Exploring Gut Fungal Profiles Using Next-Generation Sequencing Platform and Investigating the Role of Fungal Dysbiosis in Adhd

Background: Attention deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disease in children and adolescents. The recognition that the gut microbiota influences brain function and complex behaviors led to the suggestion of the concept of a “gut–brain axis”. Among the gut microbiome, fungus is a hyperdiverse group of multicellular eukaryotes and can influence hosts’ health. Internal transcribed spacer (ITS) region is the most widely sequenced DNA region in molecular ecology of fungi and has been recommended as the universal fungal barcode sequence. The next-generation sequencing (NGS) concept constructs high-throughput technology to achieve simultaneous high-speed large-scale nucleic acid sequencing. Increased intestinal permeability (also known as "leaky gut") is related to the pathophysiology of neuroimmune diseases. To assess intestinal permeability, zonulin, occludin and DEFA1 in serum can serve as markers of endothelial and epithelium tight junctions modulating both gut and blood–brain barriers. However, it remains unclear whether gut fungal community are associated with intestinal permeability and the immune system, and further involve the pathological mechanism of ADHD.Objectives: 1. To compare the differences of fungal microbial communities between ADHD patients and healthy control children by sequencing the ITS regions. 2. Investigating the relationship between fungal microbial community, intestinal permeability, immune function and ADHD performance in each intestine.Methods: In this two-year cross-sectional study, we will collect 60 children or adolescents with ADHD between the ages of 6 and 16 and 60 healthy control groups that are similar in age and sex to ADHD cases. All cases will be assessed for behavior (SNAP-IV) and neuropsychological function (WISC-IV and IVA/CPT). Fecal specimens will be collected from both groups of cases. DNA will be extracted from the fecal samples and the ITS library will constructed by two-step PCR, followed by sequencing the ITS region by the NGS platform. The fungal community will be analyzed by bioinformatics tools and the taxonomic category will be identified in ADHD and healthy control groups. ADHD cases and the control group received blood (about 10 ml). Blood will be centrifuged to extract serum and plasma for quantitative analysis of intestinal biomarkers (zonulin, occludin, and DEFA1). In addition, we will establish an in vitro cell model (epithelial and endothelial models), to characterize the role of overproduction of serum biomarkers in intestinal permeability.Expected results: This project will be a pioneering study of the distribution of intestinal fungi in ADHD children based on the NGS platform. The results of this study will be able to understand the differences in intestinal fungal distribution between ADHD cases and healthy controls, and further understand the role of fungi in ADHD. It can be understood whether intestinal fungi are important for intestinal permeability and immune function, and then involve in the pathological mechanism of ADHD. In addition to academic contributions, the research results can be applied to the establishment of microbial biological indicators or new therapeutic directions, and have important clinical application value for the treatment of ADHD.

Project ID:PC10907-0931
External Project ID:MOST109-2314-B182-045-MY2