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

Abstract

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.

Conflict of interest statement

D.D. is a consultant for Biomarin, Ichorian, and Complete Genomics. J.F. holds shares in Illumina and Spouse is Founder and Principal of Friedman Bioventure, which holds a variety of publicly traded and private biotechnology interests. D.D. is on the Scientific Advisory Board for Audentes Therapeutics. The remaining authors declare no competing interests.

Figures

Fig. 1

VARS mutations lead to a neurodevelopmental disorder with microcephaly, seizures, and cortical atrophy. a Pedigrees showing consanguinity in three families (double bars). Family 3007 showed remote consanguinity. Seven affected individuals were identified (black), and one individual was likely affected but DNA was not available for genotyping (3439-IV-1). Multiple miscarriages in family GB31 suggested fetal demise. b Available axial or midline sagittal brain MRIs demonstrated diffuse cortical atrophy (yellow star) and thinned corpus callosum (yellow arrow) compared to control. The brain MRI in 3007-II-1 was acquired at 6 months of age, and thus the patient was probably too young to observe these features. Two individuals from family 3439 had follow-up MRIs that demonstrated progressive cortical atrophy. D—days; M—months; Y—years. c Major domains of the 1264 amino acid protein, showing the GST, aminoacylation (including HIGH, Editing and KMSKS domains) and anticodon-binding domains. Disease-associated variants described in this study (red) and those from Karaca et al. (black) are noted. Variants were in the aminoacylation or in or near the anticodon-binding domains, suggesting altered function. d Sequence conservation of mutated amino acids. The variants were either precisely conserved or maintain charge/polarity, with the exception of NM_006295.2:c.2653C>T p.(Leu885Phe), which is absent in T. thermophiles

Fig. 2

Predicted mutational impact on VARS structure and tRNA binding. a Locations of the mutational sites on the structural model of human VARS-tRNAVal complex (VARS green, tRNAVal orange, mutational sites magenta). Residues Pro661, Arg947, and Arg1119 are closer to the tRNA molecule (<6 Å), while Ala692 and Arg1058 have a longer distance of >15 Å from tRNA. b–f Pair-wise comparisons between the wild-type (left) and mutant (right) residues for predicted changes in local contacts with tRNA or other amino acids. Amino acids are indicated by letter and number (e.g., Pro661), tRNA bases are indicated by letter and dash and number (e.g., A-75). Hydrogen bonds are presented as yellow dashed lines. b Residue Pro661 forms a hydrogen bond with Val670 and has a distance of ~4.5 Å from A-75 of the tRNA. c Residue Ala692 forms hydrogen bonds with adjacent residues Ala689, Arg695, Gln696, and Asp697. Mutation to proline predicts abolished contacts with Ala689 and disrupted helical structure. d Residue Arg947 is close to C-37 and predicts hydrogen-bonding interaction that is abolished by the Arg947H mutation. This mutation does not predict altered hydrogen-bonding with Ile943 and Asn951. e Residue Arg1058 forms hydrogen bonds with Asp1054, Ser1061 and Pro1090. The NM_006295.2:c.3173G>A p.(Arg1058Gln) mutation predicts elimination one of the two possible bonds with Asp1054. f The side chain of Arg1119 forms a hydrogen bond with C-39, predicted to be abolished by the mutation

Fig. 3

VARS patient cells show normal VARS protein level, but reduced enzymatic ARS activity. a Western blot of patient or control cells showing retained VARS protein level in affected individuals (2937-III-1A and 3308-III-1A), carrier parents (2937-II-1F and 3308-II-2M), and controls (3311-III-2C, 3838-III-1C, and 4936-III-1C). b VARS, LARS (leucine ARS) and RARS (arginine ARS) aminoacylation activity. VARS activity was specifically reduced in patient cells, normalized to control cells at 100% (from n = 3 replicates, error bars = S.E.M.). c VARS activity in patients compared with carrier parents and controls. Black: patients, light gray: parents of affecteds (obligate carriers of the VARS mutation), dark gray: healthy controls (from n = 3 replicates, error bars = S.E.M). Differences between patients and controls or carriers were calculated based upon the average of the group (P < 0.0001 for both comparisons, One tailed Student’s t-test). Full gel in Supplementary Fig. 2