Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr;137(4):293-303.
doi: 10.1007/s00439-018-1882-3. Epub 2018 Apr 24.

Loss of Function Mutations in VARS Encoding Cytoplasmic valyl-tRNA Synthetase Cause Microcephaly, Seizures, and Progressive Cerebral Atrophy

Affiliations
Free PMC article

Loss of Function Mutations in VARS Encoding Cytoplasmic valyl-tRNA Synthetase Cause Microcephaly, Seizures, and Progressive Cerebral Atrophy

Joshi Stephen et al. Hum Genet. .
Free PMC article

Abstract

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.

Conflict of interest statement

Compliance with ethical standards

Conflict of interest The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Pedigree and clinical features of the family. a Pedigree of the family (arrow points to the proband). b Clinical photographs of the elder sibling of the proband (A-II.1) showing severe microcephaly, low set ears, upslant of eyes, sloping forehead, micrognathia, short nose and flat nasal bridge. c, d MRI of A-II.1 at the age of 4 months showing diffuse cerebral atrophy, thin corpus callosum and normal cerebellum. e, f T2 Axial and coronal MRI images of the proband (A-II.2) at 52 days of life showing minimal diffuse brain atrophy with prominent fissures and sulci and no significant cerebellar atrophy. g, h MRI repeated at 2 years of proband (A-II.2) showing progressive worsening of cerebral and cerebellar atrophy (arrow in H) and thinning of corpus callosum
Fig. 2
Fig. 2
Sanger validation of the variants, conservation and splice analysis. a Sanger validation of identified compound heterozygous variants in the proband: the splice site variation (c.1577–2A>G) inherited from the father and the missense variation (c.3192G>A) inherited from the mother. b Conservation of amino acids around the position of missense variation (c.3192G>A; p.Met1064Ile); methionine is highly conserved up to C.elegans and Baker’s yeast. c Diagrammatic representation of different splice variants of VARS and primer designing scheme for splice analysis and qPCR. Each box represents the exonic regions of the gene; the black boxes are coding parts and the boxes with dashed line denote untranslated regions, and ‘ATG’ marks the start of the coding frame. Two splice variants (XM_017011247.1 and XM_017011246.1) lack 5′ coding regions and exon 13 compared to the longest variants (NM_006295.2 and XM_005249362.2) and have their start codon in-frame downstream. Position of primers for splice defect analysis (F1–R1, F2–R2) and qPCR (Q1F–Q1R, Q2F–Q2R, Q3F–Q3R) are also depicted. d Gel images of the PCR products of F1–R1 flanking the splice site mutation. The lower band (288 bp) which lacks exon 13 is present in both control and patient, results from the amplification of shorter splice variants that lack exon 13. e Chromatogram showing the sequencing results of F2–R2 amplifying cDNA, depicting the presence of only one allele (c.3192A) in the patient (lower panel) compared to control (upper panel), showing the absence of G allele, which harbors the splice site variation. f Schematic representation of different functional domains in the full length VARS protein (Uniprot: P26640). The splice site variation is predicted to abolish both tRNA synthetase and anticodon-binding domains while the missense variation is in a highly conserved area of the anticodon-binding domain
Fig. 3
Fig. 3
The model of human valyl-tRNA synthetase with tRNA ▸ bound. a, b The protein main chain (alpha-carbon atoms) is colored according to sequence identity with the template structure, yellow where identical (39%) and light blue where different. a Stereo view, with the tRNA backbone (phosphorus atoms) shown in red, a Val-AMP analog shown as ball and stick, bound in the aminoacylation site, and the side chain of Met 1064 shown as space filling. The location of an unmodeled region inserted in the human sequence between the KMSKS loop and the DALR motif is marked by the blue spheres representing Asp 875 and Cys 917. b Stereo view of residues neighboring Met 1064, with carbon atoms in side chains of residues identical in the model and template colored dark gray. The DALR motif and other conserved residues are labeled. For clarity, only the tip of Leu 953 is shown. c Protein-tRNA interface, with tRNA shown as ball and stick and the Val-AMP analogue shown as sticks. The possible impact of the M1064I mutation on ValRS-tRNA binding is illustrated by showing side chains as space filling for Met 1064 and Cys 950, as well as for conserved residues at or near the interface: Arg 947, Phe 949, Asn 951, Lys 952, Asn 955, Ala 956, and Phe 959. The M1064I mutation might also perturb the interface between the catalytic core domain (colored salmon) and the anticodon-binding domain (green)
Fig. 4
Fig. 4
VARS expression studies and aminoacylation activity in proband cells. a VARS mRNA expression studies using three different primer probes (see the primer designing scheme in Fig. 2c) showing 50–60% reduction of transcripts in proband, relative to control. b VARS immunoblotting using cell lysates derived from three controls and proband showing severe reduction in protein expression in proband cells. VARS overexpression lysate (OEL) produced in HEK293T cells was used as positive control and compared to empty vector lysate (EVL) (Novus Biologicals, NBP2–07645). Results were normalized to beta actin loading control. c, d Decreased aminoacylation activity in proband fibroblast cell lysate. Valylation was carried out using cell lysate containing 920 μg/mL of protein and 1.5 mg/mL full length bovine liver tRNA. c Enzyme activity of fibroblasts from control unaffected individual (closed squares) and proband II.2 (closed circles) was measured and normalized with total protein content. Mean values from three different control cell lines are shown. d The assay was optimized using mouse embryonic stem cells (wild type, closed squares; knockout, closed circles). All assays were done in triplicates and the error bars represent standard error of means

Similar articles

See all similar articles

Cited by 3 articles

MeSH terms

Feedback