A 'Negative' CFTR Result in a Child With Classic Symptoms
A 3-year-old boy has recurrent respiratory infections, steatorrhea, and failure to thrive; sweat chloride is 92 mmol/L on two occasions. A 50-variant CFTR genotyping panel returns negative (no variants detected). The referring physician concludes the child does not have cystic fibrosis and stops pancreatic enzyme replacement. The laboratory's panel uses allele-specific hybridization and does not detect copy-number variants or deep intronic variants. The child's parents are nonconsanguineous and of Northern European ancestry.
1. Explain why the negative panel does not exclude cystic fibrosis and what testing strategy should follow.
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The diagnosis of CF rests on clinical features plus evidence of CFTR dysfunction (sweat chloride ≥60 mmol/L is diagnostic in this context). A 50-variant panel interrogates only common alleles; it cannot detect the ~2,000+ described CFTR variants, large deletions/duplications, or deep intronic variants. A negative panel therefore has limited negative predictive value, and the pretest probability here is extremely high (classic phenotype plus two elevated sweat chlorides). The correct next step is CFTR full-gene sequencing with deletion/duplication analysis (MLPA or equivalent), which detects the majority of remaining alleles. If that is negative, consider CFTR-related testing for deep intronic variants or RNA analysis, and revisit the diagnosis only if CFTR dysfunction cannot be demonstrated. Discontinuing pancreatic enzymes was inappropriate.
2. If full-gene sequencing identifies one known pathogenic variant and no second variant, how would you proceed?
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Finding only one pathogenic variant in a child with a classic phenotype and diagnostic sweat chloride suggests the second allele is missed by the assay. Options: (1) confirm deletion/duplication analysis was performed and interpreted with appropriate probe coverage; (2) consider deep intronic or regulatory variants via RNA-based analysis (nasal or rectal epithelium) or extended sequencing; (3) consider that the second variant may lie in a region poorly covered or in a homologous region; (4) consider uniparental disomy or a large deletion if segregation studies show non-Mendelian inheritance. Importantly, the clinical diagnosis of CF is not contingent on identifying two variants-the sweat chloride is diagnostic-so management should continue while genetic confirmation is pursued for family counseling and carrier testing.
3. What validation limitation of an allele-specific hybridization panel is most relevant to this case, and how would you document it?
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Allele-specific hybridization/ASO panels are designed to detect predefined sequence variants and are blind to copy-number changes and to variants outside the targeted set. A key limitation is that a homozygous deletion of an exon or a large deletion removing probe-binding sites can produce a false-negative (no signal interpreted as wild type) or ambiguous result. Validation must establish: analytical sensitivity/specificity per variant, the ability to detect heterozygotes and homozygotes, and explicit documentation that CNVs and unlisted variants are not detected. The report and package insert should state the panel's scope and the residual risk, with a recommendation for reflex full-gene testing when clinical suspicion remains. This limitation should be captured in the laboratory's validation summary and communicated to ordering providers.