A non-coding variant in the Kozak sequence of RARS2 strongly decreases protein levels and causes pontocerebellar hypoplasia

Abstract

Bi-allelic variants in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been involved in early-onset encephalopathies classified as pontocerebellar hypoplasia (PCH) type 6 and in epileptic encephalopathy. A variant (NM_020320.3:c.-2A > G) in the promoter and 5'UTR of the RARS2 gene has been previously identified in a family with PCH. Only a mild impact of this variant on the mRNA level has been detected. As RARS2 is non-dosage-sensitive, this observation is not conclusive in regard of the pathogenicity of the variant.We report and describe here a new patient with the same variant in the RARS2 gene, at the homozygous state. This patient presents with a clinical phenotype consistent with PCH6 although in the absence of lactic acidosis. In agreement with the previous study, we measured RARS2 mRNA levels in patient's fibroblasts and detected a partially preserved gene expression compared to control. Importantly, this variant is located in the Kozak sequence that controls translation initiation. Therefore, we investigated the impact on protein translation using a bioinformatic approach and western blotting. We show here that this variant, additionally to its effect on the transcription, also disrupts the consensus Kozak sequence, and has a major impact on RARS2 protein translation. Through the identification of this additional case and the characterization of the molecular consequences, we clarified the involvement of this Kozak variant in PCH and on protein synthesis. This work also points to the current limitation in the pathogenicity prediction of variants located in the translation initiation region.

Keywords: Bioinformatic predictions; Kozak; Non-coding variant; Pontocerebellar Hypoplasia; RARS2.

Fig. 1

Patient brain MRI at 6 years. A Sagittal T1-weighted image showing pontocerebellar hypoplasia. B and C Coronal T1 and T2 weighted images with the "dragonfly-like" cerebellar pattern (flattening and severely reduced size of the cerebellar hemispheres with relative sparring of the vermis) indicated by the white arrows. D MR spectroscopy showing no lactate peak

Fig. 2

IGV (Integrative Genomics Viewer) track of the variation. The variant NC_000006.11:g.88299677 T > C (NM_020320.3:c.-2A > G) has been detected in a homozygous state in the Patient. The reverse complement sequence is shown

Fig. 3

RARS2 mRNA levels and protein levels on fibroblasts. A Real-time PCR performed on the RNA extracted from fibroblasts. The RARS2 mRNA level in the Patient's fibroblasts is only ~ 46% of the Control's. The statistical analysis was performed using the SciPy (version 1.9.3) package from the Python programming language (version 3.9.15). The RARS2 mRNA levels were compared to those of GAPDH by the ΔΔCt method and based on four cell culture replicates and then three technical replicates for each genotype. The Welch’s test (corrected t-test for unequal variances) was performed on the log10 of the fold change. B Quantification of the RARS2 protein levels from the Western blot in 3C and showing a 92.98% decrease in the Patient’s fibroblasts compared to Control’s. C Western blotting of RARS2 Patient and Control’s cell lysate using RARS2 (green) antibody. β Actin serves as a loading control. Two technical duplicates were loaded for the Patient’s cell lysate as well as for the three different Controls’. The Western blot displayed here has been cropped and the full-length version is presented in Supplementary Fig. 1. Pt = Patient, Ctl = Control