Abstract
Despite linkage to chromosome 16q in 1996, the mutation causing spinocerebellar ataxia type 4 (SCA4), a late-onset sensory and cerebellar ataxia, remained unknown. Here, using long-read single-strand whole-genome sequencing (LR-GS), we identified a heterozygous GGC-repeat expansion in a large Utah pedigree encoding polyglycine (polyG) in zinc finger homeobox protein 3 (ZFHX3), also known as AT-binding transcription factor 1 (ATBF1). We queried 6,495 genome sequencing datasets and identified the repeat expansion in seven additional pedigrees. Ultrarare DNA variants near the repeat expansion indicate a common distant founder event in Sweden. Intranuclear ZFHX3–p62–ubiquitin aggregates were abundant in SCA4 basis pontis neurons. In fibroblasts and induced pluripotent stem cells, the GGC expansion led to increased ZFHX3 protein levels and abnormal autophagy, which were normalized with small interfering RNA-mediated ZFHX3 knockdown in both cell types. Improving autophagy points to a therapeutic avenue for this novel polyG disease. The coding GGC-repeat expansion in an extremely G+C-rich region was not detectable by short-read whole-exome sequencing, which demonstrates the power of LR-GS for variant discovery.
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Data availability
The data that support the findings of this study are available on request from the corresponding authors (S.M.P. and O.R.). The data are not publicly available as they contain information that could compromise the privacy of research participants. Source data are provided with this paper.
Code availability
(1) Open-source tools and pipelines: (1.1) in-house developed tools and pipelines: megSAP version 2022_08 (short-read WGS data): https://github.com/imgag/megSAP, https://doi.org/10.5281/zenodo.10817663 (ref. 57); megLR v1.0.0 (long-read WGS data): https://github.com/imgag/megLR, https://doi.org/10.5281/zenodo.10820153 (ref. 58); Expander (repeat expansion detection with Nanopore data): https://github.com/caspargross/expander, https://doi.org/10.5281/zenodo.10820172 (ref. 59); SCA4 project-specific scripts (scripts for data formatting, plotting, haplotype analysis and linkage analysis): https://github.com/imgag/sca4_project; (1.2) other open-source tools for long-read WGS analysis (used by in-house pipeline): trgt v0.4.0 (repeat expansion detection with long-read WGS HiFi): https://github.com/PacificBiosciences/trgt; Straglr v1.4.0 (repeat expansion detection with long-read WGS Nanopore): https://github.com/bcgsc/straglr; pbsv v2.9.0 (structural variant detection with long-read WGS HiFi): https://github.com/PacificBiosciences/pbsv; Sniffles2 v2.0.7 (structural variant detection with long-read WGS Nanopore): https://github.com/fritzsedlazeck/Sniffles; minimap2 v2.25 (long-read WGS alignment): https://github.com/lh3/minimap2; Flye v2.9.2 (genome assembly with long-read WGS): https://github.com/fenderglass/Flye; pb-assembly v1 (genome assembly with long-read WGS HiFi): https://github.com/PacificBiosciences/pb-assembly; hapdup v0.12 (haplotype phasing): https://github.com/KolmogorovLab/hapdup; hapdiff v0.8 (phased structural variant calling): https://github.com/KolmogorovLab/hapdiff; PEPPER-Margin-DeepVariant r0.8 (variant calling with long-read WGS Nanopore): https://github.com/kishwarshafin/pepper/releases/tag/r0.8. (2) Vendor software for sequencing platforms: (2.1) PacBio HiFi software platform: PacBio WGS Variant Pipeline: https://github.com/PacificBiosciences/HiFi-human-WGS-WDL; PacBio HiFi tools: https://github.com/PacificBiosciences; (2.2) ONT software: MinKNOW with Guppy basecaller: https://community.nanoporetech.com/docs/analyse; ONT basecallers: https://github.com/nanoporetech; (2.3) Illumina software (srWGS): Illumina DRAGEN Bio-IT Platform (release 4.2): https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform.html.
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Acknowledgements
We express our gratitude to the individuals with SCA4 and their relatives and caregivers. We thank L. Huynh, J. Hübener-Schmid, Q. Mao and the University of Utah Cell Imaging Core for their contributions. This work was supported by NIH grant R35 NS127253 to S.M.P. S.M.P. was partially supported by the Rindlisbacher Endowment for Research in Neurodegeneration. O.R. was partially supported by the Solve-RD project (European Union’s Horizon 2020 547 research and innovation program under grant agreement no. 779257). S.O. received funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; OS 647/1-1). S.Z. was supported by the DFG, grant 465281508. T.B.H. was supported by the DFG, grants 418081722 and 433158657. J. Park was supported by the Else Kröner-Fresenius-Stiftung Clinician Scientist program ‘PRECISE.net’. NGS methods were performed with the support of the DFG-funded NGS Competence Center Tübingen (INST 37/1049-1). We thank our colleagues from the Clinical Long-read Genome Initiative (lonGER consortium) for valuable discussion and exchange on methodological aspects of ONT LR-GS.
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K.P.F., M. Spielmann, O.R., S.O., T.B.H. and S.M.P. jointly supervised the research. K.P.F., N.C. and S.M.P. conceptualized and designed the experiments. K.P.F., C.G., E.B.-A., S.P., M.G., T.B.H., N.K., J. Park, K.H., C.D. and A.K. performed the experiments. K.P.F. performed real-time PCR analyses. S.P. performed western blotting. M.G. produced iPS cells and iPS cell-derived neurons. M.G. and A.K. performed immunohistological stains. K.P.F., S.P., M.G., J. Park, D.R.S., S.O. and T.B.H. performed statistical tests. K.P.F., E.B.-A., S.P., M.G., N.K., M. Sturm, N.C., J.A., Y.H., J. Pozojevic, S.B., T.F. and S.O. performed data analyses. K.P.F., N.K., C.Z., Y.H., S.Z., D.T., F.E., L.H., M.Z., T.K., O.R. and S.M.P. contributed patient materials or patient data. K.P.F., E.B.-A., N.K., M. Sturm, N.C., J. Park, D.R.S., A.K., M. Spielmann, O.R., S.O., T.B.H. and S.M.P. contributed to writing the paper.
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Quantifications of histological data presented in Fig. 2.
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Full unprocessed blots associated with Fig. 3.
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Quantifications of blots presented in Fig. 5.
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Figueroa, K.P., Gross, C., Buena-Atienza, E. et al. A GGC-repeat expansion in ZFHX3 encoding polyglycine causes spinocerebellar ataxia type 4 and impairs autophagy. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01719-5
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DOI: https://doi.org/10.1038/s41588-024-01719-5