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Genetic Risk Factors for Perinatal Arterial Ischemic Stroke

      Abstract

      The cause of perinatal arterial ischemic stroke is unknown in most cases. We explored whether genetic polymorphisms modify the risk of perinatal arterial ischemic stroke. In a population-based case-control study of 1997-2002 births at Kaiser Permanente Northern California, we identified 13 white infants with perinatal arterial ischemic stroke. Control subjects included 86 randomly selected white infants. We genotyped polymorphisms in nine genes involved in inflammation, thrombosis, or lipid metabolism previously linked with stroke, and compared genotype frequencies in case and control individuals. We tested several polymorphisms: tumor necrosis factor-α −308, interleukin-6, lymphotoxin A, factor V Leiden, methyltetrahydrofolate reductase 1298 and 667, prothrombin 20210, and apolipoprotein E ε2 and ε4 alleles. Patients with perinatal arterial ischemic stroke were more likely than control subjects to demonstrate at least one apolipoprotein E ε4 allele (54% vs 25%, P = 0.03). More patients with perinatal arterial ischemic stroke carried two ε4 alleles than did control subjects (15% vs 2%, P = 0.09), although this finding lacked statistical significance. Proinflammatory and prothrombotic polymorphisms were not associated with perinatal arterial ischemic stroke. The apolipoprotein E polymorphism may confer genetic susceptibility for perinatal arterial ischemic stroke. Larger population-based studies are required to confirm this finding.

      Introduction

      Perinatal arterial ischemic stroke is a well recognized cause of cerebral palsy, epilepsy, and behavioral abnormalities in children [
      • Lee J.
      • Croen L.A.
      • Lindan C.
      • et al.
      Predictors of outcome in perinatal arterial stroke: A population-based study.
      ]. By definition, perinatal arterial ischemic stroke occurs either in utero or before age 28 days, although infants may present later in infancy [
      • Lee J.
      • Croen L.A.
      • Lindan C.
      • et al.
      Predictors of outcome in perinatal arterial stroke: A population-based study.
      ,
      • Raju T.N.
      • Nelson K.B.
      • Ferriero D.
      • Lynch J.K.
      Ischemic perinatal stroke: Summary of a workshop sponsored by the National Institute of Child Health and Human Development and the National Institute of Neurological Disorders and Stroke.
      ]. The incidence of perinatal arterial ischemic stroke involves 20 per 100,000 live births, i.e., 17 times higher than the rate of childhood ischemic stroke, and as high as the annual incidence of large vessel ischemic stroke in adults (17-23 per 100,000) [
      • Lee J.
      • Croen L.A.
      • Lindan C.
      • et al.
      Predictors of outcome in perinatal arterial stroke: A population-based study.
      ,
      • Lee J.
      • Croen L.A.
      • Backstrand K.H.
      • et al.
      Maternal and infant characteristics associated with perinatal arterial stroke in the infant.
      ]. Previously identified risk factors for perinatal arterial ischemic stroke include primiparity, infertility, preeclampsia, the prolonged rupture of membranes, emergency cesarean delivery, chorioamnionitis, neonatal prothrombotic disorders and meningitis, or other intracranial infections [
      • Lee J.
      • Croen L.A.
      • Backstrand K.H.
      • et al.
      Maternal and infant characteristics associated with perinatal arterial stroke in the infant.
      ,
      • Wu Y.W.
      • Lynch J.K.
      • Nelson K.B.
      Perinatal arterial stroke: Understanding mechanisms and outcomes.
      ]. However, the cause of perinatal arterial ischemic stroke remains unknown in most cases [
      • Miller S.P.
      • Wu Y.W.
      • Lee J.
      • et al.
      Candidate gene polymorphisms do not differ between newborns with stroke and normal controls.
      ].
      Genetic susceptibility may play a role in the pathogenesis of perinatal stroke. For instance, studies have suggested that factor V Leiden and methyltetrahydrofolate reductase mutations may be associated with an increased risk of perinatal stroke [
      • Mercuri E.
      • Cowan F.
      • Gupte G.
      • et al.
      Prothrombotic disorders and abnormal neurodevelopmental outcome in infants with neonatal cerebral infarction.
      ,
      • Hagstrom J.N.
      • Walter J.
      • Bluebond-Langner R.
      • Amatniek J.C.
      • Manno C.S.
      • High K.A.
      Prevalence of the factor V Leiden mutation in children and neonates with thromboembolic disease.
      ,
      • Del Balzo F.
      • Spalice A.
      • Ruggieri M.
      • Greco F.
      • Properzi E.
      • Iannetti P.
      Stroke in children: Inherited and acquired factors and age-related variations in the presentation of 48 paediatric patients.
      ]. On the other hand, a study examining polymorphisms of genes involved in the regulation of thrombosis and thrombolysis, and genes related to nitric oxide, cytokines, blood pressure control, and cell adhesion, did not detect any differences in patients with perinatal arterial ischemic stroke compared with control subjects [
      • Miller S.P.
      • Wu Y.W.
      • Lee J.
      • et al.
      Candidate gene polymorphisms do not differ between newborns with stroke and normal controls.
      ].
      Studies of ischemic stroke in adults revealed several single-nucleotide polymorphisms that modify an individual's risk of stroke [
      • Trompet S.
      • de Craen A.J.
      • Slagboom P.
      • et al.
      Lymphotoxin-alpha C804A polymorphism is a risk factor for stroke: The PROSPER Study.
      ,
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ,
      • Rubattu S.
      • Speranza R.
      • Ferrari M.
      • et al.
      A role of TNF-alpha gene variant on juvenile ischemic stroke: A case-control study.
      ,
      • Hoppe C.
      • Klitz W.
      • D’Harlingue K.
      • et al.
      Confirmation of an association between the TNF(−308) promoter polymorphism and stroke risk in children with sickle cell anemia.
      ]. Whether these polymorphisms are associated with risk of perinatal stroke remains unknown. This case-control study explores whether genetic polymorphisms known to be associated with ischemic stroke in adults are also associated with a risk of arterial ischemic stroke in newborn infants.

      Methods

      We performed a case-control study nested within the cohort of all 199,176 infants born from January 1, 1997 to December 31, 2002 at Kaiser Permanente of Northern California. Kaiser Permanente of Northern California is a large, integrated healthcare delivery system that provides care for approximately 30% of the population in northern California. The members of Kaiser Permanente of Northern California are demographically similar to the California population, except that the very poor and very wealthy are underrepresented [
      • Krieger N.
      Overcoming the absence of socioeconomic data in medical records: Validation and application of a census-based methodology.
      ]. All demographic data were obtained from reviews of medical records. The study procedures were approved by the institutional review boards at Kaiser Permanente of Northern California, the University of California at San Francisco, Utah State University, and the California Committee for the Protection of Human Subjects.

      Case and control identification

      The methodology used to identify infants with perinatal arterial ischemic stroke was described previously [
      • Lee J.
      • Croen L.A.
      • Backstrand K.H.
      • et al.
      Maternal and infant characteristics associated with perinatal arterial stroke in the infant.
      ]. Perinatal arterial ischemic stroke was defined as stroke that occurred in utero or up to 28 days after birth [
      • Lee J.
      • Croen L.A.
      • Backstrand K.H.
      • et al.
      Maternal and infant characteristics associated with perinatal arterial stroke in the infant.
      ]. To identify infants with perinatal arterial ischemic stroke, we searched all cranial magnetic resonance imaging and computed tomographic reports of infants within the Kaiser Permanente of Northern California birth cohort for key words indicating possible perinatal arterial ischemic stroke [
      • Lee J.
      • Croen L.A.
      • Backstrand K.H.
      • et al.
      Maternal and infant characteristics associated with perinatal arterial stroke in the infant.
      ]. Imaging studies were reviewed by a neuroradiologist to confirm the presence of an arterial-distribution ischemic infarction. Infants with both acute stroke (neurologic presentation within 28 days of age) and presumed perinatal stroke (neurologic presentation after 1 month of age, with imaging to indicate an old arterial-ischemic infarction) were included.
      Given the heterogeneity in single-nucleotide polymorphism frequencies among different ethnic groups [
      • Das K.
      • Das M.K.
      • Mastana S.S.
      Genetic diversity of serum proteins in three subpopulations of the Maria Gond tribe of Madhya Pradesh, India.
      ,
      • Roberts L.N.
      • Patel R.K.
      • Arya R.
      Venous thromboembolism and ethnicity.
      ], genetic association studies typically restrict analyses to one ethnic group [
      • Ramos E.M.
      • Lin M.T.
      • Larson E.B.
      • et al.
      Tumor necrosis factor alpha and interleukin 10 promoter region polymorphisms and risk of late-onset Alzheimer disease.
      ,
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ,
      • Stoica A.L.
      • Stoica E.
      • Constantinescu I.
      • Uscatescu V.
      • Ginghina C.
      Interleukin-6 and interleukin-10 gene polymorphism, endothelial dysfunction, and postoperative prognosis in patients with peripheral arterial disease.
      ] or stratify by ethnicity [
      • Kaiser R.
      • Li Y.
      • Chang M.
      • et al.
      Genetic risk factors for thrombosis in systemic lupus erythematosus.
      ]. We restricted our study to non-Hispanic white infants, because the small size of our study precluded meaningful analyses of other ethnic groups. Of the 37 infants identified with perinatal arterial ischemic stroke, 13 white infants constituted the cases in this study. In a previous study, we randomly selected 165 healthy control infants born during the years 1991-2002 at Kaiser Permanente [
      • Wu Y.W.
      • Croen L.A.
      • Torres A.R.
      • Van De Water J.
      • Grether J.K.
      • Hsu N.N.
      Interleukin-6 genotype and risk for cerebral palsy in term and near-term infants.
      ]. Among these previously described control infants, we selected the subset of all 86 non-Hispanic whites born during the current study period (1997-2002) as control infants for the present study.

      Blood sample retrieval

      Our methods of blood sample collection and of genomic DNA extraction were described previously [
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ]. We retrieved stored neonatal blood specimens from the newborn screening specimen archives maintained by the California Department of Public Health. Dried bloodspots have been stored for all infants born in California since 1980. Newborn blood specimens are collected on Guthrie card filter paper and dried at room temperature before submission for routine genetic and metabolic screening. Upon completion of screening tests, the remaining blood samples are stored at −15°C in a single refrigerated warehouse [
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ].

      Genomic DNA extraction from bloodspots

      Bloodspot Guthrie cards were punched with a 3-mm paper punch in a laminar flow hood under aseptic conditions. Two 3-mm punches from each subject were placed in a 96-well plate and incubated at 56°C for 1 hour in Qiagen buffer (Qiagen Inc., Gaithersburg, MD) and Proteinase K enzyme (Qiagen Inc.). Genomic DNA was isolated from the bloodspot punches, using QIAamp 96 DNA blood kits supplied by Qiagen. The procedure for multiple displacement amplification using Phi 29 polymerase was performed at 30°C for 16 hours, using RepliPHI Phi 29 Reagent Sets (Epicentre Technologies, Madison, WI), and stopped by inactivating the Phi 29 enzyme at 65°C in a water bath for 5 minutes. The amount of DNA extracted from two punches varied somewhat between samples, but an average of 235 ng of genomic DNA was acquired from two 3.2-mm punches [
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ].

      Single-nucleotide polymorphism genotyping

      Standard Taqman polymerase chain reactions were performed using 7500 Fast System AB 96-well optical plates (plates P/N 4366932; Applied Biosystems, Grand Island, NY). The reactions were designed according to the Applied Biosystems single-nucleotide polymorphism assay protocol in 10-μL volumes. Each reaction was performed in a single well because of the limiting amounts of genomic DNA. Results from all experiments were obtained from Applied Biosystems SDS software version 2.0 and Copy Caller software version 1.0 (Applied Biosystems). All genotyping was performed blind to case status and clinical histories [
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ]. We genotyped several polymorphisms previously associated with ischemic stroke in adults, i.e., tumor necrosis factor-α −308 G/A (rs1800629) [
      • Rubattu S.
      • Speranza R.
      • Ferrari M.
      • et al.
      A role of TNF-alpha gene variant on juvenile ischemic stroke: A case-control study.
      ,
      • Hoppe C.
      • Klitz W.
      • D’Harlingue K.
      • et al.
      Confirmation of an association between the TNF(−308) promoter polymorphism and stroke risk in children with sickle cell anemia.
      ], interleukin-6 −174 G/C (rs1800795) [
      • Stoica A.L.
      • Stoica E.
      • Constantinescu I.
      • Uscatescu V.
      • Ginghina C.
      Interleukin-6 and interleukin-10 gene polymorphism, endothelial dysfunction, and postoperative prognosis in patients with peripheral arterial disease.
      ,
      • Manso H.
      • Krug T.
      • Sobral J.
      • et al.
      Variants in the inflammatory IL6 and MPO genes modulate stroke susceptibility through main effects and gene-gene interactions.
      ], lymphotoxin C804A (rs1041981) [
      • Trompet S.
      • de Craen A.J.
      • Slagboom P.
      • et al.
      Lymphotoxin-alpha C804A polymorphism is a risk factor for stroke: The PROSPER Study.
      ], factor V Leiden 506 G/A (rs6025) [
      • Casas J.P.
      • Hingorani A.D.
      • Bautista L.E.
      • Sharma P.
      Meta-analysis of genetic studies in ischemic stroke: Thirty-two genes involving approximately 18,000 cases and 58,000 controls.
      ,
      • Kenet G.
      • Lutkhoff L.K.
      • Albisetti M.
      • et al.
      Impact of thrombophilia on risk of arterial ischemic stroke or cerebral sinovenous thrombosis in neonates and children: A systematic review and meta-analysis of observational studies.
      ], methyltetrahydrofolate reductase 1298 A/C (rs1801131) [
      • Rook J.L.
      • Nugent D.J.
      • Young G.
      Pediatric stroke and methylenetetrahydrofolate reductase polymorphisms: An examination of C677T and A1298C mutations.
      ] and 667 C/T (rs1801133) [
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ,
      • Rook J.L.
      • Nugent D.J.
      • Young G.
      Pediatric stroke and methylenetetrahydrofolate reductase polymorphisms: An examination of C677T and A1298C mutations.
      ], prothrombin 20210 G/A (rs1799963) [
      • Casas J.P.
      • Hingorani A.D.
      • Bautista L.E.
      • Sharma P.
      Meta-analysis of genetic studies in ischemic stroke: Thirty-two genes involving approximately 18,000 cases and 58,000 controls.
      ,
      • Kenet G.
      • Lutkhoff L.K.
      • Albisetti M.
      • et al.
      Impact of thrombophilia on risk of arterial ischemic stroke or cerebral sinovenous thrombosis in neonates and children: A systematic review and meta-analysis of observational studies.
      ], and the apolipoprotein E ε2 and ε4 alleles (rs429358 and rs7412) [
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ].

      Data analysis

      We defined single-nucleotide polymorphism genotypes such that a common homozygote contains two copies of the common allele, a heterozygote contains one copy of each allele, and a rare homozygote contains two copies of the rare allele. Using logistic regression, we determined odds ratios and 95% confidence intervals according to two genetic models: (1) rare homozygote vs common homozygote, and (2) heterozygote or rare homozygote vs common homozygote [
      • Wu Y.W.
      • Croen L.A.
      • Vanderwerf A.
      • Gelfand A.A.
      • Torres A.R.
      Candidate genes and risk for CP: A population-based study.
      ]. For apolipoprotein E analyses, to be in concordance with the literature, we compared allelic frequencies of the three apolipoprotein E alleles (ε2, ε3, and ε4) in cases vs control subjects. We performed χ2 analyses, Fisher exact tests, and the Student t test as appropriate when comparing demographic variables in the case and control groups. We used logistic regression to compare continuous variables such as birth weight and gestational age. Given the small and exploratory nature of this study, we did not correct for multiple comparisons. Given the limited number of cases, a multivariable analysis was not feasible.

      Results

      Birth weight, infant sex, and maternal age did not differ between the cases and control subjects (Table 1). In addition, rates of preeclampsia and chorioamnionitis did not differ. However, infants with perinatal arterial ischemic strokes were slightly older (mean age, 40.1 vs 39.3 weeks, respectively; P = 0.0 3) and more likely than control infants to be born by cesarean section (46% vs 19%, respectively; P = 0.0 3). A trend for more primigravida mothers among case infants was evident (75% vs 46%, respectively; P = 0.0 7). Neonatal seizures were present in five of the cases and none of the control subjects.
      Table 1Associations between clinical factors and perinatal arterial ischemic strokes
      Cases (n = 13)Control Subjects (n = 86)P Value
      Male77%55%0.22
      Maternal age (years), mean (S.D.)28 (6.6)28.6 (5.8)0.73
      Birth weight (g), mean (S.D.)
      Data missing for one control infant.
      3561 (485)3498 (529)0.69
      Gestational age (weeks), mean (SD)
      Data missing for 24 control infants.
      40.1 (0.9)39.3 (1.4)0.03
      Cesarean section
      Data missing for three control infants.
      46%19%0.03
      Prima gravida
      Data missing for one control infant.
      Data missing for one case infant.
      75%46%0.07
      Preeclampsia8%4%0.44
      Chorioamnionitis
      Data missing for one control infant.
      Data missing for three case infants.
      10%5%0.43
      Abbreviation:
      S.D. = Standard deviation
      Data missing for one control infant.
      Data missing for one case infant.
      Data missing for 24 control infants.
      § Data missing for three control infants.
      Data missing for three case infants.
      All distributions of single-nucleotide polymorphism genotypes within the control population were in Hardy-Weinberg equilibrium (Table 2). The distributions of single-nucleotide polymorphism genotypes for the cases are presented in Table 3. None of the proinflammatory or prothrombotic polymorphisms tested were significantly different between the two groups (Table 4).
      Table 2Genotype distributions among 86 white control infants without perinatal arterial ischemic stroke
      GenePolymorphismnCommon Homozygote (%)Heterozygote (%)Rare Homozygote (%)Hardy-Weinberg χ2
      All χ2 values were determined to be less than 3.84 (P < 0.05). Therefore, all allele frequencies are in Hardy-Weinberg equilibrium.
      TNF-α−308 G/A86702910.83
      IL-6G to C774839130.96
      Lymphotoxin804 C/A844050100.94
      Factor V Leiden506 G/A8594600.00
      MTHFR677 C/T843256122.34
      MTHFR1298 A/C84464952.76
      Prothrombin (F2)20210 G/A8699100.00
      Apolipoprotein Eε3/ε484661000.38
      Apolipoprotein Eε3/ε284661920.20
      Abbreviations:
      IL = Interleukin
      MTHFR = Methyltetrahydrofolate reductase
      TNF = Tumor necrosis factor
      All χ2 values were determined to be less than 3.84 (P < 0.05). Therefore, all allele frequencies are in Hardy-Weinberg equilibrium.
      Table 3Genotype distributions among 13 white control infants with perinatal arterial ischemic stroke
      GenePolymorphismnCommon Homozygote (%)Heterozygote (%)Rare Homozygote (%)
      TNF-α−308 G/A1377230
      IL-6G to C1339620
      Lymphotoxin804 C/A1339548
      Factor V Leiden506 G/A1310000
      MTHFR677 C/T1354398
      MTHFR1298 A/C13315415
      Prothrombin (F2)20210 G/A1310000
      Apolipoprotein Eε3/ε413542315
      Apolipoprotein Eε3/ε2135480
      Abbreviations:
      IL = Interleukin
      MTHFR = Methyltetrahydrofolate reductase
      TNF = Tumor necrosis factor
      Table 4Associations between polymorphisms in inflammatory and thrombotic genes, and perinatal arterial stroke
      Gene (codon)Rare Homozygote or Heterozygote Versus Common HomozygoteRare Homozygote Versus Common Homozygote
      Inflammatory
       TNF-α (−308)OR, 0.7 (95% CI, 0.2-2.7)NA
       IL-6 (−174)OR, 1.5 (95% CI, 0.4-4.9)NA
       Lymphotoxin (804)OR, 1.1 (95% CI, 0.3-3.6)OR, 0.9 (95% CI, 0.1-8.3)
      Thrombotic
       Factor V Leiden (506)NANA
       MTHFR (677)OR, 2.0 (95% CI, 0.6-6.8)OR, 4.9 (95% CI, 0.7-35.5)
       MTHFR (1298)OR, 0.4 (95% CI, 0.1-1.3)OR, 0.4 (95% CI, 0.04-3.5)
       Prothrombin (20,210)NANA
      Abbreviations:
      CI = Confidence interval
      IL = Interleukin
      MTHFR = Methyltetrahydrofolate reductase
      NA = Not applicable, i.e., the rare genotype was not present in any patients with perinatal arterial ischemic stroke; therefore, odds ratios could not be calculated
      OR = Odds ratio
      TNF = Tumor necrosis factor
      Infants with perinatal arterial ischemic strokes were more likely than control infants to manifest one or more apolipoprotein E ε4 alleles (54% vs 25%, respectively; P = 0.03). More patients with perinatal arterial ischemic stroke carried two ε4 alleles than did control subjects (15% vs 2%, respectively; P = 0.09), but this difference did not achieve statistical significance (Table 5). Case infants were significantly less likely than control infants to demonstrate at least one ε3 allele (69% vs 94%, respectively; P = 0.02). The allelic frequencies for apolipoprotein E in both groups are presented in Table 6. Compared with control children, case children demonstrated a significantly lower overall allelic frequency of ε3 (54% vs 80%, respectively; P = 0.006) and a significantly higher allelic frequency of ε4 (35% vs 14%, respectively; P = 0.02).
      Table 5Apolipoprotein E allele frequencies in infants with perinatal arterial ischemic stroke and control subjects
      Percentage of Cases (n = 13)Percentage of Control Subjects (n = 84)
      Apolipoprotein E genotypes were unavailable for two control infants.
      P Value
      Number of ε2 alleles
       077870.39
       123130.39
       2001.00
      Number of ε3 alleles
       03160.02
       131291.00
       239660.07
      Number of ε4 alleles
       046750.03
       139230.3
       21520.09
      Apolipoprotein E genotypes were unavailable for two control infants.
      Table 6Apolipoprotein E allele frequencies in infants with perinatal arterial ischemic stroke and control subjects
      Percentage of Cases (n = 13)Percentage of Control Subjects (n = 84
      Apolipoprotein E genotypes were unavailable for two control infants.
      )
      P Value
      ε435140.02
      ε354800.006
      ε21160.39
      Apolipoprotein E genotypes were unavailable for two control infants.

      Discussion

      In this exploratory study, the apolipoprotein E ε4 allele was associated with an increased risk of perinatal arterial ischemic stroke. Although the apolipoprotein E ε4 allele has been linked to cerebral palsy [
      • Kuroda M.M.
      • Weck M.E.
      • Sarwark J.F.
      • Hamidullah A.
      • Wainwright M.S.
      Association of apolipoprotein E genotype and cerebral palsy in children.
      ,
      • de Meirelles Kalil Pessoa B.
      • Rodrigues C.J.
      • de Barros T.E.
      • Bevilacqua R.G.
      Presence of apolipoprotein E epsilon4 allele in cerebral palsy.
      ] and to adult stroke [
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ], this study is the first, to our knowledge, to explore the relationship between apolipoprotein E and perinatal arterial ischemic stroke. Perinatal arterial ischemic stroke is a relatively common cause of hemiplegic cerebral palsy [
      • Wu Y.W.
      • Lindan C.E.
      • Henning L.H.
      • et al.
      Neuroimaging abnormalities in infants with congenital hemiparesis.
      ]. Therefore, a relationship between the apolipoprotein E ε4 allele and perinatal arterial ischemic stroke could explain the previously reported increase in rates of cerebral palsy among infants who carry this genetic variant.
      Apolipoprotein E is a gene involved in lipid transport and metabolism, and is highly expressed in the central nervous system [
      • Sudlow C.
      • Martinez Gonzalez N.A.
      • Kim J.
      • Clark C.
      Does apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17,965 controls.
      ,
      • Balcerzyk A.
      • Zak I.
      • Niemiec P.
      • et al.
      ApoE gene epsilon polymorphism does not determine predisposition to ischemic stroke in children.
      ]. Apolipoprotein E is secreted by astrocytes into the extracellular space, where it binds cholesterol. Neurons then take up apolipoprotein E so that cholesterol can be incorporated into cell membrane structures and myelin. These processes are critical in neurodevelopment and in neuronal repair after central nervous system injury [
      • Braga L.W.
      • Borigato E.V.
      • Speck-Martins C.E.
      • et al.
      Apolipoprotein E genotype and cerebral palsy.
      ,
      • Xu Q.
      • Bernardo A.
      • Walker D.
      • Kanegawa T.
      • Mahley R.W.
      • Huang Y.
      Profile and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with targeting of green fluorescent protein gene to the ApoE locus.
      ,
      • Lanterna L.A.
      • Biroli F.
      Significance of apolipoprotein E in subarachnoid hemorrhage: Neuronal injury, repair, and therapeutic perspectives—A review.
      ,
      • Wright R.O.
      • Hu H.
      • Silverman E.K.
      • et al.
      Apolipoprotein E genotype predicts 24-month Bayley scales infant development score.
      ]. Apolipoprotein E may also play a role in regulating central nervous system inflammation [
      • Braga L.W.
      • Borigato E.V.
      • Speck-Martins C.E.
      • et al.
      Apolipoprotein E genotype and cerebral palsy.
      ,
      • Laskowitz D.T.
      • Matthew W.D.
      • Bennett E.R.
      • et al.
      Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production.
      ].
      Apolipoprotein E contains three alleles (ε2, ε3, and ε4), yielding six possible genotypes. In most white populations, the ε3 allele is most common, appearing on more than 75% of chromosomes, making ε3ε3 the most common genotype [
      • Sudlow C.
      • Martinez Gonzalez N.A.
      • Kim J.
      • Clark C.
      Does apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17,965 controls.
      ,
      • Farrer L.A.
      • Cupples L.A.
      • Haines J.L.
      • et al.
      Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.
      ,
      • Zannis V.I.
      • Kardassis D.
      • Zanni E.E.
      Genetic mutations affecting human lipoproteins, their receptors, and their enzymes.
      ]. The ε2 and ε4 alleles demonstrate frequencies of 8% and 15%, respectively [
      • Farrer L.A.
      • Cupples L.A.
      • Haines J.L.
      • et al.
      Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.
      ,
      • Zannis V.I.
      • Kardassis D.
      • Zanni E.E.
      Genetic mutations affecting human lipoproteins, their receptors, and their enzymes.
      ,
      • Mahley R.W.
      • Rall Jr., S.C.
      Is epsilon4 the ancestral human ApoE allele?.
      ], in white populations, which is similar to the 6% and 14% frequencies of ε2 and ε4 alleles, respectively, in our control population. The ε4 form binds preferentially to triglyceride-rich lipoproteins such as very low density lipoproteins, whereas the other isoforms exhibit a greater affinity for high-density lipoproteins [
      • Mahley R.W.
      • Rall Jr., S.C.
      Is epsilon4 the ancestral human ApoE allele?.
      ,
      • Zetterberg H.
      • Palmer M.
      • Ricksten A.
      • et al.
      Influence of the apolipoprotein E epsilon4 allele on human embryonic development.
      ].
      In adults, the ε4 isoform is strongly associated with sporadic Alzheimer disease, cognitive decline, and atherosclerotic cardiovascular disease [
      • Farrer L.A.
      • Cupples L.A.
      • Haines J.L.
      • et al.
      Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.
      ,
      • Davignon J.
      • Bouthillier D.
      • Nestruck A.C.
      • Sing C.F.
      Apolipoprotein E polymorphism and atherosclerosis: Insight from a study in octogenarians.
      ,
      • Davignon J.
      • Gregg R.E.
      • Sing C.F.
      Apolipoprotein E polymorphism and atherosclerosis.
      ], whereas the ε2 allele is typically viewed as protective. Ischemic stroke risk is increased in adults with an ε4 allele, with odds ratios ranging from 1.1-2.5 [
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ,
      • Sudlow C.
      • Martinez Gonzalez N.A.
      • Kim J.
      • Clark C.
      Does apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17,965 controls.
      ,
      • McCarron M.O.
      • Delong D.
      • Alberts M.J.
      ApoE genotype as a risk factor for ischemic cerebrovascular disease: A meta-analysis.
      ]. In addition, neurologic outcomes after subarachnoid hemorrhage, traumatic brain injury, and intracerebral hemorrhage are worse in adults with an ε4 allele [
      • Lanterna L.A.
      • Biroli F.
      Significance of apolipoprotein E in subarachnoid hemorrhage: Neuronal injury, repair, and therapeutic perspectives—A review.
      ,
      • Teasdale G.M.
      • Nicoll J.A.
      • Murray G.
      • Fiddes M.
      Association of apolipoprotein E polymorphism with outcome after head injury.
      ,
      • Friedman G.
      • Froom P.
      • Sazbon L.
      • et al.
      Apolipoprotein E-epsilon4 genotype predicts a poor outcome in survivors of traumatic brain injury.
      ,
      • Martinez-Gonzalez N.A.
      • Sudlow C.L.
      Effects of apolipoprotein E genotype on outcome after ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage.
      ,
      • Gallek M.J.
      • Conley Y.P.
      • Sherwood P.R.
      • Horowitz M.B.
      • Kassam A.
      • Alexander S.A.
      ApoE genotype and functional outcome following aneurysmal subarachnoid hemorrhage.
      ].
      The effect of the apolipoprotein E ε4 allele may be different in neonates compared with adults. The presence of an ε4 allele in the fetus appears to protect against spontaneous miscarriage (odds ratio, 0.3; 95% confidence interval, 0.1-0.8) [
      • Zetterberg H.
      • Palmer M.
      • Ricksten A.
      • et al.
      Influence of the apolipoprotein E epsilon4 allele on human embryonic development.
      ]. Healthy Scottish newborns are more likely to manifest an ε4 allele than are stillborn infants (odds ratio, 1.6; 95% confidence interval, 1.1-2.3), although the ε4 allele did not protect against postnatal perinatal death [
      • Becher J.C.
      • Keeling J.W.
      • McIntosh N.
      • Wyatt B.
      • Bell J.
      The distribution of apolipoprotein E alleles in Scottish perinatal deaths.
      ]. Despite these possible benefits, the ε4 allele may confer increased risk for other perinatal complications. For instance, the ε4 allele has been associated with cerebral palsy in most [
      • Xin X.Y.
      • Song Y.Y.
      • Ma J.F.
      • et al.
      Gene polymorphisms and risk of adult early-onset ischemic stroke: A meta-analysis.
      ,
      • Kuroda M.M.
      • Weck M.E.
      • Sarwark J.F.
      • Hamidullah A.
      • Wainwright M.S.
      Association of apolipoprotein E genotype and cerebral palsy in children.
      ,
      • de Meirelles Kalil Pessoa B.
      • Rodrigues C.J.
      • de Barros T.E.
      • Bevilacqua R.G.
      Presence of apolipoprotein E epsilon4 allele in cerebral palsy.
      ,
      • McCarron M.O.
      • Delong D.
      • Alberts M.J.
      ApoE genotype as a risk factor for ischemic cerebrovascular disease: A meta-analysis.
      ,
      • Wang B.
      • Zhao H.
      • Zhou L.
      • et al.
      Association of genetic variation in apolipoprotein E and low density lipoprotein receptor with ischemic stroke in Northern Han Chinese.
      ,
      • Wu D.
      • Zou Y.F.
      • Xu X.Y.
      • et al.
      The association of genetic polymorphisms with cerebral palsy: A meta-analysis.
      ], but not all [
      • Sudlow C.
      • Martinez Gonzalez N.A.
      • Kim J.
      • Clark C.
      Does apolipoprotein E genotype influence the risk of ischemic stroke, intracerebral hemorrhage, or subarachnoid hemorrhage? Systematic review and meta-analyses of 31 studies among 5961 cases and 17,965 controls.
      ,
      • McMichael G.L.
      • Gibson C.S.
      • Goldwater P.N.
      • et al.
      Association between apolipoprotein E genotype and cerebral palsy is not confirmed in a Caucasian population.
      ], studies. In addition, the maternal presence of ε4 is associated with recurrent pregnancy loss [
      • Wu D.
      • Zou Y.F.
      • Xu X.Y.
      • et al.
      The association of genetic polymorphisms with cerebral palsy: A meta-analysis.
      ,
      • Goodman C.
      • Coulam C.
      • Jeyendran R.S.
      Association of apolipoprotein E polymorphisms and recurrent pregnancy loss.
      ,
      • Bianca S.
      • Barrano B.
      • Cutuli N.
      • et al.
      No association between apolipoprotein E polymorphisms and recurrent pregnancy loss.
      ,
      • Agarwal M.
      • Parveen F.
      • Faridi R.M.
      • Phadke S.R.
      • Das V.
      • Agrawal S.
      Recurrent pregnancy loss and apolipoprotein E gene polymorphisms: A case-control study from north India.
      ,
      • Zhang H.L.
      • Mao X.J.
      • Yang Y.
      • Wu J.
      Recurrent pregnancy loss and apolipoprotein E gene polymorphisms.
      ,
      • Zhang H.L.
      • Wu J.
      Apolipoprotein E4 allele and recurrent pregnancy loss: Is it time to draw a conclusion?.
      ].
      Apolipoprotein E is expressed by fetal genes in the placenta, where it is thought to play an active role in the metabolism of maternal lipoproteins [
      • Francoual J.
      • Audibert F.
      • Claise C.
      • et al.
      Implication of apolipoprotein E and the L-arginine-nitric oxide system in preeclampsia.
      ,
      • Descamps O.S.
      • Bruniaux M.
      • Guilmot P.F.
      • Tonglet R.
      • Heller F.R.
      Lipoprotein metabolism of pregnant women is associated with both their genetic polymorphisms and those of their newborn children.
      ]. Some cases of perinatal arterial ischemic stroke have been hypothesized to result from an embolic clot originating in the placenta [
      • Ghidini A.
      • Locatelli A.
      Diffuse placental chorioangiomatosis causing multiple fetal cerebral embolism: A case report.
      ,
      • Massachusetts General Hospital
      Case records of the Massachusetts General Hospital: Weekly clinicopathological exercises. Case 15—1997: Respiratory distress and seizure in a neonate.
      ,
      • Barmada M.A.
      • Moossy J.
      • Shuman R.M.
      Cerebral infarcts with arterial occlusion in neonates.
      ,
      • Kraus F.T.
      • Acheen V.I.
      Fetal thrombotic vasculopathy in the placenta: Cerebral thrombi and infarcts, coagulopathies, and cerebral palsy.
      ]. The ε4 allele demonstrates a greater binding affinity for low-density lipoproteins compared with the other isoforms [
      • Mahley R.W.
      • Rall Jr., S.C.
      Is epsilon4 the ancestral human ApoE allele?.
      ]. Whether placental clots are more likely to arise in the presence of an apolipoprotein E ε4 allele because of altered placental lipoprotein metabolism remains unknown.
      We did not detect an association between the apolipoprotein E ε2 allele and perinatal arterial ischemic stroke. However, in one study, the ε2 allele was associated with cerebral palsy [
      • Kuroda M.M.
      • Weck M.E.
      • Sarwark J.F.
      • Hamidullah A.
      • Wainwright M.S.
      Association of apolipoprotein E genotype and cerebral palsy in children.
      ], and has also been associated with worse behavioral outcomes in young children [
      • Gaynor J.W.
      • Nord A.S.
      • Wernovsky G.
      • et al.
      Apolipoprotein E genotype modifies the risk of behavior problems after infant cardiac surgery.
      ]. More research is required to determine whether the ε2 allele presents implications for early-life neurologic outcomes.
      Previous studies suggested that genetic thrombophilia may increase the risk of perinatal arterial ischemic stroke [
      • Mercuri E.
      • Cowan F.
      • Gupte G.
      • et al.
      Prothrombotic disorders and abnormal neurodevelopmental outcome in infants with neonatal cerebral infarction.
      ,
      • Hagstrom J.N.
      • Walter J.
      • Bluebond-Langner R.
      • Amatniek J.C.
      • Manno C.S.
      • High K.A.
      Prevalence of the factor V Leiden mutation in children and neonates with thromboembolic disease.
      ,
      • Kenet G.
      • Lutkhoff L.K.
      • Albisetti M.
      • et al.
      Impact of thrombophilia on risk of arterial ischemic stroke or cerebral sinovenous thrombosis in neonates and children: A systematic review and meta-analysis of observational studies.
      ], although not all studies have supported this notion [
      • Miller S.P.
      • Wu Y.W.
      • Lee J.
      • et al.
      Candidate gene polymorphisms do not differ between newborns with stroke and normal controls.
      ,
      • Laugesaar R.
      • Kahre T.
      • Kolk A.
      • Uustalu U.
      • Kool P.
      • Talvik T.
      Factor V Leiden and prothrombin 20210G>A [corrected] mutation and paediatric ischaemic stroke: A case-control study and two meta-analyses.
      ]. Given our small sample size, we were likely underpowered to detect anything other than large-magnitude associations. Therefore, given the potential for type 2 error, we are unable to exclude an association between perinatal arterial ischemic stroke and the other single-nucleotide polymorphisms that we studied. We included the single-nucleotide polymorphism genotype frequencies from our cases in Table 3 so that they can be included in future meta-analyses with greater power. Given that ours was a small exploratory study, we elected not to correct for multiple comparisons.

      Conclusion

      In our cohort, the apolipoprotein E ε4 allele occurred more frequently in those with perinatal arterial ischemic stroke vs control subjects. Other polymorphisms in our study were not associated with perinatal arterial ischemic stroke, although our study possessed limited power. More large, population-based studies are required to investigate more fully the potential association between apolipoprotein E ε4 and perinatal arterial ischemic stroke.
      The authors thank Judy Grether, Martin Kharrazi, and Steven Graham for assistance with bloodspot retrieval. The authors also gratefully acknowledge Andrew Van de Werf, who performed the Taqman typing, and Heather Fullerton, MD, MAS for helpful comments on an earlier version of the manuscript. This study was funded by National Institutes of Health grant K02 NS46688 , by the United Cerebral Palsy Foundation, and by the Cerebral Palsy Institute .

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