A 28-year-old woman with a family history of hereditary breast and ovarian cancer (BRCA1/BRCA2 mutations) undergoes genetic testing. The initial PCR-based screening panel is negative. Which confirmatory investigation would be most appropriate to detect large genomic rearrangements or deletions that may have been missed?

## Confirmatory Investigation for BRCA Rearrangements ### Why MLPA is the Best Confirmatory Test **Key Point:** Multiplex ligation-dependent probe amplification (MLPA) is the gold standard confirmatory test for detecting large deletions and duplications in BRCA1/BRCA2 genes because: - Standard PCR fails to amplify large deletions (>100 bp) - MLPA uses multiple probes that bind to target sequences - It detects copy number variations (CNVs) with high sensitivity - It is cost-effective and faster than alternative methods - ~10–15% of BRCA mutations are large rearrangements missed by PCR alone ### Mechanism of MLPA ```mermaid flowchart TD A[DNA Sample]:::outcome --> B[Multiple MLPA Probes Bind]:::action B --> C[Ligation of Probes if Sequence Present]:::action C --> D{Copy Number Normal?}:::decision D -->|Yes| E[Probe Amplified by PCR]:::action D -->|No Deletion| F[No Ligation, No Amplification]:::outcome D -->|Duplication| G[Extra Probe Binding, Extra PCR Product]:::outcome E --> H[Fragment Analysis by Capillary Electrophoresis]:::action H --> I[Quantification of Copy Number]:::outcome ``` ### Comparison of Techniques for Rearrangement Detection | Technique | Detects | Sensitivity for Large Deletions | Speed | Cost | |-----------|---------|--------------------------------|-------|------| | **Standard PCR** | Point mutations, small indels | Poor (<100 bp) | Fast | Low | | **MLPA** | Copy number changes, large deletions/duplications | Excellent (>100 bp) | Moderate | Moderate | | **Northern Blot** | mRNA expression | No (DNA-level detection) | Slow | High | | **SNP Array** | Genome-wide CNVs | Good but less targeted | Moderate | High | ### Clinical Pearl **High-Yield:** Approximately 10–15% of pathogenic BRCA1/BRCA2 mutations are large genomic rearrangements (deletions or duplications). These are **invisible to standard PCR** because the primers cannot amplify across the deleted region. MLPA is specifically designed to detect these copy number variations. **Mnemonic:** **MLPA = M**ultiplex **L**igation **P**robe **A**mplification — uses multiple probes to detect what PCR misses. [cite:Harrison 21e Ch 81]