Identification of GGC Repeat Expansions in ZFHX3 Among Chilean Movement Disorder Patients

Abstract

Background: Hereditary ataxias are genetically diverse, yet up to 75% remain undiagnosed due to technological and financial barriers. A pathogenic ZFHX3 GGC repeat expansion was recently linked to spinocerebellar ataxia type 4 (SCA4), characterized by progressive ataxia and sensory neuropathy, with all reported cases in individuals of Northern European ancestry.

Methods: We performed Oxford Nanopore Technologies (ONT) genome long-read sequencing (>115 GB per sample) on a total of 15 individuals from Chile; 14 patients with suspected hereditary movement disorders and one unrelated family member. Variants were identified using PEPPER-Margin-DeepVariant 0.8 (SNVs), Sniffles 2.4 (SVs), and Vamos 2.1.3 (STRs). Ancestry was inferred using GenoTools with reference data from the 1000 Genomes Project, Human Genome Diversity Project, and an Ashkenazi Jewish panel. Haplotype analysis was conducted by phasing SNVs within ZFHX3, and methylation profiling was performed with modbamtools.

Results: We identified ZFHX3 GGC repeat expansions (47-55 repeats) in four individuals with progressive ataxia, polyneuropathy, and vermis atrophy. One case presented parkinsonism-ataxia, expanding the phenotype. Longer expansions correlated with earlier onset and greater severity. Hypermethylation was detected on the expanded allele, and haplotype analysis linked ultra-rare ZFHX3 variants to distant Swedish ancestry.

Conclusion: This is the first report of SCA4 outside Northern Europe, confirming a shared founder haplotype and expansion instability. The presence of parkinsonism broadens the clinical spectrum. Comprehensive genetic testing across diverse populations is crucial, and long-read sequencing enhances diagnostic yield by detecting repeat expansions and SNVs in a single assay.

Figure 1.

a) Schematic overview of the study design. b) Waterfall plots displaying ONT long-read sequencing data for the four predicted ZFHX3 GGC repeat carriers. The red dotted line marks the pathogenic threshold. Created using BioRender.com.

Figure 2:

Haplotype analysis of ZFHX3 GGC repeat expansion carriers. Out of the six rare SNVs reported as part of the distance common founder event, four were found in our samples within the repeat region (Highlighted by the pink box) and compared to the Utah index patient from Chen et al. These SNVs are missing in the unaffected Chilean individual.

Figure 3.. Haplotype specific Differential methylation around the expansion.

a-d) Modbamtools plots of methylation frequency for the four heterozygous expansion carriers. Methylation frequency is plotted at the top where haplotype 1 (blue) represents the non-expanded allele and haplotype 2 (orange) represents the expanded allele. The ZFHX3 gene track is overlaid at the top, showing the last two exons. Haplotype-specific reads are shown at the bottom with blue sections of the reads denoting hypomethylation and red sections of the reads denoting hypermethylation. Purple box indicates repeat region. For all four carriers, the expanded allele is hypermethylated compared to the non-expanded allele. e) Modbamtools plot of an unaffected related individual, homozygous non-repeat carrier, showing that hypomethylation in this region is expected under normal conditions.

Figure 4.

a) Distribution of ZFHX3 GGC repeat lengths in the Chilean samples (n=15), read indicates pathogenic length carrier. b) Distribution of ZFHX3 GGC repeat lengths in the 1000G control cohort (n=100) comprising individuals of mixed ancestry. c) Distribution of ZFHX3 GGC repeat lengths in the NABEC/HBCC control cohort (n=338) comprising individuals of European and African and African-admixed ancestry. The red dotted line marks the pathogenic threshold.