Functionally pathogenic EARS2 variants in vitro may not manifest a phenotype in vivo

Abstract

Objective: To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC).

Methods: Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity.

Results: Whole-exome sequencing uncovered compound heterozygous mutations in EARS2, c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA (SNORD118) responsible for LCC.

Conclusions: Here, we report seemingly pathogenic EARS2 mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant.

Clinicaltrialsgov identifier: NCT00001671.

Figures

Figure 1. Patient with leukoencephalopathy brain imaging

MRIs (A–D) and CT (E) indicate white matter signal abnormalities, cysts, and calcifications throughout the cerebral hemispheres. Diffuse cerebral white matter lesions are present, which is demonstrated by hypointense signals in the sagittal (A) and axial T1-weighted (B) images and the CT (E) and by the hyperintense signals in the axial T2-weighted (C) and axial fluid-attenuated inversion recovery (FLAIR) images. The CT (E) shows extensive calcifications in the subcortical white matter and along the periventricular white matter. Large cystic lesions can be seen along the quadrigeminal plane and parieto-occipital regions (B–D).

Figure 2. Functional characterization of EARS2 variants on fibroblasts

(A) EARS2 protein amount in the patient's (Pt) and control (CT1, CT2, and CT3) fibroblasts, obtained by Western blot using an anti-EARS2 antibody. An anti-GAPDH antibody was used as a loading control. (B) Oxygen consumption analysis in the patient's (Pt) and control fibroblasts. Histograms show OCR (B.a and D.a) and MRR (B.b and D.b). OCR and MRR values (mean of 6–8 replicates) are expressed as picomoles of O2 per minute and normalized by cell number. p value obtained with 2-tailed Student t test, *p < 0.05; **p < 0.01. (C) EARS2 protein amount in the patient's (iPt) and control (iCT) immortalized fibroblasts, in basal conditions and after transduction with wt EARS2 (+EARS2); Western blot analysis was performed as described in A. (D) Oxygen consumption analysis, as reported in B, performed in patient's (iPt) and control (iCT) immortalized fibroblasts, in basal conditions and after transduction with wild-type EARS2 (+EARS2). ***p < 0.001. MRR = maximum respiration rate; OCR = oxygen consumption rate.

Figure 3. LTBL brain imaging

Axial T2-weighted (A–C) and T1-weighted (D) MRIs demonstrate T2-hyperintensities and T1-hypointensities respectively, indicating lesions in the deep cerebral white matter and periventricular white matter with sparing of the periventricular rim. Signal hyperintensities are also present in the thalami (B) and dorsal part of the midbrain (C). Modified from reference 18. LTBL = leukoencephalopathy with thalamus and brainstem involvement and high lactate.