SUMMARY
# Mechanism: C9orf72 Hexanucleotide Repeat Expansion in ALS/FTD
## Background and Rationale
This comprehensive validation study addresses one of the most significant questions in neurodegeneration: how a single genetic mutation in C9orf72 can manifest as distinct diseases (ALS, FTD, or combined phenotypes). The C9orf72 hexanucleotide repeat expansion represents the most common genetic cause of both familial ALS (~40%) and familial FTD (~25%), yet the mechanisms underlying phenotypic diversity re
METHODOLOGY NOTES
**Phase 1: Patient Cohort Assembly and Characterization (Months 1-3)**
Recruit 150 participants: 50 C9orf72+ ALS patients, 50 C9orf72+ FTD patients, 25 C9orf72+ ALS/FTD patients, and 25 healthy controls with C9orf72 mutations but no symptoms. Perform comprehensive clinical phenotyping including ALSFRS-R, CDR-FTLD, and neuropsychological testing. Extract genomic DNA and perform Southern blot analysis to quantify hexanucleotide repeat length. Establish iPSC lines from all participants using episomal reprogramming.
**Phase 2: Molecular Pathology Analysis (Months 4-8)**
Generate motor neurons and cortical neurons from iPSCs using dual SMAD inhibition protocol. Perform RNA-seq on differentiated neurons at days 21, 35, and 50 to identify disease-specific transcriptional signatures. Quantify RNA foci formation using FISH with (GGGGCC)4 and (CCCCGG)4 probes. Measure dipeptide repeat protein (DPR) accumulation via immunofluorescence and Western blot for poly-GA, poly-GP, poly-GR, poly-PA, an