Independent Validation and Structural Characterization of Expanded <scp> <i>HSF1</i> </scp> Intronic Variable Number Tandem Repeats in Essential Tremor

BACKGROUND: Recent studies have reported associations between expanded HSF1 intronic variable number tandem repeats (VNTRs) and essential tremor (ET), necessitating independent validation. METHODS: We analyzed HSF1 VNTR lengths in 720 ET patients and 507 controls using fluorescence amplicon length analysis. Sequence composition and interruption patterns were characterized using long-read sequencing (LRS) data from 26 ET patients and 29 controls harboring expanded VNTRs. RESULTS: Expanded VNTRs showed similar frequencies in ET patients and controls at both 700 base pairs (bp) (5.9% vs. 4.3%, P = 0.22) and 550 bp thresholds (13.7% vs. 10.8%, P = 0.14). LRS refined the motif pattern from (CCCCGCNCCGCCT)n/(CCNCGCCT)n to (CNCCGCNCCGCCT)n/(CNNCGCCT)n. Both patients and controls showed identical repeat configurations with predominantly CC interruptions accompanied by CCCCG interruptions (75% in patients, 41.4% in controls). Self-organizing map analysis revealed no clustering differences between groups. CONCLUSIONS: In our study, expanded HSF1 VNTRs are not enriched in ET patients, with no distinguishing sequence features between patients and controls. Further validation in additional large cohorts is warranted. © 2026 International Parkinson and Movement Disorder Society.