ARTICLE
Inherited Prothrombotic Risk Factors in Children
With Stroke, Transient Ischemic Attack, or Migraine
De´sire´e Coen Herak, BSca, Margareta Radic Antolic, BSca, Jasna Lenicek Krleza, PhDb, Marina Pavic, BScc, Slavica Dodig, PhDd, Vlasta Duranovic, MD, PhDe, Anica Basnec Brkic, MDf, Renata Zadro, PhDa
aClinical Institute of Laboratory Diagnosis andfDepartment of Pediatric Neurology, Clinical Hospital Center Zagreb and Zagreb University School of Medicine, Zagreb, Croatia;
Departments ofbLaboratory Diagnosis andeNeuropediatrics, Children’s Hospital Zagreb, Zagreb, Croatia;cDepartment of Laboratory Diagnosis, University Hospital of
Traumatology, Zagreb, Croatia;dDepartment of Clinical Laboratory Diagnosis, Special Hospital for Respiratory Diseases in Children and Adolescents, Zagreb, Croatia
The authors have indicated they have no financial relationships relevant to this article to disclose.
What’s Known on This Subject
Several inherited prothrombotic risk factors, particularly the presence of factor V G1691A, factor II G20210A, and MTHFR C677T, have been demonstrated to play a role in childhood stroke, but the role of platelets has not been well studied in children.
What This Study Adds
Factor V G1691A has an important role in susceptibility for AIS, both in the perinatal/ neonatal period and in childhood, and TIA. A minor impact of HPA polymorphisms suggests that platelet glycoprotein polymorphisms may increase the risk of TIA and migraine.
ABSTRACT
OBJECTIVE.The aim of this study was to investigate the prevalence and possible associ-ation of inherited prothrombotic risk factors in children with stroke, transient isch-emic attack, or migraine.
METHODS.We performed genotypic analysis for factor V G1691A, factor II G20210A, methylenetetrahydrofolate reductase C677T, and 4 common platelet glycoprotein polymorphisms (human platelet alloantigen-1, -2, -3, and -5) in 150 children⬍18 years of age with established diagnoses of stroke, transient ischemic attack, or migraine. Children were classified into 5 groups, namely, childhood arterial ischemic stroke (N⫽33), perinatal arterial ischemic stroke (N⫽26), hemorrhagic stroke (N⫽
20), transient ischemic attack (N⫽36), and migraine (N⫽35). The control group consisted of 112 childrenⱕ18 years of age from the same geographical region who had no history of neurologic or thromboembolic diseases.
RESULTS.Heterozygosity for factor V G1691A was associated with approximately sev-enfold increased risk for arterial ischemic stroke, perinatal arterial ischemic stroke, and transient ischemic attack. Increased risk for transient ischemic attack was found in carriers of the human platelet 2b allele, human platelet alloantigen-5a/b genotype, and combined human platelet alloantigen-2b and human platelet alloantigen-5b genotype. The presence of the human platelet alloantigen-2b allele was associated with a 2.23-fold increased risk for migraine, whereas carriers of the human platelet alloantigen-3b allele had a lower risk for arterial ischemic stroke than did carriers of the human platelet alloantigen-3a allele.
CONCLUSIONS.Factor V G1691A has an important role in susceptibility to arterial ischemic stroke, both in the perinatal/neonatal period and in childhood, as well
as transient ischemic attacks. A minor impact of human platelet alloantigen polymorphisms suggests that platelet glycoprotein polymorphisms may increase the risk of transient ischemic attacks and migraine, but this should be confirmed in larger studies. Pediatrics2009;123:e653–e660
C
EREBROVASCULAR DISORDERS ARE being increasingly recognized as important causes of death and chronic morbidity in children and represent an emerging area for clinical research.1,2Stroke in children is aheteroge-neous disorder that can result from either vascular occlusion (ie, ischemic stroke) or bleeding from a ruptured vessel (ie, hemorrhagic stroke [HS]). Arterial ischemic stroke (AIS) has also been classified according to the time of onset, as perinatal AIS (PAS) and childhood AIS. Two different neurovascular disorders can be connected with stroke, namely, transient ischemic attack (TIA) and migraine. TIA is attributed to focal cerebral ischemia and frequently precedes AIS in children.3Migraine is a complex neurovascular disorder that can induce ischemic stroke and is
considered an independent risk factor for stroke in youths.4,5
Genetic factors in children with stroke have been incompletely characterized,2and the distributions of candidate
genes for stroke risk differ in various populations.6There is increasing evidence that inherited or acquired
prothrom-www.pediatrics.org/cgi/doi/10.1542/ peds.2007-3737
doi:10.1542/peds.2007-3737
Key Words
inherited prothrombotic risk factor, children, stroke, transient ischemic attack, migraine, genetic polymorphism
Abbreviations
TIA—transient ischemic attack MTHFR—methylenetetrahydrofolate reductase
AIS—arterial ischemic stroke PAS—perinatal arterial ischemic stroke HS— hemorrhagic stroke
OR— odds ratio CI— confidence interval HPA— human platelet alloantigen
Accepted for publication Dec 23, 2008
Address correspondence to Renata Zadro, PhD, Clinical Institute of Laboratory Diagnosis, Clinical Hospital Center Zagreb and Zagreb University School of Medicine, Kispaticeva 12, Zagreb 10000, Croatia. E-mail: rzadro@mef.hr
botic disorders may be implicated in the pathogenesis of stroke in childhood and also of migraine.5–7 The
best-studied genetic polymorphisms include polymorphisms in proteins that constitute the coagulation system, such as factor V G1691A (factor V Leiden) and factor II G20210A, and the common methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism.
In addition, polymorphisms in platelet membrane re-ceptors may be associated with increased thromboem-bolic disease. Platelets are essential for maintaining nor-mal hemostasis by participating in primary hemostasis through adhesion to the site of injury, secretion, and aggregation. Platelets contain a wide range of membrane glycoproteins that mediate adhesion to the components of the subendothelial matrix, such as the main von Willebrand factor receptor glycoprotein Ib/IX/V and col-lagen receptor glycoprotein Ia/IIa, whereas the main fibrinogen receptor glycoprotein IIb/IIIa is involved in subsequent platelet aggregation.
A number of these glycoproteins are polymorphic and can be recognized as human platelet alloantigens (HPAs).8,9 The 4 most-common HPA polymorphisms,
HPA-1, -2, -3, and -5, are attributable to single-base pair substitutions resulting in amino acid replacements and are present on the major glycoprotein complexes in-volved in adhesion and aggregation, HPA-1 (Leu33Pro) on glycoprotein IIIa,10HPA-2 (Met145Thr) on
glycopro-tein Ib␣,11HPA-3 (Ile843Ser) on glycoprotein IIb,12and
HPA-5 (Glu505Lys) on glycoprotein Ia.13The aim of the
present study was to investigate the role of inherited prothrombotic risk factors in children with stroke, TIA, and migraine by determining the prevalence of factor V G1691A, factor II G20210A, MTHFR C677T, and HPA-1, -2, -3, and -5, to establish possible relationships between the investigated polymorphisms and selected disorders.
METHODS
Patients and Control Subjects
We studied 150 unrelated white children,⬍18 years of age, who were admitted to the Department of Neurope-diatrics, Children’s Hospital Zagreb, or the Department of Pediatric Neurology, University School of Medicine and Clinical Hospital Center Zagreb, between February 1998 and August 2006. All enrolled patients had an established diagnosis of stroke, TIA, or migraine.
Control subjects from the same geographical region (N⫽112; age:ⱕ18 years), with no history of neurologic or thromboembolic diseases, were recruited from chil-dren awaiting minor surgery and chilchil-dren with respira-tory diseases at routine visits. Informed consent was obtained from the parents of all children, and the hos-pital ethics committee approved the study.
Diagnosis of Stroke, TIA, and Migraine
Diagnoses were made after careful analysis of clinical history, physical, and neurologic examination findings, on the basis of clinical symptoms and signs, and were documented with ⱖ1 brain imaging technique (trans-fontanellar ultrasonography, computed tomography, or MRI/magnetic resonance angiography).
Electrocardiog-raphy, echocardiogElectrocardiog-raphy, electroencephalogElectrocardiog-raphy, and transcranial color Doppler ultrasonography were per-formed for all patients. Children with stroke were fur-ther classified into 3 groups, that is, childhood AIS, PAS, and HS. AIS was defined as an acute focal neurologic deficit persisting for ⱖ24 hours, with evidence of cere-bral infarction in an arterial distribution in brain imaging scans, manifested as hemiplegia, aphasia, visual or bal-ance disturbbal-ance, or seizures.14For newborns, AIS (PAS)
was detected after convulsions in the first days after birth or the diagnosis of presumed prenatal/perinatal AIS was made retrospectively after the clinical presentation of hemiparesis or seizures, with ultrasonographic, com-puted tomographic, or MRI/magnetic resonance angio-graphic scans showing a lesion consistent with remote vasoocclusive AIS. HS was defined as an acute focal neurologic deficit lasting⬎24 hours with neuroimaging evidence of intracranial hemorrhage not associated with ischemic infarction, with symptoms of headache, vom-iting, decreased level of consciousness, and seizures. TIA was defined as a focal deficit of acute onset lasting⬍24 hours, of presumed vascular origin not attributable to migraine or seizure and with radiologic images showing no parenchymal infarction.14 Diagnoses of migraine
were made according to the criteria of the International Headache Society.15
DNA Analysis
Genomic DNA was extracted from EDTA- or sodium citrate-treated whole-blood samples according to stan-dard procedures, by using the salting-out method.16
Fac-tor V G1691A and facFac-tor II G20210A were identified after amplification with the polymerase chain reaction, followed by digestion withMnlI17andHindIII,18
respec-tively, as reported previously. MTHFR C677T was iden-tified by using melting curve analysis with a LightCycler (Roche Diagnostics, Mannheim, Germany).19HPA-1, -2,
-3, and -5 were detected by using sequence-specific primers, according to the procedure described by Klu¨ter et al.20
Statistical Analysis
RESULTS
In this study, we analyzed data for a total of 150 children with stroke (N⫽79), TIA (N ⫽36), or migraine (N⫽
35) and 112 control subjects. Childhood AIS was estab-lished for 33 children, PAS for 26 children, and HS for 20 children. Patient characteristics, including gender, age at onset, and age at diagnosis, are shown in Table 1.
A larger proportion of boys was found among chil-dren with stroke (63.3 vs 36.7%), AIS, HS, and PAS, whereas TIA and migraine were found more frequently in girls. The mean age at testing was different from the mean age at diagnosis for AIS and PAS. PAS was diag-nosed before 28 days of life for only 5 children, whereas the diagnosis was made months later for 21 patients with PAS, after delayed presentation of clinical symptoms.
Affected vascular territories in children with AIS and PAS are presented in Table 2, whereas clinical symptoms and risk factors are shown in Table 3. Risk factors were identified for 26 children (79%) with AIS and 22 chil-dren (85%) with PAS.
One half of the children with HS had intracerebral
hemorrhage, 4 children had subarachnoid hemorrhage, 5 children had intraventricular hemorrhage, and 1 child had intracerebral hemorrhage with intraventricular ex-tension. Among children with migraine, 24 children had migraine with aura and 11 had migraine without aura. Paresthesis was the most common aura symptom (65%), followed by visual manifestations (50%) and speech disturbances (45%).
Genotypic distributions and allelic frequencies of in-vestigated polymorphisms in patient and control groups are shown in Table 4. Comparisons of genotypic distri-butions between patient groups and control subjects and calculated ORs with 95% CIs are presented in Table 5.
TABLE 1 Characteristics of the Population Studied
Characteristic Patients (Nⴝ150) Control Subjects
(Nⴝ112)
AIS (Nⴝ33) PAS (Nⴝ26) HS (Nⴝ20) TIA
(Nⴝ36)
Migraine (Nⴝ35)
Gender,n(%)
Male 24 (72.7) 14 (53.8) 12 (60.0) 13 (36.1) 15 (42.9) 78 (69.6)
Female 9 (27.3) 12 (46.2) 8 (40.0) 23 (63.9) 20 (57.1) 34 (30.4)
Age at testing
Mean⫾SD, y 8.5⫾4.1 4.3⫾4.6 6.6⫾5.7 11.9⫾3.0 11.7⫾2.7 9.8⫾4.3
Median, y 8.5 2.4 5.0 12 12 10
Range 4.33 mo to 16 y 0.60 mo to 17.75 y 1.17 mo to 17.75 y 4–17 y 6–17 y 1–18 y
Age at diagnosis
Mean⫾SD 7.8⫾4.2 y 4.1⫾3.4 mo 6.6⫾5.7 y 11.9⫾3.0 y 11.7⫾2.7 y
Median 8.0 y 3 mo 5.0 y 12 y 12 y
Range 4.33 mo to 16 y 2 d to 10 mo 2 d to 17.75 y 4–17 y 6–17 y
TABLE 3 Clinical Symptoms and Risk Factors for Children With AIS or PAS
n(%)
AIS (Nⴝ33)
PAS (Nⴝ26)
Clinical symptoms
Hemiparesis 16 (48.5) 16 (61.5)
Seizures 9 (27.3) 3 (11.5)
Hypotonia 3 (9.1) 4 (15.4)
Monoparesis 1 (3.0) 0 (0.0)
Tetraparesis 0 (0.0) 2 (7.7)
Spasticity 0 (0.0) 1 (3.9)
Headache 6 (18.2) 0 (0.0)
Ataxia 2 (6.1) 0 (0.0)
Vertigo 2 (6.1) 0 (0.0)
Vomiting 1 (3.0) 0 (0.0)
Risk factors
Infection 9 (27.3) 6 (23.1)
Cardiac disease 3 (9.1) 4 (15.4)
Vasculitis 3 (9.1) 0 (0.0)
Moyamoya disease 2 (6.1) 0 (0.0)
Head trauma 2 (6.1) 0 (0.0)
Vessel hypoplasia 1 (3.0) 3 (11.5)
Perinatal complications 0 (0.0) 6 (23.1)
Placental abruption 0 (0.0) 1 (3.8)
Lipoprotein(a) levels of⬎30 mg/dL 5 (15.1) 2 (7.7)
Iron deficiency 1 (3.0) 0 (0.0)
Unknown 7 (21.2) 4 (15.4)
Multiple 3 (9.1) 5 (19.2)
TABLE 2 Infarction Location and Affected Vascular Territory in Children With AIS or PAS
Infarction Location/Affected Vascular Territory
n(%)
AIS (Nⴝ33)
PAS (Nⴝ26)
Infarction location
Unilateral 30 (90.9) 23 (88.5)
Left 14 (42.4) 17 (65.4)
Right 16 (48.5) 6 (23.1)
Bilateral 3 (9.1) 3 (11.5)
Vascular territory involvement
Middle cerebral artery 23 (69.7) 20 (76.9)
Main branch 7 (30.4) 10 (50.0)
Cortical region 8 (34.8) 6 (30.0)
Subcortical structures 8 (34.8) 4 (20.0) Anterior cerebral artery 2 (6.1) 0 (0.0) Posterior cerebral artery 3 (9.1) 3 (11.5)
Basilar artery 1 (3.0) 0 (0.0)
Anterior and middle cerebral artery 0 (0.0) 2 (7.7) Middle and posterior cerebral artery 1 (3.0) 0 (0.0)
Heterozygosity for factor V G1691A was significantly associated with a⬎7-fold increased risk for AIS and PAS and a 6.87-fold increased risk for TIA. Among patient
groups, a greater proportion of heterozygotes for factor II G20210A was observed only for children with TIA, al-though the findings were not statistically significant.
TABLE 4 Genotypic Distributions and Allelic Frequencies of Investigated Polymorphisms in Patient and Control Groups
Polymorphism n(%)
Patients (Nⴝ150) Control Subjects
(Nⴝ112) AIS (Nⴝ33) PAS (Nⴝ26) HS (Nⴝ20) TIA (Nⴝ36) Migraine (Nⴝ35)
Factor V G1691A Genotype
GG 29 (87.9) 23 (88.5) 19 (95.0) 32 (88.9) 34 (97.1) 110 (98.2)
GA 4 (12.1) 3 (11.5) 1 (5.0) 4 (11.1) 1 (2.9) 2 (1.8)
AA 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Allele
G 62 (93.9) 49 (94.2) 39 (97.5) 68 (94.4) 69 (98.6) 222 (99.1)
A 4 (6.1) 3 (5.8) 1 (2.5) 4 (5.6) 1 (1.4) 2 (0.9)
Factor II G20210A Genotype
GG 32 (97.0) 26 (100.0) 19 (95.0) 32 (88.9) 33 (94.3) 108 (96.4)
GA 1 (3.0) 0 (0.0) 1 (5.0) 4 (11.1) 2 (5.7) 4 (3.6)
AA 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
Allele
G 65 (98.5) 52 (100.0) 39 (97.5) 68 (94.4) 68 (97.1) 220 (98.2)
A 1 (1.5) 0 (0.0) 1 (2.5) 4 (5.6) 2 (2.9) 4 (1.8)
MTHFR C677T Genotype
CC 11 (33.3) 9 (34.6) 8 (40.0) 17 (47.2) 16 (45.7) 46 (41.1)
CT 17 (51.5) 13 (50.0) 11 (55.0) 12 (33.3) 13 (37.2) 56 (50.0)
TT 5 (15.2) 4 (15.4) 1 (5.0) 7 (19.5) 6 (17.1) 10 (8.9)
Allele
C 39 (59.1) 31 (59.6) 27 (75.0) 46 (63.9) 45 (64.3) 154 (68.8)
T 27 (40.9) 21 (40.4) 13 (25.0) 26 (36.1) 25 (35.7) 70 (31.2)
HPA-1 Genotype
a/a 23 (69.7) 21 (80.8) 14 (70.0) 21 (58.3) 26 (74.3) 82 (73.2)
a/b 7 (21.2) 5 (19.2) 5 (25.0) 15 (41.7) 9 (25.7) 28 (25.0)
b/b 3 (9.1) 0 (0.0) 1 (5.0) 0 (0.0) 0 (0.0) 2 (1.8)
Allele
a 53 (80.3) 47 (90.4) 33 (82.5) 57 (79.2) 61 (87.1) 192 (85.7)
b 13 (19.7) 5 (9.6) 7 (17.5) 15 (20.8) 9 (12.9) 32 (14.3)
HPA-2 Genotype
a/a 25 (75.8) 22 (84.6) 15 (75.0) 24 (66.7) 24 (68.6) 93 (83.0)
a/b 8 (24.2) 4 (15.4) 5 (25.0) 10 (27.8) 10 (28.6) 19 (17.0)
b/b 0 (0.0) 0 (0.0) 0 (0.0) 2 (5.5) 1 (2.8) 0 (0.0)
Allele
a 58 (87.9) 48 (92.3) 35 (75.0) 58 (80.6) 58 (82.9) 205 (91.5)
b 8 (12.1) 4 (7.7) 5 (25.0) 14 (19.4) 12 (17.1) 19 (8.5)
HPA-3 Genotype
a/a 14 (42.4) 12 (46.1) 6 (30.0) 12 (33.3) 13 (37.1) 30 (26.8)
a/b 16 (48.5) 10 (38.5) 10 (50.0) 18 (50.0) 17 (48.6) 57 (50.9)
b/b 3 (9.1) 4 (15.4) 4 (20.0) 6 (16.7) 5 (14.3) 25 (22.3)
Allele
a 44 (66.7) 34 (65.4) 22 (55.0) 42 (58.3) 43 (61.4) 117 (52.2)
b 22 (33.3) 18 (34.6) 18 (45.0) 30 (41.7) 27 (38.6) 107 (47.8)
HPA-5 Genotype
a/a 28 (84.8) 22 (84.6) 13 (65.0) 22 (61.1) 29 (82.9) 92 (82.1)
a/b 5 (15.2) 4 (15.4) 7 (35.0) 14 (38.9) 6 (17.1) 18 (16.1)
b/b 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (1.8)
Allele
a 61 (92.4) 48 (92.3) 33 (82.5) 58 (80.6) 64 (91.4) 202 (90.2)
Greater proportions of homozygotes for MTHFR C677T were identified in all patient groups except for children with HS, compared with control subjects, but a statisti-cally significant difference was not found.
The frequencies of HPA genotypes were not signifi-cantly different between any of patient groups and con-trol subjects, except for HPA-5 in patients with TIA. Carriers of the HPA-5a/b genotype had an almost three-fold increased risk (P⫽.013), whereas the presence of the HPA-5b allele was associated with a 2.22-fold (95% CI: 1.07– 4.60-fold) increased risk for TIA (P⫽
.038; data not shown). Furthermore, a relatively high prevalence of combined HPA-2a/b and HPA-5a/b ge-notypes (16.7%) was found for children with TIA, com-pared with control subjects (4.4%), children with PAS (3.8%), and children with AIS (3.0%). The presence of these genotypes was associated with a 4.28-fold (95% CI: 1.22–15.00-fold) increased risk for TIA (P ⫽ .025; data not shown).
The HPA-1b/b genotype was fivefold more frequently identified in patients with AIS than in control subjects, but this difference was not significant (P⫽ .078). The presence of the HPA-2b allele was associated with a 2.23-fold (95% CI: 1.02– 4.87-fold) increased risk for migraine (P⫽.046) and a 2.6-fold (95% CI: 1.23–5.51-fold) increased risk for TIA (P⫽.016; data not shown). Children carrying the HPA-3b allele had an almost two-fold lower risk for AIS than did carriers of the HPA-3a allele (OR: 0.55 [95% CI: 0.31– 0.97];P⫽.048; data not shown).
DISCUSSION
Stroke in children has been investigated extensively. Several inherited prothrombotic risk factors, particularly
the presence of factor V G1691A, factor II G20210A, and MTHFR C677T, were demonstrated to play a role in childhood stroke in a number of studies.21–25The role of
platelets has not been well studied in children,7although
polymorphisms in platelet glycoprotein receptors con-tribute to variable platelet function and might modify platelet reactivity, predisposing individuals to a clinical event such as thrombosis. Because the distributions of genetic factors differ according to age, geographic region, and stroke type,2we performed genotypic analyses for
factor V G1691A, factor II G20210A, MTHFR C677T, and 4 common platelet glycoprotein polymorphisms (HPA-1, -2, -3, and -5) in children with stroke, TIA, and mi-graine.
The present study shows that, among the investigated polymorphisms, only factor V G1691A was significantly associated with increased risk for AIS in childhood and PAS. Our finding is in contrast to some studies26–29but in
accordance with a number of studies performed on childhood AIS21–25,30 and perinatal/neonatal AIS30–32 in
different populations. In addition, we observed twofold higher rates of homozygosity for MTHFR C677T for pa-tients with AIS and PAS, compared with control sub-jects, which is in accordance with the findings of Pren-gler et al,33Cardo et al,34and Lynch et al.35In contrast to
the findings of Nowak-Go¨ttl et al21that homozygosity for
MTHFR C677T increased the risk for AIS, we did not demonstrate any association, which is similar to most previous studies.22,27,29,30,32–36 The presence of factor II
G20210A was not associated with increased risk for AIS and PAS in our study, which is consistent with other studies22,25–30,32but in contrast to studies from Germany,21
Turkey,36and Portugal.28
In this study, polymorphisms were analyzed
sepa-TABLE 5 Comparison of Genotypic Distributions Between Patient and Control Groups and Calculated ORs
Polymorphism AIS (Nⴝ33) PAS (Nⴝ26) HS (Nⴝ20) TIA (Nⴝ36) Migraine (Nⴝ35)
Factor V G1691A
P .024a .046a .392 .031a .561
OR (95% CI) 7.59 (1.32–43.5)a 7.17 (1.13–45.41)a 2.89 (0.25–33.54) 6.87 (1.20–39.28)a 1.62 (0.14–18.41)
Factor II G20210A
P 1.000 1.000 .566 .099 .628
OR (95% CI) 0.84 (0.10–7.34) 0.46 (0.02–8.72) 1.42 (0.15–13.42) 3.37 (0.80–14.26) 1.64 (0.29–9.34) MTHFR C677T
P 0.333 0.469 0.698 0.129 0.213
OR (95% CI) 1.82 (0.58–5.77) 1.86 (0.53–6.46) 0.54 (0.07–4.44) 2.46 (0.86–7.04) 2.11 (0.71–6.30) HPA-1
P .665 .617 .788 .099 1.000
OR (95% CI) 1.19 (0.51–2.79) 0.65 (0.22–1.88) 1.17 (0.41–3.33) 1.95 (0.892–4.27) 0.95 (0.40–2.25) HPA-2
P .445 1.000 .363 .057 .091
OR (95% CI) 1.57 (0.61–4.00) 0.89 (0.27–2.88) 1.63 (0.53–5.03) 2.45 (1.04–5.73)a 2.24 (0.94–5.34)
HPA-3
P .131 .595 1.000 .638 .348
OR (95% CI) 0.35 (0.10–1.24) 0.63 (0.20–2.01) 0.87 (0.27–2.84) 0.70 (0.26–1.86) 0.58 (0.20–1.65) HPA-5
P .799 1.000 .127 .013a 1.000
OR (95% CI) 0.82 (0.28–2.39) 0.84 (0.26–2.69) 2.48 (0.88–6.99) 2.93 (1.28–6.69)a 0.95 (0.35–2.60)
ORs and 95% CIs were calculated by using a dominant model (homozygous or heterozygous variant in comparison with the homozygous wild-type) for factor V G1691A, factor II G20210A, and HPA-1, -2, and -5 and a recessive model (homozygous variant in comparison with the heterozygous variant and homozygous wild-type) for MTHFR C677T and HPA-3.
rately for children with AIS and PAS, because the cause of stroke during the perinatal/neonatal period might be different from that in childhood.22The only difference
observed between the AIS and PAS groups was in the distribution of HPA-1 genotypes and HPA-3 alleles. We identified an increased, although nonsignificant, rate of the HPA-1b/b genotype in AIS, whereas this genotype was not found among children with PAS. Moreover, the results of our study suggested a mild protective effect of the HPA-3b allele in AIS, which was not the case for PAS.
Although a number of studies have investigated the association between inherited prothrombotic risk factors and ischemic stroke in children, to our knowledge there are no published reports regarding HS, although some data are available for adults with HS.37–40 In our study,
neither of the investigated risk alleles increased the risk for the occurrence of HS in children.
In the present study, significant differences in gen-der (P ⫽.005) and age at diagnosis (P ⬍.001) were observed between children with TIA and children with AIS. TIA occurred more frequently in girls and was diagnosed in older children, compared with AIS. Whereas a male predominance for children with isch-emic stroke has been reported consistently,2,30,41to our
knowledge no information regarding gender is avail-able for children with TIA. We can only speculate that girls in and after puberty are more prone to develop TIA than are boys, similar to migraine, which occurs more frequently in boys before puberty but is more common in girls after puberty, possibly because of hormonal differences.5,42
Inherited prothrombotic risk factors have not been investigated in children with TIA as a separate group, except in 2 studies that included children with TIA in addition to children with AIS.23,33 The results of our
study demonstrated a strong association between fac-tor V G1691A and TIA, similar to that determined for AIS and PAS. Furthermore, significantly higher fre-quencies of HPA-2b and HPA-5b alleles, HPA-5a/b, and combined HPA-2a/b and HPA-5a/b genotypes were detected in children with TIA, compared with control subjects. Although TIA was diagnosed more frequently in girls in our study, HPA-2b, HPA-5b, or both alleles were more prevalent in boys than in girls (69.2% vs 47.8%).
The present data regarding an almost threefold in-creased risk for TIA among carriers of the HPA-2b allele are in accordance with those of Sonoda et al,43 who
observed significant associations of 2a/b and HPA-2b/b genotypes with the risk for TIA and lacunar stroke but not for atherothrombotic stroke in adults. Gonzales-Conejero et al44found a significant relationship between
the HPA-2b polymorphism and increased risk of cere-brovascular disease in adults, but their study group in-cluded patients with TIA, minor stroke, and cerebral infarction. Our results suggest that HPA-2 and -5 poly-morphisms, which are implicated in platelet adhesion, might have a role in the development of TIA but not stroke in children. It has been found that the HPA-2 polymorphism is located within the von Willebrand
factor-binding domain of glycoprotein Ib␣43 and that
the HPA-5 polymorphism is located in the cation-binding domain of glycoprotein Ia.45Amino acid
sub-stitutions could cause conformational variations in the structure of glycoprotein Ib␣and glycoprotein Ia that might affect ligand binding and augment initial plate-let adhesion to von Willebrand factor and collagen after exposure to vascular subendothelium under high shear rates.
In contrast to childhood stroke, inherited prothrom-botic risk factors have not been studied extensively in children with migraine. However, a possible relationship between migraine and inherited prothrombotic risk fac-tors has been suggested, because both increased platelet aggregability and plasma coagulability during migraine attacks have been described.5 In our study, factor V
G1691A and factor II G20210A were not associated with an increased risk for migraine in children, which is in agreement with the findings of other authors.5,46,47
Al-though there was a trend toward an increased risk for migraine in children homozygous for MTHFR C677T, which was even more pronounced for migraine with aura, the association was not statistically significant. Our results are in accordance with those of Bottini et al47but
different from results obtained by Bassi et al.5In contrast
to the study by Corral et al,48who found no difference in
the prevalence of HPA-1 and -2 in adults with migraine and control subjects, we noted that the presence of the HPA-2b allele was associated with a 2.23-fold increased risk for migraine.
CONCLUSIONS
Factor V G1691A was found to have an important role in susceptibility to AIS, both in the perinatal/neonatal period and in childhood, as well as TIA. These findings suggest a possible link between stroke and TIA in children. Furthermore, a minor impact of HPA morphisms suggests that platelet glycoprotein poly-morphisms may increase the risk for TIA and mi-graine, but this should be confirmed in larger studies.
ACKNOWLEDGMENTS
This study was supported by grant 0214212 from the Croatian Ministry of Science, Education, and Sports.
REFERENCES
1. Lynch JK. Cerebrovascular disorders in children.Curr Neurol Neurosci Rep.2004;4(2):129 –138
2. Sofronas M, Ichord RN, Fullerton HJ, et al. Pediatric stroke initiatives and preliminary studies: what is known and what is needed?Pediatr Neurol.2006;34(6):439 – 445
3. Nowak-Go¨ttl U, Gu¨nther G, Kurnik K, Strater R, Kirkham F. Arterial ischemic stroke in neonates, infants and children: an overview of underlying conditions, imaging methods, and treatment modalities. Semin Thromb Hemost. 2003;29(4): 405– 414
4. Merikangas KR, Febton BT, Cheng SH, Stolar MJ, Risch N. Association between migraine and stroke in a large-scale epi-demiological study of the United States. Arch Neurol. 1997; 54(4):362–368
acquired thrombophilia in a cohort of young migrainous pa-tients.J Headache Pain.2003;4(3):138 –145
6. Kirkham FJ. Is there a genetic basis for pediatric stroke?Curr Opin Pediatr.2003;15(6):547–558
7. Barnes C, deVeber G. Prothrombotic abnormalities in child-hood ischaemic stroke.Thromb Res.2006;118(1):67–74 8. Santoso S. Human platelet alloantigens. Transfus Apher Sci.
2003;28(3):227–236
9. Deckmyn H, Ulrichts H, Van de Walle G, Vanhoorelbeke K. Platelet antigens and their function.Vox Sang.2004;87(suppl 2):S105–S111
10. Newman PJ, Derbes RS, Aster RH. The human platelet alloan-tigens, PIA1and PIA2, are associated with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa and are distinguishable by DNA typing.J Clin Invest.1989;83(5): 1778 –1781
11. Kuijpers RWAM, Faber NM, Cuypers HTM, Ouwehand WH, von dem Borne AEGK. NH2-terminal globular domain of hu-man platelet glycoprotein Ib␣ has a methionine145/ threonine145amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens. J Clin Invest.1992;89(2): 381–384
12. Lyman S, Aster RH, Visentin GP, Newman PJ. Polymorphism of human platelet membrane glycoprotein IIb associated with the Baka/Bakb alloantigen system. Blood. 1990;75(12): 2343–2348
13. Santoso S, Kalb R, Walka M, Kiefel V, Mueller-Eckhardt C, Newman PJ. The human platelet alloantigens, Braand Brb, are associated with a single amino acid polymorphism on glycoprotein Ia (integrin ␣2). J Clin Invest. 1993;92(5): 2427–2432
14. Se´bire G, Fullerton H, Riou E, deVeber G. Toward the defini-tion of cerebral arteriopathies of childhood.Curr Opin Pediatr. 2004;16(6):617– 622
15. International Headache Society, Headache Classification Subcommittee. The International Classification of Headache Disorders, 2nd edition.Cephalagia.2004;24(suppl 1):1–160 16. Miller SA, Dykes DD, Polesky HF. A simple salting out
proce-dure for extracting DNA from human nucleated cells.Nucleic Acids Res.1988;16(3):1215
17. Zo¨ller B, Svensson PJ, He X, Dahlba¨ck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C.J Clin Invest. 1994;94(6):2521–2524
18. Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3⬘-untranslated region of the pro-thrombin gene is associated with elevated plasma propro-thrombin levels and an increase in venous thrombosis. Blood. 1996; 88(10):3698 –3703
19. Aslanidis C, Nauck M, Schmitz G. High-speed prothrombin G3A 20210 and methylenetetrahydrofolate reductase C3T 677 mutation detection using real-time fluorescence PCR and melting curves.Biotechniques.1999;27(2):234 –238
20. Klu¨ter H, Fehlau K, Panzer S, Kirchner H, Bein G. Rapid typing for human platelet antigen systems-1, -2, -3 and -5 by PCR amplification with sequence-specific primers.Vox Sang.1996; 71(2):121–125
21. Nowak-Go¨ttl U, Stra¨ter R, Heinecke A, et al. Lipoprotein(a) and genetic polymorphisms of clotting factor V, prothrombin and methylenetetrahydrofolate reductase are risk factors of ischemic stroke in childhood.Blood.1999;94(11):3678 –3682 22. Kenet G, Sadetzki S, Murad H, et al. Factor V Leiden and antiphospholipid antibodies are significant risk factors for isch-emic stroke in children.Stroke.2000;31(6):1283–1288 23. Hagstrom JN, Walter J, Bluebond-Langner R, Amatniek JC,
Manno CS, High KA. Prevalence of the factor V Leiden
muta-tion in children and neonates with thromboembolic disease. J Pediatr.1998;133(6):777–781
24. Akar N, Akar E, Yilmaz E, Deda G. Plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish children with ce-rebral infarct and effect on factor V 1691A mutation.J Child Neurol.2001;16(4):294 –295
25. Zenz W, Bodo Z, Plotho J, et al. Factor V Leiden and prothrom-bin gene G 20210 A variant in children with ischemic stroke. Thromb Haemost.1998;80(5):763–766
26. McColl MD, Chalmers EA, Thomas A, et al. Factor V Leiden, prothrombin 20210G3A and the MTHFR C677T mutations in childhood stroke.Thromb Haemost.1999;81(5):690 – 694 27. Barreirinho S, Ferro A, Santos M, et al. Inherited and acquired
risk factors and their combined effects in pediatric stroke. Pe-diatr Neurol.2003;28(2):134 –138
28. 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(2): 81– 86
29. Miller SP, Wu YW, Lee J, et al. Candidate gene polymorphisms do not differ between newborns with stroke and normal con-trols.Stroke.2006;37(11):2678 –2683
30. Komitopoulou A, Platokouki H, Kapsimali Z, Pergantou H, Adamtziki E, Aronis S. Mutations and polymorphisms in genes affecting hemostasis proteins and homocysteine metabolism in children with arterial ischemic stroke. Cerebrovasc Dis. 2006; 22(1):13–20
31. Gu¨nther G, Junker R, Strater R, et al. Symptomatic ischemic stroke in full-term neonates: role of acquired and genetic prothrombic risk factors.Stroke.2000;31(10):2437–2441 32. Mercuri E, Cowan F, Gupte G, et al. Prothrombic disorders
and abnormal neurodevelopmental outcome in infants with neonatal cerebral infarction. Pediatrics. 2001;107(6): 1400 –1404
33. Prengler M, Sturt N, Krywawych S, Surtees R, Liesner R, Kirkham F. Homozygous thermolabile variant of the methyl-enetetrahydrofolate reductase gene: a potential risk factor for hyperhomocysteinaemia, CVD, and stroke in childhood.Dev Med Child Neurol.2001;43(4):220 –225
34. Cardo E, Monros E, Colome C, et al. Children with stroke: polymorphism of the MTHFR gene, mild hyperhomocys-teinemia, and vitamin status. J Child Neurol. 2000;15(5): 295–298
35. Lynch JK, Han CJ, Nee LE, Nelson KB. Prothrombotic factors in children with stroke or porencephaly. Pediatrics. 2005; 116(2):447– 453
36. Akar N, Akar E, Deda G, Sipahi T, Orsal A. Factor V 1691 G–A, prothrombin 20210 G–A, and methylenetetrahydrofolate re-ductase 677 C–T variants in Turkish children with cerebral infarct.J Child Neurol.1999;14(11):749 –751
37. Corral J, Iniesta JA, Gonza´lez-Conejero R, Villalo´n M, Vi-cente V. Polymorphisms of clotting factors modify the risk of primary intracranial hemorrhage. Blood. 2001;97(10): 2979 –2982
38. Sazci A, Ergul E, Tuncer N, Akpinar G, Kara I. Methylenetet-rahydrofolate reductase gene polymorphisms are associated with ischemic and hemorrhagic stroke: dual effect ofMTHFR polymorphisms C677T and A1298C. Brain Res Bull. 2006; 71(1–3):45–50
39. Reiner AP, Kumar PN, Schwartz SM, et al. Genetic variants of platelet glycoprotein receptors and risk of stroke in young women.Stroke.2000;31(7):1628 –1633
40. Iniesta JA, Gonza´lez-Conejero R, Piqueras C, Vicente V, Corral J. Platelet GP IIIa polymorphism HPA-1 (PIA) protects against subarachnoid hemorrhage. Stroke. 2004;35(10): 2282–2286
children: ethnic and gender disparities.Neurology.2003;61(2): 189 –194
42. Lewis DW. Toward the definition of childhood migraine.Curr Opin Pediatr.2004;16(6):628 – 636
43. Sonoda A, Murata M, Ito D, et al. Association between platelet glycoprotein Ib␣genotype and ischemic cerebrovascular dis-ease.Stroke.2000;31(2):493– 497
44. Gonzalez-Conejero R, Lozano ML, Rivera J, et al. Polymor-phism of platelet glycoprotein Ib␣ associated with arterial thrombotic disease.Blood.1998;92(8):2771–2776
45. Kroll H, Gardemann A, Fechter A, Haberbosch W, Santoso S.
The impact of the glycoprotein Ia receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction.Thromb Haemost.2000;83(3):392–396 46. Soriani S, Borgna-Pignatti C, Trabetti E, Casartelli A, Montagna P, Pignatti PF. Frequency of factor V Leiden in juvenile mi-graine with aura.Headache.1998;38(10):779 –781
DOI: 10.1542/peds.2007-3737
2009;123;e653
Pediatrics
Slavica Dodig, Vlasta Duranovic, Anica Basnec Brkic and Renata Zadro
Désirée Coen Herak, Margareta Radic Antolic, Jasna Lenicek Krleza, Marina Pavic,
Ischemic Attack, or Migraine
Inherited Prothrombotic Risk Factors in Children With Stroke, Transient
Services
Updated Information &
http://pediatrics.aappublications.org/content/123/4/e653
including high resolution figures, can be found at:
References
http://pediatrics.aappublications.org/content/123/4/e653#BIBL
This article cites 48 articles, 14 of which you can access for free at:
Subspecialty Collections
sub
http://www.aappublications.org/cgi/collection/hematology:oncology_
Hematology/Oncology
following collection(s):
This article, along with others on similar topics, appears in the
Permissions & Licensing
http://www.aappublications.org/site/misc/Permissions.xhtml
in its entirety can be found online at:
Information about reproducing this article in parts (figures, tables) or
Reprints
http://www.aappublications.org/site/misc/reprints.xhtml
DOI: 10.1542/peds.2007-3737
2009;123;e653
Pediatrics
Slavica Dodig, Vlasta Duranovic, Anica Basnec Brkic and Renata Zadro
Désirée Coen Herak, Margareta Radic Antolic, Jasna Lenicek Krleza, Marina Pavic,
Ischemic Attack, or Migraine
Inherited Prothrombotic Risk Factors in Children With Stroke, Transient
http://pediatrics.aappublications.org/content/123/4/e653
located on the World Wide Web at:
The online version of this article, along with updated information and services, is
by the American Academy of Pediatrics. All rights reserved. Print ISSN: 1073-0397.
