GGC expansion in ZFHX3 causes SCA4 and impairs autophagy

Despite linkage to 16q in 1996, the mutation for spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, escaped detection for 25 years. Using long- read PacBio-HiFi and ONT-Nanopre sequencing and bioinformatic analysis, we identified expansion of a GGC DNA repeat in a >85% GC-rich region in exon 10 of the ZFHX3 gene coding for poly-glycine (polyG). In a total of 15 nuclear families from Utah and 9 from Europe, the repeat was expanded to >40 repeats in SCA4 patients accompanied by significant phenotypic variation independent of repeat size compared to the most common normal repeat size of 21 repeats. The RE event likely occurred in a frequent Swedish haplotype shared by cases from Utah and Germany. Six characteristic ultra-rare SNVs in the vicinity of the RE in cases from Utah and Lübeck (Germany) indicate a common founder event for some of the patients. In fibroblast and iPS cells, the GGC expansion leads to increased ZFHX3 protein levels, polyG aggregates, and abnormal autophagy, which normalized with ZFHX3 siRNA. Increasing autophagic flux may provide a therapeutic avenue for this novel polyG disease. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by NIH grant R35127253 to SMP. SO received funding from the German Research Foundation DFG (DFG OS 647/1-1). NGS sequencing methods were performed with the support of the DFG-funded NGS Competence Center Tuebingen (INST 37/1049-1). JP was supported by the Clinician Scientist program PRECISE.net funded by the Else Kroener-Fresenius-Stiftung. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All procedures involving human subjects were approved by the Institutional Review Board (IRB) at the University of Utah. Human subjects use was approved by the ethics committee of the Medical Faculty of the University of Tuebingen, Germany (Genome+, ClinicalTrial.gov-Nr: [NCT04315727][1]). All human subjects in the US and Europe gave written consent for inclusion in the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04315727&atom=%2Fmedrxiv%2Fearly%2F2023%2F10%2F28%2F2023.10.26.23297560.atom