectrum of LBSL and aim to improve the laboratory diagnosis of this form of leukodystrophy.

Functional enrichment analysis reveals the involvement of DARS2 in multiple biological pathways and its potential as a therapeutic target in esophageal carcinoma.

OBJECTIVE: The enzyme Aspartyl tRNA synthetase 2 (DARS2) is a crucial enzyme in the mitochondrial tRNA synthesis pathway, playing a critical role in maintaining normal mitochondrial function and protein synthesis. However, the role of DARS2 in ESCA is unclear. MATERIALS AND METHODS: Transcriptional data of pan-cancer and ESCA were downloaded from UCSC XENA, TCGA, and GEO databases to analyze the differential expression of DARS2 between tumor samples and normal samples, and its correlation with clinicopathological features of ESCA patients. R was used for GO, KEGG, and GSEA functional enrichment analysis of DARS2 co-expression and to analyze the connection of DARS2 with glycolysis and m6A-related genes. In vitro experiments were performed to assess the effects of interfering with DARS2 expression on ESCA cells. TarBase v.8, mirDIP, miRTarBase, ENCORI, and miRNet databases were used to analyze and construct a ceRNA network containing DARS2. RESULTS: DARS2 was overexpressed in various types of tumors. In vitro experiments confirmed that interfering with DARS2 expression significantly affected the proliferation, migration, apoptosis, cell cycle, and glycolysis of ESCA cells. DARS2 may be involved in multiple biological pathways related to tumor development. Furthermore, correlation and differential analysis revealed that DARS2 may regulate ESCA m6A modification through its interaction with METTL3 and YTHDF1. A ceRNA network containing DARS2, DLEU2/has-miR-30a-5p/DARS2, was successfully predicted and constructed. CONCLUSIONS: Our findings reveal the upregulation of DARS2 in ESCA and its association with clinical features, glycolysis pathway, m6A modification, and ceRNA network. These discoveries provide valuable insights into the molecular mechanisms underlying ESCA.

Mitochondrial dysfunction in lipid processing and gastrointestinal disorders.

Mitochondrial dysfunctions predominantly cause encephalomyopathies with muscle atrophy and neurodegeneration. However, their impact on other tissues, particularly the gastrointestinal tract, requires further investigation. In a recent report in Nature, Moschandrea et al. used mice deficient in the mitochondrial aminoacyl-tRNA synthetase DARS2 to investigate the role of enterocytic mitochondria in dietary lipid processing and transport. Their work sheds light on the development of gastrointestinal disorders as a result of mitochondrial dysfunction.

DARS2 promotes the occurrence of lung adenocarcinoma via the ERK/c-Myc signaling pathway.

BACKGROUND: DARS2 expression is upregulated in lung adenocarcinoma (LUAD) which correlates with tumor patient stage and prognosis. The mechanism of DARS2 involvement in LUAD still needs to be further explored. METHODS: In this study, we found that DARS2 expression in LUAD tissue was significantly higher than that in normal tissue. At the same time, the Kaplan-Meier curve showed that the survival prognosis of LUAD patients with high expression of DARS2 was significantly worse than low expression of DARS2. The expression of DARS2 was detected in LUAD and adjacent normal tissues by IHC staining, histochemical scoring and a survival curve was generated. In addition, we demonstrated that the knockdown and overexpression of DARS2 significantly affected the proliferation, invasion, and migration of LUAD cells in vitro and in vivo. Finally, western blot and rescue assay were performed on LUAD cells to further explore and verify the signaling pathway. RESULTS: DARS2 expression was significantly upregulated in LUAD tissues and cell lines. What is more, the increased expression of DARS2 was closely related to proliferation, invasion and metastasis. The tumorigenic assay in nude mice further showed that the tumorigenic ability of nude mice was significantly improved with the increase in DARS2 expression. Finally, we determined that DARS2 plays its role in LUAD by targeting the ERK/c-Myc signaling pathway. CONCLUSION: Our data revealed the oncogenic role of DARS2 in LUAD, indicating that DARS2 may be a predictive biomarker and novel therapeutic target for LUAD.

DARS2 overexpression is associated with PET/CT metabolic parameters and affects glycolytic activity in lung adenocarcinoma.

BACKGROUND: This study investigated the correlation between the expression of DARS2 and metabolic parameters of 18F-FDG PET/CT, and explored the potential mechanisms of DARS2 affecting the proliferation and glycolysis of lung adenocarcinoma (LUAD) cells. METHODS: This study used genomics and proteomics to analyze the difference in DARS2 expression between LUAD samples and control samples. An analysis of 62 patients with LUAD who underwent 18F-FDG PET/CT examinations before surgery was conducted retrospectively. The correlation between DARS2 expression and PET/CT metabolic parameters, including SUVmax, SUVmean, MTV, and TLG, was examined by Spearman correlation analysis. In addition, the molecular mechanism of interfering with DARS2 expression in inhibiting LUAD cell proliferation and glycolysis was analyzed through in vitro cell experiments. RESULTS: DARS2 expression was significantly higher in LUAD samples than in control samples (p < 0.001). DARS2 has high specificity (98.4%) and sensitivity (95.2%) in the diagnosis of LUAD. DARS2 expression was positively correlated with SUVmax, SUVmean, and TLG (p < 0.001). At the same time, the sensitivity and specificity of SUVmax in predicting DARS2 overexpression in LUAD were 88.9% and 65.9%, respectively. In vitro cell experiments have shown that interfering with DARS2 expression can inhibit the proliferation and migration of LUAD cells, promote cell apoptosis, and inhibit the glycolytic activity of tumor cells by inhibiting the expression of glycolytic related genes SLC2A1, GPI, ALDOA, and PGAM1. CONCLUSIONS: Overexpression of DARS2 is associated with metabolic parameters on 18F-FDG PET/CT, which can improve LUAD diagnosis accuracy. DARS2 may be a useful biomarker to diagnose, prognosis, and target treatment of LUAD patients.

DARS2 is a prognostic biomarker and correlated with immune infiltrates and cuproptosis in lung adenocarcinoma.

Overexpression of DARS2 may enhance the progression of hepatocellular carcinoma (HCC). However, there are few extensive reports on DARS2 function in lung adenocarcinoma (LUAD). The differential expression of DARS2 was detected by genomics and in vitro experiments, and the effect of DARS2 expression on LUAD cell activity was analyzed. Functional enrichment analysis was performed to explore possible signal pathways involved in the biological functions of DARS2 and its co-expressed genes. Utilizing TIMER and GEPIA datasets, the association between DARS2 expression and immunological infiltrating cells was analyzed. At the same time, the association between DARS2 expression pattern and LUAD m6A modification and cuproptosis was examined utilizing TCGA and GEO datasets. The level of DARS2 in LUAD increased, and inhibition of DARS2 expression could significantly inhibit the proliferation of LUAD cells. ROC curves showed that DARS2 overexpression could accurately diagnose LUAD and lead to a significant decline in the survival rates of OS, DSS, and PFI in LUAD. Enrichment analysis showed that DARS2 and its co-expressed genes were closely associated with chromosome segregation and the cell cycle. TIMER and GEPIA database analysis demonstrated that the DARS2 expression pattern was adversely correlated with the infiltration of B cells and Tfh cells. TCGA and GEO dataset examination revealed that DARS2 expression was significantly linked to four m6A-related genes and one cuproptosis-related gene. DARS2 expression is increased in LUAD patients and is closely associated with LUAD immune cell infiltration, modification of m6A, and cuproptosis. DARS2 is a potential reliable prognostic biomarker of LUAD.

A case series evaluation of comprehensive drug testing in the pediatric acute care setting.

Introduction: Drug testing typically follows a one-size-fits-all approach that is inadequate in some clinical scenarios, such as child maltreatment, neglect, and unintentional drug exposure. Results from immunoassay-based testing, which are non-specific, insensitive, and far from comprehensive, can lead to unintended consequences for children and their families. Objectives: The objective of this retrospective case series study is to evaluate the utility of real-time (0-1 day) comprehensive drug testing as an alternative to immunoassay-based testing in the pediatric acute care setting. Methods: Comprehensive drug testing results obtained by mass spectrometry testing and associated medical data for all pediatric cases (0-12 years) at one institution from 2019 to 2022 were included in the analysis. The final case series (n = 7) included all cases from patients <3 years with comprehensive drug testing results that were inconsistent with medication history and/or toxicology results by immunoassay. Results: Comprehensive drug testing by mass spectrometry was ordered for 174 urine and blood samples representing 97 patients (0-12 years) from 2019 to 2022. Of these, 76 cases were from patients <3 years old; results were consistent with medication history and confirmatory for immunoassay results (n = 34), consistent with medication history (n = 14), confirmatory for immunoassay results (n = 10), negative (n = 9), or medical history was incomplete (n = 2). The remaining 7 cases were included in the final case series. Conclusions: The cases highlight the value of real-time comprehensive drug testing in acute pediatric cases. Testing results can rule out toxic exposure from the diagnostic differential when negative, and lead to appropriate medical and social interventions when positive.

Prognostic biomarker DARS2 correlated with immune infiltrates in bladder tumor.

Background: DARS2 is a pivotal member of the Aminoacyl-tRNA synthetases family that is critical for regulating protein translation. However, the biological role of DARS2 in bladder cancer remains elusive. Methods: We analyzed the correlation between DARS2 expression and prognosis, tumor stage, and immune infiltration in bladder cancer using The Cancer Genome Atlas (TCGA) database. We validated findings in clinical samples from The First Affiliated Hospital of Nanchang University and explored the biological functions of DARS2 using cell and animal models. Results: We found DARS2 to be upregulated in bladder cancer, associated with tumor progression and poor prognosis. Immune infiltration analysis suggested that DARS2 may facilitate immune evasion by modulating PD-L1. Cell and animal experiments validated that DARS2 knockdown and overexpress can inhibit or increase cancer cell proliferation, metastasis, tumorigenesis, immune escape, and PD-L1 levels. Conclusions: Our study reveals DARS2 as a potential prognostic biomarker and immunotherapy target in BLCA.

Identification and validation of the mitochondrial function related hub genes by unsupervised machine learning and multi-omics analyses in lung adenocarcinoma.

Background: The mitochondrion and its associated genes were heavily implicated in developing and therapy tumors as the primary cellular organelle in charge of metabolic reprogramming and ferroptosis. Our work focuses on discovering new potential targets while analyzing the multi-omics data of mitochondria-related genes in lung adenocarcinoma (LUAD). Methods: The Cancer Genome Atlas (TCGA) database provided multi-omics data for LUAD patients. Based on the expression profile of the genes associated with mitochondria, the patients were grouped by the unsupervised clustering method. R was used to explore the differential expressed protein-code gene, miRNA, and lncRNA, as well as their enriched functions and ceRNA networks. Additionally, the discrepancy between immune infiltration and genetic variation was comprehensively characterized. Our clinical samples and in vitro experiments investigated the hub gene determined by LASSO and batch analysis. Results: Two clusters are distinguished using unsupervised consensus clustering based on mitochondrial heterogeneity. The integrated analysis emphasized that patients in cluster B had a worse prognosis, higher mutation frequencies, and less immune cell infiltration. The hub genes DARS2 and COX5B are identified by further analysis using LASSO penalization. In vitro experiments indicated that DARS2 and COX5B knockdown inhibited tumor cell proliferation. The specimen of our hospital cohort conducted the immunohistochemistry analysis and validated that DARS2 and COX5B's expression was significantly higher in the tumor than in adjacent normal tissue and correlated to LUAD patients' prognosis. Conclusion: Our observations implied that LUAD patients' tumors had distinct mitochondrial function heterogeneity with different clinical and molecular characteristics. DARS2 and COX5B might be critical genes involved in mitochondrial alterations and potential therapeutic targets.

New candidates in the differential diagnosis of malignant mesothelioma from benign mesothelial hyperplasia and adenocarcinoma; DARS2 and suprabasin.

OBJECTIVES: Malignant mesothelioma (MM) is a primary malignant tumour with a very bad prognosis, which develops from the mesothelial cells lining the serosal surfaces. Because MM can show a wide variety of histological patterns and its cytomorphological features are quite extensive, it is often confused with lung adenocarcinomas (LAC) and reactive mesothelial hyperplasia (RMH). The immunohistochemical examination method is the most useful method for discrimination. In this study, we aimed to determine the value of suprabasin and DARS2 markers in the differential diagnosis of RMH, MM and LAC. METHODS: Thirty MM, 30 LAC and 30 RMH samples selected from the archive of Firat University Hospital Pathology Department Laboratory were included in this study. Suprabasin and DARS2 markers were applied to the samples immunohistochemically and their place in the differential diagnosis was examined. RESULTS: Although DARS2 expression was observed in RMH, MM and adenocarcinoma samples, Suprabasin expression was only observed in adenocarcinoma. There was a significant difference between the groups in terms of DARS2 and Suprabasin expression. No suprabasin expression was detected in MM and RMH. CONCLUSION: Suprabasin and DARS2 may be proposed as new biomarkers to differentiate MM from LAC.

High expression of DARS2 indicates poor prognosis in lung adenocarcinoma.

BACKGROUND: DARS2 was overexpressed in multiple tumor types, but the biological role of DARS2 in lung adenocarcinoma (LUAD) have not been elucidated. METHODS: Firstly, the DARS2 expression in LUAD was explored using The Cancer Genome Atlas (TCGA). Then, qRT-PCR and Western blot were performed to confirm DARS2 expression in LUAD. Next, Cox regression and Kaplan-Meier methods were utilized to evaluate whether DARS2 expression can affect the overall survival. The relationships between DARS2 expression and clinicopathological characteristics were investigated by TCGA database. Moreover, we utilized Gene Set Enrichment Analysis (GSEA) to detect DARS2-related signaling pathways in LUAD. Finally, the special function of DARS2 in cell proliferation, invasion and apoptosis was assessed in vitro. RESULTS: The higher expression of DARS2 was found in LUAD compared to para-carcinoma tissues and significantly related to tumor stage, T stage, and M stage. The survival analysis indicated that DARS2 overexpression was related to poor prognosis in LUAD. Multivariate analysis suggested that DARS2 expression was a prognostic indicator. GSEA revealed that DARS2 was primarily involved in cell cycle-related pathways. In addition, upregulation of DARS2 facilitated LUAD cell proliferation, migration, invasion and inhabited apoptosis, DARS2 knockdown showed an opposite result. CONCLUSION: DARS2 modulates the proliferation, invasion and apoptosis of LUAD cells, and sever as a promising therapeutic target for LUAD.

Functional analysis of missense DARS2 variants in siblings with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation.

Biallelic pathogenic variants in the nuclear gene DARS2 (MIM# 610956), encoding the mitochondrial enzyme aspartyl-tRNA synthetase (MT-ASPRS) cause leukoencephalopathy with Brain Stem and Spinal Cord Involvement and Lactate Elevation (LBSL) (MIM# 611105), a neurometabolic disorder characterized by progressive ataxia, spasticity, developmental arrest or regression and characteristic brain MRI findings. Most patients exhibit a slowly progressive disease course with motor deterirartion that begins in childhood or adolescence, but can also occasionaly occur in adulthood. More severe LBSL presentations with atypical brain MRI findings have been recently described. Baker's yeast orthologue of DARS2, MSD1, is required for growth on oxidative carbon sources. A yeast with MSD1 knockout (msd1Δ) demonstrated a complete lack of oxidative growth which could be rescued by wild-type MSD1 but not MSD1 with pathogenic variants. Here we reported two siblings who exhibited developmental regression and ataxia with different age of onset and phenotypic severity. Exome sequencing revealed 2 compound heterozygous missense variants in DARS2: c.473A>T (p.Glu158Val) and c.829G>A (p.Glu277Lys); this variant combination has not been previously reported. The msd1Δ yeast transformed with plasmids expressing p.Glu259Lys, equivalent to human p.Glu277Lys, showed complete loss of oxidative growth and oxygen consumption, while the strain carrying p.Gln137Val, equivalent to human p.Glu158Val, showed a significant reduction of oxidative growth, but a residual ability to grow was retained. Structural analysis indicated that p.Glu158Val may interfere with protein binding of tRNAAsp, while p.Glu277Lys may impact both homodimerization and catalysis of MT-ASPRS. Our data illustrate the utility of yeast model and in silico analysis to determine pathogenicity of DARS2 variants, expand the genotypic spectrum and suggest intrafamilial variability in LBSL.

CLPP deficiency ameliorates neurodegeneration caused by impaired mitochondrial protein synthesis.

Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.

Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation: A Novel DARS2 Mutation and Intra-Familial Heterogeneity.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is characterized by slowly progressive spastic gait, cerebellar symptoms, and posterior cord dysfunction. DARS2, which encodes mitochondrial aspartyl tRNA synthase, is associated with the rare disease. CASES: The proband had gait disturbance since age 56, while her younger brother had the gait problem since his 20s and needed cane-assistance at age 45. Both cases showed typical demyelinating features of LBSL on the magnetic resonance imaging (MRI) involving the periventricular white matter, brainstem, cerebellum and spinal cord. Sequencing of both cases showed compound heterozygous mutations: c.228-16C>A and c.508C>T in DARS2. The c.228-16C>A is a common mutation in splicing site of intron 2, which causes alternative splicing defect of exon 3, while the c.508C>T at the exon 6 is novel. Our patients are unique in the relative late onset and the apparent difference in disease progression. LITERATURE REVIEW: Literatures from PubMed were reviewed. Five families showed intra-familial heterogeneity on age at onset or clinical severity. CONCLUSION: We identified a family of LBSL with compound heterozygous mutations, and c.508C>T at the exon 6 is a novel one. Clinical heterogeneity was observed in the family and other literatures. Further research for underlying mechanism is required.

Case report: 'AARS2 leukodystrophy'.

BACKGROUND: Mitochondrial alanyl-tRNA synthetase 2 gene (AARS2) related disease is a rare genetic disorder affecting mitochondrial metabolism, leading to severe cardiac disease in infants or progressive leukodystrophy in young adults. The disease is considered ultra-rare with only 39 cases of AARS2-leukodystrophy previously reported. CASE PRESENTATION: We present the case of a young man of consanguineous heritage suffering from cognitive decline and progressive spasticity as well as weakness of the proximal musculature. Utilizing MRI and whole genome sequencing, the patient was diagnosed with a homozygous AARS2 missense variant (NM_020745.3:c.650C > T; p.(Pro217Leu)) and a homozygous CAPN3 variant (NM_000070.2: c.1469G > A; p.(Arg490Gln)), both variants have previously been identified in patients suffering from AARS2 related leukodystrophy and limb-girdle muscular dystrophy, respectively. CONCLUSIONS: This case report presents a case of homozygous AARS2 leukodystrophy and serves to highlight the importance of whole genome sequencing in diagnosing rare neurological diseases as well as to add to the awareness of adult onset leukodystrophies.

Perinatal Manifestations of DARS2-Associated Leukoencephalopathy With Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL).

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a progressive disorder associated with deficiency of mitochondrial aspartyl-tRNA synthetase, a homodimer encoded by the gene DARS2. There is a wide range in age of onset of symptoms, typically from childhood to adulthood, with very few cases of infantile onset disease reported. We report a child at age 10 years with perinatal onset of symptoms evidenced by congenital microcephaly with progression to severe but non-lethal epileptic encephalopathy and spastic quadriplegia. A comprehensive epilepsy focused gene panel performed as a trio with parents detected a novel homozygous DARS2 variant. This variant is located at the dimer interface in a critical catalytic domain and is expected to result in markedly reduced enzyme activity which likely explains the severe and early onset symptoms in this case.

A New DARS2 Mutation Discovered in an Adult Patient.

We report a case of an adult patient suffering from leukoencephalopathy with brainstem and spinal cord involvement and elevated white matter lactate (LBSL) caused by a DARS2 polymorphism. DARS2 mutation was identified by combining MRI and genetic analysis. Our patient was affected by compound heterozygosity for a pathogenic mutation and a common variant, but with reduced aspartyl-tRNA synthetase activity. Brain and spinal cord magnetic resonance imaging revealed extensive white matter abnormalities; spectroscopy revealed no lactate elevation. A new compound heterozygous DARS2 variant combined with a polymorphism in the other allele in an adult patient with LBSL was identified, resulting in reduced DARS2 activity. This combination is rare and has consequences on how we should consider benign variant polymorphisms in the future.

Phenotypes and genotypes of mitochondrial aminoacyl-tRNA synthetase deficiencies from a single neurometabolic clinic.

Mitochondrial aminoacyl-tRNA synthetases play a major role in protein translation, synthesis, and oxidative phosphorylation. We reviewed all patients diagnosed with mitochondrial aminoacyl-tRNA synthetase deficiencies diagnosed in a single neurometabolic clinic. We report five patients with mitochondrial aminoacyl-tRNA synthetase deficiencies including DARS2, EARS2, PARS2, and RARS2 deficiencies. Siblings with DARS2 deficiency presented with global developmental delay within the first year of life. DARS2, EARS2, PARS2, and RARS2 deficiencies were identified by whole exome sequencing. We report coagulation factor abnormalities in PARS2 deficiency for the first time. We also report symmetric increased signal intensity in globus pallidi in FLAIR images in brain MRI in EARS2 deficiency for the first time. One patient with RARS2 deficiency had compound heterozygous variants in RARS2. One of those variants was an intronic variant. We confirmed the pathogenicity by mRNA studies. Mitochondrial aminoacyl-tRNA synthetase deficiencies are diagnosed by molecular genetic investigations. Clinically available non-invasive biochemical investigations are non-specific for the diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies. A combination of brain MRI features and molecular genetic investigations should be undertaken to confirm the diagnosis of mitochondrial aminoacyl-tRNA synthetase deficiencies.

Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.