Pitfalls in carrier testing of haemophilia A

B. Preisler1, I. Radermacher1, I. Schwarze1, R. Kuhlemann1, R. Maurer1, J. Beckedahl1, L. Rothbrust1, J. Junen1, U. Scholz2, B. Pezeshkpoor1, A. Pavlova1, J. Oldenburg1 (1Bonn, Germany, 2Leipzig, Germany)

Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: Background: Females carrier testing in haemophilia families is important for family planning and personal health. Genetic analyses are the golden standard as FVIII activity give not always reliable results. We report a case, where the initial genetic diagnosis failed to identify the second mutation.

Methods: Materials and Methods: All 26 exons and flanking regions of the F8 gene were amplified and directly sequenced from genomic DNA. MLPA was performed following the manufacturer’s instructions. Intron1/22 were analyzed by inverse PCR. X-Inactivation was performed though analyses of HUMARA locus.

Results: Results: A female patient with questionable carrier status showed a reduced FVIII:C value of 30 IU/dl and mild bleeding episodes. No male family member was available for testing at the time. Intron 22 inversion testing revealed an abnormal pattern suggesting, that an additional defect might be combined with the intron inversion. MLPA analysis showed a supplementary large deletion from exon 1 to 22. Thus, our patient carried an intron 22 inversion combined with large deletion of exons 1-22. Subsequently her father was sent for genetic analysis. He was diagnosed as mild haemophilia (FVIII:C 59 IU/dl), rarely experienced bleeding symptoms, which did not correlate with his daughter`s genetic defect. The sequence of the F8 gene revealed a missense mutation in exon 23, described in international register for a mild haemophila. Reinvestigation of the daughter’s DNA showed that she also carried the paternal mutation – in addition to the other 2 genetic defects. Although both X- chromosomes of our patient have been affected with genetic defects in the F8 gene, the FVIII:C showed relatively high values. Hence we performed skewed X chromosome analysis, which showed complete inactivation of one of the X chromosomes (99:1), most likely the one with the combined intron 22/large deletion. Both, patient’s mother and daughter carry intron 22 inversion/ large deletion defect in heterozygous state. The missense mutation was not detected.

Conclusion: Conclusions: Identification of two genetic defects in females carriers of haemophilia is of extreme importance for the genetic counselling. In our case haemophilia boy will be born either with severe (intron 22/large deletion) or mild (missense mutation) form of the disease. This will impact the risk of inhibitor development and treatment regimen.