MicroRNA profiling in body fluids of patients affected by Autism Spectrum Disorder, Tourette syndrome and Arnold-Chiari syndrome

The growing prevalence of neurodevelopmental disorders (NDDs) is leading to a significant social impact and a considerable interest in the scientific community. In the last decades, there has been an increasing need for society to invest in early diagnosis and provision of appropriate healthcare and educational services. However, the multifactorial and heterogeneous nature of NDDs makes the etiopathogenesis poorly understood and the diagnostic process challenging, especially in cases of overlapping clinical symptomatology. Consistent evidences indicate that complex and dynamic interactions among genetic, epigenetic and environmental factors influence the etiology of neurodevelopmental disorders. Morphological abnormalities of the central nervous system also represent a significant problem of child neurology, which dates to the embryonic or foetal period. However, the etiology of neural malformations is still unknown and the molecular basis has not yet been fully clarified. Using liquid biopsies could be an extremely valuable and innovative approach to strengthen the standard clinical diagnostic processes and to further explore the molecular basis of such elusive disorders, for example Autism Spectrum disorder (ASD) and Tourette syndrome (TS), as well as Arnold-Chiari (AC) syndrome. In fact, the use of serum or saliva provides a more effective and suitable alternative to the conventional tissue biopsies, which often lack or are not easily accessible to researchers for the study of neurodevelopmental anomalies. Over the past few years, circulating microRNAs (miRNAs) have attracted increasing attention for their consistent presence in all body fluids, their specific expression patterns related to both physiological and pathological conditions, as well as their remarkable stability in the extracellular environment. For all these reasons, circulating miRNAs have been progressively emerging as promising diagnostic, prognostic and predictive biomarkers in neurodevelopmental disorders. Based on these premises, we conducted two research projects aimed to profile circulating miRNA expression (i) in saliva from ASD patients and (ii) in serum of AC, TS and comorbid ACTS patients, compared to neurologically unaffected individuals. Moreover, the ASD project aimed to verify the existence of a molecular correlation between salivary miRNA dysregulation and oral microbiota alterations, as well as their potential contribution to ASD pathogenesis. Many studies proposed the use of circulating miRNAs as promising biomarkers for several pathologies, but very few studies profiled salivary miRNAs in ASD, much fewer ones explored serum miRNAs in TS context, no one in AC syndrome and the very rare comorbid condition between AC syndrome and TS (ACTS). In addition, while mounting evidences suggest a key role for the gut microbiota in ASD, the etiopathogenetic contribution of microorganisms living in the oral cavity has not been satisfactorily explored. In our saliva study, in order to investigate salivary ASD-related miRnome and microbiome alterations, we performed a combined approach of miRNA expression profiling by NanoString technology, followed by validation experiments in qPCR, and 16S rRNA microbiome analysis on saliva from 53 ASD and 27 neurologically unaffected control (NUC) children. We revealed that miR-29a-3p and miR-141-3p were upregulated, while miR-16-5p, let-7b-5p, and miR-451a were downregulated in ASD compared to NUCs. Microbiome analysis on the same subjects revealed that Rothia, Filifactor, Actinobacillus, Weeksellaceae, Ralstonia, Pasteurellaceae and Aggregatibacter increased their abundance in ASD patients, while Tannerella, Moryella and TM7-3 decreased. Neuropsychological scores related to typical ASD cognitive impairments, especially for anomalies in social interaction and communication, were statistically associated to variations of both miRNAs and microbes. Among miRNA/bacteria associations, the most relevant was the negative correlation between salivary miR-141-3p expression and Tannerella abundance. This interesting finding suggests the occurrence of a potential cross-talking between circulating miRNAs and resident bacteria in saliva of ASD patients. To evaluate the diagnostic accuracy of miRNAs and microbiome taxa, we performed univariate and multivariate ROC curve analyses. The first analysis led to moderate but significant Area Under Curves (AUCs) values, while the second one, which evaluated the combination of all miRNAs and bacteria, resulted in a remarkable increase of the predictive performance to discriminate ASD patients from NUCs. In particular, the combination of the 5 salivary miRNAs and 4 bacterial species (i.e., Filifactor, Moryella, Tannerella and TM7-3) significantly represented the best-fit performing model for ASD diagnosis, suggesting a potential application as non-invasive biomarkers. Pathway enrichment analyses were computed to investigate the potential biological impact associated with the differential expression of the miRNAs reported in this study. The computational data would suggest a functional involvement of differentially expressed (DE) salivary miRNAs in molecular signaling pathways related to the development of cognitive functions and often reported to be dysfunctional in ASD. In our serum study, we aimed to identify serum microRNA expression profiles as molecular fingerprints for AC syndrome, TS and ACTS, by using NanoString technology. For this aim, 10 AC patients, 11 ACTS patients, 6 TS patients, and 8 unaffected controls (NC) were recruited. Nine miRNAs resulted significantly differentially expressed: let-7b-5p (upregulated in ACTS vs TS); miR-21-5p (upregulated in ACTS vs AC; downregulated in AC vs TS); miR-23a-3p (upregulated in TS vs NCs; downregulated in AC vs TS); miR-25-3p (upregulated in AC vs TS and NCs; downregulated in ACTS vs AC); miR-93-5p (upregulated in AC vs TS); miR-130a-3p (downregulated in ACTS and TS vs NCs); miR-144-3p (downregulated in ACTS vs AC; upregulated in AC vs TS); miR-222-3p (upregulated in ACTS vs NCs); miR-451a (upregulated in AC vs TS and NCs; in ACTS vs NCs). The altered expression of miRNAs was statistically correlated to neuroimaging and neuropsychological anomalies, especially for low intelligent quotient scores, cranial disproportions and behavioral impairments. Furthermore, computational analyses indicated that DE miRNAs are involved in AC syndrome and TS pathomechanisms. Our liquid biopsy-based investigation approach aims to pave the way to valuable, non-invasive, unbiased molecular tools for such elusive neurodevelopmental disorders and neurological malformations and to provide some clues into their molecular basis.