The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.
Objective: We aimed to decipher the molecular genetic basis of disease in a cohort of children with a uniform clinical presentation of neonatal irritability, spastic or dystonic quadriplegia, virtually absent psychomotor development, axonal neuropathy, and elevated blood/CSF lactate. Methods:We performed whole-exome sequencing of blood DNA from the index patients. Detected compound heterozygous mutations were confirmed by Sanger sequencing. Structural predictions and a bacterial activity assay were performed to evaluate the functional consequences of the mutations. Mass spectrometry, Western blotting, and protein oxidation detection were used to analyze the effects of selenoprotein deficiency.Results: Neuropathology indicated laminar necrosis and severe loss of myelin, with neuron loss and astrogliosis. In 3 families, we identified a missense (p.Thr325Ser) and a nonsense (p.Tyr429*) mutation in SEPSECS, encoding the O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase, which was previously associated with progressive cerebellocerebral atrophy. We show that the mutations do not completely abolish the activity of SEPSECS, but lead to decreased selenoprotein levels, with demonstrated increase in oxidative protein damage in the patient brain. Conclusions:These results extend the phenotypes caused by defective selenocysteine biosynthesis, and suggest SEPSECS as a candidate gene for progressive encephalopathies with lactate elevation. Neurology ® 2015;85:306-315 GLOSSARY PCH2D 5 pontocerebellar hypoplasia type 2D; PEHO 5 progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy; RC 5 respiratory chain; SRM-MS 5 selected reaction monitoring-mass spectrometry; T 4 5 thyroxine; tRNA 5 transfer RNA; TSH 5 thyroid-stimulating hormone; T 3 5 triiodothyronine.Mitochondrial dysfunction is a frequent cause of childhood encephalopathy. Besides the typical multisystemic disorders, an increasing number of mitochondrial defects are shown to cause a CNS-specific phenotype.1-5 Lactate elevation raises suspicion of mitochondrial involvement and may be observed even in encephalopathies in which muscle biopsies show normal mitochondrial respiratory chain (RC) function. [1][2][3]6 Within our cohort of pediatric patients, we identified patients with an undefined cause of cerebellocerebral atrophy, seizures, severe spasticity, and axonal neuropathy with lactate elevation. We report that despite many of the clinical and neuropathologic signs pointing toward mitochondrial impairment, the patients had novel mutations in the SEPSECS gene, which functions in cytoplasmic transfer RNA (tRNA)-charging in the selenoprotein biosynthesis pathway. We describe the uniform clinical, neuroradiologic, and neuropathologic features of this entity and a detailed mutation *These authors contributed equally to this work.From the
Objective:To identify the molecular genetic basis of a syndrome characterized by rapidly progressing cerebral atrophy, intractable seizures, and intellectual disability.Methods:We performed exome sequencing in the proband and whole-genome single nucleotide polymorphism genotyping (copy number variant analysis) in the proband-parent trio. We used heterologous expression systems to study the functional consequences of identified mutations.Results:The search for potentially deleterious recessive or de novo variants yielded compound heterozygous missense (c.1202G>A, p.Cys401Tyr) and frameshift deletion (c.2396delG, p.Ser799IlefsTer96) mutations in ADAM22, which encodes a postsynaptic receptor for LGI1. The deleterious effect of the mutations was observed in cell surface binding and immunoprecipitation assays, which revealed that both mutant proteins failed to bind to LGI1. Furthermore, immunoprecipitation assays showed that the frameshift mutant ADAM22 also did not bind to the postsynaptic scaffolding protein PSD-95.Conclusions:The mutations identified abolish the LGI1-ADAM22 ligand-receptor complex and are thus a likely primary cause of the proband's epilepsy syndrome, which is characterized by unusually rapidly progressing cortical atrophy starting at 3–4 months of age. These findings are in line with the implicated role of the LGI1-ADAM22 complex as a key player in nervous system development, specifically in functional maturation of postnatal synapses. Because the frameshift mutation affects an alternatively spliced exon with highest expression in postnatal brain, the combined effect of the mutations is likely to be hypomorphic rather than complete loss of function. This is compatible with the longer survival of the patient compared to Lgi1−/− and Adam22−/− mice, which develop lethal seizures during the first postnatal weeks.
Progressive encephalopathy with oedema, hypsarrhythmia, and optic atrophy (PEHO) syndrome is an early childhood onset, severe autosomal recessive encephalopathy characterized by extreme cerebellar atrophy due to almost total granule neuron loss. By combining homozygosity mapping in Finnish families with Sanger sequencing of positional candidate genes and with exome sequencing a homozygous missense substitution of leucine for serine at codon 31 in ZNHIT3 was identified as the primary cause of PEHO syndrome. ZNHIT3 encodes a nuclear zinc finger protein previously implicated in transcriptional regulation and in small nucleolar ribonucleoprotein particle assembly and thus possibly to pre-ribosomal RNA processing. The identified mutation affects a highly conserved amino acid residue in the zinc finger domain of ZNHIT3. Both knockdown and genome editing of znhit3 in zebrafish embryos recapitulate the patients' cerebellar defects, microcephaly and oedema. These phenotypes are rescued by wild-type, but not mutant human ZNHIT3 mRNA, suggesting that the patient missense substitution causes disease through a loss-of-function mechanism. Transfection of cell lines with ZNHIT3 expression vectors showed that the PEHO syndrome mutant protein is unstable. Immunohistochemical analysis of mouse cerebellar tissue demonstrated ZNHIT3 to be expressed in proliferating granule cell precursors, in proliferating and post-mitotic granule cells, and in Purkinje cells. Knockdown of Znhit3 in cultured mouse granule neurons and ex vivo cerebellar slices indicate that ZNHIT3 is indispensable for granule neuron survival and migration, consistent with the zebrafish findings and patient neuropathology. These results suggest that loss-of-function of a nuclear regulator protein underlies PEHO syndrome and imply that establishment of its spatiotemporal interaction targets will be the basis for developing therapeutic approaches and for improved understanding of cerebellar development.
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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