Clinical And Molecular Genetic Analysis Of The Children With Cerebral Palsy

Part I:Analysis of Some clinical Characts And the correlative risk factors of Cerebral Palsy in ChildrenObjective:To analysis the clinical characteristics and the correlative risk factors of cerebral palsy in childrenMethods:1. The study population consisted of 374 CP patients were from centers of cerebral palsy rehabilitation in the Third Affiliated Hospital of Zhengzhou University, Zhengzhou Children's Hospital and the First Affiliated Hospital of Henan Traditional Chinese Medical College between May 1,2008 and Oct.31,2009.2.Materials of 374 children with CP were analyzed retrospectively.Results:l.The gender and age of preliminary diagnosis of cases:Of the total 374 cases,the rate of gender(male:female)was 1.9:1(245/129). The age of preliminary diagnosis of all cases were from 3months to 120months(mean age±SD:16.9±12.6 months).The frequencies of cases in six age groups(0-3 months,-6months,-12 months,-24 months,-48 months and more than 48 months) were 0%,17±17%,30%,29%,18%and 6%respectively among all cases.2. The distribution of gestational age among cases:The frequencies of cases in five gestational age groups(<30wks,～32 wks,～34 wks,～37 wks and≥37 wks) were 6%,5%,7%,13%and 69%respectively among all cases. The most dominant gestational age was more than 37 wks.3. The distribution of birth weights among cases:The frequencies of cases in five birth weights groups(< 1000g,～1500g,-2500g,～4000g and≥4000g) were 0%,2%,22%,75%and 1%respectively among all cases. The most dominant birth weights was 2500～4000g.4. The distribution of the CP type among cases:According to motor dysfuntion, patients with spastic, hypotonic, atonia, dyskinetic,mixed and cryptogenice type accounted for 211(56%),45(12%),30(8%),24(6%),47(13%) and 17(5%) respectively among all cases. Among 211 spasticity cases, it was found that the main type was quadriplegia(98)followed by diplegia(46)and hemiplegia(36). 5.Brain MRI/CT Imaging.There were 158 cases examined by imageology. According to the imageology findings, the abnormal rates of CT/MRI was 64.32%(119/158). The most dominant imageology findings was PVL followed by cerebrunl dysplasia, enchalodysplasi.6.Complications:Of the total 374 cases,170 cases had one or more complications, which the most dominant wsa mental retardation (29.7%).7. The risk factors of CP:Of the total 374 cases,133 cases (35.6%)had an correlative risk factors of CP,in which the most common risk factors were hypoxic ischemic encephalopathy(41.3%) followed by preterm delivery(28.5%) and asphyxia in laboring(7.5%)in this study.No any risk factors were found in 241cases(64.4%).Conclusions:Preterm delivery,asphyxia in laboring and hypoxia-ischemia in neonate were the most common risk factors of CP. Of 241(64.4%)cases had no any risk factors to be found. We should improve the level of perinatology technique to decrease the morbidity of CP effectively and investigate other risk factors of CP.Part II:Linkage Disequilibrium Studies of MTHFR Gene Polymorphisms in Cerebral PalsyObjective:To investigate whether MTHFR gene polymorphisms contribute to the development of cerebral palsy in Chinese infantsMethods:1. The study population consisted of 159 CP patients (50 girls 31.4%, 109 boys 68.6%, mean age±SD:16.9±14.4 months), were from centers of cerebral palsy rehabilitation in the Third Affiliated Hospital of Zhengzhou University, Zhengzhou Children's Hospital and the First Affiliated Hospital of Henan Traditional Chinese Medical College between May 1,2008 and Oct.31,2009. The 169 healthy control subjects from the Department of Child Health Care at the same hospital during the same period matched for age, sex and ethnicity were also included in the study (64 girls 37.9%,105 boys 62.1%, mean age±SD:16.4±13.8 months). 2. Genomic DNA was prepared from venous blood using AxyPrepTM Blood Genomic DNA Miniprep Kit. A total of five SNPs (rs4846049,rsl476413,rs1801131,rs1 801133 and rs9651118) were genotyped using TaqMan? technology. We conducted Hardy-Weinberg equilibrium tests, allele and genotype frequency analysis online on the SHESIS software platform (http://analysis.bio-x.cn/). and compared the discrepancies of allele and genotype frequencies on single loci between patients and controls. The program SNPSpD (http://gump.qimr.edu.au/general/daleN/matSpD/) was used to correct for multiple testing performed on each individual SNP. The numbers of observations for each haplotype were compared using x2 tests. Bonferroni correction was applied for haplotype analysis. Results:1. The chi-square goodness-of-fit test showed that the genotypic distribution of the 5 SNPs in both CP patients and controls were in Hardy-Weinberg equilibrium (p>0.05).2.There were no significant differences of allele or genotype frequencies between cerebral palsies and controls at any of the five genetic polymorphisms. Subgroup analysis found significant difference in allele and genotype frequencies between cases of CP combined MR and controls at rs4846049, rs 1476413 and rs1801131 (p values were 0.031,0.028 and 0.014 respectively after SNPSpD correction). The frequencies of the T alleles of rs2433322, rs1476413 and G allele of rs1801131 were greater in CP combined MR patients than in the controls. Haplotype analysis reported a significant global p value (p= 0.021 after Bonferroni correction) and haplotype frequency discrepancies. The haplotype TTGGT (rs4846049, rs1476413, rs1801131, rs1801133, rs9651118) was observed to be strongly associated with CP+MR (p= 0.001, OR=2.695,95%CI=1.470～4.941, p= 0.005 after Bonferroni correction). There was no difference in the allele or genotype frequencies analysis in other CP subgroups, including, gestation age, birth weight and birth asphyxiaConclusions:Our results support the notion that all five SNPs in the MTHFR did not contribute to the occurrence of CP in Chinese infants. However, MTHFR genetic polymorphisms are associated with CP combined with mental retardation.MTHFR gene polymorphism is a risk factor of cerebral palsy combined with mental retardation.Part III Linkage Disequilibrium Studies of AP4M1 Gene Polymorphisms in Cerebral PalsyObjective:To investigate whether AP4M1 gene polymorphisms contribute to the development of cerebral palsy in Chinese infantsMethods:l.The study population consisted of 159 CP patients (50 girls 31.4%,109 boys 68.6%, mean age±SD:16.9±14.4 months) were from centers of cerebral palsy rehabilitation in the Third Affiliated Hospital of Zhengzhou University, Zhengzhou Children's Hospital and the First Affiliated Hospital of Henan Traditional Chinese Medical College between May 1,2008 and Oct.31,2009. The 169 healthy control subjects from Department of Child Health Care at the same hospital during the same period matched for age, sex and ethnicity were also included in the study (64 girls 37.9%,105 boys 62.1%, mean age±SD:16.4±13.8 months).2.Genomic DNA was prepared from venous blood using AxyPrepTM Blood Genomic DNA Miniprep Kit. A total of four SNPs(rs1534310. rs4729577, rs2293479 and rs13309)were genotyped using TaqMan? technology. We conducted Hardy-Weinberg equilibrium tests, allele and genotype frequency analysis online on the SHESIS software platform (http://analysis.bio-x.cn/). and compared the discrepancies of allele and genotype frequencies on single loci between patients and controls. The program SNPSpD (http://gump.qimr.edu.au/general/daleN/matSpD/) was used to correct for multiple testing performed on each individual SNP. The numbers of observations for each haplotype were compared using x2 tests. Bonferroni correction was applied for haplotype analysis.Results:1. The chi-square goodness-of-fit test showed that the genotypic distribution of the 4 SNPs in both CP patients and controls were in Hardy-Weinberg equilibrium(p>0.05).2.There were no significant differences of allele or genotype frequencies between cerebral palsies and controls at any of the four genetic polymorphisms. Haplotype analysis found no association between the five haplotypes(A C C T,A C T T,A T C A,A T T A, T C T T) and cerebral palsies. Subgroup analysis found that there was no difference in the allele or genotype frequencies analysis in CP subgroups, including, gestation age, mental retardation, birth weight and birth asphyxiaConclusions:Our results support the notion that all four SNPs in the AP4M1 did not contribute to the occurrence of CP in Chinese infants.Part IV:Study of Whole Genome DNA Methylation of Cerebral Palsy Using Monozygotic Twin PairsObjective:To investigate effect of Whole Genome DNA methylation of Cerebral Palsy in Twin PairsMethods:1.The study population consisted of two MZ twin pairs:Twin pair I were 14-month-old boys which one was CP patient (No 1), while another (No 2) was normal. Twin pair II were 31-month-old boys which one was CP patient (No 6), while another (No 5) was normal. Two twin pairs were from centers of cerebral palsy rehabilitation in the Third Affiliated Hospital of Zhengzhou University, Zhengzhou Children's Hospital.2. In order to ascertain the zygosity of this two twin pairs by microsatellite polymorphism or STR (short string repetitive sequence),5ml EDTA-2Na blood samples were collected from each of the twin pairs.Genomic DNA was prepared from venous blood using AxyPrepTM Blood Genomic DNA Miniprep Kit. Using MBD2b protein column chromatography method enriched methylated DNA fragments. We analyzed all the methylated DNA fragments by Solexa sequencing.Results:1. Zygosity Examination:The genotype of 15 loci were completely identical in each twin pair, Therefore,the twin brothers of each twin pairs were MZ.2. Analysis for Differentially DNA methylation regions.There were differentially DNA methylation regions between CP patien and his normal brother of each twin pair. Through compairing of the hypermethylation regions of two CP patiens or two normal individuals we found that there were 10679 common methylation regions between two CP patiens, while 626 common methylation regions between two normal individuals.We selected 626 common promoter hypermethylation regions in CP patiens,41 in normal individuals. Conclusions:Through study of two MZ pairs by using method of whole genome DNA methylation analysis we found some genes with common promoter methylation regions between two CP patiens,which lay the foundation of further studies with a larger number of subjects.