🧫 Experiment Protocol
Validationproposed
SUMMARY
# TMEM106B Haplotype as Genetic Modifier in FTD — Mechanism and Therapeutic Exploitation
## Background and Rationale
Validation experiment to elucidate how the TMEM106B protective haplotype modifies FTD disease course, with implications for therapeutic target identification.
**Protocol**: (1) CRISPR-engineered iPSC lines: isogenic GRN+/- (FTD risk) with either TMEM106B risk or protective haplotype (4 lines total). Differentiate to cortical neurons, microglia, and astrocytes. (2) Multi-omic prof
METHODOLOGY NOTES
**Phase 1: Patient Cohort Assembly and Genetic Characterization (Months 1-6)**
• Recruit n=500 FTD patients with confirmed pathogenic mutations (C9orf72, MAPT, GRN) and n=500 age-matched controls
• Extract genomic DNA from peripheral blood samples using Qiagen QIAamp DNA Blood Maxi Kit
• Perform whole genome sequencing (30x coverage) on Illumina NovaSeq 6000 platform
• Genotype TMEM106B rs1990622 and rs3173615 variants using TaqMan assays in triplicate
• Construct TMEM106B haplotypes (protective vs. risk) based on linkage disequilibrium patterns
• Collect detailed clinical phenotyping including age of onset, disease duration, CDR-FTLD scores
**Phase 2: Functional Mechanism Investigation (Months 7-18)**
• Generate iPSC lines from n=24 FTD patients (12 protective haplotype, 12 risk haplotype)
• Differentiate iPSCs to cortical neurons using dual SMAD inhibition protocol (21-day protocol)
• Perform TMEM106B protein quantification via Western blot and immunofluorescence microscopy
• Measur