Clinical Variant Interpretation

Clinical Variant Interpretation

Prioritize and interpret variants for clinical significance using databases and ACMG/​AMP guidelines.

Interpretation Framework

Annotated VCF
    │
    ├── Database Lookup
    │   ├── ClinVar (clinical assertions)
    │   ├── OMIM (disease associations)
    │   └── gnomAD (population frequency)
    │
    ├── Computational Predictions
    │   ├── SIFT, PolyPhen-2
    │   ├── CADD, REVEL
    │   └── SpliceAI
    │
    ├── ACMG Classification
    │   └── Pathogenic → Likely Pathogenic → VUS → Likely Benign → Benign
    │
    └── Prioritized Variant List

ClinVar Annotation

Download ClinVar

wget https://ftp.ncbi.nlm.nih.gov/​pub/​clinvar/​vcf_GRCh38/​clinvar.vcf.gz
wget https://ftp.ncbi.nlm.nih.gov/​pub/​clinvar/​vcf_GRCh38/​clinvar.vcf.gz.tbi

Annotate with bcftools

bcftools annotate \
    -a clinvar.vcf.gz \
    -c INFO/​CLNSIG,INFO/​CLNDN,INFO/​CLNREVSTAT \
    input.vcf.gz -Oz -o with_clinvar.vcf.gz

Filter Pathogenic Variants

# Pathogenic or Likely pathogenic
bcftools view -i 'INFO/​CLNSIG~"Pathogenic" || INFO/​CLNSIG~"Likely_pathogenic"' \
    with_clinvar.vcf.gz -Oz -o pathogenic.vcf.gz

# Exclude benign
bcftools view -e 'INFO/​CLNSIG~"Benign" || INFO/​CLNSIG~"Likely_benign"' \
    with_clinvar.vcf.gz -Oz -o not_benign.vcf.gz

ClinVar Significance Levels

ClinVar Review Status

# Filter for high-confidence assertions (2+ stars)
bcftools view -i 'INFO/​CLNREVSTAT~"multiple_submitters" || \
    INFO/​CLNREVSTAT~"expert_panel" || \
    INFO/​CLNREVSTAT~"practice_guideline"' \
    with_clinvar.vcf.gz -Oz -o high_confidence.vcf.gz

InterVar (ACMG Classification)

Automated ACMG/​AMP variant classification.

Installation

git clone https://github.com/​WGLab/​InterVar.git
cd InterVar
# Download databases per documentation

Run InterVar

python Intervar.py \
    -i input.avinput \
    -o output \
    -b hg38 \
    -d humandb/ \
    --input_type=AVinput

From VCF

# Convert VCF to ANNOVAR format
convert2annovar.pl -format vcf4 input.vcf > input.avinput

# Run InterVar
python Intervar.py -i input.avinput -o intervar_results -b hg38

ACMG/​AMP Criteria

Pathogenic Criteria

Benign Criteria

Population Frequency Filtering

# Rare variants only (gnomAD AF < 0.01)
bcftools view -i 'INFO/​gnomAD_AF<0.01 || INFO/​gnomAD_AF="."' \
    input.vcf.gz -Oz -o rare.vcf.gz

# Ultra-rare for dominant diseases (AF < 0.0001)
bcftools view -i 'INFO/​gnomAD_AF<0.0001 || INFO/​gnomAD_AF="."' \
    input.vcf.gz -Oz -o ultrarare.vcf.gz

Pathogenicity Score Filtering

CADD Scores

# CADD > 20 (top 1% deleterious)
bcftools view -i 'INFO/​CADD_PHRED>20' input.vcf.gz -Oz -o cadd_filtered.vcf.gz

# CADD > 30 (top 0.1%)
bcftools view -i 'INFO/​CADD_PHRED>30' input.vcf.gz -Oz -o highly_deleterious.vcf.gz

REVEL Scores

# REVEL > 0.5 (likely pathogenic)
bcftools view -i 'INFO/​REVEL>0.5' input.vcf.gz -Oz -o revel_filtered.vcf.gz

Combined Filtering

bcftools view -i '(INFO/​CADD_PHRED>20 || INFO/​REVEL>0.5) && \
    (INFO/​CLNSIG~"Pathogenic" || INFO/​CLNSIG~"Likely" || INFO/​CLNSIG=".")' \
    input.vcf.gz -Oz -o prioritized.vcf.gz

Python: Clinical Prioritization

from cyvcf2 import VCF, Writer

def classify_variant(variant):
    clnsig = variant.INFO.get('CLNSIG', '')
    af = variant.INFO.get('gnomAD_AF', 0) or 0
    cadd = variant.INFO.get('CADD_PHRED', 0) or 0
    revel = variant.INFO.get('REVEL', 0) or 0

    # Known pathogenic
    if 'Pathogenic' in str(clnsig):
        return 'PATHOGENIC'
    if 'Likely_pathogenic' in str(clnsig):
        return 'LIKELY_PATHOGENIC'

    # Known benign
    if 'Benign' in str(clnsig) or af > 0.05:
        return 'BENIGN'

    # Computational prediction
    if cadd > 25 or revel > 0.7:
        if af < 0.0001:
            return 'LIKELY_PATHOGENIC'
        elif af < 0.01:
            return 'VUS_FAVOR_PATH'

    if cadd < 10 and revel < 0.3:
        return 'LIKELY_BENIGN'

    return 'VUS'

vcf = VCF('annotated.vcf.gz')
results = []

for variant in vcf:
    classification = classify_variant(variant)
    if classification in ('PATHOGENIC', 'LIKELY_PATHOGENIC', 'VUS_FAVOR_PATH'):
        gene = variant.INFO.get('SYMBOL', 'Unknown')
        consequence = variant.INFO.get('Consequence', 'Unknown')
        results.append({
            'chrom': variant.CHROM,
            'pos': variant.POS,
            'ref': variant.REF,
            'alt': variant.ALT[0],
            'gene': gene,
            'consequence': consequence,
            'classification': classification,
            'clnsig': variant.INFO.get('CLNSIG', '.'),
            'cadd': variant.INFO.get('CADD_PHRED', '.'),
            'af': variant.INFO.get('gnomAD_AF', '.')
        })

# Output prioritized variants
for r in results:
    print(f"{r['gene']}\t{r['chrom']}:{r['pos']}\t{r['consequence']}\t{r['classification']}")

Gene Panel Filtering

# Filter to gene panel
bcftools view -R gene_panel.bed input.vcf.gz -Oz -o panel_variants.vcf.gz

# Or by gene symbol (requires VEP annotation)
bcftools view -i 'INFO/​CSQ~"BRCA1" || INFO/​CSQ~"BRCA2"' \
    input.vcf.gz -Oz -o brca_variants.vcf.gz

Disease-Specific Resources

Reporting Template

bcftools query -f '%CHROM\t%POS\t%REF\t%ALT\t%INFO/​SYMBOL\t%INFO/​Consequence\t\
%INFO/​CLNSIG\t%INFO/​CLNDN\t%INFO/​gnomAD_AF\t%INFO/​CADD_PHRED\n' \
    prioritized.vcf.gz > clinical_report.tsv

Complete Workflow

#!/​bin/​bash
set -euo pipefail

INPUT=$1
CLINVAR=$2
OUTPUT_PREFIX=$3

echo "=== Add ClinVar annotations ==="
bcftools annotate -a $CLINVAR \
    -c INFO/​CLNSIG,INFO/​CLNDN,INFO/​CLNREVSTAT,INFO/​CLNVC \
    $INPUT -Oz -o ${OUTPUT_PREFIX}_clinvar.vcf.gz

echo "=== Filter rare variants ==="
bcftools view -i 'INFO/​gnomAD_AF<0.01 || INFO/​gnomAD_AF="."' \
    ${OUTPUT_PREFIX}_clinvar.vcf.gz -Oz -o ${OUTPUT_PREFIX}_rare.vcf.gz

echo "=== Extract pathogenic/​likely pathogenic ==="
bcftools view -i 'INFO/​CLNSIG~"athogenic"' \
    ${OUTPUT_PREFIX}_rare.vcf.gz -Oz -o ${OUTPUT_PREFIX}_pathogenic.vcf.gz

echo "=== Extract high-impact VUS ==="
bcftools view -i 'INFO/​CLNSIG~"Uncertain" && INFO/​CADD_PHRED>20' \
    ${OUTPUT_PREFIX}_rare.vcf.gz -Oz -o ${OUTPUT_PREFIX}_vus_review.vcf.gz

echo "=== Generate report ==="
bcftools query -H -f '%CHROM\t%POS\t%REF\t%ALT\t%INFO/​SYMBOL\t%INFO/​Consequence\t\
%INFO/​CLNSIG\t%INFO/​CLNDN\t%INFO/​gnomAD_AF\t%INFO/​CADD_PHRED\n' \
    ${OUTPUT_PREFIX}_pathogenic.vcf.gz > ${OUTPUT_PREFIX}_report.tsv

echo "=== Complete ==="
echo "Pathogenic: ${OUTPUT_PREFIX}_pathogenic.vcf.gz"
echo "VUS for review: ${OUTPUT_PREFIX}_vus_review.vcf.gz"
echo "Report: ${OUTPUT_PREFIX}_report.tsv"

Related Skills

• variant-calling/​variant-annotation - VEP/​SnpEff annotation
• variant-calling/​filtering-best-practices - Quality filtering
• database-access/​entrez-fetch - Download ClinVar/​OMIM data
• pathway-analysis/​go-enrichment - Gene set analysis