The Multi-omics Mechanism Research Of Gut Microbiome And Serum Lipidome In Familial Hypercholesterolemia

Background:Low-density lipoprotein cholesterol（LDL-C）is a key cause of atherosclerotic cardiovascular disease and a target for intervention.Familial Hypercholesterolemia（FH）is a co-autosomal dominant disorder of lipid metabolism characterized by elevated LDL-C levels.FH can be divided into the heterozygous and the homozygous forms based on the type of gene mutation and clinical characteristics.The homozygous FH is a severe and fatal rare disease,which can lead to severe atherosclerosis and premature coronary heart disease.Previous pedigree studies found that the gut microbiota of FH patients was significantly altered.However,the specific characteristics of the gut microbiome in hereditary and non-hereditary hypercholesterolemia and the interplay between the gut microbiome and host lipids remain to be systematically studied.Methods:We performed non-targeted lipidomics analysis on serum samples and deep metagenomic sequencing and metabolomics analysis on fecal samples from 50 patients with FH（including 35 heterozygous FH and 15 rare homozygous FH）,92 patients with primary hypercholesterolemia（PH）and 71 healthy controls,and constructed disease classifier with random forest model basing on differential multi-omics features.The characteristics of the gut microbiome were investigated in two dimensions（microbial abundance and functionality）,and an in-depth analysis of the gut virome was conducted to elucidate the ecological interactions among gut Phages,bacterial communities,and metabolites.Sequential mediation analysis was conducted to integrate multi-omics data to evaluate the correlation between microbial characteristics and fecal metabolites and their effects on host lipids.Finally,we demonstrated the microbe-host interaction in FH patients through fecal microbiota transplantation experiments.Results:In Chapter I,we investigated host clinical features and serum lipidomics,and identified a wide range of specific lipid fractions that could reflect the disease metabolic profile of the subjects.Triglyceride（40%）,ceramide,cholesteryl ester and stigmasteryl ester were the most highly expressed lipids in the HeFH group.The characteristic lipids highly expressed in HoFH group were mainly sphingomyelin,phosphatidylethanolamine,and methylphosphatidylcholine,among which PC（20:0e₁8:1）was highly expressed in both HeFH and HoFH patients.In Chapter 2,we found that compared with HC and PH subjects（i.e.,non-hereditary hypercholesterolemia）,FH patients（i.e.hereditary hypercholesterolemia）have significant differences in the structure and function of the gut microbiota,the composition of phages and fecal metabolites,and grouping is the most important factor explaining the variability of the gut microbiome（strains,phages,metabolites）.Metagenomics showed that there was gut microbial dysbiosis in FH patients,characterized by overgrowth of Faecalibacterium sp and Faecalibacterium prausnitzii.The microbial function was mainly enriched in a large number of amino acid synthesis,especially branched-chain amino acid related synthesis pathway,and the glycogen synthesis pathway was increased.The metametabolome showed that the levels of isobutyric acid,dehydroepiandrosterone and pilocarpine in feces of HeFH patients were significantly increased,while the levels of cadaverine and kmarosin were decreased.In HoFH patients,α-keto isovaleric acid was significantly increased,and pentadecanoic acid was significantly decreased.The differential metabolic enrichment pathways of the four groups were consistent with the functional pathways of intestinal microorganisms,reflecting that the changes of gut microbiome caused the corresponding changes in fecal metabolomics.Network analysis showed that gut phages and starins formed a strong and extensive positive correlation and co-occurrence relationship,while starins and fecal metabolites showed mild correlation.Faecalibacterium sp enriched in the HoFH group,Faecalibacterium prausnitzii and a large number of Faecalibacterium prausnitzii_BPhage,Faecalibacterium prausnitzii_CPhage,Faecalibacterium prausnitzii_DPhage and Faecalibacterium prausnitzii_EPhage were positively correlated.While Lachnospira eligens with high expression in HeFH group were negatively correlated with metabolite cytochalasin B levels.Based on multi-proteomics features,a disease classifier composed of eight markers including TG（6:0₁8:1₁8:2）,TG（10:0₁2:0₁8:1）,PC（20:0e₁8:1）,methyl eugenol,3-（2-hydroxyphenyl）propionic acid,gulonic acid,porphobilinogen and the bacteriophage Bacteroides B vulgatus Phage uvig₄66128 could accurately distinguish PH from FH and its different subgroups,with diagnostic rate as high as 88%.Mediation analysis showed that gut microbiota can affect lipid composition and lipid profile levels by regulating fecal metabolites,and the overgrowth of Faecalibacterium prausnitzii I can mediate LDL-C levels through eugenol methyl ether.In Chapter 3,the fecal microbiota transplantation（FMT）experiment demonstrated that the FH-disordered microbial colonization promoted the elevation of circulating cholesterol level and induced the phenotypes of metabolic syndrome including glucose and lipid metabolism disorders in the pseudo-germ free wild-type mice induced by Western diet.Dysregulation of the gut-liver axis and intestinal barrier function were identified by transcriptome analysis of the livers and colons.Furthermore,dysregulated FH-associated microbes lead to decreased intestinal tight junction protein Occludin,increased endotoxemia and pro-atherogenic inflammatory cytokines,which increase the risk of cardiometabolic diseases.Conclusion:Intestinal microbes and their metabolites are closely related to host lipids.This thesis describes a multi-omics landscape of hereditary and non-hereditary cholesterolemia and assists in the clinical differential diagnosis.It reveals that the imbalance of microbes in subjects with FH can lead to lipid metabolism disorders.This study provides a theoretical basis for the translational application of gut microbiome in clinical diagnosis and treatment of people with lipid metabolism disorders.