21 Pontocerebellar hypoplasia type 6 (PCH6) is a rare infantile-onset progressive encephalopathy 22 caused by biallelic mutations in RARS2 that encodes the mitochondrial arginine-tRNA synthetase 23 enzyme (mtArgRS). The clinical presentation overlaps that of PEHO syndrome (Progressive Encephalopathy with oedema, Hypsarrhythmia and Optic atrophy). The proband presented with 25 severe intellectual disability, epilepsy with varying seizure types, optic atrophy, axial hypotonia, 26 acquired microcephaly, dysmorphic features and progressive cerebral and cerebellar atrophy and 27 delayed myelination on MRI. The presentation had resemblance to PEHO syndrome but 28 sequencing of ZNHIT3 did not identify pathogenic variants. Subsequent whole genome sequencing 29 revealed novel compound heterozygous variants in RARS2, a missense variant affecting a highly 30 conserved amino acid and a frameshift variant with consequent degradation of the transcript 31 resulting in decreased mtArgRS protein level confirming the diagnosis of PCH6. Features 32 distinguishing the proband's phenotype from PEHO syndrome were later appearance of hypotonia 33 and elevated lactate levels in blood and cerebrospinal fluid. On MRI the proband presented with 34 more severe supratentorial atrophy and lesser degree of abnormal myelination than PEHO 35 syndrome patients. The study highlights the challenges in clinical diagnosis of patients with 36 neonatal and early infantile encephalopathies with overlapping clinical features and brain MRI 37 findings. 38 39 Keywords 40 Pontocerebellar hypoplasia type 6, RARS2, PEHO syndrome, progressive cerebellar and cerebral 41 atrophy 3 42 Introduction 43 Pontocerebellar hypoplasia (PCH) is a group of neurodegenerative disorders with autosomal 44 recessive inheritance. Up to date 11 different subtypes have been described, with 17 causative 45 genes identified (van Dijk et al., 2018). Most of the subtypes are characterized by prenatal or 46 neonatal onset, global developmental delay and intellectual disability, microcephaly, hypoplasia and variable atrophy of cerebellar cortex and/or brainstem. The specific neurological symptoms 48 and the severity of symptoms and brain loss vary between the subtypes (van Dijk et al., 2018).49 Pontocerebellar hypoplasia type 6 (PCH6; MIM 611523) is a rare form of PCH first described in 50 2007 in three patients of a consanguineous Sephardic Jewish family (Edvardson et al., 2007). Since 51 then, altogether 32 patients in 18 families have been reported in the literature (for a detailed 52 summary of the patients and phenotypes, see Supplementary Table;Edvardson et al., 2007;

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The involvement of mitochondrial dysfunction in cystatin B (CSTB) deficiency has been suggested, but its role in the onset of neurodegeneration, myoclonus, and ataxia in the CSTB-deficient mouse model (Cstb−/−) is yet unknown. CSTB is an inhibitor of lysosomal and nuclear cysteine cathepsins. In humans, partial loss-of-function mutations cause the progressive myoclonus epilepsy neurodegenerative disorder, EPM1. Here we applied proteome analysis and respirometry on cerebellar synaptosomes from early symptomatic (Cstb−/−) mice to identify the molecular mechanisms involved in the onset of CSTB-deficiency associated neural pathogenesis. Proteome analysis showed that CSTB deficiency is associated with differential expression of mitochondrial and synaptic proteins, and respirometry revealed a progressive impairment in mitochondrial function coinciding with the onset of myoclonus and neurodegeneration in (Cstb−/−) mice. This mitochondrial dysfunction was not associated with alterations in mitochondrial DNA copy number or membrane ultrastructure. Collectively, our results show that CSTB deficiency generates a defect in synaptic mitochondrial bioenergetics that coincides with the onset and progression of the clinical phenotypes, and thus is likely a contributor to the pathogenesis of EPM1.