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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. Controls included 86 randomly selected white infants. We genotyped polymorphisms in 9 genes involved in inflammation, thrombosis or lipid metabolism that have been previously linked with stroke, and compared genotype frequencies in case and control individuals. We tested the following polymorphisms: TNF-α-308, IL-6, lymphotoxin A, factor V Leiden, MTHFR 1298 and 667, prothrombin 20210, and apolipoprotein E ε2 and ε4 alleles. Patients with perinatal arterial ischemic stroke were more likely than controls to have 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 controls (15% vs. 2%, p=0.09), though this finding was not statistically significant. Proinflammatory and prothrombotic polymorphisms were not associated with perinatal arterial ischemic stroke in this small study. The apolipoprotein E polymorphism may confer genetic susceptibility for perinatal arterial ischemic stroke. Larger population-based studies are needed to confirm this finding.
Perinatal arterial ischemic stroke is a well recognized cause of cerebral palsy, epilepsy, and behavioral abnormalities in children. By definition, perinatal arterial ischemic stroke occurs either in utero or before twenty-eight days of life, although infants may present in a delayed fashion later in infancy[1, 2]. The incidence of perinatal arterial ischemic stroke is 20 per 100,000 live births, 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)[1, 3]. Previously identified risk factors for perinatal arterial ischemic stroke include primiparity, infertility, preeclampsia, prolonged rupture of membranes, emergency Cesarean delivery, chorioamnionitis, neonatal prothrombotic disorders and meningitis or other intracranial infections[3, 4]. However, the cause of perinatal arterial ischemic stroke is unknown in most cases.
Genetic susceptibility may play a role in the pathogenesis of perinatal stroke. For instance, studies have suggested that Factor V Leiden and MTHFR mutations may be associated with increased risk of perinatal stroke[6–8]. 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 find any differences in patients with perinatal arterial ischemic stroke compared to controls.
Studies of ischemic stroke in adults have revealed several single nucleotide polymorphisms (SNPs) that modify an individual’s risk of stroke[9–12]. Whether these polymorphisms are associated with risk of perinatal stroke is unknown. The objective of this case-control study was to explore whether genetic polymorphisms known to be associated with ischemic stroke in adults are also associated with risk of arterial ischemic stroke in newborn infants.
We performed a case-control study nested within the cohort of all 199,176 infants born from January 1, 1997, to December 31, 2002, in Kaiser Permanents of Northern California (KPNC). KPNC is a large integrated healthcare delivery system that provides care for approximately 30% of the population in northern California. The members of KPNC are demographically similar to the California population, except that the very poor and very wealthy are underrepresented. All demographic data were obtained from medical record review. The study procedures were approved by the institutional review boards at KPNC, the University of California, San Francisco, Utah State University and the California Committee for the Protection of Human Subjects.
The methodology used to identify the infants with perinatal arterial ischemic stroke has been described previously. Perinatal arterial ischemic stroke was defined as stroke that occurred in utero or up to twenty-eight days after birth. To identify infants with perinatal arterial ischemic stroke, we searched all brain magnetic resonance imaging (MRI) and computed tomographic (CT) reports of infants within the KPNC birth cohort for key words indicating possible perinatal arterial ischemic stroke . The 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 first twenty-eight days of life) and presumed perinatal stroke (neurologic presentation after one month of age with imaging showing an old arterial-ischemic infarction) were included.
Given the heterogeneity in SNP frequencies among different ethnic groups[14, 15], genetic association studies typically restrict analyses to one ethnic group[16–18], or stratify by ethnicity. We restricted our study to non-Hispanic white infants, since the small size of our study precluded meaningful analyses of other ethnic groups. Of the 37 infants identified with perinatal arterial ischemic stroke, the 13 white infants constituted the cases of this study. In a previous study, we randomly selected 165 healthy control infants born during the years 1991–2002 at Kaiser Permanente. 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 current study.
Our methods of blood sample collection and genomic DNA extraction have been described previously. We retrieved stored neonatal blood specimens from the newborn screening specimen archives maintained by the California Department of Public Health. Dried blood spots have been stored on all infants born in California since 1980. Newborn blood specimens are collected on Guthrie card filter paper and dry at room temperature prior to submission for routine genetic and metabolic screening. Upon completion of the screening tests, remaining blood samples are stored at −15°C in a single refrigerated warehouse.
Blood spot 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 into a 96-well plate and incubated at 56° C for one hour in Qiagen buffer and Proteinase K enzyme. Genomic DNA was isolated from the blood spot punches using QIAamp 96 DNA blood kits supplied by Qiagen. The procedure for multiple displacement amplification (MDA) using Phi 29 polymerase was performed at 30°C for 16 hours using RepliPHI™ Phi 29 Reagent Sets (Epicentre®Technologies, Madison, Wisconsin) 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 there was an average of 235ng of genomic DNA from two 3.2mm punches.
Standard Taqman PCR reactions were performed using an Applied Biosystems 7500 Fast system AB 96-well optical plates (plates P/N 4366932). The reactions were designed according to the Applied Biosystems SNP assay protocol in 10μL volumes. Each reaction was done in a single well due to limiting amounts of genomic DNA. Results from all experiments were obtained from Applied Biosystems SDS software v2.0 and Copy Caller software v1.0. All genotyping was performed blind to case status and clinical history. We genotyped the following polymorphisms because they have been previously associated with ischemic stroke in adults: tumor necrosis factor-α (TNF-α) −308 G/A (rs1800629)[11, 12]; IL-6 −174 G/C (rs1800795)[18, 21, 22]; lymphotoxin C804A (rs1041981); factor V Leiden (FVL) 506 G/A (rs6025)[23, 24]; methyltetrahydrofolate reductase (MTHFR) 1298 A/C (rs1801131) and 667 C/T (rs1801133)[10, 25]; prothrombin 20210 G/A (rs1799963)[23, 24]; and apolipoprotein E (apoE) ε2 and ε4 alleles (rs429358 and rs7412).
We defined SNP genotypes as follows: common homozygote = two copies of the common allele; heterozygote = one copy of each allele; and rare homozygote = two copies of the rare allele. With logistic regression, we determined odds ratios (OR) and 95% confidence intervals (CI) using two genetic models: 1) rare homozygote vs. common homozygote and 2) heterozygote or rare homozygote vs. common homozygote. For the apolipoprotein E analysis, to be in keeping with other literature, we compared allelic frequency of the three apolipoprotein E alleles (ε2, ε3, and ε4) in cases versus controls. We performed chi-squared analyses, Fisher’s exact tests and Student’s 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, multivariable analysis was not feasible.
Birth weight, infant sex and maternal age did not differ between the cases and controls (Table 1). Preeclampsia and chorioamnionitis rates also did not differ. However, infants with perinatal arterial ischemic stroke were slightly older (mean 40.1 vs. 39.3 weeks, p=0.03) and more likely than control infants to be born by Cesarean section (46% vs. 19%, p=0.03). There was a trend for more primigravida mothers among case infants (75% vs. 46%, p=0.07). Neonatal seizures were present in five of the cases and none of the controls.
All SNP genotype distributions within the control population were in Hardy-Weinberg equilibrium (Table 2). The SNP genotype distributions for the cases are shown in Table 3. None of the proinflammatory or prothrombotic polymorphisms tested were significantly different between the two groups (Table 4).
Infants with perinatal arterial ischemic stroke were more likely than control infants to possess one or more apoE ε4 allele (54% vs. 25%, p=0.03). More patients with perinatal arterial ischemic stroke carried two ε4 alleles than did controls (15% vs. 2%, p=0.09), but this difference did not reach statistical significance (Table 5). Case infants were significantly less likely than control infants to have at least one ε3 allele (69% vs. 94%, p=0.02). The allelic frequencies for apolipoprotein E in both groups are shown in Table 6. Compared to control children, case children had significantly lower overall allelic frequency of ε3 (54% vs. 80%, p=0.006) and significantly higher allelic frequency of ε4 (35% vs. 14%, p=0.02).
In this exploratory study, the apolipoprotein E ε4 allele was associated with increased risk of perinatal arterial ischemic stroke. Although the apoE ε4 allele has been linked to cerebral palsy[26, 27] and to adult stroke, this is the first study to our knowledge to explore the relationship between apoE and perinatal arterial ischemic stroke. perinatal arterial ischemic stroke is a relatively common cause of hemiplegic cerebral palsy. Therefore, a relationship between the apoE ε4 allele and perinatal arterial ischemic stroke could explain the previously reported increase in cerebral palsy rates 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[29, 30]. ApoE is secreted by astrocytes into the extracellular space, where it binds cholesterol. Neurons then take up the apoE so that cholesterol can be incorporated into cell membrane structures and myelin. These processes are critical in neurodevelopment as well as in neuronal repair after central nervous system injury[31–34]. ApoE may also play a role in regulating central nervous system inflammation[31, 35].
ApoE has three alleles, ε2, ε3 and ε4, yielding six possible genotypes. In most Caucasian populations the ε3 allele is most common, appearing on more than 75% of chromosomes, making ε3ε3 the most common genotype[29, 36, 37]. The ε2 and ε4 alleles have frequencies of 8% and 15% respectively[36–38] in white populations, which is similar to the 6% and 14% allelic frequencies found in our control population. The ε4 form binds preferentially to triglyceride rich lipoproteins such as very low-density lipoproteins, while the other isoforms have a higher affinity for high-density lipoproteins[38, 39].
In adults, the ε4 isoform is strongly associated with sporadic Alzheimer disease, cognitive decline and atherosclerotic cardiovascular disease[36, 40, 41], while 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 to 2.5[10, 29, 42]. In addition, neurologic outcomes after subarachnoid hemorrhage, traumatic brain injury, and intra-cerebral hemorrhage are worse in adults who have an ε4 allele[33, 43–46].
The effect of the apoE ε4 allele may be different in neonates compared to adults. The presence of an ε4 allele in the fetus appears to protect against spontaneous miscarriage (OR 0.3, 95% CI 0.1–0.8). Healthy Scottish newborns are more likely to have an ε4 allele than stillborn infants (OR 1.6, 95% CI 1.1–2.3), though the ε4 allele did not protect against post-natal perinatal death. 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[10, 26, 27, 42, 48, 49], but not all studies[29, 50]. In addition, maternal carriage of ε4 is associated with recurrent pregnancy loss[49, 51–55].
ApoE is expressed by fetal genes in the placenta, where it is thought to play an active role in the metabolism of maternal lipoproteins[56, 57]. It has been hypothesized that some cases of perinatal arterial ischemic stroke may result from an embolic clot originating from the placenta[58–61]. The ε4 allele has a higher binding affinity for low density lipoproteins compared to the other isoforms. Whether placental clots may be more likely to arise in the presence of an apoE ε4 allele because of altered placental lipoprotein metabolism is unknown.
We did not find an association between the apoE ε2 allele and perinatal arterial ischemic stroke. However, in one study the e2 allele was associated with cerebral palsy, and it has also been associated with worse behavioral outcomes in young children. More research is needed to determine if the e2 allele has implications for early-life neurologic outcomes.
Previous studies have suggested that genetic thrombophilias may increase the risk of perinatal arterial ischemic stroke[6, 7, 24], though not all studies have supported this notion[5, 63]. Given our small sample size, it is likely we were underpowered to detect anything other than large-magnitude associations. Therefore, given potential Type 2 error, we are unable to rule out an association between perinatal arterial ischemic stroke and the other SNP’s that we studied. We included the SNP genotype frequencies from our cases in Table 3 so that they can be included in future meta-analysis studies that might have higher power. Given this was a small exploratory study, we elected not to correct for multiple comparisons.
In our cohort, the apoE ε4 allele was more frequent in those with perinatal arterial ischemic stroke versus controls. Other polymorphisms in our study were not associated with perinatal arterial ischemic stroke, though the study had limited power. More large population based studies are needed to further investigate the potential association between ApoE ε4 and perinatal arterial ischemic stroke.
The authors would like to thank Judy Grether, Martin Kharrazi and Steven Graham for their assistance with blood spot retrieval. We also gratefully acknowledge Andrew Van de Werf, who performed the Taqman typing. We would like to thank Dr. Heather Fullerton for her helpful comments on an earlier version of the manuscript.
Financial disclosure: This study was funded by NIH K02 NS46688, the United Cerebral Palsy Foundation, and by the Cerebral Palsy Institute
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