Maternal Environmental Risk Factors Exposure During Pregnancy, Abnormal Transsulfuration Metabolism And Immune Dysfunction In Children With Autism Spectrum Disorder

Objective Autism spectrum disorder(ASD) is a neurological disorder with a spectrum of qualitative impairments in social interaction, communication, and restricted and stereotyped patterns of behaviors, interests, and activities. ASD affects much more males than females(approximately 4-5 males versus 1 female), and usually manifests in children before the age of 3 years. The etiology of ASD remains to be unknown despite the numerous efforts towards this subject. It has been a hotspot to explore the etiology of ASD, and it is becoming clear that the etiology of ASD involves complicated interactions between genetic predisposition and environmental exposures or triggers. It has been also a focus of attention for the role of transsulfuration metabolism and immune status in the pathogenesis of ASD. This study aimed to investigate the environmental and genetic factors in ASD and evaluated the transsulfuration metabolism and immune status in Chinese children with ASD by comparing those of typical developmental children. Furthermore, to explore the environmental and genetic factors and redox and immune status in ASD, and provide scientific basis for the etiology study of ASD.Methods 1. Qusetionnaires survey and interview were conducted among 108 children with ASD and 318 typical developmental children. Both case and control mothers completed a self-administered questionnaire with trained study staff to provide clarifications. The questionnaire covered the whole prenatal period(before conception to the end of the pregnancy) and gathered information on personal characteristics and lifestyle habits, health, pregnancy details, medication during pregnancy, and occupational or other potential harmful exposure during pregnancy. The clinical severity of ASD was evaluated with the Childhood Autism Rating Scale(CARS), and the autistic children’s present behavior was measured by the Autism Behavior Checklist(ABC). 2. The GSTM1 and GSTT1 gene polymorphism were detected by polymerase chain reaction(PCR) in 65 children with ASD and 65 typical developmental children. The difference of genotype and the variation of allele frequencies were analyzed in two groups. 3. Levels of serum homo Cysteine(Hcy), Cysteine(Cys), reduced glutathione(GSH), total glutathione(t GSH), and oxidized glutathione(GSSG) were measured using high-performance liquid chromatography(HPLC) and commercially available assay kits in 50 children with ASD and 50 typical developmental children. We analyzed the correlation between transsulfuration pathway metabolites and behavior characteristics in ASD. 4. The IL-1β-511 and IL-6-572 gene polymorphism were detected by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) in 92 children with ASD and 257 typical developmental children. The difference of genotype and the variation of allele frequencies were analyzed in two groups. 5. The two SNPs, rs73677 and rs2229864 on Reelin gene were genotyped by Taqman-PCR and adenosine deaminase(ADA) gene were genotyped by PCR-RFLP in 66 children with ASD and 69 typical developmental children. The difference of genotype and the variation of allele frequencies were analyzed in two groups. 6. Levels of serum IL-1β, IL-2R, IL-6, IL-8 and myelin basic protein(MBP) were measured using enzyme linked immunosorbent assay(ELISA) and levels of ADA were measured using UV spectrophotometry in 50 children with ASD and 50 typical developmental children. We analyzed the correlation between transsulfuration pathway metabolites and behavior characteristics in ASD. 7. All analyses were conducted with SPSS17.0 for Windows. We used χ2 test, Fisher’s exact test, logistic regression analysis, t’ test, Mann-Whitney U test, Pearson correlation, Spearman’s rho correlation and receiver operating characteristic(ROC) analysis according to the type of the data.Results 1. In part of qusetionnaires survey, there were significant differences in maternal environmental risk factors exposure and diseases during pregnancy between children with ASD and control. A logistic regression analysis showed that passive smoking (OR=2.270), maternal occupational/environmental toxicant exposure(OR=1.664), vaginal bleeding(OR=3.535), gestational diabetes(OR=4.732) and virus infection(OR=3.296) during pregnancy were significantly associated with the incidence of ASD. 2. In part of GST gene polymorphisms analysis, the GSTM1 null genotype was significantly higher in the ASD compared to the control group(P<0.05). No significant difference in GSTT1 gene distributions was found between groups. There was no significant difference in the distribution of the GSTM1 and GSTT1 null genotypes between the ASD group and control group. To investigate whether the GSTM1/GSTT1 genotype profiles are associated with ASD severity, we examined both genotypes in combination. There was a significant difference in the distribution of combinational GST genotypes between the mild to moderate ASD group and the severe ASD group(P<0.05). No significant gene-environment interactions were observed. Logistic regression did not show an increased risk for ASD in the presence of positive maternal exposure and GSTM1 and GSTT1 polymorphism. 3. In part of serum transsulfuration pathway metabolites analysis, The results indicated that Hcy and GSSG levels were significantly higher in children diagnosed with ASD, Cys, t GSH and GSH levels as well as the GSH/GSSG ratio showed remarkably lower values in ASD children compared to control subjects(P<0.05). We found a significant negative correlation between Cys levels and ABC total score(P<0.05) and language score(P<0.05). In addition, a positive correlation(P<0.05) was observed between CARS total score and serum Hcy levels in autistic children, levels of Hcy increased with increasing severity of autism as defined by the CARS total score. However, autistic behaviors measures were not correlated with any of transsulfuration metabolites. We also did not find any correlations between the transsulfuration metabolites and clinical severity measured with the CARS total score. 4. Analysis of IL-6-572 SNP revealed that there was significant difference in genotype distribution and allele frequencies between the ASD children and controls(P<0.05). Analysis of IL-1β-511 SNP revealed insignificant change in genotype distribution and allele frequencies between ASD children and controls. There was a significant difference in the distribution of IL-6-572 SNP genotypes between the mild to moderate ASD group and the severe ASD group(P<0.05). 5. In case-control association analysis, no significant difference were found in allele frequencies and genotype distribution of the RELN rs736707 SNP between ASD group and control group. Analysis of RELN rs2229846 SNP revealed that there was significant difference in genotype distribution between the ASD children and controls(P<0.05). There was no significant difference in allele frequencies between the ASD children and controls. 6. Serum levels of IL-1β, IL-2R, IL-6 and MBP were significantly higher in children with ASD compared with the corresponding values of matched control children(P<0.05). There was no significant difference in serum median level for IL-8 and ADA in children with ASD in comparison to control children. We found a significant positive correlation between MBP levels and IL-1β, IL-2R, IL-6 levels(P<0.05). 7. The results of ROC analysis indicated that Cys, GSSG, GSH/GSSG, IL-1β, IL-2R, IL-6 and MBP produced the good sensitivity and specificity for diagnosis of ASD.Conclusions 1. Passive smoking, maternal occupational/environmental toxicant exposure, vaginal bleeding, gestational diabetes and virus infection during pregnancy may be the specific risk factors associated with the incidence of ASD, indicating that the development of ASD may be influenced by both genes and environmental factors. 2. The polymorphism of GSTM1 gene is associated with the occurrence of ASD in China. Our results suggest that an abnormal transsulfuration metabolism and reduced antioxidant capacity(i.e., hyperhomo Cysteinemia and increased oxidative stress), and Hcy level appears to have a potentially negative impact on clinical severity of autistic disorder. 3. The polymorphism of IL-6-572 and RELN rs2229846 gene is associated with the occurrence of ASD in China. We demonstrate that there are significant increases in IL-1β, IL-2R, IL-6 and MBP levels in Chinese children with ASD as compared with age- and sex-matched typically developing children. Furthermore, children with ASD showed levels of these cytokine levels were positive correlation with the level of MBP. These findings suggest that there were ongoing inflammatory responses in Chinese children with ASD and MBP as a CNS antigen could significantly induces inflammatory mediators, resulting in the increased levels of cytokines. 4. The serum levels of Cys, GSSG, GSH/GSSG, IL-1β, IL-2R, IL-6 and MBP performed the good as biomarkers in the diagnosis of ASD.