Diversity Of Gut Microbiota Associated With Congenital Insensitivity To Pain With Anhidrosis

Background:Congenital insensitivity to pain with anhidrosis(CIPA),a rare autosomal inherited disorder,has been estimated to occur in 1 in 125 million individuals.The main clinical manifestations of this disease are congenital insensitivity to pain,absence of sweating,variable mental retardation,self-mutilation,recurrent high fever,and bone and joint fractures.There are many pathological changes in CIPA,including loss of sympathetic neurons,unmyelinated fibers in sensory neurons,small diameter myelinated fibers in sensory neurons,some brain neurons,bone mineral density and norepinephrine deficiency.Disorders of NGF/TrkA signaling pathway underlie all pathological changes in CIPA,however,the mechanism by which NGF/TrkA signaling pathway is involved in regulating symptoms in CIPA is not fully understood.For these pathological changes,the diagnosis of CIPA relies mainly on history,general examination,sensory nerve conduction test,quantitative sensory test,motor function assessment and genetic testing.In addition,recent studies have proposed that cardiac 123Imeta-iodobenzylguanidine scintillation detection can be used as a semi-quantitative method for the diagnosis of CIPA,however,there is still a lack of non-invasive quantitative diagnosis strategy for CIPA.In recent years,a large number of studies have shown that gut microbiota disorder can participate in the pathological mechanism of diseases through the "gut-brain axis".Meanwhile,numerous studies have used gut microbiota as a diagnostic biomarker.Studies have found that NGF/TrkA signaling pathway plays an important role in regulating the "gut-brain axis",and CIPA is a classic disease model of visceral hypoesthesia accompanied by emotional and intellectual development disorders due to NGF/TrkA signaling pathway disorder.Therefore,this study aimed to explore the pathological changes of gut microbiota dysregulation in CIPA,focusing on the structural changes of gut microbiota in CIPA,and exploring the gut microbiota related to the pathogenesis of CIPA.Methods:29 CIPA subjects and 28 HC were recruited using hospital records and social networking platforms.A 16S rRNA analysis was performed to detect the fecal samples,and the structural diversity of gut microbiota in CIPA patientswas comprehensively analyzed based on bioinformatics and statistical analysis.Based on the differential gut microbiota,the diagnostic model of CIPA was constructed by receiver operating characteristic curve,and area under the curve was used to determine the diagnostic efficacy of the model.Subsequently,gut microbiota from CIPA patients were transplanted into pseudo germ-free mice,and the effects of gut microbiota on host phenotypes were further observed by behavioral methods including mechanical pain threshold test,thermal pain threshold test,open field test and Marris water maze.Combined with molecular biology experiments and high-throughput sequencing,we further clarified the possible role of gut microbiota in the pathogenesis of CIPA.Results:A total of 29 CIPA patients and 28 healthy controls were recruited.The results showed that there was significant difference in the composition of gut microbiota between the two groups.Specifically,compared to HC group,the relative abundance of 11 gut microbiota in CIPA patients was significantly decreased,and the relative abundance of 7 gut microbiota was significantly increased.The presence of multiple differential gut microbiota associated with pain relief and cognitive impairment was consistent with the variation of relative abundance in CIPA patients.In addition,the functional pathways of bacterial toxin,phosphatidylinositol signaling system,streptomycin biosynthesis,and amino acid and nucleotide glucose metabolism pathways were significantly enriched in CIPA,and phosphatidylinositol signaling system was an important pathway in the pathogenesis of CIPA.Studies on the diagnostic efficancy of CIPA based on the differential gut microbiota showed that the diagnostic efficacy of single dominant microbiota for CIPA was low,and the diagnostic model based on the differential gut microbiota at different classification levels could greatly improve the diagnostic titer for CIPA.The differential gut microbiota at genus level(AUC=0.893)and species level(AUC=0.881)had the highest diagnostic efficancy of CIPA as a whole,which may be a new non-invasive quantitative biomarker of CIPA.In the experiment of fecal microbiota transplantation,pseudo germ-free mice that received fecal microbiota from CIPA patients showed reduced pain sensitivity,cognitive dysfunction,and normal motor ability.Meanwhile,fecal microbiota from CIPA patients reduced the expression level of inflammatory factors in the colon tissue of host mice such as TNF-α,IL-6 and IFN-γ,and damaged the synaptic structure of the hippocampal tissue of host mice.In addition,the expression levels of postsynaptic dense 95(PSD-95)and brain-derived neurotrophic factor(BDNF)in hippocampus were decreased.The analysis of differential gut microbiota further found that the changes in the microbiota associated with changes in pain degree and cognitive level were consistent with those in the recipient mice.Conclusions:In this study,we comprehensively analyzed the structure of gut microbiota in patients with CIPA,and found that multiple gut microbiota associated with pain relief,cognitive function,and inflammatory response were abnormally expressed in patients with CIPA.Moreover,multiple metabolic pathways associated with pain and neurodevelopment were significantly enriched in CIPA.These results suggest that imbalance of gut microbiota may be involved in the pathogenesis of CIPA through multiple metabolic pathways,suggesting that therapeutic strategies to manipulate the gut microbiota may help alleviate symptoms of CIPA patients.In addition,the differential gut microbiota at genus and species levels had high efficacy for the diagnosis of CIPA as a whole,respectively,and these two classification models may be potential biomarkers for the identification of CIPA.Subsequently,FMT experiments in animal model further provided evidence that the gut microbiota of CIPA patients may be involved in the regulation of pain by influencing the inflammatory state of the colon,and also in the regulation of cognitive function by disrupting the synaptic structure of the hippocampal tissue.Overall,this study comprehensively explored the possible role of gut microbiota in regulating pathogenesis of CIPA.