Associations between Apolipoprotein E(APOE) polymorphisms and Cerebral Palsy: a meta-analysis

Background Apolipoprotein E (APOE ) is one of the main apolipoproteins playing important roles in the central neuronal system. The relationship between polymorphisms and cerebral palsy (CP) is contradictory. Methods The overall experimental studies of APOE polymorphisms and CP risk were researched. We conducted eligible studies identified from Elsevier Science Direct, PubMed, Springer Link, WEB OF SCIENCE, Chinese National Knowledge Infrastructure and WanFang Data up to February 2019 to make a systematic review. Results Totally 10 eligible studies were used to meta-analysis (1570 CP cases and 1982 subjects). Significant associations with CP were observed for APOE polymorphisms in allele (ε4: P < 0.001, OR 2.05, 95% CI 1.40 to 2.99; ε2: P = 0.04, OR 1.41, 95% CI 1.01 to 1.96) and dominant (E4 carriers: P = 0.004, OR 1.90, 95% CI 1.23 to 2.92) models in overall analyses. Interestingly, subgroup analysis indicated a significant increased risk for CP in Chinese patients of CP with APOE ε4 (P<0.00001, OR 3.71, 95% CI 2.37 to 5.78) and E4 carriers(P<0.00001, OR 3.95, 95% CI 2.38 to 6.53), but not with APOE ε2 (P=0.69, OR 1.09, 95% CI 0.72 to 1.65). Conclusions Combined with the results of our analysis, we concluded that the risk of CP was significantly increased in ε4 individuals. But meta-analysis yielded an incongruent result for the APOE ε2 allele between in multi-ethnic samples and in Chinese subgroup. These conclusions should be confirmed through further studies.


Introduction
Cerebral palsy (CP) is a group of motor and posture development disorders caused by non-progressive injuries of developing fetuses or infants, resulting in a limited activity. CP is a severe disability in children, with the fact that 40 percent of children unable to walk independently, 1/3 have epilepsy, up to 1/3 are non-verbal and about 1/2 have some degree of cognitive impairment. [1][2][3][4][5][6] In recent years, evidence from several high-income countries (United States, Australia, Europe, Canada, Sweden, and Japan) have shown that the prevalence of CP has decreased (mainly in low birth weight and premature infants), but still remains at 2‰ ~ 3‰. 6 The epidemiological survey results of more than 320,000 children aged 1-6 in 12 provinces and autonomous regions in China in 2013 showed that the prevalence of CP was 2.46‰, which was consistent with the international average. 7 In the United States, children with CP are estimated to spend less than $1 million per person on health care, educational needs, social services, and lost economic opportunities. 8 The prevalence, severity, as well as the burden of CP is becoming an important public-health problem threatening children's health.
The pathogenesis of CP is multifactorial and various. The causes are premature, inflammatory, anoxic, traumatic, metabolic and genetic. Previous studies on the pathogenesis of CP have focused on the clinical etiology. In recent years, both domestic and foreign studies have found that genetic factors also involved in the etiology of CP, while apolipoprotein E (APOE) genotype as one of the most studied genetic risk factors.
Apolipoprotein E plays an important part in the distribution of lipids in peripheral tissues such as peripheral nerve, arterial wall, and brain. The human APOE gene produces three protein subtypes: APOE ε2 (112Cys/158Cys), APOE ε4 (112Arg/158Arg) and wild-type APOE ε3 (112Cys/158Arg), and six genotypes (E2/2, E2/3, E2/4, E3/3, E3/4, and E4/4), is found located on chromosome 19q13.2. 9,10 The gene polymorphism of APOE and its relationship with the risk of Parkinson disease has been assured. 11 Meanwhile, APOE ε4 allele has been reported relate to Alzheimer's disease, age-related cognitive decline and coronary heart disease (CHD). [12][13][14] The risk of CHD was increased by 42% in people with APOE ε4 allele than in people with the ε3 allele in the previous study. 15 Nowadays, whether there is a link within the APOE genotype and the risk of CP has been investigated. [16][17][18][19][20][21][22][23][24][25][26][27] However, the results in existence have been conflicted. Some studies showed that there was a highly significant association between ε2 20,22 or ε4 22,23 allele and the risk of CP, whereas others showed no association. 18,19,21 Due to the small number of samples, the complex genetic relationship may not be detected in individual studies. The purpose of this research is to comprehensively evaluate the possible relationship between APOE polymorphisms and the CP risk.

Search Strategy
We conducted a systematic study of the research articles published up to February 2019 and provided them through Elsevier Science Direct, PubMed, Springer Link, WEB OF SCIENCE, Chinese National Knowledge Infrastructure (CNKI, in Chinese) and WanFang Data (in Chinese). Two authors independently searched literature using the following keywords: (Apolipoprotein E OR APOE) AND (cerebral palsy OR CP) AND (gene OR polymorphism OR genotype OR variation OR allele). Some of the relevant literature in the review articles was reviewed to identify more additional publications. Studies that meet our eligibility standard were involved in the meta-analysis.

Inclusion Criteria
The inclusion standard provides sufficient raw data for: (a) they explained the association between APOE gene polymorphism and CP; (b) they offered enough original data of allele frequency or genotype distribution; and (c) when the same case and control subjects appeared in multiple articles, the study with the largest number of participants was included. Conference reports or summaries were not chosen.

Data extraction and Quality Assessment
Two authors (C-HY, Y-H) identified eligible articles independently in accordance with the inclusion criteria. The authors also looked up the following data independently: year of publication, first author's family name, population, study type, types of CP, gene genotyping methods, source of controls (hospital-based and population-based), APOE genotype and allele distribution. The Newcastle Ottawa Scale (NOS) was used to assess the quality of the studies in the meta-analysis. The genotype distribution reported in percentages were calculated to figures. Hardy-Weinberg equilibrium (HWE) in the control groups was evaluated by the chi-square test (p<0.05 was considered significant).
Extracted data were contrasted, if there were discrepancies, it would be resolved through discussion with the third author (Z-XW).

Meta-analysis methods and bias testing
Based on the allele and genotype frequency between the case and the control, the odd ratio (OR) was adopted to evaluate the intensity of the correlation between the APOE polymorphism and the CP susceptibility. We calculated ORs and 95% CIs to assess potential associations between APOE polymorphisms and CP in allele, dominant and recessive models based on genotypic distributions of investigated polymorphisms. The Chinese subgroup was then divided according to ethnicity. On the basis of the Q-test, We used χ 2 test to make an analysis of the heterogeneity, which was thought to be that there has statistical significance at P value <0.05. 28 In order to quantify heterogeneity, the I 2 value was calculated and clarified in the following: no heterogeneity, I 2 =0%; low heterogeneity, I 2 =25%, moderate heterogeneity, I 2 =50% and high heterogeneity, 29,30 The summary OR was derived by using the method of Mantel-Haenszel (MH) with the assumptions of a fixed-effects model as well as by using the DerSimonian and Laird method with the assumptions of a random-effects model. 31,32 And the value of OR was also evaluated using the Z test, and p value <0.05 was thought to be statistical significance.
The publication bias was evaluated by visual examination of Begg's funnel plots. An asymmetric funnel indicated a publication bias, and after that Egger's test analysis was perfomed. 33,34 We have also implemented the Duval and Tweedie nonparametric "trim and fill" process to moreover evaluate the possible impact of publication bias in our metaanalysis. 35 The whole statistical analysis were conducted in  Table 1. We used the NOS rating scale to assess the quality score of each study as shown in Table 1. The data for the frequencies of APOE alleles and genotypes in the individual studies are shown in Table 1S. The deviation from HWE in the control population was found in three study. 17,21,22 Overall analyses of the association between APOE polymorphisms and CP Susceptibility Firstly, the meta-analysis of the APOE alleles and the CP risk was analyzed. The overall 10 studies were used to evaluate the effect of APOE alleles on the CP risk. 16,17,[20][21][22][23][24][25][26][27] The comparison of the presence of ε2 vs. ε3 alleles within CP patients as well as the control group indicated heterogeneity between studies (p=0.01, χ 2 =21.37, I 2 =58%, Figure 2A). The random-effects model was adopted. The findings showed that the existence of ε2 allele conferred CP a risk (p=0.04, OR 1.41, 95% CI 1.01 to 1.96, Figure   2A). What is more, the presence of ε4 vs. ε3 alleles between CP cases and control groups was estimated. Because of the heterogeneity within the studies (p<0.00001, χ 2 =41.01, I 2 =78%, Figure 3A), the random-effects model was used. The meta-analysis showed that there was a significant positive correlation between ε4 allele and CP risk (p<0.001, OR 2.05, 95% CI 1.40 to 2.99, Figure 3A). Moreover, the pooled data supported the result that the E4 carriers showed significantly increased CP risk, contrasted with those with E3/3 genotype (p=0.004, OR 1.90, 95% CI 1.23 to 2.92, Figure 4A). The random-effect model was adopted due to heterogeneity in 10 studies (p<0.0001, χ 2 =34.68, I 2 =74%, Figure   4A). The results of dominant and recessive models for contrasts of E4, E3, and E2 genotypes were shown in Table 2. To further exclude heterogeneity, we removed studies with substantial departure from HWE among controls. This time fixed-effects model was then applied, because the heterogeneity was not significant among the pooled 7 studies (I 2 =44%, Figure 2B), 16,20,[23][24][25][26][27] and the meta-analysis showed that there was a significant positive correlation between ε2 allele and CP risk (p=0.001, OR 1.63, 95% CI 1.21 to 2.19, Figure 2B).

APOE Polymorphisms and CP Susceptibility in Chinese Subgroups
We also researched the subgroup of Chinese because we involved four Chinese studies that had never appeared in other meta-analyses. In this paper, four studies of the ε4 vs. ε3 alleles were carried out. 24-27 The summary of the data supported a significant increase in the CP risk in individuals with ε4 alleles contrasted with those with ε3 alleles (p<0.00001, OR 3.70, 95% CI 2.37 to 5.78, Figure 3C). Because there was no heterogeneity between studies (I 2 = 9%, Figure 3C), the fixed-effects model was then applied. We found that, comparison with those with ε4 alleles, individuals with ε2 alleles haven't a risk for CP development in the Chinese population (p=0.69, OR 1.09, 95% CI 0.72 to 1.65, Figure 2C). In addition, the summary data showed that those with E4 carriers had a high risk of developing CP compared with the individuals with E3/3 genotype (p<0.00001, OR 3.95, 95% CI 2.38 to 6.53, Figure 4C). Because there was no heterogeneity between studies (I 2 =13%, Figure 4C), the fixed-effects model was then used. What is more, Table 2 shows the results for comparing the dominant and recessive models of the E4, E3 and E2 genotypes.

Evaluation of Publication Bias
Firstly, Begg's funnel plots were used to evaluate the publication bias. Asymmetry and publication bias showed on funnel plots were evaluated by Egger's tests (  Figure S1A-D). Because of this, we used the trim and fill method for sensitivity analysis, which conservatively presupposes hypothetical negative unpublished studies to reflect the positive study that leads to the asymmetry of the funnel diagram. 35 The collected analysis incorporating the hypothetical studies continued to suggest both APOE ε4 and E4 carriers acts as a risk factor for CP ( Figure S1E-H).  Figure 3A and 3C). We found a highly significant association between E4 carriers and CP development risk as well, especially in the Chinese population too ( Figure 4A and 4C).
The APOE ε2 allele also appeared to be related to an increased risk of CP, but not appeared in the Chinese population (Figure 2A and 2C). However, in addition to E4 carriers, we found no significant associations between other APOE polymorphisms and the risk of CP development. The results of our study suggested that APOE ε4 is an important genetic risk factor for the development of CP.
Apolipoprotein E is one of the main apolipoproteins of the central neuronal system playing important roles in neurobiology. Between the APOE ԑ4 allele and CP, the existence of an association was defined in many studies. 16,22,[23][24][25][26][27] Disturbance in neurobehavioral functions and the brain healing process along with reduced ischemia tolerance have all been implicated with possessing the ApoE ԑ4 allele in a number of studies. 17,36 Interestingly, against poor prognosis and unfavorable clinical outcomes stemmed from ԑ4 allele, some studies suggest that having the ApoE ԑ3 allele renders a favorable response to traumatic and hypoxic injury in developing brain. 37 A meta-analysis of 2,000 adults aged 45-89 years found that APOE ε4 resulted in poor executive function in cognitive assessment. It is suggested that the efficiency of nerve cell repair is low in allele ԑ4 carriers. 38 Combined with the results of our analysis, we concluded that the risk of CP was significantly increased in ε4 individuals.
The relationship between APOE ε2 allele and CP is contradictory. BRAGA et al 20  show that this association is not an artifact of unpublished negative studies ( Figure S1).
However, this approach does not completely rule out this possibility. Fourth, although we detected an association of APOE genetic polymorphisms (ε2 vs. ε3 alleles; ε4 vs. ε3 alleles; E4 carriers vs. E3/3 genotypes) with CP, the result should be explained with caution because the number of participants was small.

Conclusions
In a word, the pooled data indicate a high correlation between APOE polymorphisms and CP. Contrasted with individuals carrying APOE ε3 allele, the risk of CP was significantly increased in individuals carrying ε4 allele. In addition, compared with individuals with APOE E3/3 genotype, E4 carriers have significantly increased risk suffering from CP.
Because of the fewer studies, further well-designed studies are still warranted to confirm whether APOE ε2 increase the susceptibility to CP. Additionally, the mechanism of apolipoprotein E involved in CP is not clear and needs to be further studied.

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download. Table S1.docx