Objective: Identification of the mutation causing SCA4.

Background: SCA4 is an autosomal dominant disease, originally described in a large Utah pedigree, characterized by sensory and cerebellar ataxia. Although it was mapped to human chromosome 16q in 1996, it has escaped identification for almost 3 decades despite the use of whole exome/genome and transcriptome sequencing.

Method: Use of multiple DNA and RNA sequencing methods including single strand, high fidelity long-read genomic sequencing (LR-GS). Analysis of an SCA4 brain and characterization of nuclear aggregates. Analysis of ZFHX3  transcript and protein abundance, analysis of autophagic flux in SCA4 fibroblast and induced pluripotent cell (iPSC) lines.

Results: Using LR-GS, we identified a heterozygous GGC-repeat expansion (RE) in a large Utah pedigree coding for poly-glycine (polyG) in ZFHX3/ATBF1. A query of 6,495 GS datasets identified the RE in a further 7 pedigrees. The most common normal alleles contained 21 repeat units that were most often interrupted twice at the DNA level, whereas SCA4 individuals had >44 uninterrupted repeats with longer repeats associated with earlier onset. Shared ultra-rare DNA variants near the RE indicate a common distant founder event in Sweden. The SCA4 phenotype extends from cerebellar and sensory ataxia, to autonomic dysfunction and chronic cough. Intranuclear  aggregates staining for ZFHX3, p62, and ubiquitin were abundant in SCA4 basis pontis neurons. We detected normal and polyG-expanded ZFHX3 in SCA4 fibroblasts consistent with a gain of function. In fibroblast and iPS cells, the GGC expansion leads to increased ZFHX3 protein levels and abnormal autophagy, which were both normalized with siRNA-mediated ZFHX3 knockdown in the two cell types.

Conclusion: Identification of the SCA4 mutation highlights the power of novel high-fidelity single-strand genomic sequencing with read lengths >10kb. The GGC-RE in an extremely GC-rich region was not detectable by short-read whole-exome sequencing and demonstrates the power of LR-GS for variant discovery. SCA4 now joins neuronal intranuclear inclusion disease, some forms of oculopharyngeal muscular dystrophy, and FXTAS as a polyglycinopathy. We describe a novel polyG cellular phenotype related to impaired autophagic flux and neuronal intranuclear inclusions. Improving autophagy by targeting ZFHX3 mRNA points to a therapeutic avenue for this novel polyG disease.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/spinocerebellar-ataxia-type-4-a-novel-polyglycine-disease-caused-by-ggc-repeat-expansion-in-zfhx3/