Key Points
- Gold standard for identifying specific genetic variants
- Multiple methodologies available based on clinical scenario
- Critical for confirming genetic diagnoses
- Essential for personalized medicine approaches
- Enables precise genetic counseling
Testing Methodologies
Sequencing Technologies
- Next-Generation Sequencing (NGS)
- Whole Genome Sequencing (WGS)
- Complete genomic coverage
- Detection of structural variants
- Identification of deep intronic variants
- Analysis of regulatory regions
- Whole Exome Sequencing (WES)
- Protein-coding regions analysis
- Cost-effective alternative to WGS
- Higher depth of coverage
- Focus on clinically relevant regions
- Targeted Gene Panels
- Disease-specific analysis
- Higher sensitivity for selected genes
- Reduced incidental findings
- Cost-effective for specific conditions
Traditional Methods
- Sanger Sequencing
- Gold standard for variant confirmation
- Single gene analysis
- Mutation-specific testing
- Family studies
- PCR-based Methods
- Allele-specific PCR
- ARMS-PCR
- Real-time PCR
- Digital PCR
Clinical Applications
Diagnostic Testing
- Monogenic Disorders
- Cystic fibrosis
- Muscular dystrophies
- Metabolic disorders
- Primary immunodeficiencies
- Complex Disorders
- Neurological conditions
- Cardiovascular diseases
- Cancer predisposition syndromes
- Developmental disorders
Screening Applications
- Presymptomatic Testing
- Family history assessment
- Risk stratification
- Early intervention planning
- Carrier Testing
- Reproductive planning
- Family studies
- Population screening
Variant Analysis and Interpretation
Variant Classification
- ACMG Guidelines
- Pathogenic
- Likely pathogenic
- Variant of uncertain significance (VUS)
- Likely benign
- Benign
- Evidence Types
- Population frequency data
- Computational predictions
- Functional studies
- Segregation analysis
- Clinical correlation
Advanced Analysis Considerations
Special Scenarios
- Mosaicism Detection
- Variant allele frequencies
- Tissue-specific analysis
- Deep sequencing approaches
- Complex Variants
- Structural variations
- Repeat expansions
- Regulatory region variants
- Splicing variants
Quality Assurance
Laboratory Standards
- Technical Parameters
- Coverage metrics
- Quality scores
- Variant calling parameters
- Confirmation protocols
- Validation Requirements
- Analytical validation
- Clinical validation
- Quality control measures
- Proficiency testing
Clinical Implementation
Testing Strategy
- Pre-test Considerations
- Clinical indication assessment
- Test selection criteria
- Cost considerations
- Turn-around time requirements
- Post-test Management
- Result interpretation
- Clinical correlation
- Follow-up recommendations
- Family implications
Limitations and Challenges
- Technical Limitations
- Coverage gaps
- Complex regions
- Structural variants
- Low-level variants
- Interpretation Challenges
- Novel variants
- Multiple variants
- Phenotype correlation
- Incidental findings
Further Reading
