A Novel Point Mutation in Severe Haemophilia A: a Further Proof of Genotype‐phenotype Correlation

Haemophilia A is a bleeding disorder resulting from deficiency of coagulation factor VIII (FVIII) because of a mutation in the FVIII gene located in the distal long arm of an X chromosome; the gene consists of 26 exons and the mature protein consists of 2332 aminoacids. Analysis of the DNA sequence shows a repeating domain structure organized in a leader peptide, three A domains, one B domain and two C domains. The mature protein is proteolitically cleaved between domains A1/A2 and between B/A3 domain. Recently, a new structural model of FVIII has located bound metal ions and glycosylation in A1 and A3 domains and A1-A2-A3-C1 domains respectively [1]. Interaction with phospholipid surface is thought to occur via C domain [2]. In the F8 HAMSTeRS mutation database, more than 800 mutations are listed. Inversion of intron 22 of FVIII gene has been found to be the cause of 40–50% of severe haemophilia A, while inversion in intron 1 is the causative mutation in 3–5% in patients with the same disease severity. The spectrum of other FVIII variations described in the remaining patients is highly heterogeneous, with predominance of private mutations and a relatively high rate of de novo sequence changes. Characterization of mutations may help to understand better the biological role of FVIII through identification of functional domains and to facilitate genetic counselling to patients families. To support characterization of genetic defects in female potential or obligate carriers of haemophilia and in the context of a national database of Italian FVIII mutations, we studied several families with severe and mild haemophilia A. Here, we confirm a single previous report of association between mutation at aminoacid 91 and mild phenotype and we describe a novel missense mutation causing severe FVIII deficiency in another family studied. Two unrelated families were studied to identify carriers for prenatal purposes. Informed consent was obtained from every member studied. Family 1: the patient (A.D.) was a 32-year-old, moderate haemophiliac A. The disease was diagnosed when he was thirty, after a pre-surgery screening on a female cousin of his, who had low FVIII levels (30%, haemophilia carrier). A.D. reported post-surgery bleedings and recurrent ankle bleeds after mild traumatic episodes. He never experienced spontaneous bleedings and had never been treated with FVIII concentrates. Family screening was extended to his sister and his uncle with his daughter. Family 2: the proband (P.A.) is the only affected person in his family. He arrived to our observation when he was 56 years old. He was diagnosed as a severe haemophilia A patient during childhood, and he had been treated with plasma first, and with plasma-derived FVIII later. He had developed multiple target joints and knee, ankle and elbow arthropathy. After informed consent had been obtained, genomic DNA was isolated by standard protocols from citrated peripheral blood samples. The entire F8 coding region, including the flanking splicing sites, was amplified using the appropriate primers as previously described [3]. The F8 promoter sequence and the polyadenylation site were analysed. All the amplified fragments of each patient were screened for the mutation by direct sequencing using a Genetic Analyser 3130 (Applied Biosystem, Foster City, CA, USA). Correspondence: Dr Alessandra Borchiellini, SSCVD Malattie Emorragiche e Trombotiche dell Adulto, Ospedale San Giovanni Battista, via Genova, 3 Turin,10126, Italy. Tel.: +39 011 633 5329; fax: +39 11 696 3737; e-mail: alesandre@libero.it