Impact of HLA alleles and cytokine polymorphisms on inhibitors development in children with severe haemophilia A.

Human Leucocyte Antigen (HLA) alleles, cytokine polymorphisms and the type of factor VIII (FVIII) gene mutation are among predisposing factors for inhibitors (inh) development in children with severe haemophilia A (HA). The aim was to investigate the correlations among (i) FVIII gene intron-22 inversion, (ii) HLA alleles and haplotypes and (iii) certain cytokine polymorphisms, with the risk for FVIII inhibitors development in 52 Greek severe HA children, exclusively treated with recombinant concentrates. We performed Long-Range PCR for detection of intron-22 inversion and PCR-SSP, PCR-SSO for genotyping of HLA-A, B, C, DRB1, DQB1 alleles and also for cytokine polymorphisms of TNF-α, TGF-ß1, IL-10, IL-6 and IFN-γ. Chi-squared test and Fischer's exact test were used for statistical analysis. A total of 28 children had developed inhibitors (Group I), 71.4% high responding, while 24 had not (Group II). No statistically increased intron-22 inversion prevalence was found in Group I compared with Group II (P = 0.5). Comparison of HLA allele frequencies between the two groups showed statistically significant differences in the following genotypes (i) promoting inhibitors development: DRB1*01(P = 0.014), DRB1*01:01(P = 0.011) and DQB1*05:01 (P = 0.005) and (ii) possibly protecting from inhibitors development: DRB1*11 (P = 0.011), DRB1*11:01 (P = 0.031), DQB1*03 (P = 0.004) and DQB1*03:01 (P = 0.014). Analysis of cytokines revealed a higher incidence of inhibitor detection only in homozygotes of the haplotypes ACC and ATA for IL-10 polymorphisms (P = 0.05). There is evidence that HLA alleles and cytokine polymorphisms play an important role in FVIII inh development. On the contrary, no statistically significant results were obtained for intron-22 inversion and its impact on FVIII inhibitors formation.

Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders.

BACKGROUND: The European Network of Rare Bleeding Disorders (EN-RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs. OBJECTIVES: To explore the relationship between coagulation factor activity level and bleeding severity in patients with RBDs. PATIENTS/METHODS: Cross-sectional study using data from 489 patients registered in the EN-RBD. Coagulation factor activity levels were retrieved. Clinical bleeding episodes were classified into four categories according to severity. RESULTS: The mean age of patients at data collection was 31 years (range, 7 months to 95 years), with an equal sex distribution. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F) X, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. The coagulation factor activity levels that were necessary for patients to remain asymptomatic were: fibrinogen, > 100 mg dL(-1); FV, 12 U dL(-1); combined FV + VIII, 43 U dL(-1); FVII, 25 U dL(-1); FX, 56 U dL(-1) ; FXI, 26 U dL(-1); FXIII, 31 U dL(-1). Moreover, coagulation factor activity levels that corresponded with Grade III bleeding were: undetectable levels for fibrinogen, FV and FXIII, < 15 U dL(-1) for combined FV + VIII; < 8 U dL(-1) for FVI; < 10 U dL(-1) for FX; and < 25 U dL(-1) for FXI. CONCLUSIONS: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. A strong association is only observed in fibrinogen, FX and FXIII deficiencies.

Assessment of the progression of haemophilic arthropathy in children.

Arthropathy is considered as an irreversible and progressive complication in patients with haemophilia, even in children on prophylaxis. To estimate the progression of haemophilic arthropathy, 85 joints of 24 boys with severe (n = 18) and moderate (n = 6) haemophilia (A: 22, B: 2) were investigated with clinical examination, X-rays and magnetic resonance imaging (MRI) at two time periods (time 0 and 1). Patients' age at time 0 was 10.5 +/- 3.6 years and time elapsed to time 1 was 3.8 +/- 1.4 years. At time 0: all investigated joints had more than three bleeds. Sixteen boys were on secondary prophylaxis for 5.4 +/- 2.8 years. Clinical score (a modification of World Federation of Haemophilia's scale): 2.0 +/- 3.6, X-ray score (Pettersson): 2.1 +/- 2.8, MRI score (Denver): 4.5 +/- 3.8. After the first evaluation, prophylaxis was intensified in 11 children and initiated in four. At time 1: clinical score: 1.5 +/- 3.1, X-ray: 1.7 +/- 2.7, MRI score: 5.1 +/- 4.1. On average, the clinical and X-ray scores showed a significant improvement (26% and 40% of the joints respectively, P < 0.01) and the number of haemarthroses evidenced a threefold reduction from time 0 to 1 (P < 0.01), findings that could be associated with the modification of prophylaxis after time 0. MRI findings showed deterioration in 34% of the joints. Conversely, 14 joints (16.5%) with mild or moderate synovitis without cartilage degradation at time 0 showed an improvement at time 1. The information carried by the three scales could be divided into information shared by the three scores and information specific to each score, thus giving a more complete picture of joint damage caused by bleedings.

Comparative study of validity of clinical, X-ray and magnetic resonance imaging scores in evaluation and management of haemophilic arthropathy in children.

To evaluate joints alterations, we performed clinical examination, X-rays and magnetic resonance imaging (MRI) (Denver score) in 165 joints of 40 children with severe (n, 32) or moderate (n, 8) haemophilia A or B. All investigated joints had a history of more than three bleeds. At evaluation, 25 of 40 haemophilic patients were on prophylaxis for the last 1-8 years (mean: 3.5 years). MRI revealed chronic synovitis in 55.4% and 50% of joints, which were diagnosed, as normal by the clinical scale and plain radiography respectively. Moreover, MRI unmasked more profound alterations than those observed by plain radiography in 70% of cases. Statistical analysis showed that the clinical and Pettersson scores in contrast to the Denver score provide an underestimation of arthritic changes. Besides, Denver score did not provide resolution in differentiating stages of arthropathy, because of its inherent nature; however, a score of 6 expressing severe synovitis seemed to be the cut-off value for the distinction of severe cases. Based on MRI findings we intensified prophylaxis in nine children and initiated it in another nine children. Five children, who were already on prophylaxis complied with our recommendations and eliminated haemorrhages. Finally, three boys with severe haemophilic arthropathy in knees underwent successful chemical synovectomy with rifampicin.

Mutations and polymorphisms in genes affecting hemostasis proteins and homocysteine metabolism in children with arterial ischemic stroke.

BACKGROUND: The pathogenesis of thrombosis in childhood seems to be multifactorial implicating genetic and environmental factors. AIM: To compare the distributions of mutations/polymorphisms in genes affecting hemostasis (factor V Leiden - FVL, FV H1298R-FVR2, FII 20210A, b-Fib 455G>A, FXIII V34L, PAI-1 4G, HPA-1b) or homocysteine metabolism (MTHFR C677T, MTHFR A1298C) among 90 children with arterial ischemic stroke (AIS) and 103 controls, and to associate the carriage of these mutations/polymorphisms with their corresponding proteins in children with AIS. RESULTS: AIS was more frequent in boys (p < 0.01). No studied mutation/polymorphism was found to be a risk factor for AIS, except for FVL [odds ratio 4.2 (95% CI 1.5-12.1)], the presence of which was even higher in 31 children with congenital AIS [odds ratio 6.82 (95% CI 2.0-22.8)]. FVL carriers had an odds ratio of 5.76 (95% CI 1.6-6.4) when FVR2 was absent. In thrombosed children, activated protein C resistance, prothrombin and fibrinogen levels were higher in the presence of FVL, FII20210A or b-Fib 455G-->A, respectively. Double heterozygotes in both MTHFR C677T and A1298T or homozygotes in one had significantly elevated homocysteine levels. CONCLUSION: Except for FVL, no definite conclusion could be reached regarding the involvement of the studied mutations/polymorphisms in childhood AIS.

Mutations and polymorphisms in genes affecting haemostasis components in children with thromboembolic events.

The distribution of mutations/polymorphisms in genes affecting haemostasis [factor V Leiden (FVL), FV H1298R (FVR(2)), FII 20210A, b-Fib 455G-->A, FXIII V34L, PAI-1 4G, HPA-1b] among 141 children with thrombosis at various sites and 103 controls was compared. Additionally, the carriage of these mutations/polymorphisms was associated with the levels of their corresponding proteins in thrombosed children. Thrombosis was more frequent in boys (p = 0.021). No studied mutation/polymorphism was found to be a risk factor for thrombosis, except for FVL (odds ratio 3.8, 95% CI 1.4-10.6). The risk of thrombosis for FVL carriers was twice as high in children with an idiopathic thrombosis (odds ratio 5.4) than in thrombosed children with an underlying disease or a triggering event (odds ratio 2.7). FVL carriers had an odds ratio of 5.9 (95% CI 1.8-19.6) when FVR(2) was absent. In thrombosed children, the activated protein C resistance ratio was significantly lower in the presence of FVL (p < 0.001). Prothrombin and fibrinogen levels, although higher in FII 20210A and b-Fib 455G-->A carriers, did not reach statistical significance.

Retrospective evaluation of long-term efficacy and safety of splenectomy in chronic idiopathic thrombocytopenic purpura in children.

AIM: To review the long-term efficacy and safety of splenectomy in children with chronic idiopathic thrombocytopenic purpura (cITP). PATIENTS AND METHODS: Data from 33 splenectomized children were retrospectively analysed (median follow up period: 18.8 y from the removal of the spleen). The median age of children at splenectomy was 12 y and the median ITP duration 3.3 y. Indications for splenectomy were: persistent severe thrombocytopenia with extensive purpura, epistaxis and/or gum bleeds, menorrhagia (n = 5) and severe or recurrent haemorrhage from various sites (n = 11). RESULTS: Eighty-five per cent of the patients showed an excellent (n = 26) or partial response to splenectomy. Five children (15%), all females, failed to respond. Of the responders, 25% experienced a transient recurrence of thrombocytopenia within 6 mo to 4 y from splenectomy. The mortality rate due to severe sepsis was 3%. However, the majority of the splenectomized patients have not so far suffered any severe or mild bacterial infection, despite incomplete vaccination and/or antibiotic prophylaxis. CONCLUSION: Splenectomy remains the only effective therapeutic modality for children with cITP, although it is associated with transient recurrence and rarely with post-splenectomy sepsis, which could be fatal. Nonetheless, splenectomy should be the last treatment option for the cITP patient, after all available therapeutic modalities have been exhausted and the child still remains profoundly thrombocytopenic and symptomatic.

Prothrombotic factors in neonates with cerebral thrombosis and intraventricular hemorrhage.

AIM: To investigate whether the factor V Leiden mutation (FVL), the prothrombin gene G20210A variant or the methylenetetrahydrofolate reductase (MTHFR) C677T genotype are risk factors for central nervous system (CNS) thrombosis or intraventricular hemorrhage (IVH) in neonates. METHODS: Thirteen full-term infants with cerebral infarct documented with magnetic resonance imaging were assessed with the whole spectrum of assays for thrombophilia including the three DNA-based prothrombotic factors. The frequency of congenital defects was compared with that observed in 38 healthy full-term infants. The genetic defects were also assessed in 55 premature neonates, gestational age <32 wk, 17 of whom developed IVH, grade II-IV. The remaining 38 premature neonates without IVH were used as controls. RESULTS: In the CNS thrombosis group: a prothrombotic factor was detected in 53% of patients and an underlying disease or a triggering event in 61.5%. The frequency of FVL in thrombosed neonates was higher (23%) than in the group of healthy full-term infants (10.5%), although it did not reach statistical significance. IVH developed in 30.9% of premature neonates. Apart from several maternal or neonatal risk factors for IVH, FII G20210A was found in a considerably higher prevalence in the cohort of neonates with IVH (12%) than in those without (2%), although the difference was not statistically significant. CONCLUSION: The pathogenesis of cerebral thrombosis or IVH in neonates is multifactorial. Along with underlying diseases or triggering events, congenital prothrombotic factors (FVL or FII G20210A) showed a trend towards a higher frequency in full-term infants with CNS thrombosis or premature neonates with IVH than in controls. However, their contribution to neonatal cerebral thrombosis or IVH remains to be determined.