Perinatal Epidemiology Research On Neural Development And ATG5/ATG7 Genetic Polymorphism In Pediatric Cerebral Palsy

IntroductionThe development of neural system is regulated by the comprehensive effort of neural generation, cell differentiation and migration and is under the influence of the environment within which the fetus spent its incubation stage. In this way, the mother’s deficient gene or other adverse elements in the environment can cause the abnormality in the fetus’ neural system. Including hydrocephalus, neural tube defects, mental retardation and cerebral palsy ect. Currently, there is a critical need for effective prevention as well as treatment strategies that can be implemented with high levels of coverage in populations with high incidence of these abnormalitiesIn china, there are 20 million neonates born every year, of which 600 thousand have congenital anomalies. China spends 200 million Yuan every year on the treatment of infants with neural tube defects. However, compare with morphological and structural abnormalities that can be easily identified, functional abnormities are potentially more dangerous and cause more economic burdens on the society because of difficulties in their early recognition.Autophagy plays an important role in the normal brain development, by which immature neurons that fail to integrate into functional network undergo discrete removal. Besides that, autophagy also helps to clean up infected or injured cells. However, over-activate autophagy causes damage to the structure of cells and leads to massive cell death. ATG5/ATG7 is a vital part in the formation of autophagosome and may greatly affect the function expression of these related genes. Research on exploring the relationship between ATG5/ATG7 gene and cerebral palsy is lacking.Cerebral palsy is the leading cause of children deformities. Currently, China has 2 million cerebral palsy cases, among which 310 thousand cases are preschoolers and this number is growing with an increase of 460 thousand per year., Cumulative evidence indicates that hereditary factors play important roles in the development of cerebral palsy.In this study, we investigated perinatal epidemiological neural development and the related risk factors, with focus on determining role of ATG5/ATG7 and its relation with neural abnormities and cerebral palsy. Our findings provided theoretical guidelines for treatment.PartⅠ: A population-based epidemiological studies of the perinatal neural developmentSection I: The epidemiological analysis of congenital anomaliesObjective:To investigate changes in the incidence and characteristics of congenital anomalies in infants in Henan Province of China over a period of 15 years, with focus on the neural tube defects. All infants between 28 weeks of gestation and within 7 days after birth were included.Methods: Population-based surveillance in Henan Province was conducted from 1997 to 2011 in 75 hospitals(40 urban districts and 35 rural counties). Basic population information was obtained from the healthcare network. All live births, intrauterine deaths after 28 weeks, and stillbirths were included. Congenital anomalies were diagnosed and reported to Henan Provincial Maternal and Pediatric Healthcare Hospital. The incidence of congenital abnormalities was analyzed using chi-square test, the incidence trend was compared using logistic regression analysis. The criterion for statistical significance was set at P < 0.05.Results: 1. Total incidence Of 1,815,920 births from 1997 to 2011, 15,660 cases of congenital anomalies were identified, resulting in an average incidence of 86.2 cases per 10,000 births. The incidence of congenital anomalies showed a significant downward trend(P < 0.0001) in rural areas and the whole province(P < 0.0001), but an increase in urban areas(P = 0.003).2. Subgroup analysis(1) The incidence in males was higher than in females, 86.4 vs 83.5 cases per 10,000 births(P = 0.035). Maternal age less than 20 years was associated with a highest incidence of congenital anomalies.(2) Among the 23 types of congenital anomalies recorded, neural tube defects were the commonest; the incidence declined from 39.3 cases per 10,000 births in 1997 to 6.1 cases per 10,000 births in 2011(χ2 = 388.853,P < 0.001).Conclusion:(1) The incidence of congenital anomalies has decreased in Henan Province over the past 15 years, displayed a significant downward in rural areas and an increase in urban areas.(2) The incidence in males was higher than that of females, due to significant reductions in rural areas and among girls. Maternal age less than 20 years had the highest incidence of giving birth to babies with congenital anomalies.(3) The incidence of neural tube defects has been significantly decreased in our province.Section II Adverse neurodevelopmental outcome in neonatesObjective: We used retrospective longitudinal study to analyze the neurodevelopmental outcome of inpatient neonates and neonatal brain damage in a provincial women’s and infants’ s hospital over the past 6 years.Methods: Data of the inpatient neonates and neonates with brain damage were collected from January 2007 to December 2012 from Henan Provincial Women’s and Infants’ s Hospital. All data was stratified by sex, birth weight, delivery type, maternal age, gestational age, and single or multiple births, pregnancy complications. The incidence of hospitalization and neonatal brain damage was calculated for each year. Main outcome measures, trends in the fundamental status of hospital-born inpatient neonates and risk factors for them and neonatal brain damage and throw follow-up visit got these related sequelaes of each neonatal brain damage infant. The incidence was analyzed by using chi-square, the risk factorswas compared using logistic regression analysis, with the criterion for statistical significance set at P < 0.05.Results. 1. General analysisOf 25871 hospital live births from 1997 to 2012 included, 5597 cases were hospitalized in NICU. The proportion of preterm births decreased from 21.4 to 20.4%( P =0.27), and cesarean deliveries decreased from 65.2 to 62.9%( P =0.029). Overall, 1695 hospitalized newborns were diagnosed as brain damage, with the incidence increased from 17.4% in 2007 to 35.1% in 2012( P <0.001), and we made follow-up records of all infants with brain damage, and the result indicated that the incidence of brain damage sequelae increased from 2.3% in 2007 to 3.97% in 2012( P =0.443).2. Risk factors(1) Newborns in NICU: The greatest risk falled in neonates with either birth weight range from 1000 to 1499g(OR = 3.185, P < 0.001),for Apgar scoring 4-7 at 1 minute(OR = 4.211, P < 0.001), etc..(2) Brain damage: GW is 34 ~ 36+6 W(OR = 1.55, P < 0.001), and cesarean delivery(OR = 1.499,P = 0.01) had the greatese risk of brain damage, etc..3. Respiratory disease accounted for the highest percentage, 34.6% of which asphyxia contributed 21.4%. Followed by jaundice, 25.9%, and the nervous system diseases 14.8%.4. Subgroup analysis(1) Cerebral palsy and mental retardation have significantly different between different imaging results( P =0.001 and 0.042)(2) There was significantly difference in the incidence of hospitalization, brain damage and sequelae between different gestational age of these 6 years(all three P <0.001).Conclusion:(1) The rates of cesarean delivery decreased continuously. However, the incidence of neonatal brain damage increased dramatically.(2) Newborn in NICU and risk factors that induced brain damage varied.(3) The incidence is different between imaging results brain damage of different GA as well as sequelae caused by brain damage.PartⅡ: The Association Between ATG5/ATG7 Polymorphisms and the Risk for Cerebral PalsyObjective: The purpose of this study was to analyze ATG5/ATG7 gene polymorphisms and to explore the relationship between the autophagy related gene and the susceptibility to cerebral palsy.Methods: A total of 658 healthy controls and 715 CP patients were studied to detect the presence of five SNPs(rs573775,rs510432,rs6568431,rs2299863 and rs3804338); in the ATG5 locus; six SNPs(rs11706903,rs2606750,rs346078,rs4684787,rs1470612 and rs2594972)) in the ATG7 locus by mass ARRAY technique. The SHEsis program was used to analyze the genotyping data. For all comparisons, multiple testing on each individual SNP was corrected by the SNPSp D program.Results: 1. General analysisThere were significant differences between the overall CP patients and normal controls in allele or genotype frequencies of ATG5(rs6568431) and ATG7(rs14706126 and rs2594972) polymorphisms(P = 0.0005, 0.0015 and 0.02,0.0004 and 0.044,0.0012 after SNPSp D correction).2. Subgroup analysis(1) Subgroup analysis found significant sex-related differences in allele and genotype frequencies in the ATG5 and ATG7 gene. Differences were found between CP and controls in males for rs6568431 and rs1470612 and rs2594972(P = 0.005,P = 0.004 after SNPSp D correction) and(P = 0.02,P = 0.0004 and P = 0.044,P = 0.0012, after SNPSp D correction);(2) For the ATG5 and ATG7 genes, differences were found between CP with HIE and controls in rs6568431 and rs2594972(P < 0.0001,P = 0.0005 after SNPSp D correction; P < 0.0001 after SNPSp D correction); and for ATG7 gene, differences was between CP with PVL and controls in rs2594972 of allele frequencies(P = 0.016 after SNPSp D correction);(3) For the ATG7 gene,differences was found between Spastic double collapsed CP and controls in rs11706903, rs1470612 and rs2594972;(4) For the ATG5 gene,differences was found between CP with PROM and controls in rs573775(P =0.015 and 0.04); for the ATG7 gene,for rs1470612 and rs2594972(all P <0.05);(5) For the ATG5 gene,differences was found between CP +MR and controls for rs6568431( P =0.015 and0.02);(6) Other subgroup analysis P > 0.05.Conclusions(1) ATG5/ATG7 genes polymorphisms are correlated with the susceptibility to CP.(2) The influence of ATG5/ATG7 genes polymorphisms is related to sex on CP.ATG5 is associated with PROM and MR,ATG7 is associated with spastic diplegia subtype and HIE.Part Ⅲ: The luciferase gene activity of ATG7 promoter in cerebral palsyObjective: Relevancies among ATG7 genetic polymorphism of cerebral palsy were analyzed in case-control study on large samples, then cloned, ATG7 gene promoter iaccording to the results of SNP. With the luciferase reporter gene carrier inserted, differences among expression quantity of the luciferase in different groups(internal control group, wild type and mutant type in positive control group) are detected to probe into its possible relationship with cerebral palsy.Methods: Intracytoplasmic dermatomes transfection and luciferase detection are adopted to amplify the ATG7 gene promoters in tool cell 293 T. With the luciferase reporter gene carrier p GL3-empty CON080 being inserted and recombinant plasmid being transfected into 293 T cell, different wild types and mutant types of different locus are chosen to measure the transcriptional activity of promoters of different locus in ATG7 gene and make a comparison with promoter activity of SV40 viral transformation. The measurement data was analyzed using T- test, the criterion for statistical significance was set at P < 0.05.Results The detection results of luciferase reporter gene:(1) Luciferase reporter genes of promoters of different locus in ATG7 gene were constructed; the constructed promoters of ATG7 gene were verified by the sequence and plasmid enzyme digestion;(2) The determination of activity indicated that reporter gene showed different promoter activity in constructing ATG7 gene in SV40 viral transfection of recombinant plasmid transfected 293 T cells and different genetic locus, presenting certain dosage effect.(3) Luciferase detection showed relative expression quantity of rs1470612 and rs2594972 in ATG7 gene decreased compared by the mutant type luciferase and there was difference between the wild type and the mutant type(the P-values of the two locus equal less than 0.0001 and 0.009 respectively).Conclusions: After the mutation of the two ATG7 locus then the luciferase of expression quantity had reduction,the polymorphism of rs1470612 and rs2594972 in ATG7 gene is related with cerebral palsy.