Spinocerebellar ataxia type 36 (SCA36) is a neurodegenerative disease caused by expanded (GGCCTG)n hexanucleotide repeat sequence in the NOP56 gene. While the expanded repeats could transcribe and form toxic RNA foci within neurons, recent evidence indicates that translation of these repeats produces dipeptide repeats (DPR) that contribute to neurotoxicity. The relative impact of hexanucleotide RNA repeats (HRR) and DPR on the neurodegeneration of SCA36 remains unclear. Here, we established a Drosophila SCA36 model to dissect the neurotoxic effects of HRR and DPR. The fly model recapitulates the cellular defects observed in SCA36 patient fibroblasts, validating its relevance for mechanistic study of SCA36. Further engineering the transgenes to express individual DPRs reveal Proline-Glycine-DPR (PG-DPR) as the most potent neurotoxin causing progressive motor and sensory dysfunction. Expressing a series of the SCA36 transgenes with varying HRR lengths demonstrates an age- and length-dependent adult-onset neurodegeneration. Interestingly, sequence modification of the transgenes to exclusively express HRR or DPR alone causes a milder phenotype, indicating both HRR and DPR contribute partially to the pathogenicity of SCA36. Therefore, this model provides a valuable platform for screening drug targeting either HRR- or DPR-mediated toxicity of SCA36. Suppression of the RNA elongation factor SUPT4H1 ortholog reduces RNA foci in cell culture. However, expression level of SUPT4H1 was not changed in SCA36 patient cells. Interestingly, knockdown of the Drosophila SUPT4H1 ortholog or 6-azauridine treatment to suppress RNA transcription aggravates the neurodegenerative phenotypes in both the fly models and patient-derived fibroblasts, highlighting the complex interplay of pathomechanisms in SCA36. These results underscore the need for carefully evaluating the potential side effects when designing therapeutic interventions for SCA36.

Spinocerebellar ataxia type 36 (SCA36) is caused by large GGCCTG repeat expansion in the NOP56 gene. The genetic diagnosis based on Southern blot is expensive and time-consuming. This study aimed to evaluate the reliability and effectiveness of whole exome sequencing (WES) for routine genetic diagnosis of suspected SCA36 patients. Pathogenic repeat expansions for SCAs including SCA36 were first analyzed based on WES data using ExpansionHunter in five probands from SCA families, then the results were confirmed by triplet repeat primed polymerase chain reaction (TP-PCR) and Southern blot. GGCCTG repeat expansion in NOP56 was indicated in all five probands by WES, then it was found in 11 SCA patients and three asymptomatic individuals by TP-PCR. The sizes of GGCCTG repeat expansions were confirmed to be 1,390-1,556 by Southern blot. The mean age at onset of the patients was 51.0 ± 9.3 (ranging from 41 to 71), and they presented slowly progressive cerebellar ataxia, atrophy and fasciculation in tongue or limb muscles. The patients were clinically and genetically diagnosed as SCA36. This study proposed that WES could be a rapid, reliable, and cost-effective routine test for the preliminarily detection of SCA36 and other ataxia diseases.

Spinocerebellar ataxias form a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia. Their prevalence varies among populations and ethnicities. Spinocerebellar ataxia 36 is caused by a GGCCTG repeat expansion in the first intron of the NOP56 gene and is characterized by late-onset ataxia, sensorineural hearing loss and upper and lower motor neuron signs, including tongue fasciculations. Spinocerebellar ataxia 36 has been described mainly in East Asian and Western European patients and was thought to be absent in the British population. Leveraging novel bioinformatic tools to detect repeat expansions from whole-genome sequencing, we analyse the NOP56 repeat in 1257 British patients with hereditary ataxia and in 7506 unrelated controls. We identify pathogenic repeat expansions in five families (seven patients), representing the first cohort of White British descent patients with spinocerebellar ataxia 36. Employing in silico approaches using whole-genome sequencing data, we found an 87 kb shared haplotype in among the affected individuals from five families around the NOP56 repeat region, although this block was also shared between several controls, suggesting that the repeat arises on a permissive haplotype. Clinically, the patients presented with slowly progressive cerebellar ataxia with a low rate of hearing loss and variable rates of motor neuron impairment. Our findings show that the NOP56 expansion causes ataxia in the British population and that spinocerebellar ataxia 36 can be suspected in patients with a late-onset, slowly progressive ataxia, even without the findings of hearing loss and tongue fasciculation.

Spinocerebellar ataxias (SCA) are often caused by expansions of short tandem repeats. Recent methodological advances have made repeat expansion detection with long-read sequencing (LRS) feasible. Our study investigated one family with SCA 36 and further summarized the genetic and clinical characteristics of the total of 161 patients across different ethnic groups reported worldwide. We enrolled a pedigree of 4 patients. The proband was a 55-year-old male. And he was screened for dynamic mutations of SCA subtypes by Tri-prime PCR (TP-PCR) and capillary electrophoresis, showing NOP56 as the candidate gene. The cosegregation was conducted by screening the NOP56 gene in his daughter and further confirmed by low-coverage (∼15 ×) LRS on the Oxford Nanopore platform. The SCA36 pedigree included a total of 4 patients. The proband showed the initial manifestation at the age of 45 years old, which was characterized by truncal ataxia. Genetic test results showed the (GGCCTG)n expansion in NOP56 gene (3/>15 and 6/>15 times respectively). To clarify the diagnosis genetically, LRS was performed in his daughter showing a large intronic insertion (chr20: 2633004 INS 7603 bp) containing (GGCCTG)n expansion of 782 units in NOP56 as the causative mutation. We identified one SCA36 pedigree by combining TP-PCR with LRS. Our study suggested LRS as an effective tool for molecular diagnosis. LRS also worked as a supplementary but necessary diagnostic tool for dynamic mutation-related SCA on the basis of repeat-primed PCR as well as capillary electrophoresis.

NOP56 belongs to a C/D box small nucleolar ribonucleoprotein complex that is in charge of cleavage and modification of precursor ribosomal RNAs and assembly of the 60S ribosomal subunit. An intronic expansion in NOP56 gene causes Spinocerebellar Ataxia type 36, a typical late-onset autosomal dominant ataxia. Although vertebrate animal models were created for the intronic expansion, none was studied for the loss of function of NOP56. We studied a zebrafish loss-of-function model of the nop56 gene which shows 70% homology with the human gene. We observed a severe neurodegenerative phenotype in nop56 mutants, characterized mainly by absence of cerebellum, reduced numbers of spinal cord neurons, high levels of apoptosis in the central nervous system (CNS) and impaired movement, resulting in death before 7 days post-fertilization. Gene expression of genes related to C/D box complex, balance and CNS development was impaired in nop56 mutants. In our study, we characterized the first NOP56 loss-of-function vertebrate model, which is important to further understand the role of NOP56 in CNS function and development.