Haemophilia A

1 High titre of inhibitors in three related patients with haemophilia A and large factor VIII gene deletion D. BELVINI, R. SALVIATO, A. ARE, E. DE BIASI, R. RISATO, P. RADOSSI, P. G. DAVOLI and G. TAGARIELLO Blood Bank and Haemophilia Centre, Castelfranco Veneto Hospital, Italy In patients with haemophilia A the formation of antibodies against the transfused coagulation factors still remains a major problem for replacement therapy. The development of inhibitors has been correlated in the past with the type and the amount of therapy as well as with the MHC status, but up to now it is not fully understood. Recently some authors have described different proportions of antibody formation in patients carrying different FVIII gene mutations. Large gene deletions, factor VIII gene inversions and stop mutations have an incidence of inhibitor formation of approximately 35%, while antibodies are present in only 5±7% of patients with missense mutations or small deletions. In our centre we con®rmed these results. In a cohort of 96 severely affected patients we found 38 inversions (39%) and 17 out of the 38 (45%) had a history of inhibitors compared with 17% of patients without an inversion. These are not yet fully characterized for mutations. Now we are screening patients for mutations by conformation sensitive gel electrophoresis (CSGE). The whole coding region of the FVIII gene is analysed by PCR from genomic DNA in 34 fragments for haemophilia A. Among the patients not carrying the inversion, we have identi®ed and characterized three related haemophiliacs (two brothers and one nephew) with a large gene deletion. This is identi®ed due to lack of ampli®cation of the region spanning from exon 2 to exon 25 of the factor VIII gene. By using the Expand long template PCR system (Boehringer) with a forward primer in exon 1 and reverse primer at the 5¢ end of exon 26, we have been able to obtain a 21 kb band which is speci®c for this mutation. All three patients now aged 45, 30 and 8 years, respectively, have a very similar story. They each developed inhibitors at a very young age, after less than 10 exposure days and with a very high and stable titre (>100 BU). In addition, in the case of the two adults, they have a very severe arthropathy. In the haemophilia A database, 42 multiple-exon large deletions are reported. For 35 patients, data on inhibitors are available and are present in 20 (57%). In conclusion we think that this large FVIII gene deletion represents a very high risk factor for development of inhibitors, but other still unknown familial factors, which could be genetic as well, may play an additional role. As in these patients the risk of developing inhibitors is very high, this should be taken into consideration for accurate genetic counselling and to evaluate the possibility of a preventive immunotolerance program at birth or, possibly, even before.