@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-variant-interpretation/SKILL.md

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+---
+name: tooluniverse-variant-interpretation
+description: Systematic clinical variant interpretation from raw variant calls to ACMG-classified recommendations with structural impact analysis. Aggregates evidence from ClinVar, gnomAD, CIViC, UniProt, and PDB across ACMG criteria. Produces pathogenicity scores (0-100), clinical recommendations, and treatment implications. Use when interpreting genetic variants, classifying variants of uncertain significance (VUS), performing ACMG variant classification, or translating variant calls to clinical actionability.
+---
+
+---
+name: tooluniverse-variant-interpretation
+description: Systematic clinical variant interpretation from raw variant calls to ACMG-classified recommendations with structural impact analysis. Aggregates evidence from ClinVar, gnomAD, CIViC, UniProt, and PDB across ACMG criteria. Produces pathogenicity scores (0-100), clinical recommendations, and treatment implications. Use when interpreting genetic variants, classifying variants of uncertain significance (VUS), performing ACMG variant classification, or translating variant calls to clinical actionability.
+---
+
+# Clinical Variant Interpreter
+
+Systematic variant interpretation skill using ToolUniverse - from raw variant calls to ACMG-classified clinical recommendations with structural impact analysis.
+
+---
+
+## Problem This Skill Solves
+
+Clinical labs and researchers face critical challenges in variant interpretation:
+
+1. **Variant classification uncertainty** - VUS (Variants of Uncertain Significance) comprise 40-60% of clinical variants
+2. **Evidence aggregation burden** - Must integrate data from 10+ databases per variant
+3. **Structural context missing** - Traditional annotation ignores 3D protein impact
+4. **Clinical actionability unclear** - How does classification translate to patient care?
+
+**This skill provides**: A systematic workflow that combines population databases, functional predictions, structural analysis (via AlphaFold2), and literature evidence into ACMG-compliant interpretations with clear clinical recommendations.
+
+---
+
+## Key Principles
+
+1. **ACMG-Guided Classification** - Follow ACMG/AMP 2015 guidelines with explicit evidence codes
+2. **Structural Evidence Integration** - Use AlphaFold2 for novel structural impact analysis
+3. **Population Context** - gnomAD frequencies with ancestry-specific data
+4. **Gene-Disease Validity** - ClinGen curation status for clinical relevance
+5. **Actionable Output** - Clear recommendations, not just classifications
+6. **English-first queries** - Always use English terms in tool calls (gene names, variant descriptions, disease names), even if the user writes in another language. Only try original-language terms as a fallback. Respond in the user's language
+
+---
+
+## Triggers
+
+Use this skill when users:
+- Ask about variant interpretation or classification
+- Have VCF data needing clinical annotation
+- Ask "what does this variant mean clinically?"
+- Need ACMG classification for variants
+- Want structural impact analysis for missense variants
+- Ask about pathogenicity of specific variants
+
+---
+
+## Workflow Overview
+
+```
+┌─────────────────────────────────────────────────────────────────┐
+│                    VARIANT INTERPRETATION                        │
+├─────────────────────────────────────────────────────────────────┤
+│                                                                  │
+│  Phase 1: VARIANT IDENTITY                                       │
+│  ├── Normalize variant notation (HGVS)                          │
+│  ├── Map to gene, transcript, protein                           │
+│  └── Get consequence type (missense, nonsense, etc.)            │
+│                                                                  │
+│  Phase 2: CLINICAL DATABASES                                     │
+│  ├── ClinVar: Existing classifications                          │
+│  ├── gnomAD: Population frequencies (all + ancestry)            │
+│  ├── OMIM: Gene-disease associations                            │
+│  ├── ClinGen: Gene validity + dosage sensitivity (ENHANCED)     │
+│  │   └─ ClinGen_search_gene_validity, ClinGen_search_dosage     │
+│  └── SpliceAI: Splice variant prediction (NEW)                  │
+│                                                                  │
+│  Phase 2.5: REGULATORY CONTEXT (NEW - for non-coding variants)  │
+│  ├── ChIPAtlas: TF binding at position                          │
+│  ├── ENCODE: Regulatory elements (enhancers, promoters)         │
+│  ├── Conservation in regulatory regions                         │
+│  └── Functional annotation of regulatory impact                 │
+│                                                                  │
+│  Phase 3: COMPUTATIONAL PREDICTIONS                              │
+│  ├── SIFT/PolyPhen: Damaging predictions                        │
+│  ├── CADD: Deleteriousness score                                │
+│  ├── SpliceAI: Splice impact (if applicable)                    │
+│  └── Conservation: Cross-species alignment                      │
+│                                                                  │
+│  Phase 4: STRUCTURAL ANALYSIS (for VUS/novel missense)          │
+│  ├── Get protein structure (PDB or AlphaFold2)                  │
+│  ├── Map variant to structure                                   │
+│  ├── Assess domain/functional site impact                       │
+│  └── Predict structural destabilization                         │
+│                                                                  │
+│  Phase 4.5: EXPRESSION CONTEXT (NEW)                            │
+│  ├── CELLxGENE: Cell-type specific expression                   │
+│  ├── Tissue relevance to phenotype                              │
+│  └── Expression validation                                       │
+│                                                                  │
+│  Phase 5: LITERATURE EVIDENCE                                    │
+│  ├── PubMed: Functional studies                                 │
+│  ├── BioRxiv/MedRxiv: Recent preprints (NEW)                   │
+│  ├── Case reports: Phenotype correlations                       │
+│  └── Segregation data (if in literature)                        │
+│                                                                  │
+│  Phase 6: ACMG CLASSIFICATION                                    │
+│  ├── Apply evidence codes (PVS1, PM2, PP3, etc.)               │
+│  ├── Calculate classification                                   │
+│  ├── Identify limiting factors                                  │
+│  └── Generate clinical recommendations                          │
+│                                                                  │
+└─────────────────────────────────────────────────────────────────┘
+```
+
+---
+
+## Phase Details
+
+### Phase 1: Variant Identity & Normalization
+
+**Goal**: Standardize variant notation and determine molecular consequence
+
+**Tools**:
+| Tool | Purpose |
+|------|---------|
+| `myvariant_query` | Get variant annotations from MyVariant.info |
+| `Ensembl_get_variant_info` | Variant effect predictor data |
+| `NCBI_gene_search` | Gene information |
+
+**Key Information to Capture**:
+- HGVS notation (c. and p.)
+- Gene symbol and Ensembl ID
+- Transcript (canonical/MANE Select)
+- Consequence type
+- Amino acid change (for missense)
+- Exon/intron location
+
+### Phase 2: Clinical Database Queries
+
+**Goal**: Aggregate existing clinical knowledge
+
+**Tools**:
+| Tool | Purpose | Key Data |
+|------|---------|----------|
+| `clinvar_search` | Existing classifications | Classification, review status, submissions |
+| `gnomad_search` | Population frequency | AF, ancestry-specific AFs, homozygotes |
+| `OMIM_search`, `OMIM_get_entry` | Gene-disease | Inheritance, phenotypes |
+| `ClinGen_gene_validity` | Curation status | Gene-disease validity level |
+| `COSMIC_search_mutations` | **Somatic mutations (NEW)** | Cancer frequency, histology |
+| `DisGeNET_search_gene` | **Gene-disease associations (NEW)** | Evidence scores, sources |
+
+### 2.1 COSMIC for Somatic Context (NEW)
+
+For cancer variants, check COSMIC for somatic mutation frequency:
+
+```python
+def get_somatic_context(tu, gene_symbol, variant_aa):
+    """Get somatic mutation context from COSMIC."""
+    
+    # Search for specific mutation
+    cosmic = tu.tools.COSMIC_search_mutations(
+        operation="search",
+        terms=f"{gene_symbol} {variant_aa}",
+        max_results=20,
+        genome_build=38
+    )
+    
+    # Get all gene mutations for context
+    gene_mutations = tu.tools.COSMIC_get_mutations_by_gene(
+        operation="get_by_gene",
+        gene=gene_symbol,
+        max_results=100
+    )
+    
+    # Determine if it's a hotspot
+    mutation_counts = Counter(m['MutationAA'] for m in gene_mutations.get('results', []))
+    is_hotspot = variant_aa in [m[0] for m in mutation_counts.most_common(10)]
+    
+    return {
+        'cosmic_hits': cosmic.get('results', []),
+        'is_somatic_hotspot': is_hotspot,
+        'cancer_types': [m['PrimarySite'] for m in cosmic.get('results', [])],
+        'total_cosmic_count': cosmic.get('total_count', 0)
+    }
+```
+
+### 2.2 OMIM Gene-Disease Context (NEW)
+
+```python
+def get_omim_context(tu, gene_symbol):
+    """Get OMIM gene-disease associations."""
+    
+    # Search OMIM for gene
+    search = tu.tools.OMIM_search(
+        operation="search",
+        query=gene_symbol,
+        limit=5
+    )
+    
+    omim_data = []
+    for entry in search.get('data', {}).get('entries', []):
+        mim = entry.get('mimNumber')
+        
+        # Get detailed entry
+        details = tu.tools.OMIM_get_entry(
+            operation="get_entry",
+            mim_number=str(mim)
+        )
+        
+        # Get clinical synopsis
+        synopsis = tu.tools.OMIM_get_clinical_synopsis(
+            operation="get_clinical_synopsis",
+            mim_number=str(mim)
+        )
+        
+        omim_data.append({
+            'mim_number': mim,
+            'title': details.get('data', {}).get('titles', {}),
+            'inheritance': synopsis.get('data', {}).get('inheritance'),
+            'clinical_features': synopsis.get('data', {})
+        })
+    
+    return omim_data
+```
+
+### 2.3 DisGeNET Gene-Disease Evidence (NEW)
+
+```python
+def get_disgenet_context(tu, gene_symbol, variant_rsid=None):
+    """Get gene-disease associations from DisGeNET."""
+    
+    # Gene-disease associations
+    gda = tu.tools.DisGeNET_search_gene(
+        operation="search_gene",
+        gene=gene_symbol,
+        limit=20
+    )
+    
+    # Variant-disease associations (if rsID available)
+    vda = None
+    if variant_rsid:
+        vda = tu.tools.DisGeNET_get_vda(
+            operation="get_vda",
+            variant=variant_rsid,
+            limit=20
+        )
+    
+    return {
+        'gene_associations': gda.get('data', {}).get('associations', []),
+        'variant_associations': vda.get('data', {}).get('associations', []) if vda else []
+    }
+```
+
+### 2.4 ClinGen Gene Validity & Dosage Sensitivity (NEW)
+
+ClinGen provides authoritative curation of gene-disease relationships:
+
+```python
+def get_clingen_evidence(tu, gene_symbol):
+    """
+    Get ClinGen gene validity and dosage sensitivity data.
+    CRITICAL for ACMG classification - establishes gene-disease validity.
+    """
+    
+    # 1. Gene-disease validity (Definitive/Strong/Moderate/Limited)
+    validity = tu.tools.ClinGen_search_gene_validity(gene=gene_symbol)
+    
+    validity_data = []
+    if validity.get('data'):
+        for entry in validity.get('data', []):
+            validity_data.append({
+                'disease': entry.get('Disease Label'),
+                'classification': entry.get('Classification'),  # Definitive, Strong, etc.
+                'inheritance': entry.get('Inheritance'),
+                'mondo_id': entry.get('Disease ID (MONDO)')
+            })
+    
+    # 2. Dosage sensitivity (haploinsufficiency, triplosensitivity)
+    dosage = tu.tools.ClinGen_search_dosage_sensitivity(gene=gene_symbol)
+    
+    dosage_data = {}
+    if dosage.get('data'):
+        for entry in dosage.get('data', []):
+            dosage_data = {
+                'haploinsufficiency_score': entry.get('Haploinsufficiency Score'),
+                'triplosensitivity_score': entry.get('Triplosensitivity Score'),
+                'disease': entry.get('Disease')
+            }
+            break  # Usually one entry per gene
+    
+    # 3. Clinical actionability (for incidental findings context)
+    actionability = tu.tools.ClinGen_search_actionability(gene=gene_symbol)
+    
+    return {
+        'gene_validity': validity_data,
+        'dosage_sensitivity': dosage_data,
+        'actionability': actionability.get('data', {}),
+        'has_definitive_validity': any(v['classification'] == 'Definitive' for v in validity_data),
+        'is_haploinsufficient': dosage_data.get('haploinsufficiency_score') == '3'
+    }
+```
+
+**ClinGen Validity Levels** (for ACMG PM1/PP4):
+| Classification | Meaning | ACMG Impact |
+|----------------|---------|-------------|
+| **Definitive** | Multiple concordant studies | Strong gene-disease support |
+| **Strong** | Extensive evidence | Moderate-strong support |
+| **Moderate** | Some evidence | Moderate support |
+| **Limited** | Minimal evidence | Weak support, use caution |
+| **Disputed** | Conflicting evidence | Do not use for classification |
+| **Refuted** | Evidence against | Gene NOT associated |
+
+**Dosage Sensitivity Scores** (for CNV interpretation):
+| Score | Meaning | Interpretation |
+|-------|---------|----------------|
+| **3** | Sufficient evidence | Haploinsufficiency/triplosensitivity established |
+| **2** | Emerging evidence | Some support, not definitive |
+| **1** | Little evidence | Minimal support |
+| **0** | No evidence | Unknown |
+
+### 2.5 SpliceAI Splice Variant Prediction (NEW)
+
+~15% of pathogenic variants affect splicing. SpliceAI is the gold standard for splice prediction:
+
+```python
+def get_spliceai_prediction(tu, chrom, pos, ref, alt, genome="38"):
+    """
+    Get SpliceAI splice effect predictions.
+    
+    Delta scores:
+    - DS_AG: Acceptor gain
+    - DS_AL: Acceptor loss  
+    - DS_DG: Donor gain
+    - DS_DL: Donor loss
+    
+    Thresholds:
+    - ≥0.8: High pathogenicity (strong PP3)
+    - 0.5-0.8: Moderate (supporting PP3)
+    - 0.2-0.5: Low (weak evidence)
+    - <0.2: Likely benign
+    """
+    
+    # Format variant for SpliceAI
+    variant = f"chr{chrom}-{pos}-{ref}-{alt}"
+    
+    # Get full splice predictions
+    result = tu.tools.SpliceAI_predict_splice(
+        variant=variant,
+        genome=genome
+    )
+    
+    if result.get('data'):
+        max_score = result['data'].get('max_delta_score', 0)
+        interpretation = result['data'].get('interpretation', '')
+        
+        # Determine ACMG support
+        if max_score >= 0.8:
+            acmg = 'PP3 (strong) - high splice impact'
+        elif max_score >= 0.5:
+            acmg = 'PP3 (supporting) - moderate splice impact'
+        elif max_score >= 0.2:
+            acmg = 'PP3 (weak) - possible splice impact'
+        else:
+            acmg = 'BP7 (if synonymous) - splice benign'
+        
+        return {
+            'max_delta_score': max_score,
+            'interpretation': interpretation,
+            'acmg_support': acmg,
+            'scores': result['data'].get('scores', [])
+        }
+    return None
+
+def quick_splice_check(tu, variant, genome="38"):
+    """Quick triage using max delta score only."""
+    
+    result = tu.tools.SpliceAI_get_max_delta(
+        variant=variant,
+        genome=genome
+    )
+    
+    return result.get('data', {})
+```
+
+**When to Use SpliceAI**:
+- **Intronic variants** near splice sites (±50bp)
+- **Synonymous variants** (may still affect splicing)
+- **Exonic variants** near splice junctions
+- **Variants creating cryptic splice sites**
+
+**Report Section for Splice Variants**:
+```markdown
+### Splice Impact Analysis (SpliceAI)
+
+| Score Type | Value | Position | Interpretation |
+|------------|-------|----------|----------------|
+| DS_AG | 0.02 | +15 | Acceptor gain unlikely |
+| DS_AL | 0.85 | -2 | **High acceptor loss** |
+| DS_DG | 0.01 | +8 | Donor gain unlikely |
+| DS_DL | 0.03 | +1 | Donor loss unlikely |
+
+**Max Delta Score**: 0.85 (DS_AL)
+**Interpretation**: High impact - likely disrupts acceptor site
+**ACMG Support**: PP3 (strong) for splice-altering effect
+
+*Source: SpliceAI via `SpliceAI_predict_splice`*
+```
+
+**ClinVar Classification Map**:
+| ClinVar | Interpretation |
+|---------|----------------|
+| Pathogenic | Disease-causing |
+| Likely pathogenic | 90%+ confidence pathogenic |
+| VUS | Uncertain significance |
+| Likely benign | 90%+ confidence benign |
+| Benign | Not disease-causing |
+| Conflicting | Multiple interpretations |
+
+**gnomAD Thresholds (for rare disease)**:
+| Frequency | ACMG Code | Interpretation |
+|-----------|-----------|----------------|
+| Absent | PM2_Supporting | Absent from controls |
+| <0.00001 | PM2_Supporting | Extremely rare |
+| <0.0001 | - | Rare (use with caution) |
+| >0.01 | BS1/BA1 | Too common for rare disease |
+
+**COSMIC Somatic Evidence (NEW)**:
+| COSMIC Finding | Interpretation | ACMG Support |
+|----------------|----------------|--------------|
+| Recurrent hotspot (>100 samples) | Known oncogenic driver | PS3 (functional) |
+| Moderate frequency (10-100) | Likely oncogenic | PM1 (hotspot) |
+| Rare somatic (<10) | Unknown significance | No support |
+
+**DisGeNET Score Interpretation (NEW)**:
+| GDA Score | Evidence Level | ACMG Support |
+|-----------|----------------|--------------|
+| >0.7 | Strong | PP4 (phenotype) |
+| 0.4-0.7 | Moderate | Supporting |
+| <0.4 | Weak | Insufficient |
+
+### Phase 2.5: Regulatory Context (NEW - for Non-Coding Variants)
+
+**Goal**: Assess regulatory impact for non-coding, intronic, and promoter variants
+
+**When to Apply**:
+- Intronic variants (not splice site)
+- Promoter variants
+- 5'UTR / 3'UTR variants
+- Intergenic variants near disease genes
+
+**Tools**:
+| Tool | Purpose | Key Data |
+|------|---------|----------|
+| `ChIPAtlas_enrichment_analysis` | TF binding at position | Bound TFs, cell types |
+| `ChIPAtlas_get_peak_data` | ChIP-seq peaks | Peak coordinates, scores |
+| `ENCODE_search_experiments` | Regulatory elements | Enhancers, promoters, DHS |
+| `ENCODE_get_experiment` | Experiment details | Assay type, targets |
+
+**Regulatory Impact Assessment**:
+
+```python
+def assess_regulatory_impact(tu, variant_position, gene_symbol):
+    """Assess regulatory impact of non-coding variant."""
+    
+    # Check TF binding at position
+    tf_binding = tu.tools.ChIPAtlas_enrichment_analysis(
+        gene=gene_symbol,
+        cell_type="all"
+    )
+    
+    # Get ChIP-seq peaks overlapping variant
+    peaks = tu.tools.ChIPAtlas_get_peak_data(
+        gene=gene_symbol,
+        experiment_type="TF"
+    )
+    
+    # Search ENCODE for regulatory annotations
+    encode_data = tu.tools.ENCODE_search_experiments(
+        assay_title="ATAC-seq",
+        biosample="all"
+    )
+    
+    # Assess if variant disrupts TF binding
+    binding_disrupted = check_motif_disruption(variant_position, peaks)
+    
+    return {
+        'tf_binding': tf_binding,
+        'regulatory_peaks': peaks,
+        'encode_annotations': encode_data,
+        'likely_regulatory': binding_disrupted
+    }
+```
+
+**Regulatory Impact Categories**:
+| Category | Criteria | ACMG Support |
+|----------|----------|--------------|
+| **High impact** | Disrupts known TF binding motif | PP3 (supporting) |
+| **Moderate impact** | In active regulatory region | Consider context |
+| **Low impact** | No regulatory annotation | No support |
+
+**Output for Report**:
+```markdown
+### 2.5 Regulatory Context (for Non-Coding Variants)
+
+| Feature | Finding | Significance |
+|---------|---------|--------------|
+| Variant location | Intron 5, 120bp from exon 6 | Not canonical splice |
+| TF binding site | CTCF binding peak (ChIPAtlas) | May affect insulation |
+| ENCODE annotation | Active enhancer (H3K27ac) | Regulatory function |
+| Conservation | PhyloP = 2.8 | Moderate conservation |
+
+**Regulatory Interpretation**: Variant overlaps CTCF binding site in active enhancer region. Potential impact on gene regulation.
+
+*Source: ChIPAtlas, ENCODE*
+```
+
+### Phase 3: Computational Predictions (ENHANCED)
+
+**Goal**: Assess in silico pathogenicity predictions using state-of-the-art models
+
+**Tools**:
+| Tool | Purpose | Score Range |
+|------|---------|-------------|
+| `CADD_get_variant_score` | **Deleteriousness score (NEW API)** | PHRED 0-99 |
+| `AlphaMissense_get_variant_score` | **DeepMind pathogenicity (NEW)** | 0-1 |
+| `EVE_get_variant_score` | **Evolutionary pathogenicity (NEW)** | 0-1 |
+| `myvariant_query` | Aggregated predictions | SIFT, PolyPhen |
+| `Ensembl_get_variant_info` | VEP predictions | SIFT, PolyPhen |
+
+### 3.1 CADD Deleteriousness Scoring (NEW)
+
+```python
+def get_cadd_score(tu, chrom, pos, ref, alt):
+    """Get CADD deleteriousness score for a variant."""
+    
+    result = tu.tools.CADD_get_variant_score(
+        chrom=str(chrom),
+        pos=pos,
+        ref=ref,
+        alt=alt,
+        version="GRCh38-v1.7"
+    )
+    
+    if result.get('status') == 'success':
+        phred = result['data'].get('phred_score')
+        return {
+            'score': phred,
+            'interpretation': result['data'].get('interpretation'),
+            'acmg_support': 'PP3' if phred >= 20 else ('BP4' if phred < 15 else 'neutral')
+        }
+    return None
+```
+
+### 3.2 AlphaMissense Pathogenicity (NEW)
+
+DeepMind's AlphaMissense provides state-of-the-art missense pathogenicity prediction:
+
+```python
+def get_alphamissense_score(tu, uniprot_id, variant):
+    """
+    Get AlphaMissense pathogenicity score.
+    variant format: 'R123H' or 'p.R123H'
+    
+    Thresholds:
+    - Pathogenic: score > 0.564
+    - Ambiguous: 0.34-0.564
+    - Benign: score < 0.34
+    """
+    
+    result = tu.tools.AlphaMissense_get_variant_score(
+        uniprot_id=uniprot_id,
+        variant=variant
+    )
+    
+    if result.get('status') == 'success' and result.get('data'):
+        score = result['data'].get('pathogenicity_score')
+        classification = result['data'].get('classification')
+        
+        # Map to ACMG
+        if classification == 'pathogenic':
+            acmg = 'PP3 (strong)'  # AlphaMissense has high accuracy
+        elif classification == 'benign':
+            acmg = 'BP4 (strong)'
+        else:
+            acmg = 'neutral'
+        
+        return {
+            'score': score,
+            'classification': classification,
+            'acmg_support': acmg
+        }
+    return None
+```
+
+### 3.3 EVE Evolutionary Prediction (NEW)
+
+EVE uses unsupervised learning on evolutionary data:
+
+```python
+def get_eve_score(tu, chrom, pos, ref, alt):
+    """
+    Get EVE evolutionary pathogenicity score.
+    
+    Threshold: >0.5 indicates likely pathogenic
+    """
+    
+    result = tu.tools.EVE_get_variant_score(
+        chrom=str(chrom),
+        pos=pos,
+        ref=ref,
+        alt=alt
+    )
+    
+    if result.get('status') == 'success':
+        eve_scores = result['data'].get('eve_scores', [])
+        if eve_scores:
+            best_score = eve_scores[0]
+            return {
+                'score': best_score.get('eve_score'),
+                'classification': best_score.get('classification'),
+                'gene': best_score.get('gene_symbol'),
+                'acmg_support': 'PP3' if best_score.get('eve_score', 0) > 0.5 else 'BP4'
+            }
+    return None
+```
+
+### 3.4 Integrated Prediction Strategy
+
+**For VUS (Variants of Uncertain Significance)**, combine multiple predictors:
+
+```python
+def comprehensive_pathogenicity_assessment(tu, variant_info):
+    """
+    Combine all prediction tools for robust classification.
+    """
+    chrom = variant_info['chrom']
+    pos = variant_info['pos']
+    ref = variant_info['ref']
+    alt = variant_info['alt']
+    uniprot_id = variant_info.get('uniprot_id')
+    aa_change = variant_info.get('aa_change')  # e.g., 'R123H'
+    
+    predictions = {}
+    
+    # 1. CADD (works for all variant types)
+    cadd = get_cadd_score(tu, chrom, pos, ref, alt)
+    if cadd:
+        predictions['cadd'] = cadd
+    
+    # 2. AlphaMissense (missense only, requires UniProt ID)
+    if uniprot_id and aa_change:
+        am = get_alphamissense_score(tu, uniprot_id, aa_change)
+        if am:
+            predictions['alphamissense'] = am
+    
+    # 3. EVE (missense only)
+    eve = get_eve_score(tu, chrom, pos, ref, alt)
+    if eve:
+        predictions['eve'] = eve
+    
+    # Consensus assessment
+    damaging_count = sum(1 for p in predictions.values() 
+                         if 'PP3' in p.get('acmg_support', ''))
+    benign_count = sum(1 for p in predictions.values() 
+                       if 'BP4' in p.get('acmg_support', ''))
+    
+    if damaging_count >= 2 and benign_count == 0:
+        consensus = 'likely_damaging'
+        acmg = 'PP3 (multiple predictors concordant)'
+    elif benign_count >= 2 and damaging_count == 0:
+        consensus = 'likely_benign'
+        acmg = 'BP4 (multiple predictors concordant)'
+    else:
+        consensus = 'uncertain'
+        acmg = 'neutral (discordant predictions)'
+    
+    return {
+        'predictions': predictions,
+        'consensus': consensus,
+        'acmg_recommendation': acmg
+    }
+```
+
+**Prediction Interpretation** (Updated):
+| Predictor | Damaging | Benign |
+|-----------|----------|--------|
+| **AlphaMissense** | >0.564 | <0.34 |
+| **CADD PHRED** | ≥20 (top 1%) | <15 |
+| **EVE** | >0.5 | ≤0.5 |
+| SIFT | <0.05 | ≥0.05 |
+| PolyPhen2 | >0.85 (probably) | <0.15 (benign) |
+
+**ACMG Application** (Enhanced):
+- **PP3**: Multiple concordant damaging predictions (AlphaMissense + CADD + EVE agreement = strong PP3)
+- **BP4**: Multiple concordant benign predictions
+- **Note**: AlphaMissense alone achieves ~90% accuracy on ClinVar pathogenic variants
+
+### Phase 4: Structural Analysis
+
+**Goal**: Assess protein structural impact (especially for VUS)
+
+**Tools**:
+| Tool | Purpose |
+|------|---------|
+| `PDB_search_by_uniprot` | Find experimental structures |
+| `NvidiaNIM_alphafold2` | Predict structure if no PDB |
+| `alphafold_get_prediction` | Get AlphaFold DB structure |
+| `InterPro_get_protein_domains` | Domain annotations |
+| `UniProt_get_protein_function` | Functional sites |
+
+**Structural Impact Categories**:
+
+| Impact Level | Description | ACMG Support |
+|--------------|-------------|--------------|
+| **Critical** | Active site, catalytic residue | PM1 (strong) |
+| **High** | Buried residue, disulfide, structural core | PM1 (moderate) |
+| **Moderate** | Domain interface, binding site | PM1 (supporting) |
+| **Low** | Surface, flexible region | No support |
+
+**Using AlphaFold2 for VUS**:
+```
+1. Get wildtype structure (PDB or AlphaFold)
+2. Identify residue location:
+   - pLDDT at position (confidence)
+   - Solvent accessibility
+   - Secondary structure
+3. Assess structural context:
+   - Distance to functional sites
+   - Interaction partners
+   - Conservation in structure
+4. Predict impact:
+   - Side chain burial
+   - Hydrogen bond disruption
+   - Charge changes in buried positions
+```
+
+### Phase 4.5: Expression Context (NEW)
+
+**Goal**: Validate gene expression in disease-relevant tissues/cells
+
+**Tools**:
+| Tool | Purpose | Key Data |
+|------|---------|----------|
+| `CELLxGENE_get_expression_data` | Cell-type specific expression | TPM per cell type |
+| `CELLxGENE_get_cell_metadata` | Cell type annotations | Tissue, disease state |
+| `GTEx_get_median_gene_expression` | Tissue expression | TPM per tissue |
+
+**Expression Validation**:
+
+```python
+def validate_expression_context(tu, gene_symbol, phenotype_tissues):
+    """Validate gene is expressed in phenotype-relevant tissues."""
+    
+    # Single-cell expression
+    sc_expression = tu.tools.CELLxGENE_get_expression_data(
+        gene=gene_symbol,
+        tissue=phenotype_tissues[0] if phenotype_tissues else "all"
+    )
+    
+    # Bulk tissue expression (GTEx)
+    gtex = tu.tools.GTEx_get_median_gene_expression(
+        gene=gene_symbol
+    )
+    
+    # Check expression in relevant tissues
+    relevant_expression = {
+        tissue: gtex.get(tissue, 0)
+        for tissue in phenotype_tissues
+    }
+    
+    return {
+        'single_cell': sc_expression,
+        'gtex': relevant_expression,
+        'expressed_in_phenotype_tissue': any(v > 1 for v in relevant_expression.values())
+    }
+```
+
+**Why it matters**:
+- Confirms gene is expressed where disease manifests
+- Supports PP4 (phenotype-specific) if highly restricted expression
+- Can challenge classification if not expressed in affected tissue
+
+**Output for Report**:
+```markdown
+### 4.5 Expression Context
+
+| Tissue | Expression (TPM) | Relevance |
+|--------|------------------|-----------|
+| Heart | 45.2 | ✓ Primary disease tissue |
+| Skeletal muscle | 38.7 | ✓ Secondary involvement |
+| Liver | 2.1 | Low expression |
+| Brain | 0.5 | Not expressed |
+
+**Single-Cell Analysis (CELLxGENE)**:
+- **Cardiomyocytes**: High expression (TPM=85)
+- **Cardiac fibroblasts**: Low expression (TPM=5)
+
+**Interpretation**: Gene highly expressed in cardiomyocytes, supporting cardiac phenotype association.
+
+*Source: GTEx, CELLxGENE Census*
+```
+
+### Phase 5: Literature Evidence (ENHANCED)
+
+**Goal**: Find functional studies, case reports, and cutting-edge preprints
+
+**Tools**:
+| Tool | Purpose | Coverage |
+|------|---------|----------|
+| `PubMed_search` | Peer-reviewed studies | Comprehensive |
+| `EuropePMC_search` | Additional literature | Europe PMC |
+| `BioRxiv_search_preprints` | Biology preprints | Recent findings |
+| `MedRxiv_search_preprints` | Clinical preprints | Clinical studies |
+| `openalex_search_works` | Citation analysis | Impact metrics |
+| `SemanticScholar_search_papers` | AI-ranked search | Relevance |
+
+**Search Strategies**:
+```python
+def comprehensive_literature_search(tu, gene, variant, phenotype):
+    """Search across all literature sources."""
+    
+    # 1. PubMed: Peer-reviewed
+    pubmed = tu.tools.PubMed_search(
+        query=f'"{gene}" AND ("{variant}" OR functional)',
+        max_results=30
+    )
+    
+    # 2. BioRxiv: Recent preprints
+    biorxiv = tu.tools.BioRxiv_search_preprints(
+        query=f"{gene} {phenotype}",
+        limit=10
+    )
+    
+    # 3. MedRxiv: Clinical preprints
+    medrxiv = tu.tools.MedRxiv_search_preprints(
+        query=f"{gene} variant {phenotype}",
+        limit=10
+    )
+    
+    # 4. Citation analysis
+    key_papers = pubmed[:5]  # Top papers
+    for paper in key_papers:
+        citations = tu.tools.openalex_search_works(
+            query=paper['title'],
+            limit=1
+        )
+        paper['citation_count'] = citations[0].get('cited_by_count', 0) if citations else 0
+    
+    return {
+        'pubmed': pubmed,
+        'preprints': biorxiv + medrxiv,
+        'key_papers_with_citations': key_papers
+    }
+```
+
+**Search Queries**:
+```
+# Gene + variant specific
+"{GENE} AND ({HGVS_p} OR {AA_change})"
+
+# Functional studies
+"{GENE} AND (functional OR functional study OR mutagenesis)"
+
+# Clinical reports
+"{GENE} AND (case report OR patient) AND {phenotype}"
+
+# Preprint-specific
+"{GENE} genetics 2024" (for recent preprints)
+```
+
+**⚠️ Preprint Warning**: Always flag preprints as NOT peer-reviewed in reports.
+
+**Evidence Types**:
+| Evidence | ACMG Code | Weight |
+|----------|-----------|--------|
+| Functional study (null) | PS3 | Strong |
+| Functional study (reduced) | PS3_Moderate | Moderate |
+| Case reports with segregation | PP1 | Supporting to Moderate |
+| Co-occurrence with pathogenic | BP2 | Supporting against |
+
+### Phase 6: ACMG Classification
+
+**Goal**: Systematic classification with explicit evidence
+
+**ACMG Evidence Codes**:
+
+**Pathogenic**:
+| Code | Strength | Description |
+|------|----------|-------------|
+| PVS1 | Very Strong | Null variant in gene where LOF is mechanism |
+| PS1 | Strong | Same amino acid change as known pathogenic |
+| PS3 | Strong | Well-established functional studies |
+| PM1 | Moderate | Mutational hot spot / functional domain |
+| PM2 | Moderate | Absent from controls |
+| PM5 | Moderate | Different missense at same residue as pathogenic |
+| PP3 | Supporting | Multiple computational predictions |
+| PP5 | Supporting | Reputable source reports pathogenic |
+
+**Benign**:
+| Code | Strength | Description |
+|------|----------|-------------|
+| BA1 | Stand-alone | MAF >5% |
+| BS1 | Strong | MAF greater than expected |
+| BS3 | Strong | Functional studies show no effect |
+| BP4 | Supporting | Multiple computational predictions benign |
+| BP7 | Supporting | Synonymous with no splice impact |
+
+**Classification Algorithm**:
+| Classification | Evidence Required |
+|----------------|-------------------|
+| Pathogenic | 1 Very Strong + 1 Strong; OR 2 Strong; OR 1 Strong + 3 Moderate |
+| Likely Pathogenic | 1 Very Strong + 1 Moderate; OR 1 Strong + 2 Moderate; OR 1 Strong + 2 Supporting |
+| Likely Benign | 1 Strong + 1 Supporting; OR 2 Supporting |
+| Benign | 1 Stand-alone; OR 2 Strong |
+| VUS | Criteria not met |
+
+---
+
+## Output Structure
+
+### Report Sections
+
+```markdown
+# Variant Interpretation Report: {GENE} {VARIANT}
+
+## Executive Summary
+- **Variant**: {HGVS notation}
+- **Gene**: {gene symbol}
+- **Classification**: {Pathogenic/Likely Pathogenic/VUS/Likely Benign/Benign}
+- **Evidence Strength**: {strong/moderate/limited}
+- **Key Finding**: {one-sentence summary}
+
+## 1. Variant Identity
+{gene, transcript, protein change, consequence}
+
+## 2. Population Data
+{gnomAD frequencies, ancestry breakdown}
+
+## 3. Clinical Database Evidence
+{ClinVar, ClinGen, OMIM}
+
+## 4. Computational Predictions
+{SIFT, PolyPhen, CADD scores}
+
+## 5. Structural Analysis
+{Domain location, functional site proximity, AlphaFold confidence}
+
+## 6. Literature Evidence
+{Functional studies, case reports}
+
+## 7. ACMG Classification
+{Evidence codes applied, classification rationale}
+
+## 8. Clinical Recommendations
+{Testing, management, family screening}
+
+## 9. Limitations & Uncertainties
+{Missing data, conflicting evidence}
+
+## Data Sources
+{All tools and databases queried}
+```
+
+---
+
+## Evidence Grading
+
+### Classification Confidence
+
+| Symbol | Classification | Evidence Level |
+|--------|----------------|----------------|
+| ★★★ | High confidence | Multiple independent lines |
+| ★★☆ | Moderate confidence | Some supporting evidence |
+| ★☆☆ | Limited confidence | Minimal evidence |
+| VUS | Uncertain | Insufficient data |
+
+### Structural Impact Confidence
+
+| pLDDT Range | Interpretation |
+|-------------|----------------|
+| >90 | Very high confidence in position |
+| 70-90 | High confidence |
+| 50-70 | Moderate (often loops) |
+| <50 | Low confidence (disorder) |
+
+---
+
+## Special Scenarios
+
+### Scenario 1: Novel Missense VUS
+
+**Additional workflow**:
+1. Check if other pathogenic variants at same residue
+2. Get AlphaFold2 structure
+3. Analyze:
+   - Is residue buried or surface?
+   - What secondary structure?
+   - Proximity to active/binding sites?
+   - Conservation across species?
+4. Apply PM1 if in functional domain
+5. Apply PP3 if predictions concordant
+
+### Scenario 2: Truncating Variant
+
+**Additional workflow**:
+1. Check if LOF is mechanism for gene
+2. Determine if escapes NMD (last exon)
+3. Check for alternative isoforms
+4. Review ClinGen LOF curation
+
+**PVS1 Application**:
+| Scenario | PVS1 Strength |
+|----------|---------------|
+| Canonical LOF gene, NMD predicted | Very Strong |
+| LOF gene, last exon | Moderate |
+| Non-LOF gene | Not applicable |
+
+### Scenario 3: Splice Variant
+
+**Additional workflow**:
+1. Check SpliceAI scores (if available)
+2. Determine canonical splice site distance
+3. Review for in-frame skipping potential
+4. Check for cryptic splice activation
+
+---
+
+## Quantified Minimums
+
+| Section | Requirement |
+|---------|-------------|
+| Population frequency | gnomAD overall + ≥3 ancestry groups |
+| Predictions | ≥3 computational predictors |
+| Literature search | ≥2 search strategies |
+| ACMG codes | All applicable codes listed |
+
+---
+
+## NVIDIA NIM Integration
+
+### When to Use AlphaFold2 for Variants
+
+**Use Case**: VUS missense variants where structural context aids interpretation
+
+**Workflow**:
+```python
+# 1. Get protein sequence
+protein_seq = tu.tools.UniProt_get_protein_sequence(accession=uniprot_id)
+
+# 2. Get/predict structure
+try:
+    pdb_hits = tu.tools.PDB_search_by_uniprot(uniprot_id=uniprot_id)
+    structure = tu.tools.PDB_get_structure(pdb_id=pdb_hits[0]['pdb_id'])
+except:
+    # Predict with AlphaFold2
+    structure = tu.tools.NvidiaNIM_alphafold2(
+        sequence=protein_seq['sequence'],
+        algorithm="mmseqs2"
+    )
+
+# 3. Analyze variant position
+# - Extract pLDDT at residue position
+# - Calculate solvent accessibility
+# - Check for nearby functional sites
+```
+
+**Structural Features to Report**:
+- pLDDT at variant position
+- Secondary structure (helix/sheet/coil)
+- Solvent accessibility (buried/exposed)
+- Distance to active site (if applicable)
+- Interactions disrupted (H-bonds, salt bridges)
+
+---
+
+## Report File Naming
+
+```
+{GENE}_{VARIANT}_interpretation_report.md
+
+Examples:
+BRCA1_c.5266dupC_interpretation_report.md
+TP53_p.R273H_interpretation_report.md
+```
+
+---
+
+## Clinical Recommendations Framework
+
+### For Pathogenic/Likely Pathogenic
+
+| Disease Context | Recommendations |
+|-----------------|-----------------|
+| Cancer predisposition | Enhanced screening, risk-reducing options |
+| Pharmacogenomics | Drug dosing adjustment |
+| Carrier status | Reproductive counseling |
+| Predictive testing | Family cascade screening |
+
+### For VUS
+
+| Action | Details |
+|--------|---------|
+| Clinical management | Do not use for medical decisions |
+| Follow-up | Reinterpret in 1-2 years |
+| Research | Functional studies if available |
+| Family | Segregation data valuable |
+
+### For Benign/Likely Benign
+
+| Action | Details |
+|--------|---------|
+| Clinical | Not expected to cause disease |
+| Family | No cascade testing needed |
+| Documentation | Include in report for completeness |
+
+---
+
+## See Also
+
+- `CHECKLIST.md` - Pre-delivery verification
+- `EXAMPLES.md` - Sample interpretations
+- `TOOLS_REFERENCE.md` - Tool parameters and fallbacks