| Objective(1) To study on risk factors and early manifestation in autism.(2) To explore whether plasma level of glutamate and gamma-aminobutyric acid were related with autism, as well as the high prevalence of epilepsy in autism.(3) To explore the relationship between Reelin gene polymorphisms and autism by detecting four single-nucleotide polymorphisms (SNPs) including rs736707,rs2229864,rs362691,rs2073559 located on RELN.Method(1) 278 autism trioes and 200 control trioes were recruited. At the screening, all the children would fulfill clinical questionnaires, including demography, conditions of pregnancy and perinatal period, postnatal development, raising patterns, previous history and family history, physical examination. Autism children were assessed with CARS and ABC and their early manifestation were recorded.(2) The plasma level of glumatic acid and gamma-aminobutyric acid was measured with high efficiency liquid chromatography in 27 autism children with epilepsy, compared with that of 30 autism children without epilepsy and 36 control children.(3) The four SNPs, rs736707,rs2229864,rs362691,rs2073559, on Reelin gene were genotyped by Taqman-PCR in 234 autistic children, 399 parents, and 286 controls in Chinese Han. And the family-based transmission disequilibrium test (TDT) and case-control association anlysis were performed at the same time.(4) To check the accuracy of the allele identified technique inTaqMan-PCR by DNA sequencing.(5) GENEHUNTER (version 2.1) was used in transmission disequilibrium test. The haplotype model was constructed with SAS and all analyses were conducted with SPSS14.0 for Windows. We used t-test, chi-square test, analysis of variance, logistic regression analysis, and odds ratio according to the type of the data.Results(1) Childhood autism probands consisted of 233 males and 45 females (male/female = 5.18/1) . The mean onset age of autism was 22.36±8.29 months. The emergence time of abnormality being detectedby their caregivers was different, ranging from 10 to 48 months.(2) There were significant difference in many pregnancy and perinatal period risk factors between autistic children and control. Logistic regression found that many items entered regression equation as the following: mother's school life (P=0.009) , father's reproductive age(P=0.001), family burden (P=0.001), dry nurse (P=0.001), pregnancy reaction (P=0.025) ,drug history during mother's pregnancy period (P=0.014), delivery modality (P=0.045 ), nurture modality (P<0.001), main instructor (P<0.001) , positive family history (P=0.002) .(3) We calculated odds ratios of six items with clinical signific -ance in regression equation: father's reproductive age more than 30years(OR=6.715), positive family history (OR=5.798 ), drug history during mother's pregnancy period (OR=4.653 ) ,un-spontaneous delivery (O R = 4.147) , mother's school life more than 11 years (OR=1.373) ,moderate /severe pregnancy reaction (OR= 1.044) .(4) There were significant differences between autism patients and control in the following items: the emergence time of early developmental sign, the total score of early manifestation, and head circumference (P<0.001) .(5) Plasma level of glutamic acid was 653.95 pmol/L (SD =545.87) in autism children (group A), 1608.81 pmol/L (SD=1478.22) in autism children with epilepsy (group B ) and 653.70 pmol/L(SD=589.86) in control group (group C). Compared with Group B, the average plasma level of glutamic acid in group A and group C was significantly lower (p<0.05) , but there was no significant difference between group A and group C (p>0.05) . In terms of plasma level of gamma-aminobutyric acid (GABA) , group A was 71.46 pmol/L(SD=65.98 ) , group B was 111.19 pmol/L ( SD=85.27 ) and group C was 70.13 pmol/L (SD=51.36) . The plasma level of GABA in group B was significantly higher than group A and group C (p<0.05 ) . But there was no significant difference between group A and Group C (p>0.05 ) .(6) No significant differences were found on family-based TDT for single-locus marker in four SNPs in autism. There was no significant transmission disequilibrium but haplotype GTCC, in which 5 haplotypes were non-transmission.All these 4 SNPs (rs736707, rs2229864, rs362691, rs2073559) among control samples met Hardy-Weinberg equilibrium. In case-control association analysis, no significant difference were found in allelic and genotypic distribution of the 4 SNPs between autism group and control group (P>0.05) .There were no significant difference in frequency of CC,CT,GC of rs362691-rs2073559 between autism and control (P>0.05 ) , but that of GT showed significant difference between the two groups ( x~2=5.805, P =0.020) . The frequency composition of haplotypes between the two groups was significantly different (x~2=9.682, P=0.021) . No significant differences were found in other multiple-locus haplotypes frequencies and frequency compositions between autism and control (P>0.05 ) .Conclusion( 1 ) Autistic children have more risk factors in duration of pregnancy and peripartum. The first three risk factors were father's reproductive age exceeding 30, positive family history, taking medicine in duration of pregnancy.(2) The global development in autism children is behind control. There are some characteristic early manifestation in the development of autistic children, which can be identified earlier.(3) The increasing of plasma level of glutamate and gamma-aminobutyric acid is not general in all autistic children but only in those with epilepsy. It suggests that disfunction of glutamatergic and GABAergic system make a role in high prevalence of epilepsy in autism.(4) Transmission disequilibrium test based on autism trios found no preferential transmission in alleles of Reelin. Case-control association study showed that only frequency of GT in rs362691-rs2073559 was significantly different between the two groups, which suggested that Reelin gene may have no association with autism.
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