Adaptive Long-Read Sequencing Unveils GGC Repeat Expansion in ZFHX3 Linked to Spinocerebellar Ataxia Type 4

In a new study published in Movement Disorders, targeted long-read DNA sequencing through adaptive sampling showed an expansion in the last coding exon of the ZFHX3 gene associated with spinocerebellar ataxia type 4 (SCA4), an autosomal dominant ataxia with invariable sensory neuropathy. Investigators concluded that adaptive long-read sequencing may be a helpful tool for deciphering causative structural variation in unsolved cases of inherited neurological disease.¹

In this study, investigators identified a heterozygous (GGC)n repeat expansion in the last coding exon of the zinc finger homeobox 3 (ZFHX3) gene that segregates with SCA4, ranging between 48 and 57 GGC repeats in affected probands. Although linkage to this region was initially described in 1996 from a fifth-generation family in Utah with Swedish ancestry (n = 38),2 this finding was replicated in the study with a separate family with SCA4 (n = 4).

“This approach allowed the accurate detection and sizing of a GC-rich structural variant, a region that we found to be very challenging to amplify through conventional polymerase chain reaction and may therefore underlie the reason for the molecular diagnosis having evaded detection. In addition, this enabled the diagnostic approach to be variant-agnostic; that is, no a priori restrictions were placed on the specific repeat motif or type of structural variant during detection,” lead author Zhongbo Chen, MD, PhD, clinical training fellow, department of neurodegenerative disease, Queen Square Institute of Neurology, University College London, and colleagues wrote.¹ “The workflow allowed all structural variants with sequence homology and overlap between the cases to be filtered. In this case, it successfully identified the segregation of the GGC repeat directly as the only plausible pathogenic structural variant candidate. This has obvious advantages to conventional approaches that rely on searching for repeats of a specific motif.”

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Inspired by pathogenic structural variation implicated in other 16q-ataxias with connection to the same locus, the investigators revisited the index SCA4 cases from the Utah family using novel technologies to assess the structural variation in the candidate region. Thus, the researchers adopted a targeted long-read sequencing approach with adaptive sampling on the Oxford Nanopore Technologies platform to detect segregating structural variants in a genomic region without a priori assumptions about any variant features.

Among the findings, Chen et al did not observe any pathogenic repeats when estimating the GGC repeat size in short-read whole genome sequencing (WGS) data. The WGS data contained information on 21,836 patients recruited to the 100,000 Genomes Project in the UK and the authors’ in-house dataset of 11,258 exomes. After evaluating the data, the study authors concluded that the variant is ultrarare.

“Although we recognize that repeat size estimation of a larger number of symptomatic individuals is needed to determine the pathogenic threshold and to assess anticipation, this clinical phenomenon would be in keeping with that of a repeat expansion disorder. Furthermore, it would suggest that different disease severities and presentations could occur within the spectrum of SCA4 with different GGC repeat sizes,” Chen et al noted.¹ “With this in mind, we speculate that this cause of ataxia and overlapping sensory neuropathy may be prevalent in Sweden, accounting for another rare cause of inherited late-onset ataxia. Certainly, its rarity within the population suggests that there is a founder haplotype associated with individuals of Swedish ancestry and that screening for this repeat expansion as a common cause of late-onset ataxia in other nonSwedish European populations may have a low yield.”

All told, the findings were limited by the quality and quantity of DNA from historically old samples. In addition, the investigators were not able to gain adequate coverage over the repeat expansion for some of the patients given the standard required for long-read sequencing. The authors noted for the future that screening for a larger number of patients would be useful to identify whether any anticipation exists.

“Finally, we note that this study adds to the association of the 16q22 region with repeat expansion ataxias. Through functional genomic annotation, this region was found to harbor a high density of naturally occurring STRs and GGC repeats. Furthermore, we found that ZFHX3 is a gene with a particularly high density of STRs. These findings provide further insights into the association of causative repeat expansions in the 16q-ataxias and provide a predictive framework for further investigation of structural variants in unsolved cohorts,” Chen et al noted.¹

REFERENCES
1. Chen Z, Gustavsson EK, Macpherson H, et al. Adaptive Long-Read Sequencing Reveals GGC Repeat Expansion in ZFHX3 Associated with Spinocerebellar Ataxia Type 4. Mov Disord. Published online January 10, 2024. doi:10.1002/mds.29704
2. Flanigan K, Gardner K, Alderson K, et al. Autosomal dominant spinocerebellar ataxia with sensory axonal neuropathy (SCA4): clinical description and genetic localization to chromosome 16q22.1. Am J Hum Genet. 1996;59(2):392-399.