GWAS of plasma pTau181 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 KCNJ3, APOE in human patients - East Asian cohort (K-ROAD). Primary outcome: plasma pTau181 levels

Description

Genome-wide association study of plasma phosphorylated tau at threonine 181 (pTau181) levels in the same K-ROAD cohort. This biomarker has been previously studied in GWAS but primarily in European populations. The study identified genome-wide significant associations at the KCNJ3 locus and strong associations at the APOE locus. The same quality control procedures and genotyping platform were used as for the pTau217 analysis.

TARGET GENE

KCNJ3, APOE

MODEL SYSTEM

human patients - East Asian cohort (K-ROAD)

ESTIMATED COST

TIMELINE

0 months

PATHWAY

tau phosphorylation, potassium channel signaling

SOURCE

extracted_from_pmid_41804841

PRIMARY OUTCOME

plasma pTau181 levels

Scoring Dimensions

📖 Wiki Pages

ApoE (Apolipoprotein E)protein APOE Lipid Metabolism Pathway in Alzheimer's Diseamechanism APOE contributes to Alzheimer's disease by regulathypothesis APOE contributes to Alzheimer's disease by regulathypothesis APOE — Apolipoprotein Egene APOE Genotyping for Neurodegenerative Disease Riskdiagnostic APOE-Expressing Astrocytescell apoe-genotype-guided-preventiontherapeutic KCNJ3 Proteinprotein GWAS Findings Hub: Alzheimer's and Parkinson's Dismechanism GWAS Findings Hubmechanism APOE Lipid Dysregulation Causal Chain in ADmechanism KCNJ3 Genegene TREM2 Variants in Alzheimer's Diseasedisease Sporadic vs Familial Alzheimer's Disease: Comprehedisease

Protocol

PHASE 1: SAMPLE COLLECTION AND DNA EXTRACTION (Week 1-4)

1.1 Study Population

Cohort: K-ROAD (Korean ROAD to Alzheimer's Disease)
Sample size: N = 3,000 East Asian participants
Inclusion: Age ≥60 years, willing to provide informed consent, East Asian ancestry (self-reported)
Exclusion: Active malignancy, end-stage renal disease, recent major surgery

1.2 Sample Collection

Fasting blood draw (10 mL EDTA whole blood, 10 mL plasma)
Centrifuge at 1,500 × g for 15 minutes at 4°C within 1 hour of collection
Aliquot plasma (500 μL × 4 tubes) and store at -80°C
Store buffy coat at -80°C for DNA extraction

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PHASE 1: SAMPLE COLLECTION AND DNA EXTRACTION (Week 1-4)

1.1 Study Population

Cohort: K-ROAD (Korean ROAD to Alzheimer's Disease)
Sample size: N = 3,000 East Asian participants
Inclusion: Age ≥60 years, willing to provide informed consent, East Asian ancestry (self-reported)
Exclusion: Active malignancy, end-stage renal disease, recent major surgery

1.2 Sample Collection

Fasting blood draw (10 mL EDTA whole blood, 10 mL plasma)
Centrifuge at 1,500 × g for 15 minutes at 4°C within 1 hour of collection
Aliquot plasma (500 μL × 4 tubes) and store at -80°C
Store buffy coat at -80°C for DNA extraction

1.3 DNA Extraction

Method: Magnetic bead-based extraction (Qiagen QIAamp DNA Blood Mini Kit or equivalent)
Equipment: QIACube Connect (Qiagen) or equivalent automated system
Reagents:
Qiagen Protease (20 mg/mL)
Buffer AL ( guanidinium hydrochloride-based)
Buffer AW1 and AW2 (ethanol-containing wash buffers)
Buffer AE (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0)
DNA concentration: Nanodrop 2000 (260/280 ratio ≥1.8, 260/230 ≥1.9)
Quantification: Qubit 3.0 Fluorometer (dsDNA BR Assay Kit)
Store DNA at 4°C (working) and -80°C (long-term)

PHASE 2: GWAS GENOTYPING (Week 5-12)

2.1 Genotyping Platform

Array: Illumina Asian筛查 Array (ASA) or Affymetrix Axiom Precision Medicine Research Array (PMRA)
Target: ~700,000 SNPs with imputation to ~10 million SNPs
Coverage: Focus on KCNJ3 region (chr4: 81,500,000-83,500,000 GRCh37) and APOE region (chr19: 44,900,000-45,100,000)

2.2 APOE Genotyping (Direct)

Method: TaqMan SNP Genotyping Assay
SNPs: rs7412 (APOE ε2), rs429358 (APOE ε4)
Reagents:
TaqMan GTXpress Master Mix (Applied Biosystems)
APOE rs7412 Assay: C____904973_10
APOE rs429358 Assay: C____3084793_20
Equipment: QuantStudio 7 Flex Real-Time PCR System
Thermal cycling: 95°C 20s → (95°C 3s, 60°C 30s) × 40 cycles

2.3 Quality Control

Sample-level QC:
Call rate <98%: exclude
Gender discrepancy: verify with XY markers
Cryptic relatedness (π > 0.2): remove one sample per pair
Heterozygosity outliers (>3 SD from mean): flag for review
SNP-level QC:
Call rate <95%: exclude
HWE p < 1×10⁻⁶: exclude (except APOE region documented)
MAF <1%: exclude for common GWAS; include for rare variant analysis

2.4 Imputation

Reference panel: 1000 Genomes Phase 3 (GRCh37) or TOPMed r2
Software: IMPUTE2 or Michigan Imputation Server
Pre-phasing: SHAPEIT2
Post-imputation filter: INFO score >0.4

PHASE 3: PLASMA pTAU181 QUANTIFICATION (Week 13-16)

3.1 Assay Platform

Method: Simoa HD-X Analyzer (Quanterix) with Homebrew assay
Alternative: Fujirebio Lumipulse G pTau181 (automated CLEIA)
Target biomarker: Phosphorylated tau at threonine 181

3.2 Simoa pTau181 Assay (Homebrew)

Capture antibody: Anti-pTau181 (Rabbit monoclonal, clone: E7A1A, Cell Signaling Technology)
Working concentration: 3 μg/mL in PBS
Coating: 50 μL/well in 96-well Quanterix plates
Incubation: overnight at 4°C, then block with 0.1% BSA/PBS
Detection antibody: Biotinylated anti-total tau (DAKO)
Working concentration: 0.3 μg/mL in assay diluent
Incubation: 30 minutes at 30°C with shaking
Enzyme: Streptavidin-β-galactosidase (SBG)
Dilution: 1:100 in SBG diluent
Incubation: 5 minutes at 30°C
Substrate: Resorufin β-D-galactopyranoside (RGP)
Concentration: 100 μM in substrate buffer
Incubation: 60 seconds at 30°C
Stop: 0.1 M EDTA, pH 8.0

3.3 Internal Quality Controls

Low QC: Pooled plasma from AD patients (target: 15-20 pg/mL)
Medium QC: Pooled plasma (target: 40-50 pg/mL)
High QC: Spiked plasma (target: 80-100 pg/mL)
Acceptance criteria: CV <20% for all QC levels
Run acceptance: ≥80% of QC samples within 2 SD of target

3.4 Sample Analysis

Run duplicate measurements per sample
Minimum sample volume: 100 μL plasma per replicate
Dilution: 1:4 in sample diluent (if needed for high samples)
Background subtraction: Average of blank wells (no sample)

PHASE 4: STATISTICAL ANALYSIS (Week 17-24)

4.1 Primary Analysis

Software: PLINK 2.0, R 4.2+ (data manipulation), SNPTest (imputed data)
Model: Linear regression with plasma pTau181 as continuous dependent variable
Covariates:
Age (continuous)
Sex (categorical)
APOE ε4 status (0, 1, or 2 copies)
PC1-PC5 (principal components for ancestry)
Batch (genotyping plate)
Additive genetic model assumed

4.2 Population Stratification

PCA: SmartPCA (EIGENSOFT 7.2)
Compute 10 ancestry PCs using LD-pruned SNPs (r² < 0.1)
Visual inspection: Q-Q plots and Manhattan plots
λ (genomic inflation factor) target: 1.0-1.05

4.3 Region of Interest Analysis

KCNJ3: ±100 kb around gene body
cis-pQTL analysis: Test all SNPs within 1 Mb of KCNJ3 transcription start site
Conditional analysis: Adjust for top GWAS hit to identify secondary signals

4.4 APOE-Stratified Analysis

Stratify by APOE ε4 status:
Group 1: ε4 non-carriers (ε2/ε2, ε2/ε3, ε3/ε3)
Group 2: ε4 heterozygotes (ε3/ε4, ε2/ε4)
Group 3: ε4 homozygotes (ε4/ε4)
Interaction analysis: SNP × APOE ε4 status

4.5 Power Calculation

Assumptions:
N = 3,000
α = 5×10⁻⁸ (genome-wide)
MAF ≥0.05
Effect size (β): 0.1-0.2 SD per allele
Target power: ≥80%

PHASE 5: FUNCTIONAL VALIDATION IN SILICO (Week 25-28)

5.1 eQTL Analysis

Datasets: Japanese MetaboBank, K-ROAD RNA-seq (if available)
Tools: Matrix eQTL, QTLtools
Test: Association between KCNJ3 SNPs and KCNJ3 mRNA expression in blood/brain

5.2 Bioinformatics Annotation

Variant effect prediction: SIFT, PolyPhen-2, CADD
Regulatory annotation: ENCODE, Roadmap Epigenomics
Brain expression: GTEx v8 (brain tissue), Allen Brain Atlas
Pathway analysis: MAGMA, DEPICT

5.3 Mendelian Randomization

Software: TwoSampleMR (R package)
Instrumental variables: Independent GWAS-significant SNPs (r² < 0.01)
Outcome: Alzheimer's disease (IGAP summary statistics)
Sensitivity: Heterogeneity, pleiotropy tests

PHASE 6: REPLICATION AND REPORTING (Week 29-36)

6.1 Replication Cohort

Internal: K-ROAD independent subset (N = 1,000)
External: East Asian cohort (e.g., Japanese-ADNI, Shanghai Aging Study)
Replication threshold: p < 0.05 with consistent direction of effect

6.2 Multi-marker Analysis

Polygenic risk score (PRS): PRSice-2
Base: East Asian AD GWAS (if available) or European AD GWAS with LD reference
Target: Plasma pTau181 levels
Clumping: r² = 0.1, kb = 250

6.3 Data Sharing

Summary statistics upload: GWAS Catalog, SSGAC
Individual-level data: Controlled access repository (dbGaP or equivalent)

Expected Outcomes

APOE ε4 effect on pTau181: APOE ε4 carriers will show plasma pTau181 levels 1.5-2.0 pg/mL higher than non-carriers (β = 0.38, SE = 0.05, p = 2.3×10⁻¹²), consistent with ~25-35% increase relative to mean.

KCNJ3 region association: At least one SNP in the KCNJ3 locus will reach suggestive significance (p < 1×10⁻⁶), with expected effect size of β = 0.15-0.25 SD per allele (approximately 0.3-0.5 pg/mL per copy of effect allele).

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Success Criteria

GWAS significance threshold: Primary analysis achieves at least 2 loci meeting genome-wide significance (p < 5×10⁻⁸) after multiple testing correction, with λGC < 1.05 indicating adequate population stratification control.
Effect size validation: Identified GWAS signals demonstrate effect sizes with 95% CI excluding the null, and β estimates are within 1.5-fold of previously reported values for the same variants.

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