Amelioration of clinical severity of similar mutations severe factor IX deficiency by coinherited thrombophilia

To the Editor: Haemophilia B is a X linked recessive bleeding disorder caused by deficiency of coagulation factor IX due to heterogeneous mutations in the factor IX gene. The mutations causing haemophilia B have been localised and characterised in more than 500 patients elucidating the diverse molecular basis of this disorder (1).The severity of bleeding associated with haemophilia B can be generally predicted by the level of residual factor IX in the plasma. Factor IX levels less than 1% (i.e. 0.01 IU ⁄mL) of normal plasma are classified as ‘severe’ and are associated with severe haemorrhagic symptoms; factor IX levels of 1–5% (i.e. 0.01–0.05 IU ⁄mL) as ‘moderate’ with mild to moderate bleeding symptoms and 6– 40% (0.06–0.40 IU ⁄mL) as ‘mild’ who hardly present with any bleeding manifestations (2). Some of the known determinant factors on the severity of haemophilia are the nature of mutations (3, 4), immune response genes or HLA loci which is specifically significant in case of haemophilia with inhibitors (5, 6) and thrombophilia genes (7) besides environmental and behavioural characteristics. More recently, attention has been given to the interaction of various thrombophilic factors in modifying the clinical severity of haemophilia. In this communication, we have studied three pairs of haemophilia B patients, two belonging to same families and the third belonging to different families, to study the effect of inherited thrombophilic genes in modifying the clinical severity in the background of a common genetic mutation. Three pairs of severe haemophilia B patients (F IX <1% of normal plasma), each pair with markedly varied clinical severity were analysed in the present study. The clinical classification of ‘severe’ and ‘mild’ was arbitrarily based on average number of bleeds and replacement therapy per year during the preceding 5 yr, joint deformity and age of diagnosis. Severe haemophilia B patients with less than 0.01 IU ⁄mL factor IX by one stage assay, who had less than 2 bleeds ⁄ factor concentrate infusions in the preceding 5 yr and absence of severe joint deformity were classified as ‘mild’ while patients with more than 10 bleeds ⁄ infusions in the preceding 5 yr and with severe joint deformity were considered ‘severe’. All the patients were above 10 yr of age with normal liver function tests and were negative for inhibitors. Number of bleeds per year in these patients were checked from a diary kept by these patients. The study was approved by the Institute Ethics Committee. Screening coagulation tests (PT, APTT, TT) and factor IX:C assays were performed using commercial reagents (Dade Behring, Marburg, Germany). Plasma samples from all the patients were analysed using the same batch of deficient plasma. Lupus anticoagulants (LA) were studied by mixing studies, Kaolin clotting time (KCT), dilute Russels Viper Venom time (DRVVT). IgG ⁄M antibodies for anticardiolipin (ACA,) beta 2 GP1 (b 2 GP1) annexin V and prothrombin were measured by enzyme-linked immunoabsorbent assay (ELISA) using commercial kits (Varelisa, Freburg, Germany). All the tests for antiphospholipid antibodies (APA) were repeated at least once with a fresh sample in a minimum time interval of 4 months. Protein C (PC), protein S (PS), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI1) and thrombin activatable fibrinolytic inhibitor (TAFI) were measured by ELISA using commercial kits (Diagnostic Stago, Asniers, France). Antithrombin (AT), plasminogen and antiplasmin were measured by chromogenic assays using commercial reagents (Diagnostica Stago, Asniers, France). DNA was extracted from citrated cell pellet using standard methods (8). Factor V Leiden (FVL), prothrombin (PT) G20210A, methylene tetrahydrofolate reductase (MTHFR) C677T, endothelial protein C receptor (EPCR) 23 bp insertion, fibrinogen gene Arg ⁄Lys and PAI – 1 4G ⁄ 5G polymorphisms were analysed by PCR with or without restriction enzyme digestion (9). All the eight exons of factor IX gene including the promoter region were amplified using ten sets of primers as described earlier (10). The clinical features of all the six patients along with the mutations detected are summarised in Table 1. The age of diagnosis in all three ‘clinically mild’ patients was considerably delayed (11–38 yr) when compared with ‘clinically severe’ patients (3–6 yr). There was no joint deformity or history of blood transfusion in the ‘clinically milder’ patients when compared with their ‘clinically severe’ counterparts. Except one patient who had an episode of gum bleeding which did not require doi:10.1111/j.1600-0609.2007.00965.x European Journal of Haematology ISSN 0902-4441