Identification of a Novel Variant in EARS2 Associated with a Severe Clinical Phenotype Expands the Clinical Spectrum of LTBL

Abstract

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient's fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient's clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.

Keywords: EARS2; LTBL; aminoacyl-tRNA synthetases; mitochondrial disorders.

Figure 1

Reverse sequence chromatograms showing the results of Sanger sequencing. (A) The missense variant c.670G>A (p.Gly224Ser), located in exon 4, was inherited from the patient’s mother and the nonsense variant c.376C>T (p.Gln126*), located in exon3, from the father. The patient harbored both variants in EARS2 in compound heterozygosity. (B) Schematic showing EARS2 domains in which all variants identified to date in leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) patients are highlighted.

Figure 2

(A) Oxygen consumption rate (OCR) measured before and after the addition of inhibitors. The Seahorse XF Cell Mito Stress Test uses compounds of respiration that target components of the electron transport chain (ETC) in the mitochondria to reveal key parameters of the metabolic functions. These modulators are ETC inhibitors (oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and a mixture of rotenone and antimycin A), which are serially injected to measure the ATP production, maximal respiration (Max Resp), nonmitochondrial respiration (Non-Mito Resp), proton leak, spare respiratory capacity (SRC), and basal respiration (Basal Resp). (B) The basal energy metabolism of each cell line was assessed by analyzing the OCR/extracellular acidification rate (ECAR) ratios following the sequential injection of the inhibitors. (C) Measurements of the enzyme activities for the different oxidative phosphorylation (OXPHOS) complexes in the patient’s fibroblasts (reference range deemed in a healthy population). CS, citrate synthase.

Figure 3

MRI images of our patient acquired at 10 (A) and 15 (B) years of age, showing complete agenesis of the corpus callosum (indicated with a red star) and cerebellar atrophy (indicated with white arrows). Note also the dilation of the fourth ventricle and enlargement of the interfolial spaces within a normal-sized posterior fossa.