Prothrombotic Factors in Children With Stroke or Porencephaly
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《小儿科》
Neuroepidemiology Branch Family Studies Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland
ABSTRACT
Objective.This study compared the frequencies of genetic and functional coagulation abnormalities in children with arterial ischemic stroke or porencephaly with frequencies in previously published studies.
Methods.A series of 59 children (age 0–18 years) with arterial ischemic stroke or porencephaly were referred to the National Institutes of Health. A blood sample, buccal smear sample, questionnaire, and pedigree were requested for each child. Blood samples were analyzed for protein C (PC); protein S; antithrombin (AT); activated PC resistance (APCR); lipoprotein (a) [Lp(a)]; lupus anticoagulant; anticardiolipin antibodies; and the methylenetetrahydrofolate reductase C677T (MTHFR), factor V G1619A, factor II G20210A (PT), plasminogen activator inhibitor-1 4G6755G, and tissue factor pathway inhibitor C536T mutations. The frequency of each coagulation abnormality was compared with published international pediatric stroke case and control rates.
Results.At least 1 prothrombotic abnormality was identified in 63% (36 of 57) of children studied, including plasminogen activator inhibitor-1 4G6755G (15 of 56), MTHFR (12 of 56), elevated Lp(a) (12 of 59), APCR (11 of 58), factor V G1619A (5 of 57), PT (3 of 57), PC deficiency (1 of 59), and AT deficiency (1 of 59). The MTHFR mutation, elevated Lp(a), the PT mutation, and AT deficiency rates were similar to rates in cases and more common than control subjects in previously published studies. The rate of children with APCR or multiple abnormalities was higher than in previous pediatric stroke studies. A family history of early thrombosis was identified in one third of the children with a prothrombotic abnormality.
Conclusions.Two thirds of children in this study had at least 1 of the prothrombotic risk factors tested, and several children had multiple risk factors. These results provide additional evidence that prothrombotic abnormalities are common among children with AIS or porencephaly.
Key Words: cerebrovascular diseases children coagulation genetic polymorphisms
Abbreviations: PC, protein C PS, protein S AT, antithrombin FVL, factor V G1619A PT, factor II G20210A PAI-1, plasminogen activator inhibitor -1 4G6755G MTHFR, methylenetetrahydrofolate reductase C677T AIS, arterial ischemic stroke APCR, activated protein C resistance Lp(a), lipoprotein (a) LA, lupus anticoagulant ACLA, anticardiolipin antibodies TFPI, tissue factor pathway inhibitor C536T Ig, immunoglobulin
During the past decade, interest has grown in the contribution of inherited coagulation and metabolic factors to the risk for stroke in adults and children. Genetic factors probably play a larger role in individuals who present with stroke at an early age as compared with onset in later years. Studies of risk factors for stroke in children, who lack the accumulated comorbidities that contribute heavily to stroke in adults, may yet provide important insights into the pathogenesis of stroke in adults. For example, clinical observations of children with homocystinuria and vascular disease led to epidemiologic studies and clinical trials that resulted in the identification of hyperhomocysteinemia as an important contributor to vascular disease in adults.1
Stroke in children is a heterogenous disorder associated with significant morbidity and mortality. Incidence has been estimated at 1 per 4000 for neonates and has ranged from 1 per 7000 to 1 per 70000 for older children (1 month to 18 years).2,3 The most frequently reported risk factors for stroke in children include cardiac disorders, prothrombotic disorders, metabolic disorders, vascular disorders, and infection.4,5 In up to two thirds of children, no specific cause has been determined.6
Many genetic and acquired prothrombotic abnormalities have been evaluated in children with cerebrovascular disease, but there has been a wide variation in the prevalence of these abnormalities among different age groups, stroke subtypes, and international populations. The most common prothrombotic factors studied include (1) deficiencies in anticoagulant proteins (protein C [PC], protein S [PS], and antithrombin [AT]), (2) antibodies to phospholipids associated with thrombosis, (3) genetic polymorphisms encoding proteins associated with thrombosis (factor V G1619A [FVL], factor II G20210A [PT], and plasminogen activator inhibitor-1 4G6755G [PAI-1]) and homocysteine metabolism (methylenetetrahydrofolate reductase C677T [MTHFR]), and (4) lipoproteins that predispose to thrombosis. Several case-control studies, using hospital-based adult and population-based child control subjects, have shown an association between many of these abnormalities and stroke,7–9 whereas other studies have been negative or too small to show a difference.10–13 The most consistently reported associations are for PC deficiency, elevated lipoprotein (a), and the FVL mutation. Recent evidence also suggests that children with specific prothrombotic abnormalities are at a greater risk for recurrent stroke.4
Currently, it is unclear to what degree these abnormalities contribute to the development of childhood stroke, either individually or in the presence of other genetic or environmental risk factors. Although a thorough evaluation for thrombophilia seems reasonable for children with stroke, the diagnostic yield is low in adults.14 There are no evidence-based guidelines for screening or treatment of children with stroke and a prothrombotic abnormality. The purpose of this pilot study was to compare the frequencies of genetic and functional coagulation abnormalities in children with arterial ischemic stroke or porencephaly with previously published population levels to plan for future genetic association studies.
METHODS
Study Children
We report an ongoing National Institute of Neurologic Disorders and Stroke study of stroke in children aged 0 to 18 years. Patients who presented to Johns Hopkins University/Kennedy Krieger Institute (Baltimore, MD), and Children's Memorial Hospital (Chicago, IL) between 1990 and 2000 were identified retrospectively through a search of hospital discharge codes and radiology records. Children with a history of stroke (International Classification of Diseases, Ninth Revision codes 430-437) or porencephalic cyst (International Classification of Diseases, Ninth Revision code 348) were contacted by mail and invited to participate in the study (n = 6). Study children were also referred from outside physicians or contacted the study investigators directly at the National Institutes of Health (n = 53). Retrospective and prospective study participants were screened according to inclusion and exclusion criteria. Participants were required to provide a buccal smear and blood sample, complete a pedigree and questionnaire, and grant permission for review of imaging and medical records. Informed consent and assent were obtained from all study children, and the study protocol was reviewed and approved by the National Institute of Neurologic Disorders and Stroke scientific and institutional review board.
Diagnosis of Stroke and Porencephaly
Arterial ischemic stroke (AIS) was defined as a new focal neurologic deficit that lasted 24 hours or longer and was presumably due to a vascular process; porencephaly was defined as a fluid-filled cavity within the cerebral hemispheres that may communicate with cerebrospinal fluid spaces. The diagnosis of AIS or porencephaly was confirmed by computed tomography or MRI for each child.
Study children were classified into 3 categories: (1) perinatal stroke, defined as systemic or neurologic symptoms with radiographic evidence of AIS or porencephaly that occurred before birth or in the first 30 days of life; (2) delayed perinatal stroke, defined as no diagnosis of stroke in the perinatal or neonatal period, focal motor dysfunction or seizures diagnosed after 2 months of age, and neuroimaging evidence of remote focal infarction, encephalomalacia, and or porencephaly; and (3) childhood stroke, defined as an acute onset of systemic or neurologic symptoms with radiographic evidence of AIS that occurred between 30 days and 18 years of age. Children with a history of cancer, heart disease, trauma, chromosomal or metabolic disorders, sickle cell disease, or central nervous system infection were excluded.
Data Abstraction
Study investigators reviewed available medical records for each child. Parents completed a questionnaire regarding maternal and birth history, stroke history, and functional status of their child with a diagnosis of stroke. Family histories were obtained by a certified genetic counselor (L.N.) through telephone interviews for a majority of the children in the study. Parents of probands were instructed to contact family members to determine a family history of a major vascular event. A positive family history was defined as a major thrombotic event (myocardial infarction, deep venous thrombosis, pulmonary embolus, or ischemic stroke) before 60 years of age in first- or second-degree relatives of the probands.
Laboratory Methods
Blood samples were collected at least 3 months after the incident stroke for each child. Blood was collected by peripheral venipuncture into plastic tubes that contained 0.105 M buffered sodium citrate (3.2%). Citrated blood was placed immediately on ice and was processed within 1 hour. Citrated blood was centrifuged at 1700g for 15 minutes at room temperature. Plasma supernatant was removed, transferred to plastic tubes, recentrifuged for 5 minutes to ensure platelet-free plasma, and then stored at –70°C. The buffy coat layer was subsequently removed and stored at –70°C before DNA extraction. Blood samples were frozen at –70°C and stored in batches until they were shipped on dry ice to the Hemostasis Reference Laboratory at the Henderson Research Centre (Hamilton, ON, Canada) for analysis.
Blood samples were analyzed for PC, PS, AT, activated PC resistance (APCR), lipoprotein (a) [Lp(a)], lupus anticoagulant (LA), anticardiolipin antibodies (ACLA), and the MTHFR, FVL, PT, PAI-1, and tissue factor pathway inhibitor C536T (TFPI) mutations. Age-related standardized ranges were used to determine normal and abnormal levels for PC, PS, AT, APCR, Lp(a), LA, and ACLA. In some cases, coagulation testing had been performed before enrollment in the study, but this information was not available to investigators at the time of screening for inclusion.
PC was measured on a BCT or BCS analyzer using a functional assay (Protein C Reagent Kit; Dade Behring, Marburg, Germany), AT was measured by chromogenic assay (Coamatic Antithrombin Assay; Diapharma, West Chester, OH), and PS and Lp(a) were measured by enzyme-linked immunosorbent assay (Asserchrom Free Protein S, Diagnostica Stago, Asnieers, France; and Tintelize Kit, Biopool, Sweden, now Trinity Biotech, County Wicklow, Ireland). APCR was assayed by Dade Actin FS aPTT reagent (Dade Behring) and activated PC (American Diagnostics, Greenwich, CT).15 LA was tested using PTT-LA reagent (Diagnostica Stago) and dilute Russell Viper Venom (American Diagnostics, Greenwich, NJ, or BioMerieux, Durham, NC). LA samples with abnormal results were repeated using a confirmatory test, either Hexogonal Phospholipid or dRVV confirm. ACLA, immunoglobulin (Ig) G, IgM, and IgA, were measured by enzyme-linked immunosorbent assay (Corgenix, Westminster, CO). The MTHFR, FVL, PT, PAI-1, and TFPI mutations were identified by amplification and polymerase chain reactions followed by digestion of amplified fragments with restriction enzymes, as previously reported.16–20
Published Population Cases and Controls
For comparisons, we identified reports, using a search of the MEDLINE database, published in English between 1995 and May 2004, that evaluated the presence of genetic coagulation abnormalities in populations of children with cerebrovascular disease. We abstracted information on type and prevalence of genetic coagulation abnormality, study design, patient and control populations, age range, and methods of analysis.
Statistical Methods
The percentages of each coagulation abnormality were compared with published pediatric stroke case and control rates. Fisher exact tests were used for internal comparisons between children with and without a family history of thrombosis and between perinatal or delayed perinatal and childhood stroke.
RESULTS
We identified 59 children with stroke of previously undetermined cause. There were 18 perinatal, 25 delayed perinatal, and 16 childhood strokes. The majority of these children were white (93%) and male (53%). The mean age at diagnosis was 23 months (median: 8 months). Neuroimaging was completed for all children: MRI for 92% (54 of 59) and computed tomography scan for 71% (42 of 59) of children. Stroke location was in the distribution of the left middle cerebral artery in 47% and right middle cerebral artery in 25%. At study entry, 76% of children had hemiparesis, 8% had monoparesis, 7% had quadriplegia, and 8% had no persisting motor disabilities (Table 1).
During a median follow-up period of 3.5 years (range: 3 months to 16 years), none of the children with a history of perinatal or delayed perinatal stroke had a recurrent stroke. Of children with childhood stroke, 19% (3 of 16) had a recurrence; all of these had a coagulation abnormality [2 PAI-1, 1 Lp(a)], and the 2 whose family history was known had a family history of thrombosis.
At least 1 prothrombotic abnormality was identified in 63% (36 of 57) of children studied, and 28% (16 of 57) had multiple abnormalities. Several prothrombotic abnormalities were identified, including PC deficiency (1 of 59), AT deficiency (1 of 59), APCR (11 of 58), elevated Lp(a) (12 of 59), MTHFR (12 of 56), PAI-1 (15 of 56), FVL (5 of 57), and PT (3 of 57). No child was identified with PS deficiency, ACLA, LA, or the TFPI mutation. Tests for prothrombotic abnormalities were incomplete for 4 children as a result of inadequate DNA samples. Of the 4 children with incomplete testing, 2 did not have an identified abnormality (Table 2).
AT deficiency, elevated Lp(a), the PT mutation, and the MTHFR mutation were similar to rates in cases and more common than control subjects in previously published studies. The rate of children with APCR or multiple abnormalities was higher than in previous pediatric stroke studies. There were no significant differences in the prevalence of a coagulation abnormality among children with perinatal stroke (56%), delayed perinatal stroke (67%), or childhood stroke (67%).
A pedigree was completed for 37 of the 59 children. Thrombosis that occurred before 60 years of age was identified in 4% of family members, none (0 of 118) of first-degree relatives of these children, and 5.7% (16 of 281) of second-degree relatives. There was a family history of thrombosis before 60 years of age in 31% (11 of 35) of children overall and among 33% (6 of 18) of children with a prothrombotic abnormality (P = 1.00).
DISCUSSION
The identification of a prothrombotic factor in a child with stroke may be helpful for determination of cause and recurrence risk and may have implications for prevention of systemic thrombosis in specific situations and for screening family members,21 yet the variability of these abnormalities among different age groups, stroke subtypes, and international populations has led to uncertainties regarding evaluation, risk estimates, and treatment in children with stroke.
The present study is the largest series of prothrombotic risk factors in American children with stroke. Two thirds of children in this study had at least 1 of the prothrombotic risk factors tested, and several children had multiple risk factors.
Prothrombotic Abnormalities and Children With Stroke
Several international case-control studies have revealed an association between prothrombotic abnormalities and stroke in children,7–9 whereas other studies were negative or too small to show a difference10–13 (Table 3). The most consistent associations have been reported for PC deficiency, elevated Lp(a), and the FVL mutation. In this study, we found that rates of AT deficiency, elevated Lp(a), the PT mutation, and the MTHFR mutation were higher than those in published rates in pediatric stroke control subjects.
Functional deficiencies in anticoagulant proteins were found in only 2 children in this study. One child was found to have AT deficiency, a rate similar to those in other series.10,11,22 AT deficiency has been reported in as many as 13% of children with stroke,21 but most studies have failed to implicate this factor in childhood stroke.9
Lp(a) is structurally homologous to plasminogen and has a similar effect on coagulation, fibrinolysis, and endothelial function.23 An increased level of Lp(a) has been reported in 3 German studies9,24,25 and 1 Portuguese26 study to be a risk factor for stroke in neonates and children. Most studies in adults have yielded similar findings.27 We found elevated Lp(a) in 20% of the children in this study, a rate much higher than in published pediatric stroke control subjects (5.5%).25
The PT mutation is a genetic variant of the 3-prime untranslated region of the PT gene that leads to increased plasma PT levels. The PT mutation is an autosomal dominant mutation that varies widely in prevalence and alone is associated with a modest increase in risk for ischemic stroke, myocardial infarction, and peripheral vascular disease in adults.28 Studies of children with stroke have yielded mixed results. Case-control studies from Turkey7 and Germany9 were positive, whereas other international studies were negative or underpowered.8,13,26 We identified the PT mutation in 5% of children in this study, a rate slightly higher than the 0.9% to 2.8% reported in published pediatric stroke control subjects.
The association between elevated homocysteine and stroke is widely recognized. The MTHFR mutation has been shown to increase the risk for stroke in children and adults. A recent meta-analysis of patients who were younger than 55 years and had AIS revealed a modest increased risk for stroke among homozygous carriers of the MTHFR mutation.28 Despite these findings in younger adults and high rates in pediatric stroke patients, only 1 case-control study in children was sufficiently powered to show an association.9 We identified homozygosity for the MTHFR mutation in 21% of the children in this study, a rate higher than that among published pediatric stroke control subjects (5.6–15.2).7,8
Almost 30% of the children in this study had >1 of the prothrombotic abnormalities evaluated. Five children had 3 or more prothrombotic abnormalities. Other studies using an extensive prothrombotic panel have reported a combination of prothrombotic abnormalities in up to 23% of children with stroke.21 A combination of prothrombotic factors substantially increases the risk for stroke in children.9,24,29 Prothrombotic combinations that have been reported with an increased risk for stroke in children include the FVL mutation and elevated Lp(a) or the MTHFR mutation.9
Recurrent Stroke and Prothrombotic Abnormalities
Recurrent stroke is rare among infants with perinatal stroke30 but in children ranges from 6% to 30%.21,31,32 Combined data from Germany, Canada, and the United Kingdom of 565 patients with AIS revealed a recurrence rate of 10% after several years of follow-up.33 The MTHFR mutation, elevated homocysteine, elevated Lp(a), and PC deficiency have been associated with recurrent stroke in infants and children.4 In this study, 19% (3 of 16) of children with childhood stroke had a recurrence, all of which had a coagulation abnormality and 2 a family history of thrombosis.
Age-Related Differences in Frequency of Prothrombotic Abnormalities
The prevalence of prothrombotic abnormalities among individuals with stroke has varied according to age and suggests that genetic risk factors are more important in individuals who present with stroke at an early age. In studies that used an extensive screen for thrombophilia, prothrombotic abnormalities were more common in neonates25,34 as compared with children12,35 or adults with stroke.36 This study found no differences in the frequency of prothrombotic factors among neonates compared with older children, but this likely reflects the small number for comparison.
Family History of Thrombosis and Stroke in Children
A family history of disease provides information on disease susceptibility reflected through common genes, behaviors, and shared environment.37 A family history of vascular disease is an independent risk factor for stroke in young adults, and the risk that it confers varies by stroke subtype.38,39 There is limited information on the risk for stroke in children and neonates with a family history of stroke or thrombosis. Case series and controlled studies of children with stroke have found a family history of thrombosis among 5% to 42% of children studied.21,26,40 A family history of thrombosis was identified in one third of the children in this study and, similar to other studies, was not predictive of the presence of a coagulation abnormality.8 None of the children in this study had a sibling with a history of AIS or porencephaly.
Treatment of Children With Stroke and a Prothrombotic Abnormality
Prothrombotic abnormalities are associated with an increased risk for stroke and recurrent stroke in children,4,31 but there are as yet no evidence-based strategies or consensus guidelines for the treatment of children with stroke and a prothrombotic abnormality. Recommendations for antithrombotic therapy in children with thrombophilia have been based on small, nonrandomized studies and expert opinion.41 Antithrombotic therapy is recommended in adults for acute events and as prophylaxis in high-risk situations and may be warranted in children with specific prothrombotic abnormalities.42 The addition of other genetic risk factors for thrombosis in symptomatic individuals probably warrants long-term therapy, but this must be weighed against the risk for anticoagulation in young children. The response and long-term complications of antithrombotic therapy are not the same in adults and children.43 Clinical trials are needed to determine the indications and best treatment for children with a prothrombotic abnormality. The screening of unaffected relatives of probands with a prothrombotic mutation has been advocated because the risk for thrombosis can be reduced by prophylaxis in high-risk situations.44
CONCLUSIONS
The evidence relating prothrombotic abnormalities and cerebrovascular disorders in children is growing, but much remains unknown. Additional case-control studies that incorporate representative cases and appropriately matched control subjects are needed to verify previous findings. Additional studies are needed to quantify the risk for a cerebrovascular event in children with a prothrombotic mutation and to determine which combination of endogenous and exogenous risk factors lead to greater rates of initial and recurrent stroke.
ACKNOWLEDGMENTS
We acknowledge Marilyn Johnston, ART, for review of the manuscript and the children, parents, and family members who participated in the study.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
PEDIATRICS (ISSN 0031 4005). Published in the public domain by the American Academy of Pediatrics.
REFERENCES
The Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA. 2002;288 :2015 –2022
Earley CJ, Kittner SJ, Feeser BR, et al. Stroke in children and sickle-cell disease: Baltimore-Washington Cooperative Young Stroke Study. Neurology. 1998;51 :169 –176
Giroud M, Lemesle M, Gouyon JB, et al. Cerebrovascular disease in children under 16 years of age in the city of Dijon, France: a study of incidence and clinical features from 1985 to 1993. J Clin Epidemiol. 1995;48 :1343 –1348
Strater R, Becker S, von Eckardstein A, et al. Prospective assessment of risk factors for recurrent stroke during childhood—a 5-year follow-up study. Lancet. 2002;360 :1540 –1545
Kirkham FJ, Prengler M, Hewes DK, et al. Risk factors for arterial ischemic stroke in children. J Child Neurol. 2000;15 :299 –307
Al Sulaiman A, Bademosi O, Ismail H, et al. Stroke in Saudi children. J Child Neurol. 1999;14 :295 –298
Akar N, Akar E, Deda G, et al. Factor V1691 G-A, prothrombin 20210 G-A, and methylenetetrahydrofolate reductase 677 C-T variants in Turkish children with cerebral infarct. J Child Neurol. 1999;14 :749 –751
Kenet G, Sadetzki S, Murad H, et al. Factor V Leiden and antiphospholipid antibodies are significant risk factors for ischemic stroke in children. Stroke. 2000;31 :1283 –1288
Nowak-Gottl U, Strater R, Heinecke A, et al. Lipoprotein (a) and genetic polymorphisms of clotting factor V, prothrombin, and methylenetetrahydrofolate reductase are risk factors of spontaneous ischemic stroke in childhood. Blood. 1999;94 :3678 –3682
Hagstrom JN, Walter J, Bluebond-Langner R, et al. Prevalence of the factor V Leiden mutation in children and neonates with thromboembolic disease. J Pediatr. 1998;133 :777 –781
Bonduel M, Sciuccati G, Hepner M, et al. Factor V Leiden and prothrombin gene G20210A mutation in children with cerebral thromboembolism. Am J Hematol. 2003;73 :81 –86
McColl MD, Chalmers EA, Thomas A, et al. Factor V Leiden, prothrombin 20210G->A and the MTHFR C677T mutations in childhood stroke. Thromb Haemost. 1999;81 :690 –694
Zenz W, Bodo Z, Plotho J, et al. Factor V Leiden and prothrombin gene G 20210 A variant in children with ischemic stroke. Thromb Haemost. 1998;80 :763 –766
Bushnell CD, Goldstein LB. Diagnostic testing for coagulopathies in patients with ischemic stroke. Stroke. 2000;31 :3067 –3078
Griffin JH, Evatt B, Wideman C, et al. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood. 1993;82 :1989 –1993
Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10 :111 –113
Bertina RM, Koeleman BP, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369 :64 –67
Poort SR, Rosendaal FR, Reitsma PH, et al. A common variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88 :3698
Kleesiek K, Schmidt M, Gotting C, et al. The 536C->T transition in the human tissue factor pathway inhibitor (TFPI) gene is statistically associated with a higher risk for venous thrombosis. Thromb Haemost. 1999;82 :1 –5
Sartori MT, Wiman B, Vettore S, et al. 4G/5G polymorphism of PAI-1 gene promoter and fibrinolytic capacity in patients with deep vein thrombosis. Thromb Haemost. 1998;80 :956 –960
deVeber G, Monagle P, Chan A, et al. Prothrombotic disorders in infants and children with cerebral thromboembolism. Arch Neurol. 1998;55 :1539 –1543
Ganesan V, Prengler M, McShane MA, et al. Investigation of risk factors in children with arterial ischemic stroke. Ann Neurol. 2003;53 :167 –173
Ariyo AA, Thach C, Tracy R. Lp(a) lipoprotein, vascular disease, and mortality in the elderly. N Engl J Med. 2003;349 :2108 –2115
Strater R, Vielhaber H, Kassenbohmer R, et al. Genetic risk factors of thrombophilia in ischaemic childhood stroke of cardiac origin. A prospective ESPED survey. Eur J Pediatr. 1999;158(suppl 3) :S122 –S125
Gunther G, Junker R, Strater R, et al. Symptomatic ischemic stroke in full-term neonates: role of acquired and genetic prothrombotic risk factors. Stroke. 2000;31 :2437 –2441
Barreirinho S, Ferro A, Santos M, et al. Inherited and acquired risk factors and their combined effects in pediatric stroke. Pediatr Neurol. 2003;28 :134 –138
Milionis HJ, Winder AF, Mikhailidis DP. Lipoprotein (a) and stroke. J Clin Pathol. 2000;53 :487 –496
Kim RJ, Becker RC. Association between factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations and events of the arterial circulatory system: a meta-analysis of published studies. Am Heart J. 2003;146 :948 –957
Heller C, Becker S, Scharrer I, et al. Prothrombotic risk factors in childhood stroke and venous thrombosis. Eur J Pediatr. 1999;158(suppl 3) :S117 –S121
Kurnik K, Kosch A, Strater R, et al. Recurrent thromboembolism in infants and children suffering from symptomatic neonatal arterial stroke: a prospective follow-up study. Stroke. 2003;34 :2887 –2892
Lanthier S, Carmant L, David M, et al. Stroke in children: the coexistence of multiple risk factors predicts poor outcome. Neurology. 2000;54 :371 –378
Chabrier S, Husson B, Lasjaunias P, et al. Stroke in childhood: outcome and recurrence risk by mechanism in 59 patients. J Child Neurol. 2000;15 :290 –294
Kirkham F, DeVeber G, Chan AK, et al. Recurrent stroke: the role of prothrombotic disorders . Ann Neurol. 2003;54 :S110
Mercuri E, Cowan F, Gupte G, et al. Prothrombotic disorders and abnormal neurodevelopmental outcome in infants with neonatal cerebral infarction. Pediatrics. 2001;107 :1400 –1404
Ganesan V, McShane MA, Liesner R, et al. Inherited prothrombotic states and ischaemic stroke in childhood. J Neurol Neurosurg Psychiatry. 1998;65 :508 –511
Hankey GJ, Eikelboom JW, van Bockxmeer FM, et al. Inherited thrombophilia in ischemic stroke and its pathogenic subtypes. Stroke. 2001;32 :1793 –1799
Yoon PW, Scheuner MT, Peterson-Oehlke KL, et al. Can family history be used as a tool for public health and preventive medicine Genet Med. 2002;4 :304 –310
Jerrard-Dunne P, Cloud G, Hassan A, et al. Evaluating the genetic component of ischemic stroke subtypes: a family history study. Stroke. 2003;34 :1364 –1369
Schulz UG, Flossmann E, Rothwell PM. Heritability of ischemic stroke in relation to age, vascular risk factors, and subtypes of incident stroke in population-based studies. Stroke. 2004;35 :819 –824
Golomb MR, MacGregor DL, Domi T, et al. Presumed pre- or perinatal arterial ischemic stroke: risk factors and outcomes. Ann Neurol. 2001;50 :163 –168
Jilma B, Kamath S, Lip GY. ABC of antithrombotic therapy: antithrombotic therapy in special circumstances. II—in children, thrombophilia, and miscellaneous conditions. BMJ. 2003;326 :93 –96
Haemostasis and Thrombosis Task Force, British Committee for Standards in Haematology. Investigation and management of heritable thrombophilia. Br J Haematol. 2001;114 :512 –528
Williams MD, Chalmers EA, Gibson BE. The investigation and management of neonatal haemostasis and thrombosis. Br J Haematol. 2002;119 :295 –309
De S, V, Rossi E, Paciaroni K, et al. Screening for inherited thrombophilia: indications and therapeutic implications. Haematologica. 2002;87 :1095 –1108
Koh S, Chen LS. Protein C and S deficiency in children with ischemic cerebrovascular accident. Pediatr Neurol. 1997;17 :319 –321
Akar N, Akar E, Ozel D, et al. Common mutations at the homocysteine metabolism pathway and pediatric stroke. Thromb Res. 2001;102 :115 –120
Nowak-Gottl U, Strater R, Kosch A, et al. The plasminogen activator inhibitor (PAI)-1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children. Childhood Stroke Study Group. Eur J Haematol. 2001;66 :57 –62
Cardo E, Monros E, Colome C, et al. Children with stroke: polymorphism of the MTHFR gene, mild hyperhomocysteinemia, and vitamin status. J Child Neurol. 2000;15 :295 –298
Debus OM, Kosch A, Strater R, et al. The factor V G1691A mutation is a risk for porencephaly: a case-control study. Ann Neurol. 2004;56 :287 –290
Prengler M, Sturt N, Krywawych S, et al. Homozygous thermolabile variant of the methylenetetrahydrofolate reductase gene: a potential risk factor for hyperhomocysteinaemia, CVD, and stroke in childhood. Dev Med Child Neurol. 2001;43 :220 –225(John Kylan Lynch, DO, MPH)
ABSTRACT
Objective.This study compared the frequencies of genetic and functional coagulation abnormalities in children with arterial ischemic stroke or porencephaly with frequencies in previously published studies.
Methods.A series of 59 children (age 0–18 years) with arterial ischemic stroke or porencephaly were referred to the National Institutes of Health. A blood sample, buccal smear sample, questionnaire, and pedigree were requested for each child. Blood samples were analyzed for protein C (PC); protein S; antithrombin (AT); activated PC resistance (APCR); lipoprotein (a) [Lp(a)]; lupus anticoagulant; anticardiolipin antibodies; and the methylenetetrahydrofolate reductase C677T (MTHFR), factor V G1619A, factor II G20210A (PT), plasminogen activator inhibitor-1 4G6755G, and tissue factor pathway inhibitor C536T mutations. The frequency of each coagulation abnormality was compared with published international pediatric stroke case and control rates.
Results.At least 1 prothrombotic abnormality was identified in 63% (36 of 57) of children studied, including plasminogen activator inhibitor-1 4G6755G (15 of 56), MTHFR (12 of 56), elevated Lp(a) (12 of 59), APCR (11 of 58), factor V G1619A (5 of 57), PT (3 of 57), PC deficiency (1 of 59), and AT deficiency (1 of 59). The MTHFR mutation, elevated Lp(a), the PT mutation, and AT deficiency rates were similar to rates in cases and more common than control subjects in previously published studies. The rate of children with APCR or multiple abnormalities was higher than in previous pediatric stroke studies. A family history of early thrombosis was identified in one third of the children with a prothrombotic abnormality.
Conclusions.Two thirds of children in this study had at least 1 of the prothrombotic risk factors tested, and several children had multiple risk factors. These results provide additional evidence that prothrombotic abnormalities are common among children with AIS or porencephaly.
Key Words: cerebrovascular diseases children coagulation genetic polymorphisms
Abbreviations: PC, protein C PS, protein S AT, antithrombin FVL, factor V G1619A PT, factor II G20210A PAI-1, plasminogen activator inhibitor -1 4G6755G MTHFR, methylenetetrahydrofolate reductase C677T AIS, arterial ischemic stroke APCR, activated protein C resistance Lp(a), lipoprotein (a) LA, lupus anticoagulant ACLA, anticardiolipin antibodies TFPI, tissue factor pathway inhibitor C536T Ig, immunoglobulin
During the past decade, interest has grown in the contribution of inherited coagulation and metabolic factors to the risk for stroke in adults and children. Genetic factors probably play a larger role in individuals who present with stroke at an early age as compared with onset in later years. Studies of risk factors for stroke in children, who lack the accumulated comorbidities that contribute heavily to stroke in adults, may yet provide important insights into the pathogenesis of stroke in adults. For example, clinical observations of children with homocystinuria and vascular disease led to epidemiologic studies and clinical trials that resulted in the identification of hyperhomocysteinemia as an important contributor to vascular disease in adults.1
Stroke in children is a heterogenous disorder associated with significant morbidity and mortality. Incidence has been estimated at 1 per 4000 for neonates and has ranged from 1 per 7000 to 1 per 70000 for older children (1 month to 18 years).2,3 The most frequently reported risk factors for stroke in children include cardiac disorders, prothrombotic disorders, metabolic disorders, vascular disorders, and infection.4,5 In up to two thirds of children, no specific cause has been determined.6
Many genetic and acquired prothrombotic abnormalities have been evaluated in children with cerebrovascular disease, but there has been a wide variation in the prevalence of these abnormalities among different age groups, stroke subtypes, and international populations. The most common prothrombotic factors studied include (1) deficiencies in anticoagulant proteins (protein C [PC], protein S [PS], and antithrombin [AT]), (2) antibodies to phospholipids associated with thrombosis, (3) genetic polymorphisms encoding proteins associated with thrombosis (factor V G1619A [FVL], factor II G20210A [PT], and plasminogen activator inhibitor-1 4G6755G [PAI-1]) and homocysteine metabolism (methylenetetrahydrofolate reductase C677T [MTHFR]), and (4) lipoproteins that predispose to thrombosis. Several case-control studies, using hospital-based adult and population-based child control subjects, have shown an association between many of these abnormalities and stroke,7–9 whereas other studies have been negative or too small to show a difference.10–13 The most consistently reported associations are for PC deficiency, elevated lipoprotein (a), and the FVL mutation. Recent evidence also suggests that children with specific prothrombotic abnormalities are at a greater risk for recurrent stroke.4
Currently, it is unclear to what degree these abnormalities contribute to the development of childhood stroke, either individually or in the presence of other genetic or environmental risk factors. Although a thorough evaluation for thrombophilia seems reasonable for children with stroke, the diagnostic yield is low in adults.14 There are no evidence-based guidelines for screening or treatment of children with stroke and a prothrombotic abnormality. The purpose of this pilot study was to compare the frequencies of genetic and functional coagulation abnormalities in children with arterial ischemic stroke or porencephaly with previously published population levels to plan for future genetic association studies.
METHODS
Study Children
We report an ongoing National Institute of Neurologic Disorders and Stroke study of stroke in children aged 0 to 18 years. Patients who presented to Johns Hopkins University/Kennedy Krieger Institute (Baltimore, MD), and Children's Memorial Hospital (Chicago, IL) between 1990 and 2000 were identified retrospectively through a search of hospital discharge codes and radiology records. Children with a history of stroke (International Classification of Diseases, Ninth Revision codes 430-437) or porencephalic cyst (International Classification of Diseases, Ninth Revision code 348) were contacted by mail and invited to participate in the study (n = 6). Study children were also referred from outside physicians or contacted the study investigators directly at the National Institutes of Health (n = 53). Retrospective and prospective study participants were screened according to inclusion and exclusion criteria. Participants were required to provide a buccal smear and blood sample, complete a pedigree and questionnaire, and grant permission for review of imaging and medical records. Informed consent and assent were obtained from all study children, and the study protocol was reviewed and approved by the National Institute of Neurologic Disorders and Stroke scientific and institutional review board.
Diagnosis of Stroke and Porencephaly
Arterial ischemic stroke (AIS) was defined as a new focal neurologic deficit that lasted 24 hours or longer and was presumably due to a vascular process; porencephaly was defined as a fluid-filled cavity within the cerebral hemispheres that may communicate with cerebrospinal fluid spaces. The diagnosis of AIS or porencephaly was confirmed by computed tomography or MRI for each child.
Study children were classified into 3 categories: (1) perinatal stroke, defined as systemic or neurologic symptoms with radiographic evidence of AIS or porencephaly that occurred before birth or in the first 30 days of life; (2) delayed perinatal stroke, defined as no diagnosis of stroke in the perinatal or neonatal period, focal motor dysfunction or seizures diagnosed after 2 months of age, and neuroimaging evidence of remote focal infarction, encephalomalacia, and or porencephaly; and (3) childhood stroke, defined as an acute onset of systemic or neurologic symptoms with radiographic evidence of AIS that occurred between 30 days and 18 years of age. Children with a history of cancer, heart disease, trauma, chromosomal or metabolic disorders, sickle cell disease, or central nervous system infection were excluded.
Data Abstraction
Study investigators reviewed available medical records for each child. Parents completed a questionnaire regarding maternal and birth history, stroke history, and functional status of their child with a diagnosis of stroke. Family histories were obtained by a certified genetic counselor (L.N.) through telephone interviews for a majority of the children in the study. Parents of probands were instructed to contact family members to determine a family history of a major vascular event. A positive family history was defined as a major thrombotic event (myocardial infarction, deep venous thrombosis, pulmonary embolus, or ischemic stroke) before 60 years of age in first- or second-degree relatives of the probands.
Laboratory Methods
Blood samples were collected at least 3 months after the incident stroke for each child. Blood was collected by peripheral venipuncture into plastic tubes that contained 0.105 M buffered sodium citrate (3.2%). Citrated blood was placed immediately on ice and was processed within 1 hour. Citrated blood was centrifuged at 1700g for 15 minutes at room temperature. Plasma supernatant was removed, transferred to plastic tubes, recentrifuged for 5 minutes to ensure platelet-free plasma, and then stored at –70°C. The buffy coat layer was subsequently removed and stored at –70°C before DNA extraction. Blood samples were frozen at –70°C and stored in batches until they were shipped on dry ice to the Hemostasis Reference Laboratory at the Henderson Research Centre (Hamilton, ON, Canada) for analysis.
Blood samples were analyzed for PC, PS, AT, activated PC resistance (APCR), lipoprotein (a) [Lp(a)], lupus anticoagulant (LA), anticardiolipin antibodies (ACLA), and the MTHFR, FVL, PT, PAI-1, and tissue factor pathway inhibitor C536T (TFPI) mutations. Age-related standardized ranges were used to determine normal and abnormal levels for PC, PS, AT, APCR, Lp(a), LA, and ACLA. In some cases, coagulation testing had been performed before enrollment in the study, but this information was not available to investigators at the time of screening for inclusion.
PC was measured on a BCT or BCS analyzer using a functional assay (Protein C Reagent Kit; Dade Behring, Marburg, Germany), AT was measured by chromogenic assay (Coamatic Antithrombin Assay; Diapharma, West Chester, OH), and PS and Lp(a) were measured by enzyme-linked immunosorbent assay (Asserchrom Free Protein S, Diagnostica Stago, Asnieers, France; and Tintelize Kit, Biopool, Sweden, now Trinity Biotech, County Wicklow, Ireland). APCR was assayed by Dade Actin FS aPTT reagent (Dade Behring) and activated PC (American Diagnostics, Greenwich, CT).15 LA was tested using PTT-LA reagent (Diagnostica Stago) and dilute Russell Viper Venom (American Diagnostics, Greenwich, NJ, or BioMerieux, Durham, NC). LA samples with abnormal results were repeated using a confirmatory test, either Hexogonal Phospholipid or dRVV confirm. ACLA, immunoglobulin (Ig) G, IgM, and IgA, were measured by enzyme-linked immunosorbent assay (Corgenix, Westminster, CO). The MTHFR, FVL, PT, PAI-1, and TFPI mutations were identified by amplification and polymerase chain reactions followed by digestion of amplified fragments with restriction enzymes, as previously reported.16–20
Published Population Cases and Controls
For comparisons, we identified reports, using a search of the MEDLINE database, published in English between 1995 and May 2004, that evaluated the presence of genetic coagulation abnormalities in populations of children with cerebrovascular disease. We abstracted information on type and prevalence of genetic coagulation abnormality, study design, patient and control populations, age range, and methods of analysis.
Statistical Methods
The percentages of each coagulation abnormality were compared with published pediatric stroke case and control rates. Fisher exact tests were used for internal comparisons between children with and without a family history of thrombosis and between perinatal or delayed perinatal and childhood stroke.
RESULTS
We identified 59 children with stroke of previously undetermined cause. There were 18 perinatal, 25 delayed perinatal, and 16 childhood strokes. The majority of these children were white (93%) and male (53%). The mean age at diagnosis was 23 months (median: 8 months). Neuroimaging was completed for all children: MRI for 92% (54 of 59) and computed tomography scan for 71% (42 of 59) of children. Stroke location was in the distribution of the left middle cerebral artery in 47% and right middle cerebral artery in 25%. At study entry, 76% of children had hemiparesis, 8% had monoparesis, 7% had quadriplegia, and 8% had no persisting motor disabilities (Table 1).
During a median follow-up period of 3.5 years (range: 3 months to 16 years), none of the children with a history of perinatal or delayed perinatal stroke had a recurrent stroke. Of children with childhood stroke, 19% (3 of 16) had a recurrence; all of these had a coagulation abnormality [2 PAI-1, 1 Lp(a)], and the 2 whose family history was known had a family history of thrombosis.
At least 1 prothrombotic abnormality was identified in 63% (36 of 57) of children studied, and 28% (16 of 57) had multiple abnormalities. Several prothrombotic abnormalities were identified, including PC deficiency (1 of 59), AT deficiency (1 of 59), APCR (11 of 58), elevated Lp(a) (12 of 59), MTHFR (12 of 56), PAI-1 (15 of 56), FVL (5 of 57), and PT (3 of 57). No child was identified with PS deficiency, ACLA, LA, or the TFPI mutation. Tests for prothrombotic abnormalities were incomplete for 4 children as a result of inadequate DNA samples. Of the 4 children with incomplete testing, 2 did not have an identified abnormality (Table 2).
AT deficiency, elevated Lp(a), the PT mutation, and the MTHFR mutation were similar to rates in cases and more common than control subjects in previously published studies. The rate of children with APCR or multiple abnormalities was higher than in previous pediatric stroke studies. There were no significant differences in the prevalence of a coagulation abnormality among children with perinatal stroke (56%), delayed perinatal stroke (67%), or childhood stroke (67%).
A pedigree was completed for 37 of the 59 children. Thrombosis that occurred before 60 years of age was identified in 4% of family members, none (0 of 118) of first-degree relatives of these children, and 5.7% (16 of 281) of second-degree relatives. There was a family history of thrombosis before 60 years of age in 31% (11 of 35) of children overall and among 33% (6 of 18) of children with a prothrombotic abnormality (P = 1.00).
DISCUSSION
The identification of a prothrombotic factor in a child with stroke may be helpful for determination of cause and recurrence risk and may have implications for prevention of systemic thrombosis in specific situations and for screening family members,21 yet the variability of these abnormalities among different age groups, stroke subtypes, and international populations has led to uncertainties regarding evaluation, risk estimates, and treatment in children with stroke.
The present study is the largest series of prothrombotic risk factors in American children with stroke. Two thirds of children in this study had at least 1 of the prothrombotic risk factors tested, and several children had multiple risk factors.
Prothrombotic Abnormalities and Children With Stroke
Several international case-control studies have revealed an association between prothrombotic abnormalities and stroke in children,7–9 whereas other studies were negative or too small to show a difference10–13 (Table 3). The most consistent associations have been reported for PC deficiency, elevated Lp(a), and the FVL mutation. In this study, we found that rates of AT deficiency, elevated Lp(a), the PT mutation, and the MTHFR mutation were higher than those in published rates in pediatric stroke control subjects.
Functional deficiencies in anticoagulant proteins were found in only 2 children in this study. One child was found to have AT deficiency, a rate similar to those in other series.10,11,22 AT deficiency has been reported in as many as 13% of children with stroke,21 but most studies have failed to implicate this factor in childhood stroke.9
Lp(a) is structurally homologous to plasminogen and has a similar effect on coagulation, fibrinolysis, and endothelial function.23 An increased level of Lp(a) has been reported in 3 German studies9,24,25 and 1 Portuguese26 study to be a risk factor for stroke in neonates and children. Most studies in adults have yielded similar findings.27 We found elevated Lp(a) in 20% of the children in this study, a rate much higher than in published pediatric stroke control subjects (5.5%).25
The PT mutation is a genetic variant of the 3-prime untranslated region of the PT gene that leads to increased plasma PT levels. The PT mutation is an autosomal dominant mutation that varies widely in prevalence and alone is associated with a modest increase in risk for ischemic stroke, myocardial infarction, and peripheral vascular disease in adults.28 Studies of children with stroke have yielded mixed results. Case-control studies from Turkey7 and Germany9 were positive, whereas other international studies were negative or underpowered.8,13,26 We identified the PT mutation in 5% of children in this study, a rate slightly higher than the 0.9% to 2.8% reported in published pediatric stroke control subjects.
The association between elevated homocysteine and stroke is widely recognized. The MTHFR mutation has been shown to increase the risk for stroke in children and adults. A recent meta-analysis of patients who were younger than 55 years and had AIS revealed a modest increased risk for stroke among homozygous carriers of the MTHFR mutation.28 Despite these findings in younger adults and high rates in pediatric stroke patients, only 1 case-control study in children was sufficiently powered to show an association.9 We identified homozygosity for the MTHFR mutation in 21% of the children in this study, a rate higher than that among published pediatric stroke control subjects (5.6–15.2).7,8
Almost 30% of the children in this study had >1 of the prothrombotic abnormalities evaluated. Five children had 3 or more prothrombotic abnormalities. Other studies using an extensive prothrombotic panel have reported a combination of prothrombotic abnormalities in up to 23% of children with stroke.21 A combination of prothrombotic factors substantially increases the risk for stroke in children.9,24,29 Prothrombotic combinations that have been reported with an increased risk for stroke in children include the FVL mutation and elevated Lp(a) or the MTHFR mutation.9
Recurrent Stroke and Prothrombotic Abnormalities
Recurrent stroke is rare among infants with perinatal stroke30 but in children ranges from 6% to 30%.21,31,32 Combined data from Germany, Canada, and the United Kingdom of 565 patients with AIS revealed a recurrence rate of 10% after several years of follow-up.33 The MTHFR mutation, elevated homocysteine, elevated Lp(a), and PC deficiency have been associated with recurrent stroke in infants and children.4 In this study, 19% (3 of 16) of children with childhood stroke had a recurrence, all of which had a coagulation abnormality and 2 a family history of thrombosis.
Age-Related Differences in Frequency of Prothrombotic Abnormalities
The prevalence of prothrombotic abnormalities among individuals with stroke has varied according to age and suggests that genetic risk factors are more important in individuals who present with stroke at an early age. In studies that used an extensive screen for thrombophilia, prothrombotic abnormalities were more common in neonates25,34 as compared with children12,35 or adults with stroke.36 This study found no differences in the frequency of prothrombotic factors among neonates compared with older children, but this likely reflects the small number for comparison.
Family History of Thrombosis and Stroke in Children
A family history of disease provides information on disease susceptibility reflected through common genes, behaviors, and shared environment.37 A family history of vascular disease is an independent risk factor for stroke in young adults, and the risk that it confers varies by stroke subtype.38,39 There is limited information on the risk for stroke in children and neonates with a family history of stroke or thrombosis. Case series and controlled studies of children with stroke have found a family history of thrombosis among 5% to 42% of children studied.21,26,40 A family history of thrombosis was identified in one third of the children in this study and, similar to other studies, was not predictive of the presence of a coagulation abnormality.8 None of the children in this study had a sibling with a history of AIS or porencephaly.
Treatment of Children With Stroke and a Prothrombotic Abnormality
Prothrombotic abnormalities are associated with an increased risk for stroke and recurrent stroke in children,4,31 but there are as yet no evidence-based strategies or consensus guidelines for the treatment of children with stroke and a prothrombotic abnormality. Recommendations for antithrombotic therapy in children with thrombophilia have been based on small, nonrandomized studies and expert opinion.41 Antithrombotic therapy is recommended in adults for acute events and as prophylaxis in high-risk situations and may be warranted in children with specific prothrombotic abnormalities.42 The addition of other genetic risk factors for thrombosis in symptomatic individuals probably warrants long-term therapy, but this must be weighed against the risk for anticoagulation in young children. The response and long-term complications of antithrombotic therapy are not the same in adults and children.43 Clinical trials are needed to determine the indications and best treatment for children with a prothrombotic abnormality. The screening of unaffected relatives of probands with a prothrombotic mutation has been advocated because the risk for thrombosis can be reduced by prophylaxis in high-risk situations.44
CONCLUSIONS
The evidence relating prothrombotic abnormalities and cerebrovascular disorders in children is growing, but much remains unknown. Additional case-control studies that incorporate representative cases and appropriately matched control subjects are needed to verify previous findings. Additional studies are needed to quantify the risk for a cerebrovascular event in children with a prothrombotic mutation and to determine which combination of endogenous and exogenous risk factors lead to greater rates of initial and recurrent stroke.
ACKNOWLEDGMENTS
We acknowledge Marilyn Johnston, ART, for review of the manuscript and the children, parents, and family members who participated in the study.
FOOTNOTES
Accepted Nov 23, 2004.
No conflict of interest declared.
PEDIATRICS (ISSN 0031 4005). Published in the public domain by the American Academy of Pediatrics.
REFERENCES
The Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: a meta-analysis. JAMA. 2002;288 :2015 –2022
Earley CJ, Kittner SJ, Feeser BR, et al. Stroke in children and sickle-cell disease: Baltimore-Washington Cooperative Young Stroke Study. Neurology. 1998;51 :169 –176
Giroud M, Lemesle M, Gouyon JB, et al. Cerebrovascular disease in children under 16 years of age in the city of Dijon, France: a study of incidence and clinical features from 1985 to 1993. J Clin Epidemiol. 1995;48 :1343 –1348
Strater R, Becker S, von Eckardstein A, et al. Prospective assessment of risk factors for recurrent stroke during childhood—a 5-year follow-up study. Lancet. 2002;360 :1540 –1545
Kirkham FJ, Prengler M, Hewes DK, et al. Risk factors for arterial ischemic stroke in children. J Child Neurol. 2000;15 :299 –307
Al Sulaiman A, Bademosi O, Ismail H, et al. Stroke in Saudi children. J Child Neurol. 1999;14 :295 –298
Akar N, Akar E, Deda G, et al. Factor V1691 G-A, prothrombin 20210 G-A, and methylenetetrahydrofolate reductase 677 C-T variants in Turkish children with cerebral infarct. J Child Neurol. 1999;14 :749 –751
Kenet G, Sadetzki S, Murad H, et al. Factor V Leiden and antiphospholipid antibodies are significant risk factors for ischemic stroke in children. Stroke. 2000;31 :1283 –1288
Nowak-Gottl U, Strater R, Heinecke A, et al. Lipoprotein (a) and genetic polymorphisms of clotting factor V, prothrombin, and methylenetetrahydrofolate reductase are risk factors of spontaneous ischemic stroke in childhood. Blood. 1999;94 :3678 –3682
Hagstrom JN, Walter J, Bluebond-Langner R, et al. Prevalence of the factor V Leiden mutation in children and neonates with thromboembolic disease. J Pediatr. 1998;133 :777 –781
Bonduel M, Sciuccati G, Hepner M, et al. Factor V Leiden and prothrombin gene G20210A mutation in children with cerebral thromboembolism. Am J Hematol. 2003;73 :81 –86
McColl MD, Chalmers EA, Thomas A, et al. Factor V Leiden, prothrombin 20210G->A and the MTHFR C677T mutations in childhood stroke. Thromb Haemost. 1999;81 :690 –694
Zenz W, Bodo Z, Plotho J, et al. Factor V Leiden and prothrombin gene G 20210 A variant in children with ischemic stroke. Thromb Haemost. 1998;80 :763 –766
Bushnell CD, Goldstein LB. Diagnostic testing for coagulopathies in patients with ischemic stroke. Stroke. 2000;31 :3067 –3078
Griffin JH, Evatt B, Wideman C, et al. Anticoagulant protein C pathway defective in majority of thrombophilic patients. Blood. 1993;82 :1989 –1993
Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10 :111 –113
Bertina RM, Koeleman BP, Koster T, et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature. 1994;369 :64 –67
Poort SR, Rosendaal FR, Reitsma PH, et al. A common variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88 :3698
Kleesiek K, Schmidt M, Gotting C, et al. The 536C->T transition in the human tissue factor pathway inhibitor (TFPI) gene is statistically associated with a higher risk for venous thrombosis. Thromb Haemost. 1999;82 :1 –5
Sartori MT, Wiman B, Vettore S, et al. 4G/5G polymorphism of PAI-1 gene promoter and fibrinolytic capacity in patients with deep vein thrombosis. Thromb Haemost. 1998;80 :956 –960
deVeber G, Monagle P, Chan A, et al. Prothrombotic disorders in infants and children with cerebral thromboembolism. Arch Neurol. 1998;55 :1539 –1543
Ganesan V, Prengler M, McShane MA, et al. Investigation of risk factors in children with arterial ischemic stroke. Ann Neurol. 2003;53 :167 –173
Ariyo AA, Thach C, Tracy R. Lp(a) lipoprotein, vascular disease, and mortality in the elderly. N Engl J Med. 2003;349 :2108 –2115
Strater R, Vielhaber H, Kassenbohmer R, et al. Genetic risk factors of thrombophilia in ischaemic childhood stroke of cardiac origin. A prospective ESPED survey. Eur J Pediatr. 1999;158(suppl 3) :S122 –S125
Gunther G, Junker R, Strater R, et al. Symptomatic ischemic stroke in full-term neonates: role of acquired and genetic prothrombotic risk factors. Stroke. 2000;31 :2437 –2441
Barreirinho S, Ferro A, Santos M, et al. Inherited and acquired risk factors and their combined effects in pediatric stroke. Pediatr Neurol. 2003;28 :134 –138
Milionis HJ, Winder AF, Mikhailidis DP. Lipoprotein (a) and stroke. J Clin Pathol. 2000;53 :487 –496
Kim RJ, Becker RC. Association between factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations and events of the arterial circulatory system: a meta-analysis of published studies. Am Heart J. 2003;146 :948 –957
Heller C, Becker S, Scharrer I, et al. Prothrombotic risk factors in childhood stroke and venous thrombosis. Eur J Pediatr. 1999;158(suppl 3) :S117 –S121
Kurnik K, Kosch A, Strater R, et al. Recurrent thromboembolism in infants and children suffering from symptomatic neonatal arterial stroke: a prospective follow-up study. Stroke. 2003;34 :2887 –2892
Lanthier S, Carmant L, David M, et al. Stroke in children: the coexistence of multiple risk factors predicts poor outcome. Neurology. 2000;54 :371 –378
Chabrier S, Husson B, Lasjaunias P, et al. Stroke in childhood: outcome and recurrence risk by mechanism in 59 patients. J Child Neurol. 2000;15 :290 –294
Kirkham F, DeVeber G, Chan AK, et al. Recurrent stroke: the role of prothrombotic disorders . Ann Neurol. 2003;54 :S110
Mercuri E, Cowan F, Gupte G, et al. Prothrombotic disorders and abnormal neurodevelopmental outcome in infants with neonatal cerebral infarction. Pediatrics. 2001;107 :1400 –1404
Ganesan V, McShane MA, Liesner R, et al. Inherited prothrombotic states and ischaemic stroke in childhood. J Neurol Neurosurg Psychiatry. 1998;65 :508 –511
Hankey GJ, Eikelboom JW, van Bockxmeer FM, et al. Inherited thrombophilia in ischemic stroke and its pathogenic subtypes. Stroke. 2001;32 :1793 –1799
Yoon PW, Scheuner MT, Peterson-Oehlke KL, et al. Can family history be used as a tool for public health and preventive medicine Genet Med. 2002;4 :304 –310
Jerrard-Dunne P, Cloud G, Hassan A, et al. Evaluating the genetic component of ischemic stroke subtypes: a family history study. Stroke. 2003;34 :1364 –1369
Schulz UG, Flossmann E, Rothwell PM. Heritability of ischemic stroke in relation to age, vascular risk factors, and subtypes of incident stroke in population-based studies. Stroke. 2004;35 :819 –824
Golomb MR, MacGregor DL, Domi T, et al. Presumed pre- or perinatal arterial ischemic stroke: risk factors and outcomes. Ann Neurol. 2001;50 :163 –168
Jilma B, Kamath S, Lip GY. ABC of antithrombotic therapy: antithrombotic therapy in special circumstances. II—in children, thrombophilia, and miscellaneous conditions. BMJ. 2003;326 :93 –96
Haemostasis and Thrombosis Task Force, British Committee for Standards in Haematology. Investigation and management of heritable thrombophilia. Br J Haematol. 2001;114 :512 –528
Williams MD, Chalmers EA, Gibson BE. The investigation and management of neonatal haemostasis and thrombosis. Br J Haematol. 2002;119 :295 –309
De S, V, Rossi E, Paciaroni K, et al. Screening for inherited thrombophilia: indications and therapeutic implications. Haematologica. 2002;87 :1095 –1108
Koh S, Chen LS. Protein C and S deficiency in children with ischemic cerebrovascular accident. Pediatr Neurol. 1997;17 :319 –321
Akar N, Akar E, Ozel D, et al. Common mutations at the homocysteine metabolism pathway and pediatric stroke. Thromb Res. 2001;102 :115 –120
Nowak-Gottl U, Strater R, Kosch A, et al. The plasminogen activator inhibitor (PAI)-1 promoter 4G/4G genotype is not associated with ischemic stroke in a population of German children. Childhood Stroke Study Group. Eur J Haematol. 2001;66 :57 –62
Cardo E, Monros E, Colome C, et al. Children with stroke: polymorphism of the MTHFR gene, mild hyperhomocysteinemia, and vitamin status. J Child Neurol. 2000;15 :295 –298
Debus OM, Kosch A, Strater R, et al. The factor V G1691A mutation is a risk for porencephaly: a case-control study. Ann Neurol. 2004;56 :287 –290
Prengler M, Sturt N, Krywawych S, et al. Homozygous thermolabile variant of the methylenetetrahydrofolate reductase gene: a potential risk factor for hyperhomocysteinaemia, CVD, and stroke in childhood. Dev Med Child Neurol. 2001;43 :220 –225(John Kylan Lynch, DO, MPH)