Clinical and genetic analyses of premature mitochondrial encephalopathy with epilepsia partialis continua caused by novel biallelic NARS2 mutations

Abstract

Biallelic NARS2 mutations can cause various neurodegenerative diseases, leading to growth retardation, intractable epilepsy, and hearing loss in early infancy and further progressing to spastic paraplegia, neurodegeneration, and even death. NARS2 mutations are associated with mitochondrial dysfunction and cause combined oxidative phosphorylation deficiency 24 (COXPD24). Relatively few cases have been reported worldwide; therefore, the pathogenesis of COXPD24 is poorly understood. We studied two unrelated patients with COXPD24 with similar phenotypes who presented with intractable refractory epilepsia partialis continua, hearing loss, and growth retardation. One patient died from epilepsy. Three novel NARS2 variants (case 1: c.185T > C and c.251 + 2T > G; case 2: c.185T > C and c.509T > G) were detected with whole-exome sequencing. c.251 + 2T > G is located at the donor splicing site in the non-coding sequence of the gene. The minigene experiment further verified that c.251 + 2T > G caused variable splicing abnormalities and produced truncated proteins. Molecular dynamics studies showed that c.185T > C and c.509T > G reduced the binding free energy of the NARS2 protein dimer. The literature review revealed fewer than 30 NARS2 variants. These findings improved our understanding of the disease phenotype and the variation spectrum and revealed the potential pathogenic mechanism of non-coding sequence mutations in COXPD24.

Keywords: COXPD24; NARS2; aminoacylation reaction; gene testing; minigene; mitochondrial disease.

FIGURE 1

Brain MRI scans of patients #1 and #2. (A–C) First brain MRI examination of patient #1. (A,B) The T2-weighted image shows a small and slightly long T2 signal shadow in bilateral internal capsules and lenticular nuclei. (C) The diffusion-weighted image shows small patches of a slightly long T2 signal shadow in the upper outer capsule and left hippocampus. (D–F) Second brain MRI examination of patient #1. (D) The T2-weighted image shows a wider T2 signal shadow in bilateral internal and external capsules with higher signal intensity. (E) The T2 signal intensities are higher in the right frontal cortex and subcortical area than in the front. (F) No obvious abnormal signal is found in bilateral hippocampal regions, and no diffusion limitation is found in the lesions. (G–I) Brain MRI examination of patient #2. (G) The diffusion-weighted image shows wider and deeper sulcus fissures in bilateral cerebral hemispheres and slightly higher cortical signal in part of the cerebral hemispheres. (H) Partial cortex of the cerebral hemisphere and the left hippocampus show slightly high signal intensities. (I) The apparent diffusion coefficient map shows a slightly low signal intensity, indicating limited dispersion.

FIGURE 2

Electroencephalograms of patients #1 and #2. (A) In patient #1, the first examination shows abundant δ-wave activity in the left and midline regions of the brain. The sharp waves in each area (significant on the left) are synchronized or not synchronized with a few more sharp waves. The second examination shows a slowed background rhythm and more δ-wave activities in the right and midline regions of the brain. The frontal, central, parietal, occipital, and temporal areas (significant on the right) and the midline show numerous sharp slow waves, consistent with epilepsia partialis continua (EPC). (B) In patient #2, the right upper limb twitches rhythmically 1–2 times/s. The electroencephalogram synchronizes with the low to medium amplitude spike wave rhythm in the parietal, occipital, middle, and posterior temporal regions (significant on the right) in the background of diffuse rhythm, which can affect the central region, and the twitches are synchronized with spike waves, consistent with EPC.

FIGURE 3

NARS2 mutation information. (A) Including the three mutations in this study (red font), 26 NARS2 mutations have been reported, mainly concentrated in the categorical domain, with the highest proportion of missense mutations. NARS2 of patient #1 has compound heterozygous mutations NM_024678: c.185T > C/p.Leu62Pro and c.251 + 2T > G. The p.Leu62Pro is inherited from the father, and c.251 + 2T > G is inherited from the mother. Patient #2 also has compound heterozygous mutations NM_024678: c.185T > C/p.Leu62Pro and c.509T > G/p.Phe170Cys. The p.Leu62Pro is inherited from the father, and p.Phe170Cys is inherited from the mother. Sanger sequencing confirms the existence of mutations. (B) The homology analysis shows that Leu62 and Phe170 of the NARS2 protein are conservative among different species.

FIGURE 4

Protein structure prediction analysis. (A) The human NARS2 protein is composed of anticodon-binding and catalytic domains and functions as a homodimer. Wild-type Leu62 (in brown) is located in a pocket, surrounded by a hydrophobic group in the deep part of the protein, and forms a hydrophobic interaction with surrounding residues. Mutant Leu62Pro (green) may weaken the hydrophobic interaction with Val92. Another mutant Phe170Cys (black) is located in the pocket structure surrounded by hydrophobic groups, possibly weakening the interaction between Lys171 and Glu161 in the homodimer. (B) Molecular dynamics simulations predict that the mutation is affected the stability of the dimer structure. In the wild-type homodimers, Gln47 and Trp49 have formed stable interactions with the dimer Phe265, with distances of 4.3 and 4.9 angstroms (Å), respectively, while mutant Leu62Pro has weakened the interaction force, with distances of 5.3 and 6.8 Å, respectively. In the wild-type homodimer, Lys171 forms a hydrogen bond with the dimer Glu161, with a distance of 2.3 Å. In mutant Phe170Cys, the distance is 4.3 Å.

FIGURE 5

Minigene experiment results of the NARS2 mutation c.251 + 2T > G. (A) Electrophoresis results of RT-PCR products of wild-type and mutant NARS2. The mutation plasmid c.251 + 2T > G produced three mRNA products, which were differentiated by cloning and sequencing because of the close distance between bands; (B) Sanger sequencing results of corresponding bands of wild-type and three mutant products.