Correlation Of Gut Microbiome Between ASD Children And Mothers And Potential Biomarkers For Risk Assessment

Background:Autism spectrum disorders(ASDs)are considered a heterogeneous set of neurobehavioral disorders that are characterized by social deficits,repetitive behaviors,and cognitive inflexibility in early childhood,which present a substantial challenge to diagnosis and treatment.The incidence of ASD is steadily increasing in various countries.According to the study by the Centers for Disease Control and Prevention,it's one in 59,more than childhood cancer,diabetes and AIDS combined,putting a heavy burden on individuals,families,and the society.However,the etiology of ASD is still unknown,and there is no effective and targeted treatment and intervention method.The past decade has seen good progress in identifying genetic risk factors for ASD that point to specific mechanisms and pathways for related behavioral deficits.Epidemiological investigation shows that genetic factors play an important role in the incidence of ASD,but the incidence of simple genetic diseases is relatively stable,the rapid rise of ASD cannot be explained by genetic analysis alone,nor does it conform to the law of the incidence of genetic diseases.Genetic studies have revealed that there is a strong genetic influence on the development of autism,however,their risk effects are highly variable,and often related to factors besides autism.Studies on the etiology of ASD currently mainly believe that genetic and environmental factors interact and play a role in the occurrence and development of ASD.The gut flora is called the"second genome",with the continuous deepening studies of the collections between gut microbiome and ASD and the development of microbial sequencing technology in recent years,more and more studies have found that children with ASD have gastrointestinal diseases and intestinal flora disorders.Intestinal flora affects brain development and function through the nervous system,endocrine system and immune system,thereby affecting human cognition and behavior,they interact with each other to form the microbiota-gut-brain axis.In recent years,there has been a lot of epidemiological and biological evidence that prenatal factors trigger a more active immune state in the mother,which is associated with the development of autism.Epidemiological and animal-based studies have suggested that inflammation-induced maternal immune activation,prenatal exposure to immune challenges,and maternal obesity,stress,and gastrointestinal symptoms during pregnancy play roles in perinatal neurodevelopmental brain damage and contribute to an increased risk of subsequent neuropsychiatric disorders,such as ASD.Multiple animal studies have also indicated that the maternal gut microbiota may play an essential role in the occurrence of autism in offspring during gestation,and exhibit continuous correlations and long-term pathological consequences during development.Altering the gut microbiota and providing gut commensal bacteria(microbial reconstitution)have been shown to reverse maternal factor induced social and synaptic deficits in offspring and have beneficial effects on ASD behaviors in both mice and humans.Evidence for the link between ASD and abnormalities in gut microbial function has been accumulating,however,to the best of our knowledge,no study has investigated the gut microbiome profiles of mother-child pairs of ASD children and evaluated their correlations at the same time.And,epidemiological and animal studies alone may not be sufficient enough to determine the actual correlations and mechanisms in humans.Thus,it is still unclear how the gut microbiome varies between mothers of ASD children and those of healthy children and whether maternal gut bacterial communities are associated with the gut microbiome profiles of ASD children.Additionally,the unique features of the gut microbiome of ASD children in comparison with their mothers or healthy children have not yet been identified.Objective:To examine the relationships between gut microbiome profiles(diversity?structure and biomarkers)of ASD children and those of their mothers,evaluated the clinical discriminatory power of discovered bacterial biomarkers of ASD children,to explore the early diagnosis and guidance of ASD children by the unique bacterial biomarker of intestinal flora,to achieve the purpose of early diagnosis and early intervention of ASDMethods:All samples of ASD were obtained from Qilu Children's Hospital of Shandong University diagnosed by the Institute of Child Health Care and training in the autism rehabilitation training center lasted six months.The healthy control group were medical staff and their children in Qilu Children's Hospital of Shandong University.We enrolled 89 faecal samples of children and their mothers who met the inclusion criteria were included in the study.59 ASD children(ASD-Cs)and their mothers(ASD-Ms),together with 30 matched healthy(neurotypical)children(H-Cs)and their mothers(H-Ms),for the current gut microbiome study.The average age of ASD-Cs and HCs at the time of sample collection was 4(range,2-7)and 5(range,2-10)years old,respectively.The average age of ASD-Ms and H-Ms at the time of sample collection was 33(range,26-38)and 31(range,27-42)years old,respectively.To analyse the microbial populations of ASD children and those of their mothers,amplification of the variable region V1-V2 of the 16 S rRNA gene was performed.The LEfSe was used to explore potential bacterial biomarkers associated with different groups.The selected biomarkers were classified and analyzed by SVM classifier of R package e1071,The ROCR R package was used to calculate the ROC curve and the AUC value.In the subsequent results and analyses,we defined the groups as ASD-C,ASD-M,H-C,H-M,ASD-M+C(for mother-child pairs of ASD children),and H-M+C(for mother-child pairs of healthy children).Results:In our study,ASD children harbored an altered gut microbiome in the Chinese Cohort.For characterization of the gut microbiome associated with ASD,we compared the alpha diversity between the ASD-C and H-C groups.We found significant increases in bacterial richness(P<0.01)in ASD-Cs and nonsignificant difference in bacterial diversity between two groups(P=0.13).The total distribution of bacterial taxonomy showed no significant variations in the bacterial communities between ASD-Cs and H-Cs at the phylum level,as characterized by a similar Firmicutes/Bacleroidetes ratio(P>0.05);however,a significant increase in the relative abundance of Proteobacteria was observed in the ASD-C group compared with the H-C group(P<0.01).Analysis of the beta diversity based on the unweighted UniFrac distances showed that the microbiome of the ASD-C group was distinct from that of the H-C group.We further performed an analysis of similarities(ANOSIM),and the results indicated that the structure of the gut microbiome of the ASD-C group was significantly different from that of the H-C group(ANOSIM,r=0.197,P<0.01,unweighted UniFrac).LEfSe analysis between the ASD-C and H-C groups revealed the signature microbiome profiles and predominant bacterial biomarkers of ASD children.LEfSe analysis between the ASD-C and H-C groups revealed the signature microbiome profiles and predominant bacterial biomarkers of ASD children.Significant increases in the relative abundance of Enhydrobacter?Chryseobacterium?Streptococcus and Acinetobacter(at the genus level),as well as Acinetobacter rhizosphaerae and Acinetobacter johnsonii(at the species level),in addition to a significant reduction in Prevotella melaninogenica(at the species level),were observed in the ASD-C group in comparison with the H-C group.To identify the differences in gut microbiome between mothers of ASD-Cs and H-Cs,we compared the alpha and beta diversities between ASD-Ms and H-Ms.Distinct gut microbiome profiles were revealed.Analysis of the alpha diversity showed a significant increase in bacterial richness of ASD-Ms(P<0.05)and nonsignificant difference in bacterial diversity(P=0.35).Then,the analysis of beta diversity based on the unweighted UniFrac distances revealed that the microbiome of ASD-Ms was significantly different from that of H-Ms(ANOSIM,r=0.248,P<0.01,unweighted UniFrac)LEfSe analysis further confirmed these significant differences.A significant increase in the relative abundances of Moraxellaceae and Enterobacteriaceae(at the family level)and Acinetobacter(at the genus level)and a significant reduction in Faecalibacterium were observed in the ASD-M group,in comparison with the H-M group(LDA score>3).For characterization of the gut microbiota between mother-child pairs,we compared the alpha and beta diversities between ASD-M+C and H-M+C groups,and again revealed distinct gut microbiome profiles.Analysis of alpha diversity showed that the sequence-based boxplot based on the PD_whole_tree was nearly asymptotic,and Wilcoxon ranksum tests demonstrated significant differences in diversity in the ASD-M+C and H-M+C groups(P<0.01).Abundance-based coverage estimator(ACE)indexes also confirmed these findings.However,there was no significant difference in Shannon's index.Analysis of beta diversity based on the unweighted UniFrac distances revealed that the microbiome of the ASD-M+C group clustered apart from that of the H-M+C group(ANOSIM,r=0.191,P<0.01).We then assessed the correlation between the two groups of gut microbiomes using selected bacterial biomarkers,and the results showed that the gut microbiomes of children and mothers showed significant clustering.We further analyzed the different gut microbiome structures between ASD children and their mothers and found that Alcaligenaceae,Clostridium,Haemophilus and Wautersiella only increased the relative abundance of the intestinal flora of ASD-C,while Prevotella and Ruminococcus only decreased the relative abundance of the gut microbiome of ASD-C.Furthermore,the number and identity of the shared operational taxonomic unit(OTU)were evaluated through Venn diagrams.The similarity of the gut microbiome in the ASD-M+C group was higher than that in the H-M+C group.The results showed that Epulopiscium?Sphingobium?xenophagum,Anaeroplasma?Adlercreutzia?Solirubrobacterales?Mesorhizobium?Hydroophilus?Salinicoccus and Promicromonosporaceae were only found in the ASD-C group.Bacteroides ovatus and Abiotrophia were found in the H-C group.Since the gut microbiome of ASD-C and H-C is significantly different,we further searched for bacterial biomarkers that can distinguish the two groups.According to stringent criteria for adjusted P value(P<0.01,Wilcoxon rank sum test)and LEfSe analysis(LDA score>3),96 OTUs with significant differences were selected.Candidate biomarkers were selected to predict the risk of disease in children with ASD;candidate biomarkers were also selected to distinguish between mothers of ASD children and healthy children.Five bacterial biomarkers were found between the ASD-C and H-C groups:Betaproteobacteria?Burkholderiales?Pseudomonadales?Moraxellaceae and Acinetobacter;six bacterial biomarkers were found between the ASD-M and H-M groups:Flavobacteriia?Gammaproteobacteria?Flavobacteriales?Weeksellaceae?Enterobacteriaceae and Enterobacteriales.To explore the potential value of the identified bacterial biomarkers for two levels of clinical discrimination(ASD-C vs.H-C,and ASD-M vs.H-M),we constructed receiver operating characteristic(ROC)curves and computed the area under the curve(AUC)values.For ASD-C vs.H-C,the highest AUC value was 0.944(4-fold,95%confidence interval[CI]:84%-100%)with 83.33%sensitivity and 91.67%specificity.For ASD-M vs.H-M,the highest AUC value was 0.986(4-fold and 5-fold,95%CI:94%-100%),with100%sensitivity and 100%specificity.Further evaluation found that age,gender,and history of gastrointestinal(GI)problem had no significant effect on five candidate biomarkers for the ASD-C and H-C groups,and six candidate biomarkers for ASD-M and H-M groups.Conclusion:1.We found significant differences in the composition of intestinal bacteria between ASD children and healthy children,and there were potential pathogens;2.There were significant differences in the composition of intestinal bacteria between the mother of ASD and the mother of healthy children;3.The gut microbiome of ASD children was closely associated with that of their mothers;4.Children with ASD had unique bacterial biomarkers;5.Candidate biomarkers discovered in this study may have potential value in risk assessment for ASD.