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.

Rituximab in the treatment of high responding inhibitors in severe haemophilia A.

The development of antibodies to factor VIII (FVIII) in severely affected haemophilia A patients is a serious complication associated with increased morbidity and mortality. Bypassing agents are used to treat acute bleeding episodes; however, elimination of the inhibitors can only be achieved with immune tolerance therapy (ITT) in 60-80% of cases. High responding (HR) inhibitors are more likely to respond to ITT if the titre is decreased to <5 BU over time or in selected cases after the administration of immunosuppressive drugs, plasmapheresis or immunoabsorption, techniques difficult to apply in children. Anti-CD20 (rituximab), a monoclonal antibody, was given as an alternative treatment in two haemophilic children with HR inhibitors and impaired quality of life, due to recurrent haemarthrosis. Rituximab was given at the dose of 375 mg m(-2), once weekly for four consecutive weeks. Both patients showed a partial response to rituximab reducing the inhibitor titre to <5 BU, thus facilitating ITT initiation; however, only the older patient eradicated the inhibitor within 21 days after application of ITT. The second patient, despite depletion of B cells, did not respond to ITT. No long-term side effects have been observed in both patients for a follow-up period of 20 and 18 months respectively. In conclusion, rituximab appears to be an alternative effective therapy to rapidly reduce or eliminate the inhibitor in selected cases of severely affected haemophiliacs before further proceeding to ITT. However, the dose and appropriate schedule, as well as long-term side effects need further investigation.

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.

Pharmacokinetics of activated recombinant coagulation factor VII (NovoSeven) in children vs. adults with haemophilia A.

To establish the pharmacokinetic profile of activated recombinant coagulation factor VII (rFVIIa; NovoSeven in children with haemophilia A, and to compare it with the pharmacokinetic profile in adults with haemophilia A. Twelve children (2-12 years) received one single dose of rFVIIa 90 and 180 micrograms kg(-1) in randomized order separated by a washout period of 48 h to 1 month. Six adults (18-55 years) received a single dose of rFVIIa 90 micrograms kg(-1). The pharmacokinetic analyses were based on a non-compartmental method. In children, the plasma level of FVII increased proportionally with the dose. The total body clearance normalized for body weight was significantly faster in children than in adults (FVII:C, 58 vs. 39 mL kg(-1) h(-1) and FVIIa, 78 vs. 53 mL kg(-1) h(-1), P < 0.05). A trend towards a larger volume of distribution at steady-state in children than in adults was observed (P > 0.05). Dose proportionality was established for plasma concentrations of FVII in children with haemophilia A at the dose levels investigated (90 and 180 micrograms kg(-1) rFVIIa). Following administration of rFVIIa 90 micrograms kg(-1), significantly faster clearance was observed in children compared with adults, suggesting that higher doses of rFVIIa may be needed to achieve the same plasma levels as in adults.

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.

Indications of coagulation and/or fibrinolytic system activation in healthy and sick very-low-birth-weight neonates.

A combined hemostatic defect consisting of a reduction in certain procoagulants, anticoagulants (antithrombin III-ATIII-, protein C-PC-) and components of the fibrinolytic system (plasminogen-Plg-) was demonstrated in very-low-birth-weight infants (VLBW <1,500 g) with gestational age 26-32 weeks. Sixteen of them were healthy, 28 were suffering from RDS and 24 from septicemia. The hemostatic defect was more profound in the RDS group, nevertheless increased TAT (thrombin + ATIII complex) and/or PAP values (plasmin + a2-antiplasmin complex) was a more frequent finding in the septicemic group of infants (91.8 vs. 17.9%). Moderate-to-severe thrombocytopenia was detected in a higher percentage in the septicemic (70.8%) than in the RDS group (50%), and increased D-dimers were demonstrated in 34.8 and 28.6% of the infants, respectively. Elevated TAT or PAP values were not always associated with gross coagulation abnormalities, and advanced disseminated intravascular coagulation (DIC) was only documented in 16.7% of the septicemic and 7.1% of the RDS infants. None of the VLBW neonates presented with clinical evidence of thrombosis, although hemorrhagic manifestations were apparent in 34.8 and 14.3% of the neonates with septicemia or RDS, respectively, mainly due to DIC or severe thrombocytopenia. In conclusion, increased TAT and/or PAP values are good indicators of the in vivo activation of the hemostatic system, but still their impact on sick neonates morbidity and mortality remains unknown.

Diffuse splenic and visceral hemangiomas complicated by chronic consumption coagulopathy.

The case of a 7-year-old girl with a 2 year history of easy bruising associated with thrombocytopenia is reported. On admission she presented with ecchymoses, abdominal distention and splenomegaly. Hemostasis investigation revealed a consumption coagulopathy. Several radiological studies failed to confirm the diagnosis of diffuse splenic and visceral hemangiomatosis, which was eventually established by an explorative laparotomy. Platelet count and the other coagulation abnormalities progressively returned to normal after splenectomy, although the remaining hemangiomas were extensive.

Haemostatic variables in phenylketonuric children under dietary treatment.

Classical phenylketonuria (PKU) (McKusick 261600) is an inborn error of metabolism treated by a controlled low-phenylalanine (Phe) diet started as soon as possible in the first days of life. Such a diet can be achieved with vegetable protein and can be considered non-atherogenic because of the reduction of animal products. Thirty patients with PKU were classified into two groups according to their annual mean Phe levels. Their daily protein intake was largely replaced by PKU2 Milupa which contains a mixture of amino acids. The product has no phenylalanine or fat of any kind. Thirty-eight (38) individuals of comparable age were used as controls. Group A (n = 15) had good compliance with the special diet (Phe mean 192 +/- 115 mumol/L); group B (n = 15) did not strictly adhere to the diet (Phe mean 595 +/- 263 mumol/L). Certain haemostatic components (factors I, VII, VIII, and X, antithrombin III, protein C, and plasminogen) and lipid variables (cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, very-low-density lipoprotein) as well as Phe levels were estimated. All the haemostatic factors studied were found within the normal range with the exception of a significant reduction in protein C in both groups of PKU patients. Furthermore, a statistically significant reduction in factor VII and X concentrations was observed in patients on strict diet. Cholesterol and low-density lipoprotein concentrations were significantly lower in PKU children compared to normal controls. It is suggested that even though the special diet of PKU children, especially in group A, is rich in vegetables, the reduced fat intake might have impaired the absorption of vitamin K and its delivery to the site of synthesis of vitamin K-dependent haemostatic factors.

Prevalence of inhibitor formation in a cohort of haemophilic children exposed to several products of various purities.

To evaluate the frequency and potency of inhibitor formation based on the product used, we retrospectively reviewed the records of 99 children with various types and severity of haemophilia (haemophilia A 82, severe 46; haemophilia B 10, severe 6; vWD 7) treated for the last 20 years. After a mean observation period of 8 years an overall of 23 patients (23.2'/0) developed an inhibitor (haernophilia A 26.8%; severe 4O%, moderate 20%, mild 3.8%). None of the haemophilia B patients presented with an inhibitor, and only one child with bevere vWD (1/7, 14.3%) showed a transient inhibitor under cryoprecipitate therapy. Inhibitor titre was low (< 5 BU) in most cases (91.3%) and in only two patients (8.75%) was 6 and 8 BU respectively. Antibodies to FVIII were transient (detected only once) in four (17.4%) and intermittent in 19 patients (82.6%). By the age of 12 years, 17/23 patients (73.9%) had demonstrated an inhibitor. The inhibitor detection seemed to be higher in the groups of patients exposed to monoclonal (3115, 20%), SID-treated (10159, 16.9%) or H/T FVIII concentrates (6/41, 14.6%), compared to groups of patients who received cryo/plasma (9.5%) or unmodified concentrates (5.1%); nevertheless the differences were not statistically significant. Surprisingly, none of the 52 patients who received a S/D + chromatography-treated factor VIIl concentrate developed an inhibitor after a mean observation period of 1.7 years (range 0.2-2 years). The overall prevalence of inhibitor formation in previously untreated haemophiliacs was 14.3% (4/28), irrespective of the product used. Our data indicate that a high proportion of our haemophilic children exposed to several products of various purities have developed a low-titre inhibitor which in most cases was transient or intermittent. However, despite the presence of the antibody, none of the patients needed a change in the mode of treatment.

Seventeen years of experience with chronic idiopathic thrombocytopenic purpura in childhood. Is therapy always better?

Between 1975 and 1992 450 children with idiopathic thrombocytopenic purpura (ITP) were diagnosed, and of those 100 (22%) developed the chronic form of the disease. Approximately half the patients with chronic ITP presented with mild to moderate hemorrhagic manifestations at the onset of purpura (30 cases) and/or later during the course of the disease (25 cases). The incidence of intracranial hemorrhage was 1%, and the mortality rate due to overwhelming septicemia after splenectomy was also 1%. Overall one-third of the patients received no therapy; two-thirds of them went into spontaneous remission within 8 months to 8 years from the onset of ITP. Steroids given in conventional or high doses (51 cases) achieved a transient (if any) rise in platelet count, but in no case were steroids curative. Remission related to intravenous immune globulin (IVIG) therapy was noticed in 38.5% of the children (10 of 26) after variable courses. The response rate to splenectomy was 95.0%. Ultimately the long-term outcome in children with chronic ITP was as follows: remission, 58 cases (spontaneous, 30; after IVIG therapy, 10; after splenectomy, 18); hemostatic platelet values, 22 cases (spontaneous, 16; after IVIG, 5; after splenectomy, 1). Thirteen children were lost in follow-up, and 7 remain thrombocytopenic but asymptomatic. These data indicate that chronic ITP in childhood runs a benign course in most cases and may remit with or without therapy even several years from onset. Therefore, therapeutic intervention has to be individualized, and splenectomy, which is not always safe, should be reserved for problematic cases that fail to respond to conventional therapeutic modalities.

Subclinical neuropathy in children with inherited haemostasis disorders.

Motor and sensory conduction of the right peroneal and sural nerves was studied in 28 children (17 HIV seropositive) with inherited hemostasis disorders, without any symptoms of neuropathy. The amplitude ratio of the evoked muscle potential (EMP) at distal stimulation to that at proximal stimulation at the right peroneal nerve was also studied. Thirty healthy aged-matched children were used as controls. There was no statistically significant difference in the distal latency, amplitude and conduction velocity of motor and sensory nerves between patients and controls. On the contrary, a great diminution of amplitude of the EMP during proximal stimulation of nerve was observed in patients, statistically very significant, as compared to controls. This difference was independent of patients' age, severity of hemostasis defect or HIV status. In 9 patients the amplitude was within normal limits. Intraneural oozing due to trivial trauma is postulated as a possible mechanism of peroneal nerve lesion.

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA.

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.

Moderately severe hemophilia A resulting from Glu----Gly substitution in exon 7 of the factor VIII gene.

To define the molecular basis of a TaqI site alteration in the factor VIII gene of a patient with moderately severe hemophilia A, we used a combination of genomic amplification followed by direct sequencing and oligonucleotide hybridization, to demonstrate an A-to-G substitution in exon 7 (codon 291) of this gene. This mutation generates a Gly in place of Glu at amino acid 272 of the mature factor VIII protein. The mutation arose de novo in a germ cell of the patient's mother.

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene.

Hemophilia A, an X-linked disease caused by deficiency of factor VIII, is characterized by variation in clinical severity and coagulation activity. This variation is though to reflect heterogeneity of mutations in the factor VIII gene. Here we describe a CG-to-CA mutation within a potential cryptic donor splice site in intron 4 of the factor VIII gene from a patient with mild disease. This mutation makes the cryptic sequence resemble more closely the consensus sequence for donor splice sites. We infer that the mutation activates the cryptic donor splice site, which in turn causes a defect in RNA processing.

Characterization of five partial deletions of the factor VIII gene.

Hemophilia A is an X-linked disorder of coagulation caused by a deficiency of factor VIII. By using cloned DNA probes, we have characterized the following five different partial deletions of the factor VIII gene from a panel of 83 patients with hemophilia A: (i) a 7-kilobase (kb) deletion that eliminates exon 6; (ii) a 2.5-kb deletion that eliminates 5' sequences of exon 14; (iii) a deletion of at least 7 kb that eliminates exons 24 and 25; (iv) a deletion of at least 16 kb that eliminates exons 23-25; and (v) a 5.5-kb deletion that eliminates exon 22. The first four deletions are associated with severe hemophilia A. By contrast, the last deletion is associated with moderate disease, possibly because of in-frame splicing from moderate disease, possibly because of in-frame splicing from adjacent exons. None of those patients with partial gene deletions had circulating inhibitors to factor VIII. One deletion occurred de novo in a germ cell of the maternal grandmother, while a second deletion occurred in a germ cell of the maternal grandfather. These observations demonstrate that de novo deletions of X-linked genes can occur in either male or female gametes.