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They can be present at birth with hypotonia, lethargy and poor sucking but can also appear during the first months of life, and even though epilepsy is often present, it is not always the first symptom. In the former case, the main clinical features include hypotonia, microcephaly, developmental delay, atrophy of cerebellum and cerebrum as well as high lactate blood-levels.

There are two main overlapping clinical conditions associated with biallelic pathogenic RARS2 variants: (i) PCH6 that was initially described and (ii) early onset epileptic encephalopathy without typical PCH on MRI. Splice site, nonsense, or missense variants on RARS2 gene have been reported in more than 50 cases, with PCH estimated to be present in half of them. RARS2 protein is involved in the addition of arginine to tRNA, thus allowing translation of mitochondrial proteins. PCH6 is usually considered as a mitochondrial disease due to variants in the gene encoding for mitochondrial arginyl-transfer RNA (tRNA) synthetase ( RARS2). Hence, defects in RNA metabolism or protein translation can be considered as a common mechanistic factor behind most PCH subtypes. Further, RNA processing defects due to variants in EXOSC3, EXOSC8 and EXOSC9 contribute to pathogenesis of PCH1. TSEN54, TSEN2, TSEN34, and CLP1 genes encode members or associated factors of this TSEN complex and variants in those genes have been associated with 6 PCH subtypes: PCH2A (OMIM #277470), PCH2B (OMIM #608753), PCH2C (OMIM #608754), PCH4 (OMIM #225753), PCH5 (OMIM #610204) and PCH10 (OMIM #615803). Among the genetic causes, variants in t-RNA splicing endonuclease (TSEN) complex genes have been prevalently reported. These subtypes have an autosomal recessive inheritance, with typical restricted development thereby leading to severe intellectual and motor function impairments, epilepsy and frequently death during childhood. PCH is further classified into 17 subtypes currently based on the underlying neuropathological features and genetic causes and new subtypes are regularly described. Pontocerebellar hypoplasia (PCH) is a class of rare neurodegenerative disorders with a pre-natal onset and characterized by degeneration or hypoplasia of the pons and the cerebellum. This work also points to the current limitation in the pathogenicity prediction of variants located in the translation initiation region. 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. 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. Therefore, we investigated the impact on protein translation using a bioinformatic approach and western blotting.

Importantly, this variant is located in the Kozak sequence that controls translation initiation. 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. This patient presents with a clinical phenotype consistent with PCH6 although in the absence of lactic acidosis. We report and describe here a new patient with the same variant in the RARS2 gene, at the homozygous state. As RARS2 is non-dosage-sensitive, this observation is not conclusive in regard of the pathogenicity of the variant. Only a mild impact of this variant on the mRNA level has been detected. 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. 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.

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