Loss-of-function alanyl-tRNA synthetase mutations cause an autosomal-recessive early-onset epileptic encephalopathy with persistent myelination defect

Abstract

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.

Figure 1

MRI Findings in Individuals with AARS Mutations (A–D) Sagittal T1-weighted images show varying degrees of parenchymal volume loss (B–D) manifesting different degrees of thinning of the corpus callosum, enlarged sulci/fissures, and ventriculomegaly along with relative hypointensity of the corpus callosum reflecting hypomyelination. Cerebral volume was normal in LD_0857.0 at day 0 (A) with progressive cerebral atrophy, severe at 14 months (B). The vermis evolved from being hypoplastic (A) to atrophic (B) and the brainstem showed no interval growth (B). LD_0115.0A (C) had moderate cerebral atrophy and mild cerebellar volume loss by 14 months, and LD_0115.0B (D) had mild cerebral and cerebellar volume loss at 17 months. Disproportionate lateral ventriculomegaly reflects superimposed hydrocephalus from prior intraventricular hemorrhage (D). (E–H) Axial T2-weighted images demonstrate variable degrees of cerebral volume loss/atrophy (F–H) and hypomyelination (E–H). Generalized, excessive-for-age white matter hyperintensity reflects hypomyelination. At birth, the normal hypointense PLIC signal is not visible in LD_0857.0 (E). By 14 months, only mild myelination progress has occurred with myelination similar to that of a normal 3 month old; the optic radiations and PLIC are now hypointense. Similar findings are present at 14 months in LD_0115.0A (G) and 17 months in LD_0115.0B (H). (I) Axial DWI image from LD_0857.0 at 14 months shows hyperintense signal consistent with restricted diffusion and myelin edema in the optic radiations (arrows) and superior cerebellar peduncle decussations (arrowhead). Similar myelin edema was present in the occipitofrontal fascicles, central tegmental tracts, and globi pallidi (not shown). (J) SV MRS (144 ms echo time) over the left basal ganglia reveals elevation of citrate at 2.6 ppm (arrowhead), glycine at 3.5 ppm (long arrow), and creatine at 3 ppm and 3.9 ppm (short arrows).

Figure 2

Pathogenic Mutations in Human AARS (A) The p.Lys81Thr mutation and p.Arg751Gly mutations mapped to the known functional domains of human AARS, showing the aminoacylation domain, the tRNA recognition domain, the editing domain, and the C-terminal domain. (B) Multiple-species sequence alignment of AARS showing the p.Lys81Thr mutation and p.Arg751Gly mutation along with the flanking protein sequences in multiple and evolutionarily diverse species. (C) Structure of an E. coli alanyl-tRNA synthetase in complex with alanine analog. Arrow indicates the conserved residue homologous to human p.Lys81. (D) Structure of archaea A. fulgidus alanyl-tRNA synthetase in complex with tRNA and an alanine analog. Arrow indicates the conserved residue homologous to human p.Arg751. In (C) and (D), the AARS domains are colored as shown in (A).

Figure 3

Analysis of the Hydrolytic Editing Activity of AARS Deacylation of the incorrectly charged Ser-tRNA^Ala was monitored over time by the wild-type enzyme (black) and the p.Arg751Gly mutant (red). The uncatalyzed deacylation reaction (the no-enzyme reaction, indicated in blue) was run in parallel as a control for background hydrolysis. Values represent the average of two independent experiments and error bars show the standard deviation. The substrate Ser-tRNA^Ala for analysis of post-transfer editing was prepared by ³²P-labeling of the QA A76 nucleotide in the transcript of human tRNA^Ala with the CCA enzyme, followed by aminoacylation with chemically synthesized Ser-DBE using the dFx flexizyme. Deacylation was monitored in 50 mM HEPES (pH 7.5), 20 mM KCl, 4 mM DTT, and 10 mM MgCl₂ at 37°C with 5 nM of WT or p.Arg751Gly human AARS and a mixture of 2.8 μM Ser-tRNA^Ala and 17.2 μM tRNA^Ala. At the indicated time points, aliquots of a deacylation reaction were quenched with a buffer containing 200 mM NaOAc (pH 5.0) and treated with S1 nuclease for 20 min at 37°C to digest the tRNA to 5′-phosphate mononucleotides. The aliquots were then subjected to thin layer chromatography through a PEI cellulose matrix with 0.1 M NH₄Cl and 5% acetic acid to separate [³²P]seryl-AMP from [³²P]AMP. Quantification of the two products by a phosphorimager and analysis of the ratio of (ser-AMP) over (AMP) determined the extent of Ser-tRNA^Ala remaining after the editing reaction.

Figure 4

Yeast Complementation Studies Yeast complementation assays were performed as previously described. Haploid Δala1 yeast strains carrying a wild-type ALA1 on a URA3-bearing vector were transformed with a LEU2-bearing vector containing wild-type ALA1, p.Lys85Thr ALA1 (orthologous to human p.Lys81Thr AARS), p.Arg747Gly ALA1 (orthologous to human p.Arg751Gly AARS), or no insert (pRS315 Empty). Single colonies from two independent transformations were grown to saturation in SD -leu-ura liquid media (Teknova) then spotted (undiluted or diluted 1:10 and 1:100 in H₂O) on plates containing 0.1% 5-fluoroorotic acid (5-FOA) or SD -leu-ura growth media (Teknova) and incubated at 30°C for 48 hr. Survival was determined by visual inspection of growth. Culture dilutions are indicated on the left and media conditions are indicated on the right.