GWAS of plasma pTau217 in East Asian cohort

Exploratory Score: 0.950 Price: $0.50 Alzheimer's disease human patients - East Asian cohort (K-ROAD) Status: proposed

What This Experiment Tests

Exploratory experiment designed to discover new patterns targeting DACT1, DAAM1, APOE in human patients - East Asian cohort (K-ROAD). Primary outcome: plasma pTau217 levels

Description

Genome-wide association study of plasma phosphorylated tau at threonine 217 (pTau217) levels in 1,972 individuals from the Korea-Registries to Overcome and Accelerate Dementia Research (K-ROAD) cohort. This study aimed to identify genetic variants associated with pTau217, an emerging AD-specific biomarker that correlates with amyloid and tau PET positivity, disease stage, and cognitive decline. Genomic DNA was genotyped using the Illumina Asian Screening Array platform. Quality control procedures included exclusion of samples with low genotype call rates, related individuals, and variants with low minor allele frequency or Hardy-Weinberg equilibrium deviations. Principal component analysis was performed to identify population outliers. The study identified genome-wide significant associations at the DACT1-DAAM1 locus and strong associations at the APOE locus.

TARGET GENE

DACT1, DAAM1, APOE

MODEL SYSTEM

human patients - East Asian cohort (K-ROAD)

ESTIMATED COST

TIMELINE

0 months

PATHWAY

tau phosphorylation, amyloid pathway

SOURCE

extracted_from_pmid_41804841

PRIMARY OUTCOME

plasma pTau217 levels

Scoring Dimensions

📖 Wiki Pages

APOE — Apolipoprotein Egene APOE Lipid Metabolism Pathway in Alzheimer's Diseamechanism APOE - Apolipoprotein Escidex_docs APOE contributes to Alzheimer's disease by regulathypothesis APOE contributes to Alzheimer's disease by regulathypothesis APOE Genotyping for Neurodegenerative Disease Riskdiagnostic APOE-Expressing Astrocytescell DNA Damage-Accumulating Neurons in Neurodegeneraticell PET Imaging in Neurodegenerationdiagnostic DNA Damage Repair Deficiency Hypothesis in Parkinshypothesis DNA Methylationentity DNA Methylation Biomarkers in Neurodegenerationbiomarker PET Imaging Combined with Fluid Biomarkersevent DNA Damage and Repair in Neuronscell DNA Damage Response in Corticobasal Syndromemechanism

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

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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

Expected Outcomes

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)

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

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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 <

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Related Hypotheses (5)

Selective APOE4 Degradation via Proteolysis Targeting Chimeras (PROTACs)0.795

Competitive APOE4 Domain Stabilization Peptides0.784

APOE4-Specific Proteolytic Fragment Inhibition Therapy0.777

APOE4 Allosteric Rescue via Small Molecule Chaperones0.765

APOE Isoform Expression Across Glial Subtypes0.743

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