Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder marked by progressive loss of upper and lower motor neurons. While 90–95% of cases are sporadic (sALS), 5–10% are familial (fALS), with many linked to hexanucleotide repeat expansions in C9ORF72. However, features such as incomplete penetrance, somatic mosaicism, and intermediate repeat lengths blur the distinction between familial and sporadic ALS, suggesting that tandem repeat (TR) variation may contribute more broadly to disease susceptibility and heterogeneity. Despite this, the genome-wide landscape and regulatory impact of TR variation in ALS remain poorly characterized.

Methods: Here, we systematically identified and analyzed TR expansions using 1,294 PCR-free whole-genome sequencing datasets from European ancestry ALS patients and matched controls from the NYGC ALS consortium. We detected 34,798 TR loci, of which 140 showed significantly longer repeat lengths and higher prevalence in ALS cases (enriched expansions, EEs). Applying stricter thresholds, we identified 15 rare ALS-associated enriched TR expansions (AREEs), all of which replicated in both case and control replication studies. Notably, we identified novel associations, including an AG repeat expansion in ESR1.

Conclusion: We also performed integrative analyses assessing their effects on gene expression and alternative splicing. Using expression and splicing quantitative trait locus mapping, we identified significant associations between TR size variation and transcriptomic changes. We further applied downstream analyses—including fine-mapping, colocalization, and mediation analyses—to elucidate the regulatory mechanisms linking TR-associated eGenes to ALS. Together, these results highlight novel candidate biomarkers and provide new insights into ALS biology, opening avenues for future therapeutic exploration.