@bgicli/bgicli 2.1.1 → 2.2.0

package/data/workflows/gwas-to-function-twas/references/therapeutic_interpretation_guide.md

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+## Therapeutic Interpretation Guide for TWAS Directionality
+
+**Purpose:** This guide explains how to interpret TWAS directionality to
+determine whether genes should be inhibited or activated for therapeutic
+benefit, with frameworks for confidence assessment and target validation.
+
+**Last Updated:** 2026-01-28
+
+---
+
+## Table of Contents
+
+1. [Understanding TWAS Directionality](#understanding-twas-directionality)
+2. [Multi-Layer Directional Analysis](#multi-layer-directional-analysis)
+3. [Confidence Assessment Framework](#confidence-assessment-framework)
+4. [Causal Inference with Mendelian Randomization](#causal-inference-with-mendelian-randomization)
+5. [Real-World Success Stories](#real-world-success-stories)
+6. [Common Pitfalls and Red Flags](#common-pitfalls-and-red-flags)
+7. [Decision Framework for Drug Modality](#decision-framework-for-drug-modality)
+
+---
+
+## Understanding TWAS Directionality
+
+### What is TWAS Directionality?
+
+TWAS (Transcriptome-Wide Association Study) identifies genes whose genetically
+regulated expression is associated with a trait. The **direction** of this
+association tells us whether higher expression increases or decreases the trait
+value.
+
+**Key concept:** TWAS Z-score (or effect size) indicates direction:
+
+- **Positive Z-score (+)**: Higher predicted expression → Higher trait value
+- **Negative Z-score (−)**: Higher predicted expression → Lower trait value
+
+### Translating Direction to Therapeutic Strategy
+
+The therapeutic strategy depends on both the TWAS direction **and** the trait
+nature:
+
+#### For Risk Traits (Higher is Bad)
+
+Examples: Disease risk, LDL cholesterol, blood pressure
+
+| TWAS Direction   | Interpretation                              | Therapeutic Strategy    |
+| ---------------- | ------------------------------------------- | ----------------------- |
+| **Positive (+)** | Higher expression → Higher risk             | **INHIBIT** expression  |
+| **Negative (−)** | Higher expression → Lower risk (protective) | **ACTIVATE** expression |
+
+**Example 1: IL6R and Cardiovascular Disease**
+
+- TWAS Z = +4.5 (positive)
+- Higher IL6R expression → Higher CAD risk
+- **Therapeutic strategy: INHIBIT IL6R** (Tocilizumab, IL-6 receptor antagonist)
+
+**Example 2: APOE and Alzheimer's Disease**
+
+- TWAS Z = −4.2 (negative)
+- Higher APOE expression → Lower AD risk
+- **Therapeutic strategy: ACTIVATE APOE** (challenging - gene therapy,
+  enhancers)
+
+#### For Protective/Beneficial Traits (Higher is Good)
+
+Examples: HDL cholesterol, bone density, cognitive function
+
+| TWAS Direction   | Interpretation                                | Therapeutic Strategy    |
+| ---------------- | --------------------------------------------- | ----------------------- |
+| **Positive (+)** | Higher expression → Higher beneficial outcome | **ACTIVATE** expression |
+| **Negative (−)** | Higher expression → Lower beneficial outcome  | **INHIBIT** expression  |
+
+#### For Quantitative Traits (Context-Dependent)
+
+Examples: Height, gene expression levels, metabolite levels
+
+- Strategy depends on clinical context
+- Determine if increasing or decreasing the trait is therapeutically desired
+
+---
+
+## Multi-Layer Directional Analysis
+
+### Why Multi-Layer Analysis?
+
+TWAS alone shows association between predicted expression and trait, but doesn't
+confirm the full causal pathway. Multi-layer analysis validates directionality
+by checking consistency across:
+
+1. **eQTL layer**: Genetic variant → Gene expression
+2. **TWAS layer**: Predicted expression → Trait
+3. **Combined pathway**: Genetic variant → Expression → Trait
+
+### Three-Layer Pathway Validation
+
+#### Layer 1: eQTL Direction (Variant → Expression)
+
+**Question:** Does the risk allele increase or decrease gene expression?
+
+- Extract lead eQTL variant for the gene
+- Determine which allele is associated with trait risk (from GWAS)
+- Check if risk allele increases or decreases expression (from eQTL)
+
+**Example (IL6R):**
+
+```
+Lead eQTL SNP: rs2228145
+Risk allele: C (increases CAD risk)
+eQTL effect: C allele → Increased IL6R expression
+eQTL direction: Risk allele INCREASES expression
+```
+
+#### Layer 2: TWAS Direction (Expression → Trait)
+
+**Question:** Does higher predicted expression increase or decrease the trait?
+
+- From TWAS Z-score or effect size
+- Positive Z = higher expression → higher trait value
+- Negative Z = higher expression → lower trait value
+
+**Example (IL6R):**
+
+```
+TWAS Z-score: +4.5
+TWAS direction: Higher expression INCREASES CAD risk
+```
+
+#### Layer 3: Combined Pathway
+
+**Question:** Is the full causal pathway consistent?
+
+Combine layers 1 and 2 to determine the full pathway:
+
+```
+Risk Allele → Expression Change → Trait Change
+```
+
+**Consistency patterns:**
+
+| eQTL Direction | TWAS Direction | Pathway                       | Consistency     | Therapeutic Strategy |
+| -------------- | -------------- | ----------------------------- | --------------- | -------------------- |
+| ↑ Expression   | ↑ Trait        | Risk allele → ↑ Expr → ↑ Risk | ✅ Consistent   | **INHIBIT**          |
+| ↓ Expression   | ↑ Trait        | Risk allele → ↓ Expr → ↑ Risk | ✅ Consistent   | **ACTIVATE**         |
+| ↑ Expression   | ↓ Trait        | Risk allele → ↑ Expr → ↓ Risk | ⚠️ Paradoxical  | Investigate          |
+| ↓ Expression   | ↓ Trait        | Risk allele → ↓ Expr → ↓ Risk | ❌ Inconsistent | Recheck alleles      |
+
+**IL6R Complete Example:**
+
+```
+Layer 1 (eQTL): Risk allele (rs2228145-C) → ↑ IL6R Expression
+Layer 2 (TWAS): ↑ IL6R Expression → ↑ CAD Risk
+Combined Pathway: Risk allele → ↑ IL6R → ↑ CAD Risk
+Consistency: ✅ Fully consistent
+Therapeutic Strategy: INHIBIT IL6R expression
+Confidence: HIGH
+```
+
+### Interpreting Paradoxical or Inconsistent Patterns
+
+**Paradoxical pattern (opposite directions across layers):**
+
+Possible explanations:
+
+1. **Feedback regulation**: Gene expression is subject to compensatory
+   mechanisms
+2. **Tissue-specific effects**: eQTL in one tissue, TWAS in another
+3. **Developmental timing**: Expression effects vary across lifespan
+4. **Complex regulation**: Gene has multiple regulatory pathways with opposing
+   effects
+
+**Action:** Requires deeper investigation before therapeutic recommendation.
+
+**Inconsistent pattern (directions don't make biological sense):**
+
+Likely causes:
+
+1. **Allele harmonization error**: Check effect allele alignment between
+   datasets
+2. **LD artifact**: TWAS hit is not the causal gene (check colocalization)
+3. **Pleiotropy**: Gene affects trait through multiple pathways
+4. **Statistical artifact**: Spurious association
+
+**Action:** Resolve technical issues before proceeding with therapeutic
+interpretation.
+
+---
+
+## Confidence Assessment Framework
+
+### Three-Tier Confidence System
+
+#### **High Confidence**
+
+Requirements:
+
+- ✅ Strong TWAS association (p < genome-wide significance)
+- ✅ Strong colocalization (PP.H4 > 0.8)
+- ✅ Consistent directionality across eQTL + TWAS layers
+- ✅ (Optional) MR causal evidence (p < 0.05, no pleiotropy)
+
+**Interpretation:** Strong evidence for causal gene-trait relationship. Proceed
+with therapeutic strategy.
+
+**Example:** IL6R → CAD (TWAS p=1e-6, PP.H4=0.92, consistent pathway, MR p=1e-5)
+
+#### **Medium Confidence**
+
+Requirements:
+
+- ✅ Significant TWAS association (p < 0.001)
+- ⚠️ Moderate colocalization (PP.H4 = 0.5-0.8) OR missing colocalization
+- ✅ Directional consistency
+- ❌ MR not performed or inconclusive
+
+**Interpretation:** Likely causal relationship, but some uncertainty remains.
+Consider as candidate target pending additional validation.
+
+**Example:** SORT1 → LDL (TWAS p=5e-4, PP.H4=0.65, consistent)
+
+#### **Low Confidence**
+
+Requirements:
+
+- ⚠️ Nominally significant TWAS (p < 0.05)
+- ❌ Poor colocalization (PP.H4 < 0.5) OR not assessed
+- ❌ Inconsistent directionality OR not assessed
+- ❌ Weak MR instruments (F < 10) OR MR shows pleiotropy
+
+**Interpretation:** Association may be spurious or due to LD. Not recommended
+for therapeutic targeting without further validation.
+
+**Action:** Deprioritize unless biological rationale is very strong.
+
+### Confidence Scoring Matrix
+
+| Evidence Type          | Weight | Scoring                                      |
+| ---------------------- | ------ | -------------------------------------------- |
+| **TWAS significance**  | 30%    | −log₁₀(p-value) / 10, capped at 1.0          |
+| **Colocalization**     | 30%    | PP.H4 value (0-1 scale)                      |
+| **MR causal evidence** | 20%    | −log₁₀(MR p-value) / 10, capped at 1.0       |
+| **Druggability**       | 20%    | Protein class score (see druggability guide) |
+
+**Composite confidence score:**
+
+```
+Confidence = 0.3 × TWAS_score + 0.3 × Coloc_score + 0.2 × MR_score + 0.2 × Drug_score
+```
+
+**Interpretation:**
+
+- **Score ≥ 0.7**: High confidence target
+- **Score 0.5-0.7**: Medium confidence target
+- **Score < 0.5**: Low confidence target
+
+---
+
+## Causal Inference with Mendelian Randomization
+
+### Why MR Strengthens Directionality Evidence
+
+TWAS identifies associations, but associations can be:
+
+1. **Causal**: Gene expression directly influences trait
+2. **Reverse causation**: Trait affects gene expression
+3. **Confounding**: Shared factors affect both expression and trait
+
+**Mendelian Randomization (MR)** uses genetic variants as instrumental variables
+to test causality, addressing confounding and reverse causation.
+
+### MR Assumptions for Valid Inference
+
+**Three core assumptions:**
+
+1. **Relevance**: Genetic variants (eQTLs) are strongly associated with gene
+   expression
+   - Check: F-statistic > 10 (strong instruments)
+
+2. **Independence**: Genetic variants are independent of confounders
+   - Assumed true due to random inheritance (Mendel's laws)
+
+3. **Exclusion restriction**: Genetic variants affect trait **only** through
+   gene expression (no horizontal pleiotropy)
+   - Check: MR-Egger intercept test (p > 0.05 = no pleiotropy)
+
+### Interpreting MR Results
+
+#### Significant MR Causal Effect
+
+**IVW p-value < 0.05** suggests gene expression causally affects trait.
+
+**Direction interpretation:**
+
+- **Positive MR beta**: ↑ Expression causally increases trait
+- **Negative MR beta**: ↑ Expression causally decreases trait
+
+**Effect size interpretation:**
+
+```
+MR beta = 0.15 for CAD risk
+Interpretation: 1 SD increase in gene expression causes 15% increase in CAD risk
+Therapeutic implication: Inhibiting expression by 1 SD could reduce CAD risk by 15%
+```
+
+#### Checking MR Assumption Violations
+
+**Pleiotropy tests:**
+
+1. **MR-Egger intercept test**
+   - Null hypothesis: No directional pleiotropy (intercept = 0)
+   - If p < 0.05: Pleiotropy detected, IVW estimate may be biased
+   - Action: Use MR-Egger slope (less powerful) or MR-PRESSO to remove outliers
+
+2. **Heterogeneity test (Cochran's Q)**
+   - Tests whether causal estimates vary across instruments
+   - If Q p-value < 0.05: Heterogeneity present
+   - Action: Use random-effects IVW or investigate heterogeneity sources
+
+3. **Method consistency**
+   - Compare IVW, weighted median, MR-Egger, weighted mode
+   - If all methods agree on direction: Strong evidence
+   - If methods disagree: Assumption violations likely, interpret cautiously
+
+#### MR Sensitivity Analysis Example
+
+```
+Gene: PCSK9
+Outcome: LDL cholesterol
+
+IVW:             beta = +0.22, p = 1e-8   ✅ Significant, positive
+Weighted Median: beta = +0.19, p = 2e-5   ✅ Consistent direction
+MR-Egger:        beta = +0.18, p = 0.03   ✅ Consistent (weaker signal)
+MR-Egger intercept: 0.01, p = 0.45        ✅ No pleiotropy detected
+Cochran's Q:     p = 0.12                 ✅ No heterogeneity
+
+Interpretation: Strong causal evidence that PCSK9 expression increases LDL.
+Therapeutic strategy: INHIBIT PCSK9 (validated by Evolocumab, Alirocumab success)
+Confidence: HIGH
+```
+
+### When MR is Essential vs. Optional
+
+**MR is ESSENTIAL for:**
+
+- High-stakes drug development decisions (large investment)
+- Genes with weak colocalization (PP.H4 < 0.8)
+- Conflicting signals from other evidence sources
+- Genes with known pleiotropic effects
+
+**MR is OPTIONAL (TWAS + colocalization sufficient) for:**
+
+- Exploratory target discovery
+- Strong colocalization already present (PP.H4 > 0.9)
+- Rapid screening of many targets
+- Limited resources (MR is time-intensive)
+
+---
+
+## Real-World Success Stories
+
+### Example 1: IL6R and Inflammation
+
+**GWAS Finding:**
+
+- rs2228145 (Asp358Ala missense variant) associated with lower CRP and lower CAD
+  risk
+
+**TWAS Analysis:**
+
+- Higher IL6R expression → Higher inflammation markers
+- TWAS Z = +4.5, p = 1e-6
+
+**Directionality:**
+
+- Layer 1 (eQTL): Protective allele → Increased soluble IL6R (competitive
+  inhibitor)
+- Layer 2 (TWAS): Higher IL6R signaling → Higher inflammation
+- Pathway: Protective allele → ↑ sIL6R → ↓ IL6 signaling → ↓ Inflammation
+- Strategy: **INHIBIT IL6R signaling**
+
+**Clinical Validation:**
+
+- **Tocilizumab** (IL-6 receptor antagonist)
+- Approved for rheumatoid arthritis, giant cell arteritis
+- Validated TWAS-based therapeutic strategy
+
+**Confidence:** VERY HIGH (genetic + clinical evidence)
+
+---
+
+### Example 2: PCSK9 and LDL Cholesterol
+
+**GWAS Finding:**
+
+- Loss-of-function variants in PCSK9 → Lower LDL cholesterol, lower CAD risk
+
+**TWAS Analysis:**
+
+- Higher PCSK9 expression → Higher LDL cholesterol
+- TWAS Z = +3.8, p = 2e-5
+- Colocalization PP.H4 = 0.88
+
+**Directionality:**
+
+- Layer 1 (eQTL): Risk allele → Higher PCSK9 expression
+- Layer 2 (TWAS): Higher PCSK9 → Higher LDL
+- Pathway: Risk allele → ↑ PCSK9 → ↑ LDL degradation → ↑ LDL levels
+- Strategy: **INHIBIT PCSK9**
+
+**Mendelian Randomization:**
+
+- IVW: beta = +0.22 SD LDL per SD PCSK9, p = 1e-8
+- No pleiotropy detected (MR-Egger intercept p = 0.45)
+
+**Clinical Validation:**
+
+- **Evolocumab, Alirocumab** (PCSK9 inhibitors)
+- Blockbuster drugs approved for hypercholesterolemia
+- Reduce LDL by 50-60%, reduce CVD events by 15-20%
+
+**Confidence:** VERY HIGH (genetic + MR + clinical evidence)
+
+---
+
+### Example 3: HMGCR and Statins
+
+**TWAS Analysis:**
+
+- Higher HMGCR expression → Higher LDL cholesterol
+- TWAS Z = +3.2, p = 5e-5
+
+**Directionality:**
+
+- HMGCR encodes HMG-CoA reductase (rate-limiting enzyme in cholesterol
+  synthesis)
+- Higher expression → More cholesterol production → Higher LDL
+- Strategy: **INHIBIT HMGCR enzyme**
+
+**Clinical Validation:**
+
+- **Statins** (Atorvastatin, Simvastatin, etc.)
+- Most prescribed cardiovascular drugs worldwide
+- HMGCR inhibitors reduce LDL by 30-50%
+
+**Confidence:** VERY HIGH (validated mechanism)
+
+---
+
+## Common Pitfalls and Red Flags
+
+### Pitfall 1: Ignoring Colocalization
+
+**Problem:** Up to 50% of TWAS hits are LD artifacts where GWAS and eQTL signals
+have distinct causal variants in the same region.
+
+**Red flag:** TWAS association present, but PP.H4 < 0.5
+
+**Solution:** Always perform colocalization testing. Only trust TWAS hits with
+PP.H4 > 0.8.
+
+---
+
+### Pitfall 2: Allele Harmonization Errors
+
+**Problem:** Effect alleles not properly aligned between eQTL and GWAS data,
+leading to incorrect directionality.
+
+**Red flag:** Directionality is inconsistent across layers, or pathway doesn't
+make biological sense.
+
+**Solution:**
+
+- Carefully harmonize effect alleles (flip beta signs when alleles are reversed)
+- Remove ambiguous SNPs (A/T, G/C) that can't be strand-aligned
+- Use automated harmonization tools (e.g., TwoSampleMR R package)
+
+---
+
+### Pitfall 3: Weak MR Instruments
+
+**Problem:** eQTL associations are weak (F-statistic < 10), leading to weak
+instrument bias in MR.
+
+**Red flag:** F-statistic < 10, very wide confidence intervals in MR
+
+**Solution:**
+
+- Use more liberal p-value threshold for instrument selection (e.g., p < 5e-5
+  instead of 5e-8)
+- Use larger eQTL studies (GTEx v8 preferred over smaller cohorts)
+- Consider using TWAS predicted expression as instrument instead of individual
+  SNPs
+
+---
+
+### Pitfall 4: Horizontal Pleiotropy
+
+**Problem:** eQTL variants affect the trait through pathways other than gene
+expression, violating MR assumptions.
+
+**Red flag:**
+
+- MR-Egger intercept p < 0.05
+- Large heterogeneity (Cochran's Q p < 0.05)
+- IVW and weighted median/MR-Egger show opposite directions
+
+**Solution:**
+
+- Use MR-PRESSO to detect and remove outlier SNPs
+- Report MR-Egger and weighted median as sensitivity analyses
+- If pleiotropy persists, acknowledge limitation and interpret cautiously
+
+---
+
+### Pitfall 5: Tissue Mismatch
+
+**Problem:** eQTL data from non-disease-relevant tissue may not reflect
+expression changes in target tissue.
+
+**Red flag:** TWAS signal only present in tissue unrelated to disease
+pathophysiology.
+
+**Example:** Liver eQTL used for brain disease → expression regulation may
+differ in brain
+
+**Solution:**
+
+- Use disease-relevant tissues (e.g., brain eQTL for neurological diseases)
+- If tissue-specific eQTL unavailable, use cross-tissue meta-analysis
+  (S-MultiXcan)
+- Validate expression directionality in disease-relevant tissue with
+  experimental data
+
+---
+
+### Pitfall 6: Reverse Causation
+
+**Problem:** Disease affects gene expression, not the other way around.
+
+**Red flag:** eQTL was measured in case-control study (disease status may affect
+expression).
+
+**Solution:**
+
+- Use eQTL from healthy populations (GTEx is healthy donors)
+- MR naturally addresses reverse causation (genetic variants precede disease)
+- If eQTL from cases, acknowledge potential for reverse causation
+
+---
+
+## Decision Framework for Drug Modality
+
+### Inhibition Strategies (Easier)
+
+**When to use:** TWAS shows higher expression increases disease risk (positive
+Z, risk trait)
+
+**Drug modalities:**
+
+1. **Small molecule inhibitors**
+   - Best for: Enzymes, kinases, GPCRs, ion channels
+   - Pros: Oral bioavailability, easier development
+   - Cons: Off-target effects if selectivity is poor
+   - **Example:** Statins (HMGCR inhibitors)
+
+2. **Monoclonal antibodies**
+   - Best for: Secreted proteins, cell surface receptors
+   - Pros: High specificity, long half-life
+   - Cons: IV/SC administration, expensive
+   - **Example:** Tocilizumab (anti-IL6R), Evolocumab (anti-PCSK9)
+
+3. **RNA interference (RNAi) / Antisense oligonucleotides (ASO)**
+   - Best for: Liver-expressed genes, "undruggable" targets
+   - Pros: Target any gene, high specificity
+   - Cons: Delivery challenges, expensive
+   - **Example:** Inclisiran (PCSK9 siRNA)
+
+4. **CRISPR/gene editing (future)**
+   - Best for: Permanent gene knockdown
+   - Pros: One-time treatment, permanent effect
+   - Cons: Delivery, off-target editing, regulatory hurdles
+
+**Druggability ranking:** Kinases/Enzymes > GPCRs > Ion Channels > Secreted
+Proteins > Transcription Factors
+
+---
+
+### Activation Strategies (Harder)
+
+**When to use:** TWAS shows higher expression decreases disease risk (negative
+Z, risk trait)
+
+**Drug modalities:**
+
+1. **Small molecule agonists/activators**
+   - Best for: GPCRs, nuclear receptors, enzymes
+   - Pros: Oral bioavailability
+   - Cons: Fewer druggable targets for activation
+   - **Example:** GLP-1 agonists (activate GLP1R for diabetes)
+
+2. **Gene therapy / overexpression**
+   - Best for: Loss-of-function diseases, monogenic conditions
+   - Pros: Permanent expression increase
+   - Cons: Delivery challenges, immunogenicity, very expensive
+   - **Example:** Factor IX gene therapy for hemophilia B
+
+3. **Protein replacement therapy**
+   - Best for: Secreted proteins, enzymes
+   - Pros: Direct supplementation
+   - Cons: Frequent dosing, expensive, immunogenicity
+   - **Example:** Enzyme replacement for lysosomal storage diseases
+
+4. **Indirect activation (pathway agonism)**
+   - Best for: When direct activation is not feasible
+   - Pros: May be easier than direct activation
+   - Cons: Less specific, potential for off-target effects
+   - **Example:** Increase APOE by activating LXR pathway
+
+**Activation is generally harder than inhibition** because:
+
+- Fewer druggable mechanisms for increasing function
+- Risk of toxicity from over-activation
+- Delivery challenges for gene/protein therapy
+
+**Strategic consideration:** If TWAS suggests activation, also consider:
+
+1. Can we inhibit a **negative regulator** of the gene instead?
+2. Can we target a **downstream pathway** that the gene activates?
+3. Is the gene's function **enzyme-like** (easier to supplement)?
+
+---
+
+## Summary: Therapeutic Interpretation Workflow
+
+```
+┌─────────────────────────────────────────────────────────────────┐
+│  TWAS Hit Identified                                            │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 1: Check Colocalization                                   │
+│  → PP.H4 > 0.8?  Yes → Proceed    No → Likely LD artifact      │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 2: Multi-Layer Directional Analysis                      │
+│  → Extract eQTL direction (risk allele → expression)            │
+│  → Extract TWAS direction (expression → trait)                  │
+│  → Check consistency across layers                              │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 3: Determine Therapeutic Strategy                        │
+│  → Positive TWAS + Risk trait → INHIBIT                         │
+│  → Negative TWAS + Risk trait → ACTIVATE                        │
+│  → Check biological plausibility                                │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 4 (Optional): Mendelian Randomization                    │
+│  → Establish causal directionality                              │
+│  → Check F-statistic > 10 (strong instruments)                  │
+│  → Test pleiotropy (MR-Egger intercept)                         │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 5: Assess Confidence Level                               │
+│  → High: Strong TWAS + Coloc + Consistent + (MR)                │
+│  → Medium: Moderate evidence, needs validation                  │
+│  → Low: Weak evidence, deprioritize                             │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Step 6: Druggability Assessment                               │
+│  → Protein class (kinase, GPCR, enzyme?)                        │
+│  → Known drugs in class?                                        │
+│  → Inhibition vs activation feasibility                         │
+└─────────────────────────────────────────────────────────────────┘
+                           │
+                           ▼
+┌─────────────────────────────────────────────────────────────────┐
+│  Final Output: Therapeutic Target Report                       │
+│  → Gene                                                          │
+│  → Therapeutic strategy (Inhibit/Activate)                      │
+│  → Confidence level (High/Medium/Low)                           │
+│  → Drug modality recommendations                                │
+│  → Priority score                                               │
+└─────────────────────────────────────────────────────────────────┘
+```
+
+---
+
+## References
+
+1. Nelson MR, et al. (2015) The support of human genetic evidence for approved
+   drug indications. _Nat Genet_ 47:856-860.
+2. King EA, et al. (2019) Are drug targets with genetic support twice as likely
+   to be approved? _PLoS Genet_ 15:e1008489.
+3. Gusev A, et al. (2016) Integrative approaches for large-scale
+   transcriptome-wide association studies. _Nat Genet_ 48:245-252.
+4. Hemani G, et al. (2018) The MR-Base platform supports systematic causal
+   inference across the human phenome. _eLife_ 7:e34408.
+5. Giambartolomei C, et al. (2014) Bayesian test for colocalisation between
+   pairs of genetic association studies. _PLoS Genet_ 10:e1004383.
+6. Swerdlow DI, et al. (2016) Selecting instruments for Mendelian randomization
+   in the wake of genome-wide association studies. _Int J Epidemiol_
+   45:1600-1616.
+
+---
+
+**Document Version:** 1.0 **Last Updated:** 2026-01-28 **Maintainer:** Claude
+Code Workflow Development