Our study demonstrates the successful use of PacBio HiFi long-read whole genome sequencing to identify a pathogenic TTTTA/TTTCA pentanucleotide repeat expansion in intron 1 of the MARCHF6 gene, establishing a molecular diagnosis of Familial Adult Myoclonic Epilepsy type 3 (FAME3) in a previously undiagnosed proband and her affected relatives. This is the first report to diagnose FAME3 using a commercially available LRS program. The findings provide compelling genetic evidence supporting autosomal dominant mode of inheritance, with expansion size increasing with one generational transmission.

Our results are consistent with prior reports describing genotype-phenotype relationships in FAME3 [1,2,3,4]. As previously shown, the presence of expanded TTTCA motifs, rather than TTTTA repeats alone, appears to be the critical pathogenic element [2, 10]. TTTTA-only expansions are often observed in unaffected individuals and are not considered pathogenic in isolation. Instead, disease seems to arise when TTTCA repeats occur in tandem with TTTTA motifs, suggesting a composite structure that contributes to mRNA-mediated toxicity or genomic instability—rather than altered gene expression or protein dysfunction—as the underlying mechanism of pathogenesis. Our case further supports this model: although the repeat expansions in affected individuals varied in size and composition, all shared the presence of pathogenic TTTCA motifs. This highlights the diagnostic importance of assessing both repeat length and motif composition when evaluating suspected repeat expansion disorders.

Our findings demonstrate that repeat mosaicism is a prominent and likely characteristic feature of MARCHF6 expansions in FAME3. Using TRGT-instability, we visualized a range of repeat sizes within individual alleles—direct evidence of somatic repeat instability that complements prior reports in other repeat expansion disorders [10]. To our knowledge, this is the first report to directly visualize MARCHF6 repeat mosaicism at the single-read level in a clinically diagnosed FAME3 family. Our observation of mosaicism raises questions about the threshold effects for pathogenicity, intergenerational instability, and the marked clinical variability often seen among those who are mosaic MARCHF6 repeat expansion variants.

From a clinical perspective, our findings underscore the transformative potential of LRS in resolving complex or undiagnosed neurological cases. Conventional short-read methods frequently fail to detect non-coding repeat expansions due to alignment and assembly limitations. By contrast, long-read sequencing provides the resolution needed to accurately detect and characterize these complex variants. Incorporating this technology into clinical workflows could substantially improve diagnostic yield, inform more accurate genetic counseling, and enable earlier interventions for affected individuals and at-risk family members.

Future efforts should focus on integrating LRS into routine clinical diagnostics and developing guidelines to identify patients most likely to benefit from this technology. As accessibility and cost-effectiveness improve, LRS may become a frontline tool in the evaluation of rare and undiagnosed genetic conditions.