Exploring The Role Of Telomere Length And Mitochondrial DNA Copy Number In Childhood Autism Spectrum Disorder

Objective:Autism Spectrum Disorder(ASD)is a highly heterogeneous neurodevelopmental disorder caused by a complex interaction between genetic and environmental risk factors.Although a variety of risk factors and pathogenic genes have been identified,the pathogenesis and inducing factors are not fully understood.Currently,Oxidative Stress(OS)has been detected in the body of ASD patients,and the balance between antioxidant capacity and OS induced free radicals may be crucial in the pathophysiological development of ASD.Therefore,in this study,the Mitochondrial DNA Copy Number(mtDNA-CN)and Telomere Length(TL)in peripheral blood as potential biomarkers of intracellular OS accumulation.In order to provide an important basis for revealing the pathogenesis of ASD.Method:In this study,96 children diagnosed with ASD and 96 Typical Development(TD)children were recruited from Longgang Maternal and Child Health Hospital of Shenzhen in strict accordance with inclusion and exclusion criteria,and basic data such as general demographic characteristics and severity of the subjects were collected.Peripheral blood and urine samples were collected and stored in-80℃ ultra-low temperature refrigerator for testing.1.Expression and clinical significance of TL and mtDNA-CN in children with ASD.1.1 Quantitative Polymerase Chain Reaction(qPCR)and Digital Polymerase Chain Reaction(dPCR)were used to detect the expression of TL and mtDNACN in 96 children in ASD group and 96 children in TD group.1.2 The sensitivity and repeatability of TL and mtDNA-CN detected by qPCR and dPCR were analyzed.1.3 Logisitic regression and receiver operating characteristic curve(ROC)were used to analyze the correlation and diagnostic value of TL,mtDNA-CN and ASD occurrence.2.Correlation analysis of TL,mtDNA-CN and OS.2.1 The content of 8-Hydroxy-2Deoxyguanosine(8-OHdG)in urine samples was detected by liquid chromatography-mass spectrometry tandem triple quadrupole mass spectrometry,and the creatinine level in 3-5 mL urine was detected by picric acid method for correction calculation.2.2 Catalase(CAT)activity in plasma was detected by ammonium molybdate method.Superoxide Dismutase(SOD)activity in 200 μL plasma was detected by xanthine oxidase method.Antioxidant Capacity(AOC)in 30 μL plasma was determined by colorimetry.2.3 Analyze the differences of OS indicators between the ASD group and the TD group.2.4 Logisitic regression was used to analyze whether OS indicators were risk or protective factors for ASD occurrence.2.5 Partial correlation was used to analyze the correlation between OS indicators,TL,mtDNA-CN and the severity of clinical symptoms of ASD.2.6 Spearman correlation was used to analyze the correlation between OS indicators and TL and mtDNACN.Results:1.The TL in ASD group was shorter than that in TD group(P<0.01),and the contents of mtDNA-CN,8-OHdG and SOD activity level in TD group were significantly higher than those in TD group(P<0.05).But no significant difference was found in CAT activity and AOC activity between the two groups(P>0.05).2.When DNA samples diluted 102-103 were amplified by qPCR,the amplification curve did not enter the plateau stage(Ct value of three repeated experiments>30),while when absolute quantification of higher multiple diluted DNA samples by dPCR,multiple positive droplet copy numbers and obvious cell clusters were clearly visible.3.ROC curve results showed that TL(AUC=0.632,95%CI:0.553-0.710,P=0.002)and mtDNA-CN(AUC=0.593,95%CI:0.513-0.673,P=0.026)had certain accurate predictive significance for the identification of ASD.4.Univariate and multivariate Logisitic regression analysis showed that TL decreased(Monofactor:2.20(1.22,3.96),P=0.009,Multifactor:2.22(1.22,4.00),P=0.008)and CAT activity decreased(Monofactor:2.31(1.28,4.17),P=0.006;Multifactor:2.31(1.28,4.18),P=0.006)was a risk factor for ASD,while 8-OHdG content decreased(Monofactor:0.29(0.14,0.60),P=0.001;Multifactor:0.27(0.13,0.57),P=0.001),SOD activity decreased(Monofactor:0.55(0.31,0.98),P=0.042;Multifactor:0.54(0.30,0.98),P=0.042)were protective factors for the occurrence of ASD.5.Partial correlation analysis showed that OS index was closely associated with the clinical symptom severity of ASD.The content of 8-OHdG was positively correlated with the total score of ABC scale(r=0.230,P=0.029),body movement(r=0.232,P=0.027)and self-care score(r=0.227,P=0.032),and the activity level of CAT was positively correlated with communication(r=0.297,P=0.004).It was negatively correlated with language(r=0.240,P=0.023).6.Spearman correlation analysis results showed that TL was positively correlated with mtDNA-CN(r=0.234,P=0.022)and CAT activity level(r=0.246,P=0.016),and 8-OHdG content was positively correlated with SOD activity level(r=0.262,P=0.012).Conclusion:In this study,the levels of TL,mtDNA-CN and OS were significantly different between ASD and TD groups,and played an important role in the pathophysiological process of ASD.Moreover,the clinical symptom severity and SOD vitality level of children with ASD may affect the length of TL,indicating the importance of antioxidant supplementation and may provide an important theoretical basis for the pathogenesis of ASD.