GWAS of plasma pTau217 in East Asian cohort

PRIMARY OUTCOME

plasma pTau217 levels

EXPECTED OUTCOMES

1. **APOE ε4 dose-response with pTau217**: ε4/ε4 carriers will show 2.1× higher baseline plasma pTau217 compared to ε3/ε3 homozygotes (ε4/ε4: 0.42 ± 0.08 pg/mL vs. ε3/ε3: 0.20 ± 0.05 pg/mL; mean ± SD, p < 1×10⁻¹²) 2. **DACT1 cis-pQTL identification**: A lead SNP (rsXXXXXXXX) in high linkage disequilibrium with DACT1 expression will achieve genome-wide significance (β = -0.18 pg/mL per allele, SE = 0.03, p = 2.3×10⁻⁹) and explain 1.2% of pTau217 variance 3. **DAAM1 nominally associated locus**: At least one DAAM1 SNP will show suggestive association (p < 1×10⁻⁶) with pTau217, with effect size of β = 0.09 pg/mL per minor allele (SE = 0.015) 4. **siRNA DACT1 knockdown efficacy**: DACT1 siRNA will reduce DACT1 mRNA by 78 ± 6% (p < 0.001 vs. scramble) and decrease secreted pTau217 by 31 ± 7% at Day 7 5. **siRNA DAAM1 knockdown efficacy**: DAAM1 siRNA will reduce DAAM1 mRNA by 72 ± 8% (p < 0.001 vs. scramble) with corresponding 24 ± 9% reduction in pTau217 6. **Heritability estimate**: Narrow-sense heritability (h²) of plasma pTau217 in East Asian cohort estimated at 0.18 ± 0.04 (GREML method), consistent with prior European studies (h² ≈ 0.15-0.22) 7. **Trans-ancestry meta-analysis**: Fixed-effects meta-analysis will identify ≥2 genome-wide significant loci, with correlation between discovery and replication β coefficients of r = 0.89 (95% CI: 0.78-0.95) ---

SUCCESS CRITERIA

- **Primary endpoint**: Identification of ≥1 variant reaching genome-wide significance (p < 5×10⁻⁸) in or near DACT1 or DAAM1 after multiple testing correction (Bonferroni for 2 genes, α = 0.025) - **Replication requirement**: Index SNP must replicate in independent cohort with p < 0.05/Number_of_SNPs and consistent direction of effect (β correlation > 0) - **Effect size threshold**: Identified SNPs must explain ≥0.5% of phenotypic variance in plasma pTau217 (partial R² ≥ 0.005) - **Functional validation**: siRNA knockdown of DACT1 or DAAM1 must produce ≥25% change in secreted pTau217 with p < 0.01 (two-sample t-test, n=3 biological replicates) - **Heritability confirmation**: SNP-heritability estimate (GREML) must exceed h² > 0.10 with standard error < 0.05 - **AD relevance**: Significant SNPs must show nominal association (p < 0.05) with clinical AD diagnosis in the K-ROAD cohort - **Quality thresholds**: Genotype call rate > 98%, sample call rate > 98%, heterozygosity F statistic between -0.05 and 0.05, and no significant population stratification (λ_GC < 1.05)

PROTOCOL

### Phase 1: Cohort Recruitment & Baseline Assessment (Month 1-6) **Timepoints**: Screening (V1), Enrollment (V2, baseline) **Methods**: - Recruit 3,000 East Asian participants from K-ROAD cohort (Korean Regional Open Cohort for Alzheimer's Disease) - Inclusion: age 60-90, East Asian ancestry (self-reported + genetic principal component verification), cognitively normal or MCI - Exclusion: prior stroke, Parkinson's disease, current anti-amyloid therapy - Collect baseline plasma via venipuncture (K2-EDTA tubes, 10 mL), centrifuge at 2,000×g for 10 min at 4°C within 1 hour of collection - Store plasma aliquots (500 μL) at -80°C with protease inhibitors (cOmplete EDTA-free, Roche) - Obtain APOE genotyping via TaqMan assay (Applied Biosystems, cat# 4351379): rs429358 (C130T) and rs7412 (T1767C) - Administer cognitive assessments: MMSE, CDR, and Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog13) ### Phase 2: Plasma pTau217 Quantification (Month 4-12) **Timepoints**: Baseline, Month 6, Month 12 **Methods**: - Measure plasma pTau217 using Simoa HD-X Analyzer (Quanterix) with pTau217 Advantage Kit (cat# 103714) - Assay sensitivity: LLoD < 0.019 pg/mL, CV < 5% intra-assay, < 10% inter-assay - Sample preparation: thaw plasma on ice, dilute 4× in sample diluent - Run all samples in duplicate; include 2 quality controls per plate (low: ~0.5 pg/mL, high: ~15 pg/mL) - Accept plates with QC CV < 10% and calibration curve R² > 0.99 - Normalize pTau217 to baseline using within-patient z-scores ### Phase 3: Genotyping & GWAS (Month 6-18) **Timepoints**: Single extraction point **Methods**: - Extract genomic DNA from peripheral blood (QIAamp DNA Blood Kit, Qiagen) - Genotype using Illumina Global Screening Array v3 (GSAMD-24v3-0) with custom content for DACT1, DAAM1, and APOE loci - Impute to TOPMed Reference Panel (build GRCh38) - Perform principal component analysis (PCA) using PLINK 2.0 to identify ancestry outliers (>4 SD from mean on PC1-PC10) - Quality control: remove samples with call rate < 98%, heterozygosity deviation > 0.1, sex discrepancies - Variant QC: remove SNPs with MAF < 1%, HWE p < 1×10⁻⁶, call rate < 95% - GWAS association: linear mixed model (LMM) using SAIGE with pTau217 as continuous outcome, adjusting for age, sex, education, APOE ε4 status, and 10 PCs - Regional association plots for DACT1 (chr14:59.5-60.5 Mb), DAAM1 (chr14:67.5-68.5 Mb), and APOE region (chr19:44.5-46.5 Mb) ### Phase 4: Functional Validation — siRNA Knockdown in iPSC-derived Neurons (Month 12-24) **Timepoints**: Day 0 (transfection), Day 3, Day 7 **Methods**: - Obtain iPSC lines from 3 K-ROAD participants (2 ε4/ε4 carriers, 1 ε3/ε3 non-carrier) - Differentiate to cortical neurons using STEMdiff Cerebral Organoid Kit (cat# 08580) - Transfect neurons (DIV 21) with DACT1 siRNA (Santa Cruz, cat# sc-106762), DAAM1 siRNA (Santa Cruz, cat# sc-106775), or negative control siRNA (Ambion, cat# AM4611) using Lipofectamine RNAiMAX - siRNA concentration: 30 pmol per well (24-well plate) - Collect conditioned media at Day 3 and Day 7 for pTau217 measurement (Simoa) - Harvest cells for western blot: lyse in RIPA buffer with Phosphatase Inhibitor Cockset 2/3 (Sigma) - Primary antibodies: Anti-DACT1 (1:500, Abcam ab239459), Anti-DAAM1 (1:500, Abcam ab125133), Anti-pTau217 (1:1000, Eli Lilly cos Yvonne), Anti-total tau (1:1000, DAKO A0024), Anti-β-actin (1:5000, Sigma A5441) - Secondary: HRP-conjugated anti-rabbit (1:5000) or anti-mouse (1:5000) - Image acquisition: ChemiDoc MP, quantify with ImageLab ### Phase 5: Epigenetic & Expression Analysis (Month 18-30) **Timepoints**: Single point **Methods**: - Extract RNA from iPSC-derived neurons (RNeasy Mini Kit, Qiagen) - Perform qRT-PCR: TaqMan Gene Expression Assays for DACT1 (Hs00212304_m1), DAAM1 (Hs00370504_m1), MAPT (Hs00213491_m1), and GAPDH (Hs99999905_m1) - Run on QuantStudio 7 Flex (Applied Biosystems), calculate ΔΔCt relative to GAPDH - ATAC-seq: perform assay on 50,000 neurons per condition (Active Motif cat# 53150) - Library prep: Illumina TruePrep DNA Library Prep Kit - Sequencing: NovaSeq 6000, 2×150 bp, target 50M reads per sample - Bioinformatic analysis: call peaks with MACS3, annotate with HOMER, pathway enrichment via GREAT ### Phase 6: Replication & Meta-Analysis (Month 24-36) **Timepoints**: Cross-sectional **Methods**: - Replicate significant SNPs (p < 5×10⁻⁸) in independent East Asian cohort (Japanese AD cohort, n=1,500 from J-ADNI) - Include non-East Asian replication cohort (UK Biobank, n=5,000 European ancestry) for trans-ancestry comparison - Fixed-effects meta-analysis using METAL (inverse-variance weighting) - Calculate β coefficients, standard errors, and p-values across cohorts - Cochran's Q-test for heterogeneity; I² statistic ---

LINKED HYPOTHESES