A 32-year-old woman with a family history of cystic fibrosis (CF) is undergoing genetic screening. Initial mutation analysis by PCR detected the ΔF508 mutation in one allele. To identify the second mutation and confirm compound heterozygosity, which investigation is most appropriate?

## Investigation of Choice for Identifying Unknown CFTR Mutations ### Clinical Context The patient has one known mutation (ΔF508) but requires identification of the second allele to confirm compound heterozygosity and establish definitive CF diagnosis. ### Why DNA Sequencing is Optimal **Key Point:** DNA sequencing (Sanger or next-generation) is the gold standard for identifying unknown point mutations, insertions, deletions, and splice site variants across the entire CFTR coding sequence and regulatory regions. **High-Yield:** When one mutation is known but the second is unknown, direct sequencing of the CFTR gene is the most comprehensive and efficient approach—it detects all classes of mutations (point mutations, small indels, splice variants) in a single assay. ### Comparison of Techniques | Technique | Best For | Limitation in This Case | |-----------|----------|-------------------------| | **DNA Sequencing** | Identifying unknown mutations; detects all variant types | None—gold standard for unknown alleles | | Southern Blot | Detecting large deletions, rearrangements | Requires prior knowledge of probe location; poor resolution for point mutations | | qRT-PCR | Measuring mRNA expression levels | Detects quantity, not sequence; cannot identify mutations | | RFLP | Detecting known restriction sites | Requires known restriction enzyme site; misses mutations outside recognition sites | ### Mechanism 1. PCR amplifies CFTR exons (or whole gene via long-range PCR) 2. Sanger sequencing reads the DNA sequence directly 3. Comparison to reference sequence identifies all variants 4. Confirms compound heterozygosity (two different mutations) **Clinical Pearl:** In CF screening, once one mutation is identified, sequencing the entire CFTR gene is faster and more cost-effective than sequential testing with multiple targeted assays. **Mnemonic: CFTR Mutation Detection — "SEQUENCE for UNKNOWN"** - **S**equencing = gold standard for unknown variants - **E**xons amplified by PCR first - **Q**uantitative methods (qRT-PCR) measure expression, not mutations - **U**nknown alleles require comprehensive approach - **E**very mutation type (point, indel, splice) detected - **N**o prior knowledge of variant needed - **C**onfirms compound heterozygosity - **E**fficient single-step identification