The association between sex-related interleukin-6 gene polymorphisms and the risk for cerebral palsy

The study population consisted of 713 CP patients (219 girls (30.7%) and 494 boys (69.3%) with a mean age ± SD of 19.1 ± 14.9 months) chosen from centers for CP rehabilitation in the Third Affiliated Hospital of Zhengzhou University and Zhengzhou Children’s Hospital from 1 July 2010 to 31 May 2012 (Table 1). Of these, 87 CP patients (54 boys (62.1%) and 33 girls (37.9%) with a mean age ± SD of 19.9 ± 14.1 months) were selected for the serum IL-6 assay (Table 2). The 753 healthy control participants (262 girls (34.8%) and 491 boys (65.2%) with a mean age ± SD of 19.6 ± 18.5 months) were chosen from the Child Healthcare Departments at the same hospitals during the same period. Of these healthy controls, 77 (63 boys (81.8%) and 14 girls (18.2%) with a mean age ± SD of 19.8 ± 12.4 months) were selected for the serum IL-6 assay. Blood samples were collected into tubes containing EDTA by skilled nurses on the second day of being hospitalized. Plasma was separated by centrifugation (1,500 × g for 15 min) at room temperature within 2 hours after being collected. DNA was obtained from the remaining blood components from the same sample. All the prepared samples were immediately stored at −70°C. All subjects were Han Chinese from the Henan Province, and written informed consent to participate in this study was provided by the children’s parents. Child neurologists diagnosed and classified the CP by clinical examination or by using medical records, including brain imaging, according to the guidelines proposed by the “Surveillance of CP in Europe” network [14]. Children in either the CP or control group with myopathy or metabolic anomalies were excluded from the analysis. For the serum IL-6 assay, the age of the selected children ranged from 5 months to 36 months. Children with cough, fever, acute respiratory illness, or any other any indications of infection within the past 3 months were excluded. Because of the genetic factors and familial factors that are associated with CP, we ensured that the controls had no familial relationships with the patients and did not have neurological symptoms. Approval for the study was obtained from the ethics committee of Zhengzhou University and the Medical Academy of Henan Province (201201002) in accordance with the Declaration of Helsinki.

The database of medical records contains information on CP risk factors, such as birth asphyxia, symptoms concomitant with CP, such as mental retardation (MR), neonatal complications, such as PVL, and on hypoxic-ischemic encephalopathy (HIE). In addition, the records contain information on maternal factors such as premature rupture of membrane (PROM), pregnancy-induced hypertension (PIH), and threatening premature labor (TPL). Asphyxia was diagnosed using the criteria of the American Academy of Pediatrics and the American College of Obstetricians and Gynecologists, and newborns were included when at least three of the four criteria were met [15]. MR was identified based on a score of less than 70 on the Bayley Scales measurement of mental development index [16]. PVL was defined as parenchymal densities or lucencies around the ventricles during ultrasound examination of the head performed any time after 14 days post-birth [17], and this was further confirmed by MRI. Diagnosis of HIE required a combination of parameters that are indicative of metabolic acidosis within the first hours after birth. These include low umbilical cord blood pH (<7.0), a base deficit of over 12 mEq/L, and evidence of a need for respiratory support starting in the first minutes after birth with low Apgar scores at and beyond 5 min after birth. The diagnosis of PROM was based on pooling of amniotic fluid in the vagina, amniotic fluid ferning patterns, and a positive nitrazine test [18]. PIH was diagnosed and classified according to the criteria recommended by the American Congress of Obstetricians and Gynecologists, including a systolic blood pressure of 140 mmHg or higher or a diastolic blood pressure of 90 mmHg or higher on two occasions at least 6 h apart occurring after 20 weeks of gestation in a pregnant woman with previously normal blood pressure and without detectable urinary protein [19]. TPL was based on at least one of the following having occurred during pregnancy: the mother was admitted to the hospital prenatally with an episode of TPL, the mother received antenatal steroids or tocolytics, or a note was made on the delivery room chart that there was a history of TPL during the pregnancy [20].

A total of five SNPs (rs1800796, rs2069837, rs2066992, rs2069840, and rs10242595) of the IL-6 gene whose minor allele frequencies in the Chinese Han population are more than 0.1 were selected from the dbSNP database (http://​www.​ncbi.​nlm.​nih.​gov/​SNP) and the hapmap human SNP database (http://​www.​hapmap.​org). All of rs2069837, rs2066992 in the second intron, and rs2069840 in the third intron are tag SNPs. rs1800796 and rs10242595 are located in the upstream and downstream regions of the IL-6 gene, respectively.

Genomic DNA was prepared from venous blood using the AxyPrep Blood Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions. Probes and primers were designed by the SEQUENOM online tools (https://​www.​mysequenom.​com) and the sequences are available upon request. After the amplification of polymorphism-spanning fragments by multiplex polymerase chain reaction (PCR), the genotyping was performed with the Sequenom MassARRAY SNP genotyping platform (Sequenom, San Diego, CA, USA). The person who analyzed the genotype results was blinded to the clinical data.

For gene analysis, the Hardy-Weinberg equilibrium (HWE) test was performed on the allele and genotype frequency analysis using the SHEsis online software platform (http://​analysis.​bio-x.​cn). Linkage disequilibrium was measured using standardized D’, and the discrepancies in allele and genotype frequencies at single loci between patients and controls were compared using a Monte Carlo simulation strategy. The numbers of observations for each haplotype were compared using χ² tests. The relative risk was approximated by the estimate of the odds ratio (OR). All ORs were adjusted for age and sex using logistic regression models. For each OR, a 95% confidence interval (CI) was calculated. For IL-6 cytokine analysis, the Student’s unpaired t-test was used. Data groups with unequal variances were analyzed using the Mann-Whitney U-test. All reported P values were two-tailed and statistical significance was set at P <0.05. The Statistical Package for the Social Sciences (SPSS version 19.0) and Graphpad Prism 6.0 software package (version 6.0 for Windows, Graphpad, La Jolla, CA, USA) were used for all statistical analyses. For all comparisons, multiple testing on each individual SNP was corrected by the SNPSpD program (http://​gump.​qimr.​edu.​au/​general/​daleN/​matSpD/​), which is based on the linkage disequilibrium information.

The genotypic distribution of the five selected SNPs in the controls was in HWE. There were no differences in allele or genotype frequencies between the total population of CP patients (n = 713) and total population of controls (n = 753) for any of the five genetic polymorphisms (Table 3). Computation of D’ indicated a strong linkage disequilibrium between these SNP markers (D’ >0.8) (Table 4). A haplotype analysis of all five SNPs between the CP and control subjects was performed (those with a haplotype frequency <0.03 were excluded from the analysis), and three common haplotypes were built. No differences in the haplotypes were found between CP cases and controls (data not shown).

Subgroup analysis of SNPs was performed according to sex, gestational age, birth weight, birth asphyxia, subtypes of CP, pregnancy complications, and maternal factors. Significant differences in allele frequencies were observed between CP patients (n = 494) and controls (n = 491) in males for rs2069837 (OR = 1.334, 95% CI = 1.073–1.659, P = 0.009, P = 0.027 after SNPSpD correction) (Table 5), but there were no differences in these SNP allele/genotype frequencies between CP patients and controls in females (data not shown).

Spastic CP was the main type of CP seen in this study population. Differences in allele frequencies were observed between spastic CP patients (n = 456) and controls (n = 753) for rs2069837, but the difference disappeared after SNPSpD correction (data not shown). Further analysis according to the sex of the CP patients revealed significant differences in allele and genotype frequencies between male spastic CP patients (n = 325) and controls (n = 491) for rs1800796, rs2069837, and rs2066992, but the differences for rs1800796 and rs2066992 disappeared after SNPSpD correction (Table 6), which was similar to our previous analysis. Haplotype analysis for rs1800796, rs2069837, and rs2066992 showed that the haplotypes “CAT” and “GGG” (rs1800796, rs2069837, and rs2066992) were strongly associated with male spastic CP patients (haplotypes with a frequency <0.03 were excluded from the analysis) (Table 7).

PVL is common in preterm brain injury and is related to the development of CP. In this study, we found differences in genotype frequencies between CP patients with PVL (n = 56) and controls (n = 491) in males at rs1800796 and rs2066992 (Table 8). The haplotype analysis of all five analyzed SNPs did not show significant differences between CP patients with PVL and control subjects in either males or females.

According to gestational age at birth, the CP patients and controls were stratified as term or preterm CP cases. A difference in allele and genotype frequencies for the rs2069837 site was observed between term CP patients (n = 338) and term controls (n = 491) in males (Table 9), but no difference was found between preterm CP patients (n = 156) and preterm controls in males or between term and preterm CP patients in either males or females.

Differences in genotype frequencies were observed between CP patients with maternal PROM (n = 88) and controls (n = 753) for rs2066992 and rs1800796 (Table 10). Further analysis according to the sex of the CP patients showed differences between CP patients with maternal PROM (n = 88) and controls (n = 491) in males for rs2066992 and rs1800796, but the differences disappeared after SNPSpD correction.

The mean plasma IL-6 concentration was higher in the CP patients than in the controls, and this difference was more pronounced in males (Figure 1A). Subgroup analysis showed that the IL-6 level was higher in the spastic CP group compared to the control group (Figure 1B). Furthermore, the IL-6 level in the CP patients was related to gestational age and a significant increase was seen in the full-term birth CP patients (Figure 1C). However, the IL-6 level was not changed compared to controls in the CP patients with PVL (Figure 1D). Further statistical analyses stratified by birth weight, birth asphyxia, MR, HIE, and maternal complications such as PIH, TPL, and PROM were also performed but no differences were found (data not shown). Furthermore, the plasma IL-6 levels were different between CP patients with genotypes CC and those with GG at rs1800796 (P = 0.033) (Figure 2A), and the plasma IL-6 levels of genotype CC at rs1800796 (Figure 2A), AA at rs2069837 (Figure 2B), and TT at rs2066992 (Figure 2C) in CP patients were higher than in their respective controls. Therefore, we have sufficient evidence to assume that there is a positive association of the IL-6 gene with the etiology of CP.