In a study of non-Hispanic white infants born at or near term, we found an increase in the frequency of the apoE ε4 and iNOS -231 T alleles in children with CP that was of borderline significance. These associations had small effect sizes, and after adjusting for multiple comparisons in an attempt to avoid false positive findings, we found that these associations were no longer statistically significant.
Genotype-phenotype associations have been observed in many complex diseases; yet most findings have been difficult to replicate (26
). Cerebral palsy is a complex and heterogeneous condition, and it is not surprising that genetic association studies have produced inconsistent results. Exploratory studies evaluating a large panel of SNP’s often involve numerous statistical analyses stratified by CP subtype, gender, ethnicity and gestational age. Such studies also employ multiple control groups, and perform a variety of genetic comparisons. Thus, in the absence of statistical adjustment for multiple comparisons, exploratory studies testing between 93 and 720 hypotheses (4
) would be expected to yield about 5 to 30 genetic associations from chance alone.
Other factors that might contribute to the inconsistent results across studies include small sample size, population heterogeneity, differences in gene-environment interactions and publication bias (3
). It is also important to remember that CP is a heterogeneous disorder that results from numerous causal pathways leading to a variety of different types of brain injury, and that lumping all patients with CP into one group further limits our ability to discern meaningful genotype-phenotype associations. Our study is limited by the inclusion of only white infants born at or near term who have spastic or dyskinetic CP. Therefore, our study is not a direct replication of all previous reports, since several past studies have included preterm and non-white infants. Furthermore, we did not perform haplotype analyses or evaluate the presence of viral infections that might modify the relationship between genetic variants and CP (9
The weak statistical associations identified in our cohort did not persist after adjustment for multiple comparisons. Given the large number of hypotheses that we tested, it was important to perform statistical adjustment to avoid type 1 error. Although we conservatively used the Bonferroni test, any method of adjustment for multiple comparisons would have rendered our findings insignificant, given the P values of 0.04 for the uncorrected genetic associations. However, the weak associations found between the apoE ε4 and iNOS -231 T alleles and CP are similar to previous findings in other populations (6
), and therefore deserve further discussion.
The relationship between the apoE ε4 allele and CP is controversial. Two studies have found a modest increased risk of CP among children who carry at least one ε4 allele (15
), but a recent meta-analysis suggests that no significant association exists (3
). Apolipoprotein E is a lipid transport protein widely expressed in the brain. Carriage of at least one copy of the ε4 allele is associated with Alzheimer disease (30
) and with ischemic stroke in adults (31
). Children with the ε4 allele have been shown to exhibit worse neurobehavioral performance and higher birth complication rates (33
). We found that children with CP had a higher frequency of carrying an apoE ε4 allele, but further large-scale studies will be necessary to determine whether this trend reflects a biologically meaningful relationship.
Similar to previous studies, we found that children with CP were more likely to have a copy of the iNOS -231 T allele, though this finding was not statistically significant after adjustment for multiple comparisons. The iNOS gene was first evaluated in relation to CP because of its role in cardiovascular regulation and ischemic and inflammatory brain injury (2
). Over-expression of iNOS in the brain has been reported in newborn periventricular white matter injury (35
), as well as in adult stroke (36
). In a rat model of newborn brain injury caused by intrauterine infection, iNOS was found to be a key mediator of oligodendrocyte injury (34
). Studies comparing iNOS production in patients with and without CP may help us better understand whether genetic variation in the iNOS gene is related to CP risk.
The IL-6 -174 polymorphism has been linked with several adverse perinatal neurologic outcomes including CP (4
), periventricular white matter injury (37
) and reduced gray matter volume (38
). We reported in a single candidate gene study that the IL-6 -174 C allele was associated with a 2.5-fold elevated risk of CP (5
), suggesting that an altered fetal inflammatory response due to genetic variation in inflammatory genes could contribute to a higher risk of CP (4
). However, none of the other cytokine gene variants in the current study were significantly associated with CP.
Given the limited sample size, it is possible that our negative results reflect type 2 error due to lack of statistical power, rather than the absence of a true genetic association. Our power analyses suggest that we had adequate power to detect previously described effect sizes for 6 of the polymorphisms studied (s: eNOS, MBL 52, MTHFR 677, MTHFR 1298, and apoE E2 and E4 alleles), and thus our negative findings for these genes, excepting the apoE E4 allele, are relatively robust. Our study had only marginal or poor power to detect previously described associations in all other genetic polymorphisms. Therefore, these other negative findings should be interpreted with caution pending further evaluation in other populations.
Despite the growing number of studies evaluating genetic risk factors for CP, the contribution of genetic factors to CP is likely to be small. The risk of recurrent isolated CP following a first affected child was 0.5% in a US population (39
). Among children with CP not due to a brain malformation, genetic syndrome or neurometabolic disease, the recurrence risk is likely to be even smaller. Given the relatively common population occurrence of genetic polymorphisms, and the relatively rare occurrence of CP in term infants, it is unlikely that successful preventative measures will be developed based on genetic risk factors alone, unless we can identify strong gene-environment interactions that confer a significantly increased risk of CP, or we can identify combinations of genetic variants that together signify a particularly high risk of CP. Our data support the evidence that the contribution of genetic factors to CP is likely to be small. Very large-scale studies of genetic, environmental and obstetric factors will be needed before effective preventative strategies can be devised