Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes

Abstract

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment.

Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays.

Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals.

Conclusions: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.

Figure 1. Representative cases for novel KARS mutation with leukoencephalopathy

Characteristics of patients for novel KARS mutations with leukoencephalopathy. (A,B) Brain imaging of Patient 1 showed bilateral FLAIR and DWI signal hyperintensity in the white matter of the frontal lobe. (C) The affacted brother of Patient 1 has also abnormality in the white matter. (D) Brain MRI of Patient 4 showed bilateral T2 signal hyperintensity in the white matter of periventricular area. (E) The MRS showed reduced NAA and elevated lactate peak in the right frontal lesion of Patient 1. (F) The MRS showed the presence of NAA and lactate in the normal white matter of Patient 1.

Figure 2. Summary of KARS mutations

(A) Schematic representation of the KARS gene and the distribution of published mutations (black, above) and mutations found in our patient cohort (red, below). (B) Ribbon diagram of the complex structure model of human lysyl-tRNA synthetase (PDB ID: 3BJU) and mapping the missense mutations onto the structure model. One monomer colored in cyan and the other one in green. The mutation is drawn as a ball-and-stick model and colored in red. (C) Close-up view of the in silico analysis for mutation p.Arg505His. (D) View for mutation p.Pro533Ser. (E) View for mutation p.Thr587Met. (F) Cross species sequence alignment of amino acids. The corresponding positions are indicated in red text.

Figure 3. Summary of the mutations identified in KARS and their effects on tRNA charging

The column “ratio to WT” indicates the decrease in tRNA charging relative to the WT enzyme for each mutant. All these mutations have a deleterious effect, ranging from 13- to nearly 10⁷-fold.

Figure 4. Yeast complementation analysis of mutant KRS1 alleles

(A) A haploid yeast strain deleted for endogenous KRS1 was transformed with a LEU2-bearing pRS315 vector containing wild-type KRS1, the indicated mutant form of KRS1, or no insert (“Empty”). Cultures for each strain (labeled along the top) were grown for 2 days in liquid medium and spotted on solid medium containing 5-FOA to determine whether the KRS1 alleles complement loss of KRS1 at 30°C. (B) Cultures for each indicated strain (labeled at right) were grown for 2 days in liquid medium and then diluted in liquid medium containing 5-FOA to determine whether the KRS1 alleles complement loss of KRS1 at 30°C. The optical density (OD₆₀₀, y-axis) was evaluated for each culture at the indicated time points (x-axis), and error bars indicate SD. 5-FOA = 5-fluoroorotic acid.