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ORIGINAL ARTICLE
Ahead of print publication
 

Hypothesis and preliminary results on the role of MUC1 and MUC2 in relationship to autism etiology


1 Biochemical Department, Milan University, Milan, Italy
2 Pharmaceutical Researcher, Frosinone, Italy

Date of Submission30-Aug-2020
Date of Acceptance29-Dec-2020
Date of Web Publication12-Jul-2021

Correspondence Address:
Menicagli Roberto,
Martiri della Libertà 6°a, 20060 Mediglia.
Italy
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpn.JPN_224_20

 

   Abstract 

Background: Autism is a neurological disorder with either genetic or environmental component. Autism generally presents changes of intestinal permeability to produce alteration of metabolism in the gastrointestinal tract. The intestinal macrobiota produces metabolites, opioid-like peptides, that show properties experimentally associated with autism. The aim of this study is to understand the cause of intestinal permeability’s alteration. Materials and Methods: We determined intestinal MUC2 on stool samples of 12 patients and healthy controls with Fecal Mucin Assay and analyzed the results with Mann–Whitney U-test calculator. Results: The results of the dosage of MUC2’s concentration in autistic people decrease when compared with those of healthy control groups: this result is statistically significant: the p-value is 0.00124. Discussion: The results show an increase of MUC2, perhaps due to genetic origin. We hypnotize a probable over expression on highly hypoglycosylated MUC1. All this prevents a regular linkage of MUC2 to MUC1, so as to form many channels in mucosa. The free MUC2 relies on themselves and joins together with hydrogen bridge bonds, leaving the highly glycosylated end parts to become the starting point for abnormal growth of the bacteria, which in turn produce opioid such as peptides, that pass into the channels of the mucosa layer.


Keywords: Autism, intestine, mucins, neuropeptide, opioids



How to cite this URL:
Roberto M, Marta L. Hypothesis and preliminary results on the role of MUC1 and MUC2 in relationship to autism etiology. J Pediatr Neurosci [Epub ahead of print] [cited 2021 Nov 28]. Available from: https://www.pediatricneurosciences.com/preprintarticle.asp?id=321164





   Introduction Top


Previous studies have focussed, for the etiology of autism spectrum disorder (ASD), on genetic causes, immune system abnormality, inflammation, exposure to environmental toxins, and alterations in the microbiota of the intestine.[1] The percentage of genetic heredity of ASD is about 50% among Swedish children, suggesting that not only genetic factors play an important role to develop the ASD, but also environmental factors.[2],[3] Many experimental evidence shows that gastrointestinal symptoms, such as abdominal pain, gastric cancer, diarrhea, constipation, and flatulence, are common in patients with ASD.[4] A recent study[5] identified constipation as the most common symptom (85%) in children with ASD according to parental reports and assessments by pediatric gastroenterologists. The percentage of gastrointestinal symptoms varies from 23% to 70% in children with ASD.[4] In addition, gastrointestinal symptoms show a relationship with ASD severity.[4],[6] Although these studies have not shown a cause–effect relationship between gastrointestinal symptoms and ASD, the results suggest that the intestine plays an important role in the etiology of ASD. Recent studies[7],[8] have shown that changes in microbiomes can leave one or more microbial ‘fingerprints’ (Metabolomics) in their biological activities. Metabolites thus become chemical compounds that can characterize people with autism and can serve as markers for diagnostic purposes. More recent studies show the important role of the intestinal barrier and its permeability in the formation of many neurological diseases, because the alteration of macrobiota is probably the consequence of the variation of intestinal permeability. Gastrointestinal symptoms are a common comorbidity in patients with autism spectrum disorder (ASD), but the underlying mechanisms are unknown. It is anyway important to note however that the latest research has shown that autism is also linked to a possible abnormal excitation related to compounds derived from metabolic cycles involved in neuronal transmissions. Recent hypotheses suggest that some phenomena may occur as a result of an altered intestinal permeability, with a consequent incorrect metabolism of some foods. This metabolic process involves the formation of bioactive peptides in the gastrointestinal tract. Recent studies[9] have confirmed that casein forms a compound, beta casomorphin-7, which is an opioid-like peptide. Some studies show that the production of these opioids-like peptides is experimentally associated with autism. These peptides pass through the brain barrier to bind receptors and prevent their smooth function. Our hypothesis is that the main aspect of this phenomenon may result from an alteration of intestinal permeability due to an imbalance of the intra- and extra-cellular mucin layer. The purpose of this study is to show the possible imbalance of the two mucins amount, and with literature data, the study makes a reasonable hypothesis for its relationship with opioids-like peptides realise


   Materials and Methods Top


This study followed a procedure in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2000. The independent Ethics Committee of Roma Biomed Research Lab, Milan, Italy obtained informed consent for children aged over 7 years participating in the research. To determine intestinal MUC2’s concentration, we tested stool samples of 12 ASD children and healthy controls, both with age between 8 and 15 years. We used Fecal Mucin Assay Kit to extract and fluorometrically find the amount of mucin content in feces. Fecal mucin amount can be a measure of index of intestinal barrier function. Step 1: Extraction and partial purification of mucin from feces. Step 2: Determination of mucin O-glycosidically linked oligosaccharide chains is β-eliminated by diluted alkali, and reducing end of sugar chain is formed. Reducing carbohydrates are fluorescence-labeled at high temperature to produce intensity fluorescent condensate.

Working calibrator: N-acetylglucosamine 250 μg/mL. Create a standard curve by serial dilution as indicated in [Figure 1]. The remaining undiluted calibrator should be stored at 2–10°C. Diluted calibrator is stable and should be stored at 2–10°C for 1 month. We statistically analyzed the results with Mann–Whitney U-calculator.
Figure 1: Standard curve for MUC2 determination

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   Results Top


The results of the dosage of MUC2 in ASD and healthy controls [see [Table 1]] show that the amount of MUC2 is lower in ASD when compared with the healthy control group, and the difference is statistically significant: the p-value is 0.00124.
Table 1: Intestinal MUC2 values in autistic and healthy people

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   Discussion Top


Many genetic and environmental components can change the normal structure of intestinal mucin. In any case, whatever the cause, this changes the structure of the mucous layer and its permeability. Our study suggested two different but complementary approaches to the problem. The first is an analysis of the metabolomic data present in the literature, which can actually prove the changes in the intestinal macrobiota to formulate a hypothesis of the relationship between autism and alterations of the defective intestinal layer. The second step is to decide the concentration of intestinal MUC2, because in all major intestinal diseases, including irritable bowel, there is a change in the intestinal permeability. This has always shown a decrease in mucus fractions in the layer structure. Research study[9] has shown that under these conditions bacterial enzymes can directly produce metabolites such as D-lactic acid and ammonia and can exert neurotoxicity with hormones and neurotransmitters identical to human hormones.[10] Metabolomic studies[11] also show that most of the discriminating metabolites in ASD, in a possible relationship, with altered intestinal permeability, belong to the metabolic pathways of tryptophan and purine. In addition, vitamin B6, riboflavin, biosynthesis tyrosine-tyrosine-tryptophan, pantothenate and CoA, pyrimidine metabolism, dimethylamine, hippuric acid, and phenylacetylglutamine differ much in autism compared with controls.[12] There is also a decrease in the plasma levels of p-hydroxyphenyl acetate, a metabolite associated with bifidobacteria and lactobacilli known to serve as an antioxidant both in circulation and in tissues.[13] This study also shows that the production of opioid peptides, such as casomorphin-7, or gliadinomorphine, is experimentally associated with autism[14]: these peptides pass through the brain barrier and bind to receptors and inhibit reuptake of the normal neuronal transmitter. All these results, according to our hypothesis, show a possible relationship with a modification of the intestinal microbe, depending on the change in the intestinal mucosa layer. A recent study[15] shows that as strains of particular bacteria normally present in the microbe, the intestine can proliferate abnormally, having as substrate the glycosylated spine and mucins, the main constituents of the protective layer. The loss of the barrier provides a gateway for bacterial spread, together with the increased potential for systemic transmission of antigens, virulence factors, other pathogens, and bacterial metabolites.[13] These factors can therefore influence brain functions and group vs. inflammatory processes. This process can obviously occur when, for reasons not yet clear, these glycosylated proteins, mainly transmembrane mucins, increase in concentration, or the mucins that form the MUC2 gel, do not sufficiently protect the epithelial mucosa and expose the terminal glycoside residue of the latter to the enzymatic action of the bacteria. We think that the latter hypothesis is very likely in autism. The experimental results of this study, see [Table 1], show that MUC2’s concentration in autistic people increases when compared with healthy control groups. This result is statistically significant: the p-value is 0.00124 and and can very well prove the hypothesis of a lack of defense of the intestinal epithelial mucosa. We hypnotized that this abnormality is a consequence of an MUC2 mucin (gel formation) and over expression. As in many bowl inflammatory processes, there is a simultaneous decrease of MUC1 transmembrane mucins. This effectively prevents MUC2 from attaching MUC1 to low glycosylate MUC1, so that the former reclines on themselves and join in the ninth place with hydrogen bridge bonds. The result is the formation of an incorrect protective layer with channels and exposure of the highly glycosylated MUC2 to bacterial attack. This fact is prone to an increase in intestinal bacterial flora with the growth of commensal strains that then go to degrade proteins such as casein or gluten in an incomplete way. From a rheological point of view, the imbalance between the transmembrane mucin fractions and the gelatinous mucins that form the mucins external to the intestinal epithelial cells modifies the spinnbarkeit value that measures the adhesion of the mucin and modifies its normal continuous layer, in a succession of bubbles, separated by channels The final result is that the intestinal mucosa layer’s alteration with a macrobiota forms opioids-like peptides These can finally enter the gut-brain axis through the channels formed in the intestinal mucosa. We can represent the consequence of this phenomenon as we can see in [Figure 2].
Figure 2: Hypothesis on the role of MUC1 and MUC2 in relationship to autism etiology

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   Conclusions Top


The intestinal microbiome is a part of human physiology; changes in the gut microbiota can modulate gastrointestinal physiology, immune function, and even behavior. Links between specific bacteria from the indigenous gut microbiota and phenotypes relevant to ASD raise the important question of whether microbial dysbiosis plays a role in the development or presentation of ASD symptoms. The genes of this human macrobiotic complex are capable of producing opioid-like peptides by certain foods that, in presence of an abnormal intestinal mucosal layer, play an important role in autism disorders. The use of enzymatic products such as plant-based proteases can begin to breakdown wheat and milk proteins while they are still in the stomach. This process facilitates not only an increase in the cleavage of proteins introduced with food, but also the degradation of peptide bonds present in opioids like peptides eventually formed.

Author contributions

Menicagli Roberto: concept, design, definition of intellectual content, manuscript preparation, manuscript editing and manuscript review. ORCID 0000-0001-9535-6859. Limodio Marta: concept, design, definition of intellectual content, manuscript preparation, manuscript editing and manuscript review. ORCID 0000-0003-3047-8374.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Gevi F, Zolla L, Gabriele S, Antonio M. Urinary metabolomics of young Italian autistic children supports abnormal tryptophan and purine metabolism. Mol Autism 2016;7:47.  Back to cited text no. 11
    
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Kałużna-Czaplińska J, Jóźwik-Pruska J, Chirumbolo S, Bjørklund G. Tryptophan status in autism spectrum disorder and the influence of supplementation on its level. Metab Brain Dis 2017;32:1585-93.  Back to cited text no. 12
    
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Srikantha P, Hasan Mohajeri M. The possible role of the microbiota-gut-brain-axis in autism spectrum disorder. Int J Mol Sci 2019;20:2115.  Back to cited text no. 13
    
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Lázaro CP, Pondé MP, Rodrigues LEA. Opioid peptides and gastrointestinal symptoms in autism spectrum disorders. Rev Bras Psiquiatr2016;38:243-6.  Back to cited text no. 14
    
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