Mitochondrial related genome-wide Mendelian randomization identifies putatively causal genes for multiple cancer types.

BACKGROUND: Mitochondrial dysfunction is a hallmark of cancer. However, it is unclear whether it is a cause of cancer. This two-sample Mendelian randomization (MR) analyses, uses genetic instruments to proxy the exposure of mitochondrial dysfunction and cancer summary statistics as outcomes, allowing for causal inferences. METHODS: Summary statistics from 18 common cancers (2107-491,974 participants), gene expression, DNA methylation and protein expression quantitative trait loci (eQTL, mQTL and pQTL, respectively, 1000-31,684 participants) on individuals of European ancestry, were included. Genetic variants located within or close to the 1136 mitochondrial-related genes (in cis) and robustly associated with the mitochondrial molecular alterations were used as instrumental variables, and their causal associations with cancers were examined using summary-data-based MR (SMR) analyses. An additional five MR methods were used as sensitivity analyses to confirm the casual associations. A Bayesian test for colocalization between mitochondrial molecular QTLs and cancer risk loci was performed to provide insights into the potential regulatory mechanisms of risk variants on cancers. FINDINGS: We identified potential causal relationships between mitochondrial-related genes and breast, prostate, gastric, lung cancer and melanoma by primary SMR analyses. The sensitivity and the colocalization analyses further refined four genes that have causal effects on three types of cancer. We found strong evidence of positive association of FDPS expression level with breast cancer risk (OR per SD, 0.66; 95% CI, 0.49-0.83; P = 9.77 × 10-7), NSUN4 expression level with both breast cancer risk (OR per SD, 1.05; 95% CI, 1.03-1.07; P = 5.24 × 10-6) and prostate cancer risk (OR per SD, 1.06; 95% CI, 1.03-1.09; P = 1.01 × 10-5), NSUN4 methylation level with both breast and prostate cancer risk, and VARS2 methylation level with lung cancer risk. INTERPRETATIONS: This data-driven MR study demonstrated the causal role of mitochondrial dysfunction in multiple cancers. Furthermore, this study identified candidate genes that can be the targets of potential pharmacological agents for cancer prevention. FUNDING: This work was supported by Styrelsen för Allmänna Sjukhusets i Malmö Stiftelse för bekämpande av cancer (20211025).

Epigenetic signatures relating to disease-associated genotypic burden in familial risk of bipolar disorder.

Environmental factors contribute to risk of bipolar disorder (BD), but how environmental factors impact the development of psychopathology within the context of elevated genetic risk is unknown. We herein sought to identify epigenetic signatures operating in the context of polygenic risk for BD in young people at high familial risk (HR) of BD. Peripheral blood-derived DNA was assayed using Illumina PsychArray, and Methylation-450K or -EPIC BeadChips. Polygenic risk scores (PRS) were calculated using summary statistics from recent genome-wide association studies for BD, major depressive disorder (MDD) and cross-disorder (meta-analysis of eight psychiatric disorders). Unrelated HR participants of European ancestry (n = 103) were stratified based on their BD-PRS score within the HR-population distribution, and the top two quintiles (High-BD-PRS; n = 41) compared against the bottom two quintiles (Low-BD-PRS; n = 41). The High-BD-PRS stratum also had higher mean cross-disorder-PRS and MDD-PRS (ANCOVA p = 0.035 and p = 0.024, respectively). We evaluated DNA methylation differences between High-BD-PRS and Low-BD-PRS strata using linear models. One differentially methylated probe (DMP) (cg00933603; p = 3.54 × 10-7) in VARS2, a mitochondrial aminoacyl-tRNA synthetase, remained significantly hypomethylated after multiple-testing correction. Overall, BD-PRS appeared to broadly impact epigenetic processes, with 1,183 genes mapped to nominal DMPs (p < 0.05); these displayed convergence with genes previously associated with BD, schizophrenia, chronotype, and risk taking. We tested poly-methylomic epigenetic profiles derived from nominal DMPs in two independent samples (n = 54 and n = 82, respectively), and conducted an exploratory evaluation of the effects of family environment, indexing cohesion and flexibility. This study highlights an important interplay between heritable risk and epigenetic factors, which warrant further exploration.

VARS2 Depletion Leads to Activation of the Integrated Stress Response and Disruptions in Mitochondrial Fatty Acid Oxidation.

Mutations in mitochondrial aminoacyl-tRNA synthetases (mtARSs) have been reported in patients with mitochondriopathies: most commonly encephalopathy, but also cardiomyopathy. Through a GWAS, we showed possible associations between mitochondrial valyl-tRNA synthetase (VARS2) dysregulations and non-ischemic cardiomyopathy. We aimed to investigate the possible consequences of VARS2 depletion in zebrafish and cultured HEK293A cells. Transient VARS2 loss-of-function was induced in zebrafish embryos using Morpholinos. The enzymatic activity of VARS2 was measured in VARS2-depleted cells via northern blot. Heterozygous VARS2 knockout was established in HEK293A cells using CRISPR/Cas9 technology. BN-PAGE and SDS-PAGE were used to investigate electron transport chain (ETC) complexes, and the oxygen consumption rate and extracellular acidification rate were measured using a Seahorse XFe96 Analyzer. The activation of the integrated stress response (ISR) and possible disruptions in mitochondrial fatty acid oxidation (FAO) were explored using RT-qPCR and western blot. Zebrafish embryos with transient VARS2 loss-of-function showed features of heart failure as well as indications of CNS and skeletal muscle involvements. The enzymatic activity of VARS2 was significantly reduced in VARS2-depleted cells. Heterozygous VARS2-knockout cells showed a rearrangement of ETC complexes in favor of complexes III2, III2 + IV, and supercomplexes without significant respiratory chain deficiencies. These cells also showed the enhanced activation of the ISR, as indicated by increased eIF-2α phosphorylation and a significant increase in the transcript levels of ATF4, ATF5, and DDIT3 (CHOP), as well as disruptions in FAO. The activation of the ISR and disruptions in mitochondrial FAO may underlie the adaptive changes in VARS2-depleted cells.

Improvement of substrate recognition in branched-chain aminoacyl-tRNA synthetases from Escherichia coli under conditions of pyrophosphate amplification.

Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l-isoleucine, l-leucine, and l-valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5'-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l-amino acid substrates (50 µM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l-valine, l-lysine, and l-threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p1, p4-di (adenosine) 5'-tetraphosphate. We also observed a strong positive correlation (R = 0.99) between the amount of pyrophosphate produced and the initial concentration of l-amino acid with 5 and 50 µM l-isoleucine, l-leucine, and l-valine. Our results suggest that amino acid assays using IleRS, LeuRS, and ValRS are promising methods to accurately measure l-valine, l-isoleucine, and l-leucine in food products and plasma.

Mutations in bdcA and valS Correlate with Quinolone Resistance in Wastewater Escherichia coli.

Single mutations can confer resistance to antibiotics. Identifying such mutations can help to develop and improve drugs. Here, we systematically screen for candidate quinolone resistance-conferring mutations. We sequenced highly diverse wastewater E. coli and performed a genome-wide association study (GWAS) to determine associations between over 200,000 mutations and quinolone resistance phenotypes. We uncovered 13 statistically significant mutations including 1 located at the active site of the biofilm dispersal gene bdcA and 6 silent mutations in the aminoacyl-tRNA synthetase valS. The study also recovered the known mutations in the topoisomerases gyrase (gyrA) and topoisomerase IV (parC). In summary, we demonstrate that GWAS effectively and comprehensively identifies resistance mutations without a priori knowledge of targets and mode of action. The results suggest that mutations in the bdcA and valS genes, which are involved in biofilm dispersal and translation, may lead to novel resistance mechanisms.

Genome-wide association study identifies novel risk variants from RPS6KA1, CADPS, VARS, and DHX58 for fasting plasma glucose in Arab population.

Consanguineous populations of the Arabian Peninsula, which has seen an uncontrolled rise in type 2 diabetes incidence, are underrepresented in global studies on diabetes genetics. We performed a genome-wide association study on the quantitative trait of fasting plasma glucose (FPG) in unrelated Arab individuals from Kuwait (discovery-cohort:n = 1,353; replication-cohort:n = 1,196). Genome-wide genotyping in discovery phase was performed for 632,375 markers from Illumina HumanOmniExpress Beadchip; and top-associating markers were replicated using candidate genotyping. Genetic models based on additive and recessive transmission modes were used in statistical tests for associations in discovery phase, replication phase, and meta-analysis that combines data from both the phases. A genome-wide significant association with high FPG was found at rs1002487 (RPS6KA1) (p-discovery = 1.64E-08, p-replication = 3.71E-04, p-combined = 5.72E-11; ß-discovery = 8.315; ß-replication = 3.442; ß-combined = 6.551). Further, three suggestive associations (p-values < 8.2E-06) with high FPG were observed at rs487321 (CADPS), rs707927 (VARS and 2Kb upstream of VWA7), and rs12600570 (DHX58); the first two markers reached genome-wide significance in the combined analysis (p-combined = 1.83E-12 and 3.07E-09, respectively). Significant interactions of diabetes traits (serum triglycerides, FPG, and glycated hemoglobin) with homeostatic model assessment of insulin resistance were identified for genotypes heterozygous or homozygous for the risk allele. Literature reports support the involvement of these gene loci in type 2 diabetes etiology.

Mechanism of discrimination of isoleucyl-tRNA synthetase against nonproteinogenic α-aminobutyrate and its fluorinated analogues.

Isoleucyl-tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200-fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α-aminobutyrate (Abu) is activated 30-fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM . To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary-related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu-tRNAIle with a rate of 40 s-1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. ENZYMES: Isoleucyl-tRNA synthetase (EC 6.1.1.5), leucyl-tRNA synthetase (EC 6.1.1.4), valyl-tRNA synthetase (EC 6.1.1.9).

A novel VARS2 gene variant in a patient with epileptic encephalopathy.

Background: Mitochondrial disorders are heterogeneous clinical syndromes caused by defective activity in the mitochondrial respiratory chain, resulting in a faulty oxidative phosphorylation system. These inherited disorders are individually rare, and furthermore they are phenotypic variables. The genetically characterized mitochondrial disorders are rarely associated with epileptic encephalopathies.Case presentation: We present the clinical phenotype, biochemical analysis, and electrographic and neuro-radiological features of a 5-month-old girl with epileptic encephalopathy, microcephaly, severe psychomotor delay, hypertrophic cardiomyopathy, and abnormal MRI scan. Using whole-genome sequencing technique, compound heterozygous mutations of the VARS2 gene were revealed, with one previously unreported frameshift mutation.Conclusion: Our report extends the phenotypic spectrum of VARS2-related disorders with an initial presentation of epileptic encephalopathy and early death due to malignant arrhythmia.

A combination of two novel VARS2 variants causes a mitochondrial disorder associated with failure to thrive and pulmonary hypertension.

The VARS2 gene encodes a mitochondrial valyl-transfer RNA synthetase which is used in mitochondrial translation. To date, several patients with VARS2 pathogenic variants have been described in the literature. These patients have features of lactic acidosis with encephalomyopathy. We present a case of an infant with lactic acidosis, failure to thrive, and severe primary pulmonary hypertension who was found to be a compound heterozygote for two novel VARS2 variants (c.1940C>T, p.(Thr647Met) and c.2318G>A, p.(Arg773Gln)). The patient was treated with vitamin supplements and a carbohydrate-restricted diet. The lactic acidosis and failure to thrive resolved, and he showed good growth and development. Functional studies and molecular analysis employed a yeast model system and the VAS1 gene (yeast homolog of VARS2). VAS1 genes harboring either one of two mutations corresponding to the two novel variants in the VARS2 gene, exhibited partially reduced function in haploid yeast strains. A combination of both VAS1 variant alleles in a diploid yeast cell exhibited a more significant decrease in oxidative metabolism-dependent growth and in the oxygen consumption rate (reminiscent of the patient who carries two mutant VARS2 alleles). Our results demonstrate the pathogenicity of the biallellic novel VARS2 variants. KEY MESSAGES: • A case of an infant who is a compound heterozygote for two novel VARS2 variants. • This infant displayed lactic acidosis, failure to thrive, and pulmonary hypertension. • Treatment of the patient with a carbohydrate-restricted diet resulted in good growth and development. • Studies with the homologous yeast VAS1 gene showed reduced function of corresponding single mutant in haploid yeast strains. • A combination of both VAS1 variant alleles in diploid yeast exhibited a more significant decrease in function, thereby confirming the pathogenicity of the biallellic novel VARS2 variants.

Pneumonia: host susceptibility and shared genetics with pulmonary function and other traits.

Pneumonia is a common and severe infectious lung disease. Host genetics, together with underlying medical and lifestyle conditions, determine pneumonia susceptibility. We performed a secondary analysis of the results of two genome-wide studies for pneumonia in 23andMe participants (40 600 cases/90 039 controls) (Tian et al., 2017) and UK Biobank (BB) participants (12 614 cases/324 585 controls) (via the Global Biobank Engine) and used the GTEx database to correlate the results with expression quantitative trait loci (eQTLs) data in lung and whole blood. In the 23andMe pneumonia single nucleotide polymorphism (SNP) set, 177 genotyped SNPs in the human leukocyte antigen (HLA) region satisfied the genome-wide significance level, P ≤ 5·0E-08. Several target genes (e.g. C4A, VARS2, SFTA2, HLA-C, HLA-DQA2) were unidirectionally regulated by many HLA eSNPs associated with a higher risk of pneumonia. In lung, C4A transcript was up-regulated by 291 pneumonia risk alleles spanning the half the HLA region. Among SNPs correlated with the expression levels of SFTA2 and VARS2, approximately 75% overlapped: all risk alleles were associated with VARS2 up-regulation and SFTA2 down-regulation. To find shared gene loci between pneumonia and pulmonary function (PF), we used data from the Global Biobank Engine and literature on genome-wide association studies (GWAS) of PF in general populations. Numerous gene loci overlapped between pneumonia and PF: 28·8% in the BB data set and 49·2% in the 23andMe data set. Enrichment analysis within the database of Genotypes and Phenotypes (dbGaP) and National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) Catalog of pneumonia and pneumonia/PF gene sets identified significant overlap between these gene sets and genes related to inflammatory, developmental, neuropsychiatric and cardiovascular and obesity-related traits.

VARS2-linked mitochondrial encephalopathy: two case reports enlarging the clinical phenotype.

BACKGROUND: Mitochondrial respiratory chain consists of five complexes encoded by nuclear and mitochondrial genomes. Mitochondrial aminoacyl-tRNA synthetases are key enzymes in the synthesis of such complexes. Bi-allelic variants of VARS2, a nuclear gene encoding for valyl-tRNA (Val-tRNA) synthetase, are associated to several forms of mitochondrial encephalopathies or cardiomyoencephalopathies. Among these, the rare homozygous c.1100C > T (p.Thr367Ile) mutation variably presents with progressive developmental delay, axial hypotonia, limbs spasticity, drug-resistant epilepsy leading, in some cases, to premature death. Yet only six cases, of which three are siblings, harbouring this homozygous mutation have been described worldwide. CASE PRESENTATION: Hereby, we report two additional cases of two non-related young girls from Sardinia, born from non-consanguineous and healthy parents, carrying the aforesaid homozygous VARS2 variant. At onset both the patients presented with worsening psychomotor delay, muscle hypotonia and brisk tendon reflexes. Standard genetic tests were normal, as well as metabolic investigations. Brain MRI showed unspecific progressive abnormalities, such as corpus callosum hypoplasia (patient A) and cerebellar atrophy (patient A and B). Diagnosis was reached by adopting massive parallel next generation sequencing. Notably clinical phenotype of the first patient appears to be milder compared to previous known cases. The second patient eventually developed refractory epilepsy and currently presents with severe global impairment. Because no specific treatment is available as yet, both patients are treated with supporting antioxidant compounds along with symptomatic therapies. CONCLUSIONS: Given the paucity of clinical data about this very rare mitochondrial encephalopathy, our report might contribute to broaden the phenotypic spectrum of the disorder. Moreover, noteworthy, three out of five pedigrees so far described belong to the Northern Sardinia ethnicity.

Biallelic mutations in valyl-tRNA synthetase gene VARS are associated with a progressive neurodevelopmental epileptic encephalopathy.

Aminoacyl-tRNA synthetases (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes.

Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish.

Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.

The nuclear background influences the penetrance of the near-homoplasmic m.1630 A > G MELAS variant in a symptomatic proband and asymptomatic mother.

In this study, we report the metabolic consequences of the m.1630 A > G variant in fibroblasts from the symptomatic proband affected with the mitochondrial encephalomyopathy lactic acidosis and stroke-like episode Syndrome and her asymptomatic mother. By long-range PCR followed by massively parallel sequencing of the mitochondrial genome, we accurately measured heteroplasmy in fibroblasts from the proband (89.6%) and her mother (94.8%). Using complementary experimental approaches, we show a functional correlation between manifestation of clinical symptoms and bioenergetic potential. Our mitochondrial morphometric analysis reveals a link between defects of mitochondrial cristae ultrastructure and symptomatic status. Despite near-homoplasmic level of the m.1630A > G variant, the mother's fibroblasts have a normal OXPHOS metabolism, which stands in contrast to the severely impaired OXPHOS response of the proband's fibroblasts. The proband's fibroblasts also exhibit glycolysis at near constitutive levels resulting in a stunted compensatory glycolytic response to offset the severe OXPHOS defect. Whole exome sequencing reveals the presence of a heterozygous nonsense VARS2 variant (p.R334X) exclusively in the proband, which removes two thirds of the VARS2 protein containing key domains interacting with the mt-tRNAval and may play a role in modulating the penetrance of the m.1630A > G variant despite similar near homoplasmic levels. Our transmission electron microscopy study also shows unexpected ultrastructural changes of chromatin suggestive of differential epigenomic regulation between the proband and her mother that may explain the differential OXPHOS response between the proband and her mother. Future study will decipher by which molecular mechanisms the nuclear background influences the penetrance of the m.1630 A > G variant causing MELAS.

The role of translation elongation factor eEF1 subunits in neurodevelopmental disorders.

The multi-subunit eEF1 complex plays a crucial role in de novo protein synthesis. The central functional component of the complex is eEF1A, which occurs as two independently encoded variants with reciprocal expression patterns: whilst eEF1A1 is widely expressed, eEF1A2 is found only in neurons and muscle. Heterozygous mutations in the gene encoding eEF1A2, EEF1A2, have recently been shown to cause epilepsy, autism, and intellectual disability. The remaining subunits of the eEF1 complex, eEF1Bα, eEF1Bδ, eEF1Bγ, and valyl-tRNA synthetase (VARS), together form the GTP exchange factor for eEF1A and are ubiquitously expressed, in keeping with their housekeeping role. However, mutations in the genes encoding these subunits EEF1B2 (eEF1Bα), EEF1D (eEF1Bδ), and VARS (valyl-tRNA synthetase) have also now been identified as causes of neurodevelopmental disorders. In this review, we describe the mutations identified so far in comparison with the degree of normal variation in each gene, and the predicted consequences of the mutations on the functions of the proteins and their isoforms. We discuss the likely effects of the mutations in the context of the role of protein synthesis in neuronal development.

A novel compound heterozygous mutation in VARS2 in a newborn with mitochondrial cardiomyopathy: a case report of a Chinese family.

BACKGROUND: Genetic defects in the mitochondrial aminoacyl-tRNA synthetase are important causes of mitochondrial disorders. VARS2 is one of the genes encoding aminoacyl-tRNA synthetases. Recently, an increasing number of pathogenic variants of VARS2 have been reported. CASE PRESENTATION: We report the novel compound heterozygous pathogenic VARS2 mutations c.643 C > T (p. His215Tyr) and c.1354 A > G (p. Met452Val) in a female infant who presented with poor sucking at birth, poor activity, hyporeflexia, hypertonia, persistent pulmonary hypertension of newborn (PPHN), metabolic acidosis, severe lactic acidosis, expansion and hypertrophic cardiomyopathy. These heterozygous mutations were carried individually by the proband's parents and elder sister; the two mutations segregated in the family and were the cause of the disease in the proband.The c.643 C > T (p. His215Tyr) mutation was not described in the ExaC, GNomAD and 1000 Genomes Project databases, and the frequency of c.1354 A > G (p. Met452Val) was < 0.001 in these gene databases.The two mutated amino acids were located in a highly conserved region of the VARS2 protein that is important for its interaction with the cognate tRNA. The two missense mutations were predicted by online tools to be damaging and deleterious. CONCLUSIONS: Our report expands the spectrum of known pathogenicVARS2 variants associated with mitochondrial disorders in humans.VARS2 deficiency may cause a severe neonatal presentation with structural cardiac abnormalities.

Biallelic variants in VARS in a family with two siblings with intellectual disability and microcephaly: case report and review of the literature.

Two male siblings ages 15 and 10 yr old had similar features of intellectual disability, developmental delay, severe speech impairment, microcephaly, prematurity, and transient elevation of liver enzymes in infancy. Exome sequencing revealed one novel (c.65C>A; p.Ala22Asp) and one ultra-rare (c.3214T>C; p.Phe1072Leu) predicted damaging missense variant in trans in the gene encoding cytoplasmic valyl-tRNA synthetase (VARS). Biallelic variants in VARS have previously been associated with a neurodevelopmental disorder characterized by microcephaly, seizures, and cortical atrophy (NDMSCA; MIM #617802). Although our patients have no history of seizures or cortical atrophy, we suggest that the biallelic variants in VARS p.Ala22Asp and p.Phe1072Leu in this family are likely pathogenic and associated with NDMSCA, expanding the clinical phenotype of the condition.

Loss of function mutations in VARS encoding cytoplasmic valyl-tRNA synthetase cause microcephaly, seizures, and progressive cerebral atrophy.

Progressive microcephaly and neurodegeneration are genetically heterogenous conditions, largely associated with genes that are essential for the survival of neurons. In this study, we interrogate the genetic etiology of two siblings from a non-consanguineous family with severe early onset of neurological manifestations. Whole exome sequencing identified novel compound heterozygous mutations in VARS that segregated with the proband: a missense (c.3192G>A; p.Met1064Ile) and a splice site mutation (c.1577-2A>G). The VARS gene encodes cytoplasmic valyl-tRNA synthetase (ValRS), an enzyme that is essential during eukaryotic translation. cDNA analysis on patient derived fibroblasts revealed that the splice site acceptor variant allele led to nonsense mediated decay, thus resulting in a null allele. Three-dimensional modeling of ValRS predicts that the missense mutation lies in a highly conserved region and could alter side chain packing, thus affecting tRNA binding or destabilizing the interface between the catalytic and tRNA binding domains. Further quantitation of the expression of VARS showed remarkably reduced levels of mRNA and protein in skin derived fibroblasts. Aminoacylation experiments on patient derived cells showed markedly reduced enzyme activity of ValRS suggesting the mutations to be loss of function. Bi-allelic mutations in cytoplasmic amino acyl tRNA synthetases are well-known for their role in neurodegenerative disorders, yet human disorders associated with VARS mutations have not yet been clinically well characterized. Our study describes the phenotype associated with recessive VARS mutations and further functional delineation of the pathogenicity of novel variants identified, which widens the clinical and genetic spectrum of patients with progressive microcephaly.

Clinical, biochemical, and genetic features associated with VARS2-related mitochondrial disease.

In recent years, an increasing number of mitochondrial disorders have been associated with mutations in mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), which are key enzymes of mitochondrial protein synthesis. Bi-allelic functional variants in VARS2, encoding the mitochondrial valyl tRNA-synthetase, were first reported in a patient with psychomotor delay and epilepsia partialis continua associated with an oxidative phosphorylation (OXPHOS) Complex I defect, before being described in a patient with a neonatal form of encephalocardiomyopathy. Here we provide a detailed genetic, clinical, and biochemical description of 13 patients, from nine unrelated families, harboring VARS2 mutations. All patients except one, who manifested with a less severe disease course, presented at birth exhibiting severe encephalomyopathy and cardiomyopathy. Features included hypotonia, psychomotor delay, seizures, feeding difficulty, abnormal cranial MRI, and elevated lactate. The biochemical phenotype comprised a combined Complex I and Complex IV OXPHOS defect in muscle, with patient fibroblasts displaying normal OXPHOS activity. Homology modeling supported the pathogenicity of VARS2 missense variants. The detailed description of this cohort further delineates our understanding of the clinical presentation associated with pathogenic VARS2 variants and we recommend that this gene should be considered in early-onset mitochondrial encephalomyopathies or encephalocardiomyopathies.

Recessive VARS2 mutation underlies a novel syndrome with epilepsy, mental retardation, short stature, growth hormone deficiency, and hypogonadism.

BACKGROUND: Most mitochondrial and cytoplasmic aminoacyl-tRNA synthetases (aaRSs) are encoded by nuclear genes. Syndromic disorders resulting from mutation of aaRSs genes display significant phenotypic heterogeneity. We expand aaRSs-related phenotypes through characterization of the clinical and molecular basis of a novel autosomal-recessive syndrome manifesting severe mental retardation, ataxia, speech impairment, epilepsy, short stature, microcephaly, hypogonadism, and growth hormone deficiency. RESULTS: A G>A variant in exon 29 of VARS2 (c.3650G>A) (NM_006295) was identified in the index case. This homozygous variant was confirmed by Sanger sequencing and segregated with disease in the family studied. The c.3650G>A change results in alteration of arginine to histidine at residue 1217 (R1217H) of the mature protein and is predicted to be pathogenic. CONCLUSIONS: These findings contribute to a growing list of aaRSs disorders, broadens the spectrum of phenotypes attributable to VARS2 mutations, and provides new insight into genotype-phenotype correlations among the mitochondrial synthetase genes.