Deleterious mutation in the mitochondrial arginyl-transfer RNA synthetase gene is associated with pontocerebellar hypoplasia

Abstract

Homozygosity mapping was performed in a consanguineous Sephardic Jewish family with three patients who presented with severe infantile encephalopathy associated with pontocerebellar hypoplasia and multiple mitochondrial respiratory-chain defects. This resulted in the identification of an intronic mutation in RARS2, the gene encoding mitochondrial arginine-transfer RNA (tRNA) synthetase. The mutation was associated with the production of an abnormally short RARS2 transcript and a marked reduction of the mitochondrial tRNA(Arg) transcript in the patients' fibroblasts. We speculate that missplicing mutations in mitochondrial aminoacyl-tRNA synthethase genes preferentially affect the brain because of a tissue-specific vulnerability of the splicing machinery.

Figure 1.

Brain MRI of patient II-2 (a–e) and patient II-5 (f–h). a, Sagittal T1-weighted image of patient II-2 obtained at age 3 d, showing hypoplasia of the cerebellum and the vermis, with progression at 5 mo (d). Brain volume was relatively preserved at age 3 d (a) but decreased at age 5 mo (b) and at age 9 mo (c), mainly because of white-matter depletion (panels b and c are axial T2-weighted images). e, Axial T2-weighted image of patient II-2 obtained at age 9 mo, showing severe pontine atrophy. Midsagittal (f) and coronal (g) T1-weighted images of patient II-5 obtained at age 3 mo show hypoplasia of the cerebellum and the vermis. h, Axial T2-weighted images disclosing some parenchymal loss, mostly of the white matter, with relative sparing of the basal ganglia and cortex.

Figure 2.

The family members’ haplotype along the critical region on chromosome 6. The polymorphic microsatellite markers and their chromosomal locations (in Mb) are given in the upper left panel. The SNPs bordering the homozygous region are given above and below the panel. The linked region is bordered by bold lines.

Figure 3.

IVS2+5 a→g mutation, which causes exon 2 skipping. RARS2 exon 2–IVS2 splice-junction sequence of the patient (a) and his mother (b). The mutation site is indicated by an arrow. c, RARS2 cDNA showing exon 2 skipping at the patient sample. Exons 1 and 3 are indicated. d, RARS2 cDNA of the mother showing heterozygosity for the wild-type and the mutant transcripts. e, PCR amplification products of RARS2 cDNA encompassing the first three exons. Lane 1, patient; lane 2, mother; lane 3, control. A 100-bp ladder was loaded in the left lane.

Figure 4.

Quantitative and aminoacylation analysis of tRNA^Arg in fibroblasts. a, Quantitative analysis of tRNA^Arg and tRNA^Leu in the patient and control (C2.5) by northern hybridization. Equal amounts of control and patient RNA were analyzed. b, Aminoacylation: results of the hybridization after separation in acid conditions. AA= aminoacylated form; DA= deacylated form. The control of fully deacylated tRNA (daControl) was obtained by incubation of the RNA at 50°C (pH 9.0) for 10 min.