F8 gene dosage defects in atypical patients with severe haemophilia A.

We performed molecular analysis of the factor 8 gene (F8) in 272 unrelated Spanish patients with haemophilia A (HA) and detected a mutation by routine analysis in 267 of them (98.1%). No mutation was detected in the remaining five patients despite clinical and laboratory confirmation of HA. The aim is to describe the molecular alterations in F8 discovered by gene dosage methodologies in three of these patients. For methodology, F8 sequencing, intragenic marker analysis, multiplex ligation-dependent probe amplification and quantitative real time-PCR were followed. One patient had Klinefelter syndrome (47,XXY) and a large deletion spanning exons 1-12 masked by the other F8 allele; the second patient showed a large duplication spanning exons 2-10 and the third patient revealed a non-contiguous double duplication of exons 14 and 23-25. The remaining two patients had mild HA and dosage results were normal. The application of gene dosage methods is useful to define haemophilic patients in whom mutations are not detected using other routine methods. Nevertheless, in a small percentage of patients (<1%), no molecular pathology can be identified after testing several genetic methodologies.

Severe haemophilia A in a female resulting from an inherited gross deletion and a de novo codon deletion in the F8 gene.

Haemophillia A (HA) is an X-linked bleeding disorder caused by mutations in the F8 gene. While the disease affects 1 in 5000 males, phenotypic expression of haemophilia A is rare in females, similar to other X-linked recessive disorders. We describe a 5-year-old female with severe haemophilia A. We determined the underlying molecular defect in the F8 genes of the proband and her closest family members by direct DNA sequencing, marker analysis and quantitative real-time polymerase chain reaction. The patient showed two different mutations in the F8 gene: the paternal copy of the F8 gene had a de novo p.Phe652/653 deletion in exon 13 while the maternally inherited gene showed a large deletion encompassing exons 1 to 22. The structural analysis of residues Phe652/Phe653 based on a three-dimensional model of activated factor VIII provides evidence of the impact of the mutant factor VIII protein in the clinical manifestations of the patient. This unusual finding highlights the need to perform a thorough molecular analysis including sequencing, marker and quantitative analyses to identify compound heterozygous females with HA.

Application of intron 9 and intron 25 dinucleotide repeats of the factor VIII gene for carrier diagnosis in haemophilia A.

We describe the usefulness of two dinucleotide repeats located in intron 9 and in intron 25 of the factor VIII gene for carrier diagnosis of haemophilia A. We analyzed 100 unrelated Spanish women and 34 women from haemophilia A (HA) families in whom known intragenic markers were unhelpful in determining their carrier status. The heterozygosity rate of intron 9 and intron 25 markers in the 100 control women was lower (0.28 and 0.38, respectively) than the values obtained with common markers routinely used in our laboratory. However, the application of intron 9 and intron 25 markers was effective in identifying the at-risk X chromosome in 11 of 34 (32%) of the uninformative women from HA families. The combined use of these repeats with current markers may facilitate the identification of the X chromosome in HA families for application in carrier, prenatal and pre-implantation diagnoses.

Utility of a (GT) dinucleotide repeat in intron 1 of the factor 8 gene for haemophilia A carrier diagnosis.

We report the usefulness of a dinucleotide GT repeat in intron 1 of the factor 8 gene in carrier diagnosis of haemophilia A (HA). We analyzed 47 women from HA families in which the mutation was not identified in the index case and in which the common intragenic polymorphic markers were uninformative. The intron 1 GT repeat was useful to identify the X chromosome with the mutation in 19 of them. The analysis of this easily detectable marker in conjunction with other current markers may facilitate X chromosome identification in a large proportion of HA families.