@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-rare-disease-diagnosis/SKILL.md

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+---
+name: tooluniverse-rare-disease-diagnosis
+description: Provide differential diagnosis for patients with suspected rare diseases based on phenotype and genetic data. Matches symptoms to HPO terms, identifies candidate diseases from Orphanet/OMIM, prioritizes genes for testing, interprets variants of uncertain significance. Use when clinician asks about rare disease diagnosis, unexplained phenotypes, or genetic testing interpretation.
+---
+
+# Rare Disease Diagnosis Advisor
+
+Systematic diagnosis support for rare diseases using phenotype matching, gene panel prioritization, and variant interpretation across Orphanet, OMIM, HPO, ClinVar, and structure-based analysis.
+
+**KEY PRINCIPLES**:
+1. **Report-first approach** - Create report file FIRST, update progressively
+2. **Phenotype-driven** - Convert symptoms to HPO terms before searching
+3. **Multi-database triangulation** - Cross-reference Orphanet, OMIM, OpenTargets
+4. **Evidence grading** - Grade diagnoses by supporting evidence strength
+5. **Actionable output** - Prioritized differential diagnosis with next steps
+6. **Genetic counseling aware** - Consider inheritance patterns and family history
+7. **English-first queries** - Always use English terms in tool calls (phenotype descriptions, gene names, disease names), even if the user writes in another language. Only try original-language terms as a fallback. Respond in the user's language
+
+---
+
+## When to Use
+
+Apply when user asks:
+- "Patient has [symptoms], what rare disease could this be?"
+- "Unexplained developmental delay with [features]"
+- "WES found VUS in [gene], is this pathogenic?"
+- "What genes should we test for [phenotype]?"
+- "Differential diagnosis for [rare symptom combination]"
+
+---
+
+## Critical Workflow Requirements
+
+### 1. Report-First Approach (MANDATORY)
+
+1. **Create the report file FIRST**:
+   - File name: `[PATIENT_ID]_rare_disease_report.md`
+   - Initialize with all section headers
+   - Add placeholder text: `[Researching...]`
+
+2. **Progressively update** as you gather data
+
+3. **Output separate data files**:
+   - `[PATIENT_ID]_gene_panel.csv` - Prioritized genes for testing
+   - `[PATIENT_ID]_variant_interpretation.csv` - If variants provided
+
+### 2. Citation Requirements (MANDATORY)
+
+Every finding MUST include source:
+
+```markdown
+### Candidate Disease: Marfan Syndrome
+- **ORPHA**: ORPHA:558
+- **OMIM**: 154700
+- **Phenotype match**: 85% (17/20 HPO terms)
+- **Inheritance**: AD
+- **Gene**: FBN1
+
+*Source: Orphanet via `Orphanet_558`, OMIM via `OMIM_get_entry`*
+```
+
+---
+
+## Phase 0: Tool Verification
+
+**CRITICAL**: Verify tool parameters before calling.
+
+### Known Parameter Corrections
+
+| Tool | WRONG Parameter | CORRECT Parameter |
+|------|-----------------|-------------------|
+| `OpenTargets_get_associated_diseases_by_target_ensemblId` | `ensemblID` | `ensemblId` |
+| `ClinVar_get_variant_by_id` | `variant_id` | `id` |
+| `MyGene_query_genes` | `gene` | `q` |
+| `gnomAD_get_variant_frequencies` | `variant` | `variant_id` |
+
+---
+
+## Workflow Overview
+
+```
+Phase 1: Phenotype Standardization
+├── Convert symptoms to HPO terms
+├── Identify core vs. variable features
+└── Note age of onset, inheritance hints
+    ↓
+Phase 2: Disease Matching
+├── Orphanet phenotype search
+├── OMIM clinical synopsis match
+├── OpenTargets disease associations
+└── OUTPUT: Ranked differential diagnosis
+    ↓
+Phase 3: Gene Panel Identification
+├── Extract genes from top diseases
+├── Cross-reference expression (GTEx)
+├── Prioritize by evidence strength
+└── OUTPUT: Recommended gene panel
+    ↓
+Phase 3.5: Expression & Tissue Context (NEW)
+├── CELLxGENE: Cell-type specific expression
+├── ChIPAtlas: Regulatory context (TF binding)
+├── Tissue-specific gene networks
+└── OUTPUT: Expression validation
+    ↓
+Phase 3.6: Pathway Analysis (NEW)
+├── KEGG: Metabolic/signaling pathways
+├── Reactome: Biological processes
+├── IntAct: Protein-protein interactions
+└── OUTPUT: Biological context
+    ↓
+Phase 4: Variant Interpretation (if provided)
+├── ClinVar pathogenicity lookup
+├── gnomAD population frequency
+├── Protein domain/function impact
+├── ENCODE/ChIPAtlas: Regulatory variant impact
+└── OUTPUT: Variant classification
+    ↓
+Phase 5: Structure Analysis (for VUS)
+├── NvidiaNIM_alphafold2 → Predict structure
+├── Map variant to structure
+├── Assess functional domain impact
+└── OUTPUT: Structural evidence
+    ↓
+Phase 6: Literature Evidence (NEW)
+├── PubMed: Published studies
+├── BioRxiv/MedRxiv: Preprints
+├── OpenAlex: Citation analysis
+└── OUTPUT: Literature support
+    ↓
+Phase 7: Report Synthesis
+├── Prioritized differential diagnosis
+├── Recommended genetic testing
+├── Next steps for clinician
+└── OUTPUT: Final report
+```
+
+---
+
+## Phase 1: Phenotype Standardization
+
+### 1.1 Convert Symptoms to HPO Terms
+
+```python
+def standardize_phenotype(tu, symptoms_list):
+    """Convert clinical descriptions to HPO terms."""
+    hpo_terms = []
+    
+    for symptom in symptoms_list:
+        # Search HPO for matching terms
+        results = tu.tools.HPO_search_terms(query=symptom)
+        if results:
+            hpo_terms.append({
+                'original': symptom,
+                'hpo_id': results[0]['id'],
+                'hpo_name': results[0]['name'],
+                'confidence': 'exact' if symptom.lower() in results[0]['name'].lower() else 'partial'
+            })
+    
+    return hpo_terms
+```
+
+### 1.2 Phenotype Categories
+
+| Category | Examples | Weight |
+|----------|----------|--------|
+| **Core features** | Always present in disease | High |
+| **Variable features** | Present in >50% | Medium |
+| **Occasional features** | Present in <50% | Low |
+| **Age-specific** | Onset-dependent | Context |
+
+### 1.3 Output for Report
+
+```markdown
+## 1. Phenotype Analysis
+
+### 1.1 Standardized HPO Terms
+
+| Clinical Feature | HPO Term | HPO ID | Category |
+|------------------|----------|--------|----------|
+| Tall stature | Tall stature | HP:0000098 | Core |
+| Long fingers | Arachnodactyly | HP:0001166 | Core |
+| Heart murmur | Cardiac murmur | HP:0030148 | Variable |
+| Joint hypermobility | Joint hypermobility | HP:0001382 | Core |
+
+**Total HPO Terms**: 8
+**Onset**: Childhood
+**Family History**: Father with similar features (AD suspected)
+
+*Source: HPO via `HPO_search_terms`*
+```
+
+---
+
+## Phase 2: Disease Matching
+
+### 2.1 Orphanet Disease Search (NEW TOOLS)
+
+```python
+def match_diseases_orphanet(tu, symptom_keywords):
+    """Find rare diseases matching symptoms using Orphanet."""
+    candidate_diseases = []
+    
+    # Search Orphanet by disease keywords
+    for keyword in symptom_keywords:
+        results = tu.tools.Orphanet_search_diseases(
+            operation="search_diseases",
+            query=keyword
+        )
+        if results.get('status') == 'success':
+            candidate_diseases.extend(results['data']['results'])
+    
+    # Get genes for each disease
+    for disease in candidate_diseases:
+        orpha_code = disease.get('ORPHAcode')
+        genes = tu.tools.Orphanet_get_genes(
+            operation="get_genes",
+            orpha_code=orpha_code
+        )
+        disease['genes'] = genes.get('data', {}).get('genes', [])
+    
+    return deduplicate_and_rank(candidate_diseases)
+```
+
+### 2.2 OMIM Cross-Reference (NEW TOOLS)
+
+```python
+def cross_reference_omim(tu, orphanet_diseases, gene_symbols):
+    """Get OMIM details for diseases and genes."""
+    omim_data = {}
+    
+    # Search OMIM for each disease/gene
+    for gene in gene_symbols:
+        search_result = tu.tools.OMIM_search(
+            operation="search",
+            query=gene,
+            limit=5
+        )
+        if search_result.get('status') == 'success':
+            for entry in search_result['data'].get('entries', []):
+                mim_number = entry.get('mimNumber')
+                
+                # Get detailed entry
+                details = tu.tools.OMIM_get_entry(
+                    operation="get_entry",
+                    mim_number=str(mim_number)
+                )
+                
+                # Get clinical synopsis (phenotype features)
+                synopsis = tu.tools.OMIM_get_clinical_synopsis(
+                    operation="get_clinical_synopsis",
+                    mim_number=str(mim_number)
+                )
+                
+                omim_data[gene] = {
+                    'mim_number': mim_number,
+                    'details': details.get('data', {}),
+                    'clinical_synopsis': synopsis.get('data', {})
+                }
+    
+    return omim_data
+```
+
+### 2.3 DisGeNET Gene-Disease Associations (NEW TOOLS)
+
+```python
+def get_gene_disease_associations(tu, gene_symbols):
+    """Get gene-disease associations from DisGeNET."""
+    associations = {}
+    
+    for gene in gene_symbols:
+        # Get diseases associated with gene
+        result = tu.tools.DisGeNET_search_gene(
+            operation="search_gene",
+            gene=gene,
+            limit=20
+        )
+        
+        if result.get('status') == 'success':
+            associations[gene] = result['data'].get('associations', [])
+    
+    return associations
+
+def get_disease_genes_disgenet(tu, disease_name):
+    """Get all genes associated with a disease."""
+    result = tu.tools.DisGeNET_search_disease(
+        operation="search_disease",
+        disease=disease_name,
+        limit=30
+    )
+    return result.get('data', {}).get('associations', [])
+```
+
+### 2.4 Phenotype Overlap Scoring
+
+| Match Level | Score | Criteria |
+|-------------|-------|----------|
+| **Excellent** | >80% | Most core + variable features match |
+| **Good** | 60-80% | Core features match, some variable |
+| **Possible** | 40-60% | Some overlap, needs consideration |
+| **Unlikely** | <40% | Poor phenotype fit |
+
+### 2.5 Output for Report
+
+```markdown
+## 2. Differential Diagnosis
+
+### Top Candidate Diseases (Ranked by Phenotype Match)
+
+| Rank | Disease | ORPHA | OMIM | Match | Inheritance | Key Gene(s) |
+|------|---------|-------|------|-------|-------------|-------------|
+| 1 | Marfan syndrome | 558 | 154700 | 85% | AD | FBN1 |
+| 2 | Loeys-Dietz syndrome | 60030 | 609192 | 72% | AD | TGFBR1, TGFBR2 |
+| 3 | Ehlers-Danlos, vascular | 286 | 130050 | 65% | AD | COL3A1 |
+| 4 | Homocystinuria | 394 | 236200 | 58% | AR | CBS |
+
+### DisGeNET Gene-Disease Evidence
+
+| Gene | Associated Diseases | GDA Score | Evidence |
+|------|---------------------|-----------|----------|
+| FBN1 | Marfan syndrome, MASS phenotype | 0.95 | ★★★ Curated |
+| TGFBR1 | Loeys-Dietz syndrome | 0.89 | ★★★ Curated |
+| COL3A1 | vascular EDS | 0.91 | ★★★ Curated |
+
+*Source: DisGeNET via `DisGeNET_search_gene`*
+
+### Disease Details
+
+#### 1. Marfan Syndrome (★★★)
+
+**ORPHA**: 558 | **OMIM**: 154700 | **Prevalence**: 1-5/10,000
+
+**Phenotype Match Analysis**:
+| Patient Feature | Disease Feature | Match |
+|-----------------|-----------------|-------|
+| Tall stature | Present in 95% | ✓ |
+| Arachnodactyly | Present in 90% | ✓ |
+| Joint hypermobility | Present in 85% | ✓ |
+| Cardiac murmur | Aortic root dilation (70%) | Partial |
+
+**OMIM Clinical Synopsis** (via `OMIM_get_clinical_synopsis`):
+- **Cardiovascular**: Aortic root dilation, mitral valve prolapse
+- **Skeletal**: Scoliosis, pectus excavatum, tall stature
+- **Ocular**: Ectopia lentis, myopia
+
+**Diagnostic Criteria**: Ghent nosology (2010)
+- Aortic root dilation/dissection + FBN1 mutation = Diagnosis
+- Without genetic testing: systemic score ≥7 + ectopia lentis
+
+**Inheritance**: Autosomal dominant (25% de novo)
+
+*Source: Orphanet via `Orphanet_get_disease`, OMIM via `OMIM_get_entry`, DisGeNET*
+```
+
+---
+
+## Phase 3: Gene Panel Identification
+
+### 3.1 Extract Disease Genes
+
+```python
+def build_gene_panel(tu, candidate_diseases):
+    """Build prioritized gene panel from candidate diseases."""
+    genes = {}
+    
+    for disease in candidate_diseases:
+        for gene in disease['genes']:
+            if gene not in genes:
+                genes[gene] = {
+                    'symbol': gene,
+                    'diseases': [],
+                    'evidence_level': 'unknown'
+                }
+            genes[gene]['diseases'].append(disease['name'])
+    
+    return genes
+```
+
+### 3.1.1 ClinGen Gene-Disease Validity Check (NEW)
+
+**Critical**: Always verify gene-disease validity through ClinGen before including in panel.
+
+```python
+def get_clingen_gene_evidence(tu, gene_symbol):
+    """
+    Get ClinGen gene-disease validity and dosage sensitivity.
+    ESSENTIAL for rare disease gene panel prioritization.
+    """
+    
+    # 1. Gene-disease validity classification
+    validity = tu.tools.ClinGen_search_gene_validity(gene=gene_symbol)
+    
+    validity_levels = []
+    diseases_with_validity = []
+    if validity.get('data'):
+        for entry in validity.get('data', []):
+            validity_levels.append(entry.get('Classification'))
+            diseases_with_validity.append({
+                'disease': entry.get('Disease Label'),
+                'mondo_id': entry.get('Disease ID (MONDO)'),
+                'classification': entry.get('Classification'),
+                'inheritance': entry.get('Inheritance')
+            })
+    
+    # 2. Dosage sensitivity (critical for CNV interpretation)
+    dosage = tu.tools.ClinGen_search_dosage_sensitivity(gene=gene_symbol)
+    
+    hi_score = None
+    ts_score = None
+    if dosage.get('data'):
+        for entry in dosage.get('data', []):
+            hi_score = entry.get('Haploinsufficiency Score')
+            ts_score = entry.get('Triplosensitivity Score')
+            break
+    
+    # 3. Clinical actionability (return of findings context)
+    actionability = tu.tools.ClinGen_search_actionability(gene=gene_symbol)
+    is_actionable = (actionability.get('adult_count', 0) > 0 or 
+                     actionability.get('pediatric_count', 0) > 0)
+    
+    # Determine best evidence level
+    level_priority = ['Definitive', 'Strong', 'Moderate', 'Limited', 'Disputed', 'Refuted']
+    best_level = 'Not curated'
+    for level in level_priority:
+        if level in validity_levels:
+            best_level = level
+            break
+    
+    return {
+        'gene': gene_symbol,
+        'evidence_level': best_level,
+        'diseases_curated': diseases_with_validity,
+        'haploinsufficiency_score': hi_score,
+        'triplosensitivity_score': ts_score,
+        'is_actionable': is_actionable,
+        'include_in_panel': best_level in ['Definitive', 'Strong', 'Moderate']
+    }
+
+def prioritize_genes_with_clingen(tu, gene_list):
+    """Prioritize genes using ClinGen evidence levels."""
+    
+    prioritized = []
+    for gene in gene_list:
+        evidence = get_clingen_gene_evidence(tu, gene)
+        
+        # Score based on ClinGen classification
+        score = 0
+        if evidence['evidence_level'] == 'Definitive':
+            score = 5
+        elif evidence['evidence_level'] == 'Strong':
+            score = 4
+        elif evidence['evidence_level'] == 'Moderate':
+            score = 3
+        elif evidence['evidence_level'] == 'Limited':
+            score = 1
+        # Disputed/Refuted get 0
+        
+        # Bonus for haploinsufficiency score 3
+        if evidence['haploinsufficiency_score'] == '3':
+            score += 1
+        
+        # Bonus for actionability
+        if evidence['is_actionable']:
+            score += 1
+        
+        prioritized.append({
+            **evidence,
+            'priority_score': score
+        })
+    
+    # Sort by priority score
+    return sorted(prioritized, key=lambda x: x['priority_score'], reverse=True)
+```
+
+**ClinGen Classification Impact on Panel**:
+| Classification | Include in Panel? | Priority |
+|----------------|-------------------|----------|
+| **Definitive** | YES - mandatory | Highest |
+| **Strong** | YES - highly recommended | High |
+| **Moderate** | YES | Medium |
+| **Limited** | Include but flag | Low |
+| **Disputed** | Exclude or separate | Avoid |
+| **Refuted** | EXCLUDE | Do not test |
+| **Not curated** | Use other evidence | Variable |
+
+### 3.2 Gene Prioritization Criteria
+
+| Priority | Criteria | Points |
+|----------|----------|--------|
+| **Tier 1** | Gene causes #1 ranked disease | +5 |
+| **Tier 2** | Gene causes multiple candidates | +3 |
+| **Tier 3** | ClinGen "Definitive" evidence | +3 |
+| **Tier 4** | Expressed in affected tissue | +2 |
+| **Tier 5** | Constraint score pLI >0.9 | +1 |
+
+### 3.3 Expression Validation
+
+```python
+def validate_expression(tu, gene_symbol, affected_tissue):
+    """Check if gene is expressed in relevant tissue."""
+    # Get Ensembl ID
+    gene_info = tu.tools.MyGene_query_genes(q=gene_symbol, species="human")
+    ensembl_id = gene_info.get('ensembl', {}).get('gene')
+    
+    # Check GTEx expression
+    expression = tu.tools.GTEx_get_median_gene_expression(
+        gencode_id=f"{ensembl_id}.latest"
+    )
+    
+    return expression.get(affected_tissue, 0) > 1  # TPM > 1
+```
+
+### 3.4 Output for Report
+
+```markdown
+## 3. Recommended Gene Panel
+
+### 3.1 Prioritized Genes for Testing
+
+| Priority | Gene | Diseases | Evidence | Constraint (pLI) | Expression |
+|----------|------|----------|----------|------------------|------------|
+| ★★★ | FBN1 | Marfan syndrome | Definitive | 1.00 | Heart, aorta |
+| ★★★ | TGFBR1 | Loeys-Dietz 1 | Definitive | 0.98 | Ubiquitous |
+| ★★★ | TGFBR2 | Loeys-Dietz 2 | Definitive | 0.99 | Ubiquitous |
+| ★★☆ | COL3A1 | EDS vascular | Definitive | 1.00 | Connective tissue |
+| ★☆☆ | CBS | Homocystinuria | Definitive | 0.00 | Liver |
+
+### 3.2 Panel Design Recommendation
+
+**Minimum Panel** (high yield): FBN1, TGFBR1, TGFBR2, COL3A1
+**Extended Panel** (+differential): Add CBS, SMAD3, ACTA2
+
+**Testing Strategy**:
+1. Start with FBN1 sequencing (highest pre-test probability)
+2. If negative, proceed to full connective tissue panel
+3. Consider WES if panel negative
+
+*Source: ClinGen via gene-disease validity, GTEx expression*
+```
+
+---
+
+## Phase 3.5: Expression & Tissue Context (ENHANCED)
+
+### 3.5.1 Cell-Type Specific Expression (CELLxGENE)
+
+```python
+def get_cell_type_expression(tu, gene_symbol, affected_tissues):
+    """Get single-cell expression to validate tissue relevance."""
+    
+    # Get expression across cell types
+    expression = tu.tools.CELLxGENE_get_expression_data(
+        gene=gene_symbol,
+        tissue=affected_tissues[0] if affected_tissues else "all"
+    )
+    
+    # Get cell type metadata
+    cell_metadata = tu.tools.CELLxGENE_get_cell_metadata(
+        gene=gene_symbol
+    )
+    
+    # Identify high-expression cell types
+    high_expression = [
+        ct for ct in expression 
+        if ct.get('mean_expression', 0) > 1.0  # TPM > 1
+    ]
+    
+    return {
+        'expression_data': expression,
+        'high_expression_cells': high_expression,
+        'total_cell_types': len(cell_metadata)
+    }
+```
+
+**Why it matters**: Confirms candidate genes are expressed in disease-relevant tissues/cells.
+
+### 3.5.2 Regulatory Context (ChIPAtlas)
+
+```python
+def get_regulatory_context(tu, gene_symbol):
+    """Get transcription factor binding for candidate genes."""
+    
+    # Search for TF binding near gene
+    tf_binding = tu.tools.ChIPAtlas_enrichment_analysis(
+        gene=gene_symbol,
+        cell_type="all"
+    )
+    
+    # Get specific binding peaks
+    peaks = tu.tools.ChIPAtlas_get_peak_data(
+        gene=gene_symbol,
+        experiment_type="TF"
+    )
+    
+    return {
+        'transcription_factors': tf_binding,
+        'regulatory_peaks': peaks
+    }
+```
+
+**Why it matters**: Identifies regulatory mechanisms that may be disrupted in disease.
+
+### 3.5.3 Output for Report
+
+```markdown
+## 3.5 Expression & Regulatory Context
+
+### Cell-Type Specific Expression (CELLxGENE)
+
+| Gene | Top Expressing Cell Types | Expression Level | Tissue Relevance |
+|------|---------------------------|------------------|------------------|
+| FBN1 | Fibroblasts, Smooth muscle | High (TPM=45) | ✓ Connective tissue |
+| TGFBR1 | Endothelial, Fibroblasts | Medium (TPM=12) | ✓ Vascular |
+| COL3A1 | Fibroblasts, Myofibroblasts | Very High (TPM=120) | ✓ Connective tissue |
+
+**Interpretation**: All top candidate genes show high expression in disease-relevant cell types (connective tissue, vascular cells), supporting their candidacy.
+
+### Regulatory Context (ChIPAtlas)
+
+| Gene | Key TF Regulators | Regulatory Significance |
+|------|-------------------|------------------------|
+| FBN1 | TGFβ pathway (SMAD2/3), AP-1 | TGFβ-responsive |
+| TGFBR1 | STAT3, NF-κB | Inflammation-responsive |
+
+*Source: CELLxGENE Census, ChIPAtlas*
+```
+
+---
+
+## Phase 3.6: Pathway Analysis (NEW)
+
+### 3.6.1 KEGG Pathway Context
+
+```python
+def get_pathway_context(tu, gene_symbols):
+    """Get pathway context for candidate genes."""
+    
+    pathways = {}
+    for gene in gene_symbols:
+        # Search KEGG for gene
+        kegg_genes = tu.tools.kegg_find_genes(query=f"hsa:{gene}")
+        
+        if kegg_genes:
+            # Get pathway membership
+            gene_info = tu.tools.kegg_get_gene_info(gene_id=kegg_genes[0]['id'])
+            pathways[gene] = gene_info.get('pathways', [])
+    
+    return pathways
+```
+
+### 3.6.2 Protein-Protein Interactions (IntAct)
+
+```python
+def get_protein_interactions(tu, gene_symbol):
+    """Get interaction partners for candidate genes."""
+    
+    # Search IntAct for interactions
+    interactions = tu.tools.intact_search_interactions(
+        query=gene_symbol,
+        species="human"
+    )
+    
+    # Get interaction network
+    network = tu.tools.intact_get_interaction_network(
+        gene=gene_symbol,
+        depth=1  # Direct interactors only
+    )
+    
+    return {
+        'interactions': interactions,
+        'network': network,
+        'interactor_count': len(interactions)
+    }
+```
+
+### 3.6.3 Output for Report
+
+```markdown
+## 3.6 Pathway & Network Context
+
+### KEGG Pathways
+
+| Gene | Key Pathways | Biological Process |
+|------|--------------|-------------------|
+| FBN1 | ECM-receptor interaction (hsa04512) | Extracellular matrix |
+| TGFBR1/2 | TGF-beta signaling (hsa04350) | Cell signaling |
+| COL3A1 | Focal adhesion (hsa04510) | Cell-matrix adhesion |
+
+### Shared Pathway Analysis
+
+**Convergent pathways** (≥2 candidate genes):
+- TGF-beta signaling pathway: FBN1, TGFBR1, TGFBR2, SMAD3
+- ECM organization: FBN1, COL3A1
+
+**Interpretation**: Candidate genes converge on TGF-beta signaling and extracellular matrix pathways, consistent with connective tissue disorder etiology.
+
+### Protein-Protein Interactions (IntAct)
+
+| Gene | Direct Interactors | Notable Partners |
+|------|-------------------|------------------|
+| FBN1 | 42 | LTBP1, TGFB1, ADAMTS10 |
+| TGFBR1 | 68 | TGFBR2, SMAD2, SMAD3 |
+
+*Source: KEGG, IntAct, Reactome*
+```
+
+---
+
+## Phase 4: Variant Interpretation (If Provided)
+
+### 4.1 ClinVar Lookup
+
+```python
+def interpret_variant(tu, variant_hgvs):
+    """Get ClinVar interpretation for variant."""
+    result = tu.tools.ClinVar_search_variants(query=variant_hgvs)
+    
+    return {
+        'clinvar_id': result.get('id'),
+        'classification': result.get('clinical_significance'),
+        'review_status': result.get('review_status'),
+        'conditions': result.get('conditions'),
+        'last_evaluated': result.get('last_evaluated')
+    }
+```
+
+### 4.2 Population Frequency
+
+```python
+def check_population_frequency(tu, variant_id):
+    """Get gnomAD allele frequency."""
+    freq = tu.tools.gnomAD_get_variant_frequencies(variant_id=variant_id)
+    
+    # Interpret rarity
+    if freq['allele_frequency'] < 0.00001:
+        rarity = "Ultra-rare"
+    elif freq['allele_frequency'] < 0.0001:
+        rarity = "Rare"
+    elif freq['allele_frequency'] < 0.01:
+        rarity = "Low frequency"
+    else:
+        rarity = "Common (likely benign)"
+    
+    return freq, rarity
+```
+
+### 4.3 Computational Pathogenicity Prediction (ENHANCED)
+
+Use state-of-the-art prediction tools for VUS interpretation:
+
+```python
+def comprehensive_vus_prediction(tu, variant_info):
+    """
+    Combine multiple prediction tools for VUS classification.
+    Critical for rare disease variants not in ClinVar.
+    """
+    predictions = {}
+    
+    # 1. CADD - Deleteriousness (NEW API)
+    cadd = tu.tools.CADD_get_variant_score(
+        chrom=variant_info['chrom'],
+        pos=variant_info['pos'],
+        ref=variant_info['ref'],
+        alt=variant_info['alt'],
+        version="GRCh38-v1.7"
+    )
+    if cadd.get('status') == 'success':
+        predictions['cadd'] = {
+            'score': cadd['data'].get('phred_score'),
+            'interpretation': cadd['data'].get('interpretation'),
+            'acmg': 'PP3' if cadd['data'].get('phred_score', 0) >= 20 else 'neutral'
+        }
+    
+    # 2. AlphaMissense - DeepMind pathogenicity (NEW)
+    if variant_info.get('uniprot_id') and variant_info.get('aa_change'):
+        am = tu.tools.AlphaMissense_get_variant_score(
+            uniprot_id=variant_info['uniprot_id'],
+            variant=variant_info['aa_change']  # e.g., "E1541K"
+        )
+        if am.get('status') == 'success' and am.get('data'):
+            classification = am['data'].get('classification')
+            predictions['alphamissense'] = {
+                'score': am['data'].get('pathogenicity_score'),
+                'classification': classification,
+                'acmg': 'PP3 (strong)' if classification == 'pathogenic' else (
+                    'BP4 (strong)' if classification == 'benign' else 'neutral'
+                )
+            }
+    
+    # 3. EVE - Evolutionary prediction (NEW)
+    eve = tu.tools.EVE_get_variant_score(
+        chrom=variant_info['chrom'],
+        pos=variant_info['pos'],
+        ref=variant_info['ref'],
+        alt=variant_info['alt']
+    )
+    if eve.get('status') == 'success':
+        eve_scores = eve['data'].get('eve_scores', [])
+        if eve_scores:
+            predictions['eve'] = {
+                'score': eve_scores[0].get('eve_score'),
+                'classification': eve_scores[0].get('classification'),
+                'acmg': 'PP3' if eve_scores[0].get('eve_score', 0) > 0.5 else 'BP4'
+            }
+    
+    # 4. SpliceAI - Splice variant prediction (NEW)
+    # Use for intronic, synonymous, or exonic variants near splice sites
+    variant_str = f"chr{variant_info['chrom']}-{variant_info['pos']}-{variant_info['ref']}-{variant_info['alt']}"
+    splice = tu.tools.SpliceAI_predict_splice(
+        variant=variant_str,
+        genome="38"
+    )
+    if splice.get('data'):
+        max_score = splice['data'].get('max_delta_score', 0)
+        interpretation = splice['data'].get('interpretation', '')
+        
+        if max_score >= 0.8:
+            splice_acmg = 'PP3 (strong) - high splice impact'
+        elif max_score >= 0.5:
+            splice_acmg = 'PP3 (moderate) - splice impact'
+        elif max_score >= 0.2:
+            splice_acmg = 'PP3 (supporting) - possible splice effect'
+        else:
+            splice_acmg = 'BP7 (if synonymous) - no splice impact'
+        
+        predictions['spliceai'] = {
+            'max_delta_score': max_score,
+            'interpretation': interpretation,
+            'scores': splice['data'].get('scores', []),
+            'acmg': splice_acmg
+        }
+    
+    # Consensus for PP3/BP4
+    damaging = sum(1 for p in predictions.values() if 'PP3' in p.get('acmg', ''))
+    benign = sum(1 for p in predictions.values() if 'BP4' in p.get('acmg', ''))
+    
+    return {
+        'predictions': predictions,
+        'consensus': {
+            'damaging_count': damaging,
+            'benign_count': benign,
+            'pp3_applicable': damaging >= 2 and benign == 0,
+            'bp4_applicable': benign >= 2 and damaging == 0
+        }
+    }
+```
+
+### 4.4 ACMG Classification Criteria
+
+| Evidence Type | Criteria | Weight |
+|---------------|----------|--------|
+| **PVS1** | Null variant in gene where LOF is mechanism | Very Strong |
+| **PS1** | Same amino acid change as established pathogenic | Strong |
+| **PM2** | Absent from population databases | Moderate |
+| **PP3** | Computational evidence supports deleterious (AlphaMissense, CADD, EVE, SpliceAI) | Supporting |
+| **BA1** | Allele frequency >5% | Benign standalone |
+
+**Enhanced PP3 Evidence** (NEW):
+- **AlphaMissense pathogenic** (>0.564) = Strong PP3 support (~90% accuracy)
+- **CADD ≥20** + **EVE >0.5** = Multiple concordant predictions
+- Agreement from 2+ predictors strengthens PP3 evidence
+
+### 4.5 Output for Report
+
+```markdown
+## 4. Variant Interpretation
+
+### 4.1 Variant: FBN1 c.4621G>A (p.Glu1541Lys)
+
+| Property | Value | Interpretation |
+|----------|-------|----------------|
+| Gene | FBN1 | Marfan syndrome gene |
+| Consequence | Missense | Amino acid change |
+| ClinVar | VUS | Uncertain significance |
+| gnomAD AF | 0.000004 | Ultra-rare (PM2) |
+
+### 4.2 Computational Predictions (NEW)
+
+| Predictor | Score | Classification | ACMG Support |
+|-----------|-------|----------------|--------------|
+| **AlphaMissense** | 0.78 | Pathogenic | PP3 (strong) |
+| **CADD PHRED** | 28.5 | Top 0.1% deleterious | PP3 |
+| **EVE** | 0.72 | Likely pathogenic | PP3 |
+
+**Consensus**: 3/3 predictors concordant damaging → **Strong PP3 support**
+
+*Source: AlphaMissense, CADD API, EVE via Ensembl VEP*
+
+### 4.3 ACMG Evidence Summary
+
+| Criterion | Evidence | Strength |
+|-----------|----------|----------|
+| PM2 | Absent from gnomAD (AF < 0.00001) | Moderate |
+| PP3 | AlphaMissense + CADD + EVE concordant | Supporting (strong) |
+| PP4 | Phenotype highly specific for Marfan | Supporting |
+| PS4 | Multiple affected family members | Strong |
+
+**Preliminary Classification**: Likely Pathogenic (1 Strong + 1 Moderate + 2 Supporting)
+
+*Source: ClinVar, gnomAD, AlphaMissense, CADD, EVE*
+```
+
+---
+
+## Phase 5: Structure Analysis for VUS
+
+### 5.1 When to Perform Structure Analysis
+
+Perform when:
+- Variant is VUS or conflicting interpretations
+- Missense variant in critical domain
+- Novel variant not in databases
+- Additional evidence needed for classification
+
+### 5.2 Structure Prediction (NVIDIA NIM)
+
+```python
+def analyze_variant_structure(tu, protein_sequence, variant_position):
+    """Predict structure and analyze variant impact."""
+    
+    # Predict structure with AlphaFold2
+    structure = tu.tools.NvidiaNIM_alphafold2(
+        sequence=protein_sequence,
+        algorithm="mmseqs2",
+        relax_prediction=False
+    )
+    
+    # Extract pLDDT at variant position
+    variant_plddt = get_residue_plddt(structure, variant_position)
+    
+    # Check if in structured region
+    confidence = "High" if variant_plddt > 70 else "Low"
+    
+    return {
+        'structure': structure,
+        'variant_plddt': variant_plddt,
+        'confidence': confidence
+    }
+```
+
+### 5.3 Domain Impact Assessment
+
+```python
+def assess_domain_impact(tu, uniprot_id, variant_position):
+    """Check if variant affects functional domain."""
+    
+    # Get domain annotations
+    domains = tu.tools.InterPro_get_protein_domains(accession=uniprot_id)
+    
+    for domain in domains:
+        if domain['start'] <= variant_position <= domain['end']:
+            return {
+                'in_domain': True,
+                'domain_name': domain['name'],
+                'domain_function': domain['description']
+            }
+    
+    return {'in_domain': False}
+```
+
+### 5.4 Output for Report
+
+```markdown
+## 5. Structural Analysis
+
+### 5.1 Structure Prediction
+
+**Method**: AlphaFold2 via NVIDIA NIM
+**Protein**: Fibrillin-1 (FBN1)
+**Sequence Length**: 2,871 amino acids
+
+| Metric | Value | Interpretation |
+|--------|-------|----------------|
+| Mean pLDDT | 85.3 | High confidence overall |
+| Variant position pLDDT | 92.1 | Very high confidence |
+| Nearby domain | cbEGF-like domain 23 | Calcium-binding |
+
+### 5.2 Variant Location Analysis
+
+**Variant**: p.Glu1541Lys
+
+| Feature | Finding | Impact |
+|---------|---------|--------|
+| Domain | cbEGF-like domain 23 | Critical for calcium binding |
+| Conservation | 100% conserved across vertebrates | High constraint |
+| Structural role | Calcium coordination residue | Likely destabilizing |
+| Nearby pathogenic | p.Glu1540Lys (Pathogenic) | Adjacent residue |
+
+### 5.3 Structural Interpretation
+
+The variant p.Glu1541Lys:
+1. **Located in cbEGF domain** - These domains are critical for fibrillin-1 function
+2. **Glutamate → Lysine** - Charge reversal (negative to positive)
+3. **Calcium binding** - Glutamate at this position coordinates Ca2+
+4. **Adjacent pathogenic variant** - p.Glu1540Lys is classified Pathogenic
+
+**Structural Evidence**: Strong support for pathogenicity (PM1 - critical domain)
+
+*Source: NVIDIA NIM via `NvidiaNIM_alphafold2`, InterPro*
+```
+
+---
+
+## Phase 6: Literature Evidence (NEW)
+
+### 6.1 Published Literature (PubMed)
+
+```python
+def search_disease_literature(tu, disease_name, genes):
+    """Search for relevant published literature."""
+    
+    # Disease-specific search
+    disease_papers = tu.tools.PubMed_search_articles(
+        query=f'"{disease_name}" AND (genetics OR mutation OR variant)',
+        limit=20
+    )
+    
+    # Gene-specific searches
+    gene_papers = []
+    for gene in genes[:5]:  # Top 5 genes
+        papers = tu.tools.PubMed_search_articles(
+            query=f'"{gene}" AND rare disease AND pathogenic',
+            limit=10
+        )
+        gene_papers.extend(papers)
+    
+    return {
+        'disease_literature': disease_papers,
+        'gene_literature': gene_papers
+    }
+```
+
+### 6.2 Preprint Literature (BioRxiv/MedRxiv)
+
+```python
+def search_preprints(tu, disease_name, genes):
+    """Search preprints for cutting-edge findings."""
+    
+    # BioRxiv search
+    biorxiv = tu.tools.BioRxiv_search_preprints(
+        query=f"{disease_name} genetics",
+        limit=10
+    )
+    
+    # ArXiv for computational methods
+    arxiv = tu.tools.ArXiv_search_papers(
+        query=f"rare disease diagnosis {' OR '.join(genes[:3])}",
+        category="q-bio",
+        limit=5
+    )
+    
+    return {
+        'biorxiv': biorxiv,
+        'arxiv': arxiv
+    }
+```
+
+### 6.3 Citation Analysis (OpenAlex)
+
+```python
+def analyze_citations(tu, key_papers):
+    """Analyze citation network for key papers."""
+    
+    citation_analysis = []
+    for paper in key_papers[:5]:
+        # Get citation data
+        work = tu.tools.openalex_search_works(
+            query=paper['title'],
+            limit=1
+        )
+        if work:
+            citation_analysis.append({
+                'title': paper['title'],
+                'citations': work[0].get('cited_by_count', 0),
+                'year': work[0].get('publication_year')
+            })
+    
+    return citation_analysis
+```
+
+### 6.4 Output for Report
+
+```markdown
+## 6. Literature Evidence
+
+### 6.1 Key Published Studies
+
+| PMID | Title | Year | Citations | Relevance |
+|------|-------|------|-----------|-----------|
+| 32123456 | FBN1 variants in Marfan syndrome... | 2023 | 45 | Direct |
+| 31987654 | TGF-beta signaling in connective... | 2022 | 89 | Pathway |
+| 30876543 | Novel diagnostic criteria for... | 2021 | 156 | Diagnostic |
+
+### 6.2 Recent Preprints (Not Yet Peer-Reviewed)
+
+| Source | Title | Posted | Relevance |
+|--------|-------|--------|-----------|
+| BioRxiv | Novel FBN1 splice variant causes... | 2024-01 | Case report |
+| MedRxiv | Machine learning for Marfan... | 2024-02 | Diagnostic |
+
+**⚠️ Note**: Preprints have not undergone peer review. Use with caution.
+
+### 6.3 Evidence Summary
+
+| Evidence Type | Count | Strength |
+|---------------|-------|----------|
+| Case reports | 12 | Supporting |
+| Functional studies | 5 | Strong |
+| Clinical trials | 2 | Strong |
+| Reviews | 8 | Context |
+
+*Source: PubMed, BioRxiv, OpenAlex*
+```
+
+---
+
+## Report Template
+
+**File**: `[PATIENT_ID]_rare_disease_report.md`
+
+```markdown
+# Rare Disease Diagnostic Report
+
+**Patient ID**: [ID] | **Date**: [Date] | **Status**: In Progress
+
+---
+
+## Executive Summary
+[Researching...]
+
+---
+
+## 1. Phenotype Analysis
+### 1.1 Standardized HPO Terms
+[Researching...]
+### 1.2 Key Clinical Features
+[Researching...]
+
+---
+
+## 2. Differential Diagnosis
+### 2.1 Ranked Candidate Diseases
+[Researching...]
+### 2.2 Disease Details
+[Researching...]
+
+---
+
+## 3. Recommended Gene Panel
+### 3.1 Prioritized Genes
+[Researching...]
+### 3.2 Testing Strategy
+[Researching...]
+
+---
+
+## 4. Variant Interpretation (if applicable)
+### 4.1 Variant Details
+[Researching...]
+### 4.2 ACMG Classification
+[Researching...]
+
+---
+
+## 5. Structural Analysis (if applicable)
+### 5.1 Structure Prediction
+[Researching...]
+### 5.2 Variant Impact
+[Researching...]
+
+---
+
+## 6. Clinical Recommendations
+### 6.1 Diagnostic Next Steps
+[Researching...]
+### 6.2 Specialist Referrals
+[Researching...]
+### 6.3 Family Screening
+[Researching...]
+
+---
+
+## 7. Data Gaps & Limitations
+[Researching...]
+
+---
+
+## 8. Data Sources
+[Will be populated as research progresses...]
+```
+
+---
+
+## Evidence Grading
+
+| Tier | Symbol | Criteria | Example |
+|------|--------|----------|---------|
+| **T1** | ★★★ | Phenotype match >80% + gene match | Marfan with FBN1 mutation |
+| **T2** | ★★☆ | Phenotype match 60-80% OR likely pathogenic variant | Good phenotype fit |
+| **T3** | ★☆☆ | Phenotype match 40-60% OR VUS in candidate gene | Possible diagnosis |
+| **T4** | ☆☆☆ | Phenotype <40% OR uncertain gene | Low probability |
+
+---
+
+## Completeness Checklist
+
+### Phase 1: Phenotype
+- [ ] All symptoms converted to HPO terms
+- [ ] Core vs. variable features distinguished
+- [ ] Age of onset documented
+- [ ] Family history noted
+
+### Phase 2: Disease Matching
+- [ ] ≥5 candidate diseases identified (or all matching)
+- [ ] Phenotype overlap % calculated
+- [ ] Inheritance patterns noted
+- [ ] ORPHA and OMIM IDs provided
+
+### Phase 3: Gene Panel
+- [ ] ≥5 genes prioritized (or all from top diseases)
+- [ ] Evidence level for each gene (ClinGen)
+- [ ] Expression validation performed
+- [ ] Testing strategy recommended
+
+### Phase 4: Variant Interpretation (if applicable)
+- [ ] ClinVar classification retrieved
+- [ ] gnomAD frequency checked
+- [ ] ACMG criteria applied
+- [ ] Classification justified
+
+### Phase 5: Structure Analysis (if applicable)
+- [ ] Structure predicted (if VUS)
+- [ ] pLDDT confidence reported
+- [ ] Domain impact assessed
+- [ ] Structural evidence summarized
+
+### Phase 6: Recommendations
+- [ ] ≥3 next steps listed
+- [ ] Specialist referrals suggested
+- [ ] Family screening addressed
+
+---
+
+## Fallback Chains
+
+| Primary Tool | Fallback 1 | Fallback 2 |
+|--------------|------------|------------|
+| `Orphanet_search_by_hpo` | `OMIM_search` | PubMed phenotype search |
+| `ClinVar_get_variant` | `gnomAD_get_variant` | VEP annotation |
+| `NvidiaNIM_alphafold2` | `alphafold_get_prediction` | UniProt features |
+| `GTEx_expression` | `HPA_expression` | Tissue-specific literature |
+| `gnomAD_get_variant` | `ExAC_frequencies` | 1000 Genomes |
+
+---
+
+## Tool Reference
+
+See [TOOLS_REFERENCE.md](TOOLS_REFERENCE.md) for complete tool documentation.