Factor V Leiden Mutation and Other Thrombophilia Markers in Childhood Ischemic Stroke

Transcription

1 Clin Appl Thrombosis/Hemostasis 11(1):83 88, Westminster Publications, Inc., Glen Head, NY Factor V Leiden Mutation and Other Thrombophilia Markers in Childhood Ischemic Stroke Rıdvan Duran, MD,* Betül Biner, MD,* Muzaffer Demir, MD, Coşkun Çeltik, MD,* and Serap Karasalihoğlu, MD* Departments of *Pediatrics and Hematology/Internal Medicine, Trakya University School of Medicine, Edirne, Turkey Summary: The aim of this study was to evaluate the association between ischemic childhood stroke and thrombophilia. The prevalence of thrombophilia risk factors in 30 unrelated children with ischemic stroke were compared with 33 age-matched control subjects. Patients and control group were tested for the presence of activated protein C (APC) resistance, antiphospholipid antibodies (APLA), increased factor VIII levels, and for the deficiency of protein C (PC), protein S (PS), and antithrombin. When APCR was detected in patients or in controls, factor V Leiden (FVL) mutation was also tested. Seventeen of 30 patients (56.6%) had at least one thrombophilia marker compared with only 5 of 33 control subjects (15.1%). Three children with ischemic stroke (10%) were affected with a combination of two or more thrombophilia markers whereas none of the children in the control group had a combination of risk factors. Seven of 30 children with ischemic stroke (23.3%) and one of 33 control subjects (3.03%) had APC resistance and in all of them FVL mutation were found. The prevalence of FVL mutation was higher among pediatric stroke patients than among control subjects (p < 0.05). None of the patients but one child from the control group (3.03%) had PS deficiency. Antithrombin and PC deficiencies and the presence of APLA and increased factor VIII levels were more frequent in the pediatric stroke patients than in controls but the difference was not statistically significant (p > 0.05). These data confirm that stroke in children is commonly associated with a combination of multiple risk factors and especially the prevalence of FVL mutation is increased in children with ischemic stroke compared with control subjects. Key Words: Ischemic stroke Thrombophilia Factor V Leiden Child. This work was supported by a grant from Trakya University Research Foundation (TÜBAP-426). Address correspondence and reprint requests to Betül Biner, MD, Trakya Universitesi Tıp Fakultesi, Cocuk Saglıgı ve Hastalıkları Anabilim Dalı, Edirne, Turkey; betulbiner@hotmail.com. Although pediatric stroke is significantly less common than stroke in adults, it is more common than previously thought (1). The annual incidence rate of 2.5 to 3.1 per 100,000 children has been estimated (2,3) with 44% to 61% are believed to be ischemic and the remainder hemorrhagic stroke (3 5). Ischemic stroke in children frequently results from cardiac embolism, nonarteriosclerotic vasculopathies, and prothrombotic states. The overall incidence of prothrombotic states in children with ischemic strokes is reported to be 10% to 50% (6,7). Prothrombotic state is an impairment of the normal hemostatic system in which the balance has shifted toward thrombosis. This is commonly caused by an abnormality or impairment of the vascular endothelium, the coagulation cascade, the fibrinolytic system, or the platelets. Several prothrombotic disorders such as deficiencies of protein C (PC), protein S (PS), antithrombin (AT), or plasminogen and the presence of antiphospholipid antibodies (APLA) are also associated with stroke in children (8 11). Activated PC (APC) resistance because of the prothrombotic polymorphism of the substitution of arginine with glutamine at amino acid residue 506 in coagulation factor V (factor V Leiden [FVL]) has been reported as a predisposing condition for ischemic stroke in children and young adults (12 17). These data, however, were not confirmed by other studies (18,19). 83

2 84 R. DURAN ET AL Increased factor VIII levels, which might be familial, were found to be a risk factor for thrombosis in adults (20,21). Few studies were done to assess the association between childhood stroke and increased factor VIII levels (22). As the association of genetic prothrombotic conditions with pediatric ischemic stroke is inconclusive, in this study we evaluated the prevalence of AT, PC, and PS deficiencies as well as the presence of APLA, APCR, and increased factor VIII levels, in 30 children with ischemic stroke. FVL mutation was tested in subjects with APCR. Although prothrombin gene mutation (G20210A) and hyperhomocysteinemia are also frequently recognized causes of thrombophilia, these were not evaluated because of technical reasons. MATERIALS AND METHODS Patients This retrospective study included 30 children with ischemic stroke who were diagnosed and being observed in a center for mentally and physically handicapped children between January 2000 and December Neonatal stroke and patients presenting with transient symptoms were excluded. The age of the patients was between 1.5 and 15 years (7.1 ± 4.6 years). The definition of ischemic stroke included the presence of an acute thrombotic cerebrovascular event that manifested as hemiplegia, aphasia, visual or balance disturbance, or seizures. In all patients the diagnosis of cerebral ischemic stroke was confirmed with computed tomography (CT) and/or magnetic resonance imaging (MRI) scans. Data collected included age at diagnosis, gender, medical history, family history, signs and symptoms at diagnosis, radiologic findings, treatment, and outcome. Control Subjects Thirty-three healthy control subjects from the pediatrics outpatient clinic, ages between 6 months and 15 years (6.8 ± 3.2 years) were enrolled in the age-matched control group. Patients with acute febrile illness, coagulation abnormalities, neurologic disease, and chronic disease were excluded. Coagulation Tests Blood samples were obtained from all patients on referral at 1.5 to 15 years after the acute stroke. Nine parts of blood were drawn into one part of 3.8% sodium citrate. Citrated blood was centrifuged within 30 minutes at 2000g for 20 minutes and plasma aliquots were stored at 80 C. PC, free PS, and factor VIII activities were measured by the coagulometric assay (Diagnostica Stago, France). AT level was measured by nephelometric assay (Beckman Coulter, Ireland). Patients were diagnosed with PC, PS, or AT deficiency if the value of the determined protein was less than 2 SD of the mean age-adjusted level (PC <30.6%, PS <65.7%, AT <5.84 mg/dl) (23). Patients were diagnosed with increased factor VIII levels if its value was greater than 2 SD of the mean age-adjusted level of control subjects (>200%). APCR was measured by using FV-depleted plasma, aptt-based coagulometric assay (Diagnostica Stago, France). The diagnosis of APC resistance was established if the APC sensitivity ratio (APC-SR) was less than 2 SD of mean APC- SR (<1.81). Immunologic Test Cardiolipin (IgA, IgG and IgM) antibodies were measured with enzyme-linked immunosorbent assay (Diagnostica Stago, France). The presence of anticardiolipin antibodies was established when the values were greater than 2 SD of the mean values (>15.96 PL units/ml). DNA Polymorphism Additional 4 ml EDTA-anticoagulated blood samples were obtained from the patients and the control subjects who were diagnosed with APCR. DNA was extracted from these blood samples through standard methods. FVL was detected with polymerase chain reaction (PCR) amplification of a 267-bp fragment and MnlI digestion, as previously described (24). Statistical Analysis Demographic characteristics between patients and healthy control subjects were compared with chi square test for categorical variables. Fisher s exact test was used to compare the prevalence of combination of factors between patients and control subjects. A p value less than 0.05 was considered to be statistically significant. RESULTS The demographic data for 30 pediatric ischemic stroke patients and 33 control subjects

3 THROMBOPHILIA MARKERS IN CHILDHOOD ISCHEMIC STROKE 85 are shown in Table 1. No significant differences in gender and ethnic origin and the time of testing were observed between the patients and the control subjects (p>0.05). Twelve children (40%) of 33 patients with ischemic stroke had a family history acceptable with primer thrombophilia such as myocardial infarction, stroke, or deep vein thrombosis at an age less than 50 years of age. Because none of the control subjects had a similar family history, the difference was statistically significant (p<0.05). When stroke occurred, none of the patients had a condition predisposing to stroke such as immobilization, sepsis, surgery, dehydration, trauma, or malignancy. The prevalence of thrombophilia markers and their levels among the pediatric stroke patients and the control subjects are shown in Tables 2 and 3. Seventeen of 30 patients with ischemic stroke (56.6%) and five of 33 control subjects (15.1%) were found to have at least one thrombophilia marker. The prevalence of thrombophilia markers was higher among patients than among control subjects (p<0.05). At least two thrombophilia markers were detected in three of 30 patients (10%) whereas no combinations were observed in the control subjects (Table 4) but the difference was not statistically significant (p>0.05). None of the patients, but one of 33 control subjects (3.03%) had PS deficiency. The prevalence of PC and AT deficiency was higher among patients, but the difference was not statistically significant (p>0.05). Seven children of 30 stroke patients (23.3%) and one of 33 control subjects (3.03%) had APC resistance, and the difference was statistically significant (p<0.05). All children with APC resistance had FVL mutation, and the prevalence of FVL mutation was statistically significantly higher among stroke patients than control subjects (p<0.05). Five stroke patients had heterozygous FVL mutation; two stroke patients and one control subject had homozygous FVL mutation (Table 2). Five stroke patients and two control subjects had APLA but the difference between patients and controls was not statistically significant (p>0.05). Four patients and one control subject were diagnosed with increased factor VIII levels, and this difference between groups was not statistically significant (p>0.05). TABLE 1. Demographic Data in Pediatric Stroke Patients and Control Subjects Patients (N=30) Control Subjects (N=33) n (%) n (%) P (Chi-Square Test) Gender Female 10 (33.3) 15 (45.5) N.S. Male 20 (66.7) 18 (54.5) Origin Trakya region 20 (66.7) 23 (69.7) Anatolia 2 (6.7) 5 (15.1) Imigrants from Greece 5 (16.7) 3 (9.1) N.S. Imigrants from Bulgaria 3 (10) 2 (6.1) Age at testing Mean±SD, years 7.1± ±3.2 N.S. Range Age at diagnosis Mean±SD, years 2.5±3.2 Range Family history Positive 12 (40) 0 (0) <0.05 Negative 18 (60) 33 (100)

4 86 R. DURAN ET AL TABLE 2. Prevalence of Thrombophilia Markers in Pediatric Stroke Patients and Control Subjects Patients (N = 30) Control Subjects (N = 33) Thrombophilia Markers n (%) n (%) P (Fisher s Exact Test) APC resistance 7 (23.3) 1 (3.03) <0.05 FVL 7 (23.3) 1 (3.03) <0.05 Heterozygote 5 (16.7) 0 (0) Homozygote 2 (6.6) 1 (3.03) APLA 5 (16.7) 2 (6.1) N.S. AT deficiency 2 (6.7) 0 (0) N.S. PC deficiency 3 (10) 0 (0) N.S. PS deficiency 0 (0) 1 (3.03) N.S. Increased FVIII levels 4 (13.3) 1 (3.03) N.S. APC: activated protein C, FVL: factor V Leiden, APLA: antiphospholipid antibodies, AT: antithrombin, PC: protein C, PS: protein S, FVIII: factor VIII. TABLE 3. Levels of Thrombophilia Markers in Pediatric Stroke Patients and Control Subjects Patients (N=30) Control Subjects (N=33) Mean±SD Mean±SD Thrombophilia Markers (Min.-Max.) (Min.-Max.) P (Chi-Square Test) APC-SR 2.46 ± 0.64 ( ) 2.73 ± 0.46 ( ) N.S. APLA (PL U/mL) 7.84 ± 6.87 (0 25.5) 6.46 ± 4.75 ( ) N.S. AT (mg/dl) 7.90 ± 1.17 ( ) 8.3 ± 1.23 ( ) N.S. PC (% activity) 79.3 ± 36.3 ( ) 72.4 ± 20.9 ( ) N.S. PS Aktivitesi (% activity) ± 22.9 ( ) ± 21.4 ( ) N.S. FVIII (% activity) ± 66.7 ( ) ± 54.2 ( ) N.S. APC: Activated protein C, APLA: Antiphospholipid antibodies, AT: Antithrombin, PC: Protein C, PS: Protein S, FVIII: Factor VIII, Min: Minimum, Max: Maximum. A combination of more than one thrombophilia marker was observed only among the patient group: a combination of FVL mutation and AT deficiency was diagnosed in one patient; another patient had FVL mutation and increased factor VIII levels. Furthermore, one patient had three thrombophilia markers, FVL, AT deficiency, and increased factor VIII levels (Table 4). DISCUSSION This study is a retrospective, case-control analysis of the risks exerted by hereditary and acquired thrombophilia markers in childhood ischemic stroke. Among the various causes for ischemic stroke in children, inherited defects of coagulation are being increasingly recognized (8 17). APC resistance, which is not only inherited as autosomal dominant with variable penetrance but also is an acquired situation, was first described by Dahlback and colleagues (25) in The most frequent cause of APC resistance is a point mutation in the factor V gene (FVL) with a prevalence of 3% to 5% of heterozygous carriers among the white healthy population (26). The mutation is a result of the replacement of Arg506

5 THROMBOPHILIA MARKERS IN CHILDHOOD ISCHEMIC STROKE 87 TABLE 4. Prevalence of Combinations Thrombophilia Markers in Pediatric Stroke Patients and Control Subjects Patients (N=30) Control Subjects (N=33) Marker n (%) n (%) P (Fisher s Exact Test) FVL AT deficiency 1 (3.3) 0 (0) N.S. FVL Increased FVIII levels 1 (3.3) 0 (0) N.S. FVL AT deficiency increased FVIII levels 1 (3.3) 0 (0) N.S. FVL: factor V Leiden, AT: antithrombin, FVIII: factor VIII. by Gln and the subsequent loss of the cleavage site for APC, then factor V is inactivated at a reduced rate (25). FVL has been reported to be the most common genetic risk factor for venous thromboembolism both in adults (27,28) and children (14,15). But in most studies with ischemic stroke in adults, FVL has not been demonstrated to be a risk factor (28 30). However in contrast to findings in adults and for unclear reasons, most of the thrombotic events in children associated with APC resistance and FVL are of arterial origin (12 17). Moreover in some reports these data were not confirmed (18,19,31). The difference among these studies can reflect the role of ethnicity, the small number of patients, and the diverse etiologies of childhood stroke and unidentified acquired thrombotic risk factors. In this study, the prevalence of FVL mutation in controls (3.03%) is in accordance with the distribution in general population in the Trakya region of Turkey (5.40%) (32), but in pediatric ischemic stroke patients FVL mutation was higher (23.3%, p<0.05). The present data on FVL concur with other similar case-control studies (7,13,15,33). In the literature, APLA was determined to be the most frequent risk factor in ischemic stroke in children (6,9). In a prospective blood bank survey, APLA were detected in approximately 6.5% of normal subjects (34). The children who have high levels of APLA have been reported to have a significantly increased risk for arterial and venous thrombosis, 50% of which occur in the central nervous system (6). De Veber and colleagues (6) reported an eightfold increase in the prevalence of APLA in children with stroke compared with the control subjects. However in this study, the prevalence of APLA in stroke patients is not found to be higher than that in control subjects. These data are consistent with those of the study of McColl and colleagues (18). In studies that demonstrate APLA as a significant risk factor, APLA were tested with three different tests (one immunologic and two coagulation-based), thereby increasing the sensitivity of detection of antibodies (6,35). In this study APLA was evaluated with only an immunologic test; therefore, the diagnosis of APLA could have been missed in some patients. Similar to other case-control studies (18,35), no association between the rare defects of hereditary PC, PS, and AT deficiencies and childhood stroke is established in this study. However there are reports in which such an association was found (6,8). Therefore, the role of these thrombophilia factors in ischemic pediatric stroke is inconclusive. In recent years, increased factor VIII levels were found in adults with recurrent thrombosis (21). However any genetic mutation that resulted with increased factor VIII levels has not been yet described. In this study, increased factor VIII levels are not identified more common in stroke patients. However, increased factor VIII levels are included in combinations of thrombophilia markers in our stroke patients whereas no combinations of thrombophilia markers are detected in control subjects. Kenet and colleagues (29) reported that the combinations of thrombophilia markers increased the risk of ischemic stroke. In conclusion, childhood stroke emerges as a condition associated with a combination of multiple risk factors and especially the presence of FVL mutation is a predisposing factor. However, the high prevalence of FVL mutation in the population and the low incidence of childhood stroke, points that some other yet undetermined factors must have a significant role in this event. Our results also justify the screening of children with ischemic stroke for thrombophilia markers. Identification of etiologic factors is mandatory to prevent recurrences.

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