Novel (ovario) leukodystrophy related to AARS2 mutations

Abstract

Objectives: The study was focused on leukoencephalopathies of unknown cause in order to define a novel, homogeneous phenotype suggestive of a common genetic defect, based on clinical and MRI findings, and to identify the causal genetic defect shared by patients with this phenotype.

Methods: Independent next-generation exome-sequencing studies were performed in 2 unrelated patients with a leukoencephalopathy. MRI findings in these patients were compared with available MRIs in a database of unclassified leukoencephalopathies; 11 patients with similar MRI abnormalities were selected. Clinical and MRI findings were investigated.

Results: Next-generation sequencing revealed compound heterozygous mutations in AARS2 encoding mitochondrial alanyl-tRNA synthetase in both patients. Functional studies in yeast confirmed the pathogenicity of the mutations in one patient. Sanger sequencing revealed AARS2 mutations in 4 of the 11 selected patients. The 6 patients with AARS2 mutations had childhood- to adulthood-onset signs of neurologic deterioration consisting of ataxia, spasticity, and cognitive decline with features of frontal lobe dysfunction. MRIs showed a leukoencephalopathy with striking involvement of left-right connections, descending tracts, and cerebellar atrophy. All female patients had ovarian failure. None of the patients had signs of a cardiomyopathy.

Conclusions: Mutations in AARS2 have been found in a severe form of infantile cardiomyopathy in 2 families. We present 6 patients with a new phenotype caused by AARS2 mutations, characterized by leukoencephalopathy and, in female patients, ovarian failure, indicating that the phenotypic spectrum associated with AARS2 variants is much wider than previously reported.

Figure 1. Biochemical and genetic features

(A) Morphologic analysis of muscle biopsy from patient P1: Gomori trichrome (GT) staining and cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histoenzymatic double staining (COX/SDH). Inset shows the reaction in an age-matched control biopsy. (B) Biochemical activities of mitochondrial respiratory chain complexes in patients P1 and P6 muscle homogenates. All enzymatic activities are normalized for citrate synthase activity and indicated as percentages relative to the mean control value. (C) Genomic structure of AARS2 with exons coding for the aminoacylation (blue) and editing (red) domains. The arrows indicate the position of mutations identified in this study (black) or previously reported (gray). ^aThe 2 missense variants p.Arg199Cys and p.Val730Met are on the same allele; the latter (rs35623954) is reported to have a frequency >1% in control populations, suggesting that the former is the pathogenic variant.

Figure 2. Brain MRI

(A) MRI in P1 at age 28. The sagittal T1-weighted image shows serious cerebellar atrophy and 2 strips of abnormal signal in the splenium (arrows in image 1). The axial T2-weighted images show inhomogeneous areas of abnormal signal in the periventricular white matter. The areas on the left and right are connected signal abnormalities in the corpus callosum (arrows in images 2–4). (B) MRI in P2 at age 14 (images 1 and 2), age 21 (images 3 and 4), and age 23 (images 5–8). At age 14, a lesion is seen in the splenium of the corpus callosum (arrow in image 1) and in the right frontal periventricular white matter. The diffusion-weighted images suggest the presence of multiple small areas of restricted diffusion in the abnormal white matter (arrows in image 3), confirmed by low signal of the corresponding areas on the apparent diffusion coefficient map (arrows in image 4). The most recent MRI shows multiple segments of abnormal signal in the corpus callosum (image 5 and arrows in image 6). More extensive signal abnormalities are seen in the periventricular white matter, especially on the right (images 6 and 7). Signal abnormalities extend downward through the posterior limb of the internal capsule and the pyramidal tracts in the brainstem on the right (arrows in images 7 and 8). (C) MRI in patient 3 at age 35. The midsagittal image shows that the anterior part of the corpus callosum is abnormal, whereas only a strip of signal abnormality is seen in the splenium (arrows in image 1). Images 2 and 3 illustrate that the frontal and parietal white matter is abnormal, whereas the central white matter in between is normal. The tract involvement is evident (arrows in images 2 and 4). The axial fluid-attenuated inversion recovery image shows that the affected white matter is rarefied (arrow in image 5). The axial T2-weighted images illustrate the involvement of the anterior limb of the internal capsule (image 6) and the frontopontine tracts going down into the brainstem (arrows in images 7 and 8).

Figure 3. Yeast studies

(A) Oxygen consumption rate of the ala1^L-16X strain transformed with the ala1^wt allele, the empty vector, and the mutant alleles ala1^F22C, ala1^V500X, and ala1^L125R. Respiratory rates were normalized to the strain transformed with ala1^wt, in which the respiratory rate was 81.1 nmol min⁻¹ mg⁻¹ at 28°C and 33.4 nmol min⁻¹ mg⁻¹ at 37°C. Values are the mean of 3 independent experiments, each with an independent clone. Two-tailed paired t test was applied for statistical significance. ***p < 0.001. (B) In vivo mitochondrial protein synthesis of the ala1^L-16X strain transformed with the ala1^wt allele, the empty vector, and the mutant alleles ala1^F22C, ala1^V500X, and ala1^L125R. Mitochondrial gene products were labeled with [³⁵S]-methionine in whole cells in the presence of cycloheximide for 10 minutes at 28°C or 37°C. (C) Mitochondrial tRNA^Ala loading of the ala1^L-16X strain transformed with the ala1^wt allele, the empty vector, and the mutant alleles ala1^F22C, ala1^V500X, and ala1^L125R. Signals were quantified with Quantity 1 (Bio-Rad, Hercules, CA). For each strain, the ratio between the charged tRNA^Ala and the uncharged one was calculated and normalized to the ala1^wt strain. Atp = ATP synthase; Cob = cytochrome b; Cox = cytochrome c oxidase; ns = not significant; Var1 = small mitochondrial ribosome subunit.