Cystic fibrosis risk after a “negative” carrier panel
A family with cystic fibrosis. Jonas (29) and Mira (28) attend genetic counselling before a planned pregnancy. Jonas's younger sister has cystic fibrosis (CF); his parents are unaffected. Mira has no family history. In their population the carrier frequency of CFTR variants is 1 in 25.
Both undergo a first-tier panel that detects the 32 most common CFTR variants and covers about 85 % of carrier alleles in this population. Both results are negative.
Jonas later brings a report issued to his affected sister by another laboratory. It states that F508del was excluded because “the PCR products of 302 bp (wild type) and 299 bp (F508del) were separated on a 2 % agarose gel and only a single band at 302 bp was seen”.
His sister is now 31 and has a son. The boy was born with meconium ileus and has a sweat chloride of 84 mmol/L (reference: < 30 normal, 30–59 intermediate, ≥ 60 diagnostic).
1. Calculate Jonas's and Mira's residual carrier risks after the negative panel, then the probability that a child of theirs would be affected. Show every step and state the assumptions.
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Step 1 - Jonas's prior risk. His parents are unaffected but have an affected child, so both are obligate carriers and, of the unaffected offspring, two-thirds are carriers: 2/3 ≈ 0.667.
Step 2 - adjust for the negative panel. A test that detects 85 % of carrier alleles misses 15 % of true carriers, so the residual risk is the prior multiplied by the miss rate: 2/3 × 0.15 = 0.10 (10 %).
Step 3 - Mira's prior risk is the population carrier frequency, 1/25 = 0.04. Residual after the same panel: 0.04 × 0.15 = 0.006 (0.6 %).
Step 4 - affected child. Both must be carriers and both must pass on the variant: 0.10 × 0.006 × 1/4 = 0.00015, i.e. about 1 in 6,700 (0.015 %).
Assumptions that must be stated: (i) the panel's 85 % detection applies to this population and to Jonas's family variant; (ii) the parents' variants are independent of Mira's - there is no consanguinity; (iii) no de novo or somatic mosaicism. A strong answer adds the practical point: the estimate is a probability, not a reassurance, and it could be converted to near certainty by identifying the sister's two variants and testing Jonas directly for them.
2. Evaluate the other laboratory's use of a 2 % agarose gel to exclude F508del. Is the conclusion valid? What method would be appropriate, and what are the consequences of the error?
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The conclusion is not valid. F508del is a 3 bp deletion, so the amplicons differ by 3 bp out of ~300 - a 1 % length difference. On a 2 % agarose gel, fragments of this size are resolved with a practical resolution of roughly 10–20 bp at best; a 3 bp difference would not be separated, and the two bands would co-migrate as a single band in the 300 bp position. Seeing one band therefore says nothing about the presence or absence of the deletion. Calling it “302 bp wild type” overstates the precision of the method.
Appropriate methods: allele-specific (ARMS) PCR, capillary electrophoresis with fluorescent sizing, restriction digest only if the variant genuinely creates or abolishes a site, or Sanger sequencing of the relevant exon. For a deletion this small, sequencing or a sizing method with single-base resolution is the standard.
Consequences: the sister's genotype remains unknown, and any relative counselled on the basis of that report has been given false reassurance. The error is not trivial - the sister is affected, so she carries two CFTR variants by definition and one of them may well be F508del. The correct action is to re-test with a validated method, document the discrepancy, and inform the family that the earlier report cannot be used for reproductive decisions.
3. The nephew's sweat chloride is 84 mmol/L and he had meconium ileus. Explain how this finding should change the counselling of Jonas and Mira, and why it is more useful than another panel test.
Read the worked answer
The nephew's findings are diagnostic of CF: meconium ileus is a classic neonatal presentation, and a sweat chloride of 84 mmol/L lies clearly above the ≥ 60 mmol/L diagnostic threshold (30–59 would be intermediate and would require further testing such as CFTR sequencing or nasal potential difference). This confirms that the family's CF is real and that the sister carries two pathogenic variants; it does not, by itself, tell us which ones.
The crucial point is what it enables. The nephew has two known pathogenic variants, one from his mother (Jonas's sister) and one from his father. Sequencing the nephew identifies the maternal variant with certainty. Jonas can then be tested for that specific variant - a targeted test with essentially 100 % sensitivity, not an 85 % panel. Two outcomes:
- Jonas negative for the family variant → his carrier risk falls from 0.10 to the de novo rate, effectively negligible, and the couple's risk becomes dominated by Mira's 0.6 %.
- Jonas positive → he is an obligate carrier and the couple's risk is 0.006 × 0.25 ≈ 0.15 % until Mira is fully sequenced.
This is why the diagnosis is more useful than repeating a panel: it converts a probabilistic residual risk into a near-definite genotype, and it also allows Mira's test to be interpreted against the actual family variant rather than a population average.