Molecular Ecology Of Intestinal Microbes In Juvenile-onset Adult Diabetes Type 2 Patients

Part1 Investigation of gut microbiota structural shifts in patients with maturity-onset diabetes of the young type 2ObjectiveMaturity-onset diabetes of the young?MODY?is a clinically and genetically heterogeneous monogenic disorders.Mutation in genes encoding glucokinase?GCK-MODY2?is one of the most common MODY subtypes.Long-term follow-up studies show that MODY2 patients have lower prevalence of microvascular and macrovascular complications than patients with T2DM.However,the underlying mechanism remains unknown.In recent years,a large number of evidence indicates that gut microbiota dysbiosis is associated with diabetes and diabetic complications,and genetic factor plays important roles in defining the structural of gut microbiota.The present study aims to investigate the compositional characteristics of the gut microbiota in patients MODY2,T2DM and healthy subjects,and further analyze the relationship between gut microbiota and clinical parameters.Our study contributes to clarifying the role of GCK gene in defining the structural of gut microbiota and provides new insight into the underlying mechanism of lower complications risk in patients with MODY2.MethodsFresh faecal samples from 26 MODY2 patients,18 age-matched subjects with T2DM and 21 healthy controls were collected.GCK mutations were analyzed by Sanger sequencing.The levels of FGF-21 and serotonin were measured with ELISA,and gut microbiota was analyzed using the Illumina MiSeq Sequencing System.Results1.T2DM patients exhibited with higher levels of total cholesterol,triglyceride,low density lipoprotein,hypersensitivity C-reactive protein,uric acid,HOMA-IR and glucose metabolism traits?including fasting and 2h-postprandial plasma glucose,glycated albumin,and glycated hemoglobin?than MODY2 patients and healthy controls?p<0.05?.MODY2 patients presented with lower levels of triglyceride and homocysteine?p<0.05?.2.MODY2 patients presented with higher levels of FGF-21 than T2DM patients and healthy controls?p<0.05?.The levels of serotonin in T2DM patients were significantly higher than MODY2 patients and healthy controls?p<0.05?.3.A total of 633 different OTUs were identified in this study.Among them,33 OTUs,48 OTUs and 26 OTUs were unique to MODY2 patients,T2DM patients and healthy controls,respectively.4.a diversity analysis showed that the observed_species indices and PD_whole_tree indices were significantly lower in MODY2 patients than T2DM patients.5.PCA and PCoA analysis showed that the overall compositions of the gut microbiota were significantly different among MODY2 patients,T2DM patients and healthy controls,and a clear separation was observed.6.The abundance of Firmicutes was increased,whereas the abundance of Bacteroidetes,Bacteroidia and Bacteroidales were decreased in T2DM group,compared to MODY2 patients and healthy controls.The proportions of Enterobacteriales,Enterobacteriaceae and Veillonellaceae were increased in T2DM group compared to healthy controls.7.33 OTUs differed significantly in abundance between MODY2 patients and healthy controls.14 OTUs including OTU₂92?Bacteroides?,OTU₄7?Romboutsia?,OTU₈5?Peptoclostridium?,OTU₁9?Escherichia_coli?,OTU₁03?Flavonifractor?and OTU₁3?Bacteroides?were increased in MODY2 group.19 OTUs including OTU₁1?Roseburia?and OTU₅3?Alistipes?were increased in healthy controls.8.54 OTUs differed significantly in abundance between MODY2 patients and T2DM patients,11 OTUs including OTU₂92,OTU₄7,OTU₈5 and OTU₄47?Bacteroidales?were enriched in MODY2 patients.The other 43 OTUs including OTU₁90?Parabacteroides?,OTU₂02?Desulfovibrio?,OTU₃1?Megasphaera?,OTU₃71?Cloacibacillus?,OTU₄51?Megamonas?,OTU₅3?Alistipes?and OTU₅7?Collinsella?were enriched in T2DM group.9.44 OTUs differed significantly in abundance between T2DM patients and healthy controls.36 OTUs including OTU₁9,OTU₂02,OTU₃71,OTU₄51,OTU₄9?Dorea?,OTU₅7 and OTU₄59?Comamonas?were enriched in T2DM group.8 OTUs including OTU₃?Prevotella₉?and OTU₆02?Prevotella₉?were enriched in healthy controls.10.OTU₂92 was negatively associated with the levels of fasting insulin and HOMA-IR.OTU₄7 was negatively associated with the levels of triglyceride.Most OTUs enriched in T2DM group were positively associated with the levels of fasting plasma glucose and glycated hemoglobin,as well as one of lipids metabolism traits including total cholesterol,triglyceride and low density lipoprotein.11.OTU₄47,OTU₁03 and OTU₁3 which were enriched in MODY2 group were negatively associated with the levels of FGF-21.OTU₃1 was positively associated with the levels of FGF-21.OTU₄9,OTU₃71 and OTU₄5?Eubacterium_hallii_group?which were enriched in T2DM group were positively associated with the levels of serotonin.OTU₃ was negatively associated with the levels of serotonin.12.The inferred metagenomic information showed that T2DM patients significantly enriched for carbohydrate metabolism and membrane transport,healthy controls significantly enriched for energy metabolism,amino acid metabolism and metabolism of cofactors and vitamins.Glycan biosynthesis and metabolism and cellular processes and signaling were significantly over represented in MODY2 patients.ConclusionsA distinct gut microbiota composition was present among MODY2 patients,T2DM patients and healthy controls.MODY2 patients were enriched in SCFAs producing bacteria in the gut,while the abundance of opportunistic pathogen was elevated in T2DM patients.Moreover,we identified a large number of OTUs that were significantly associated with metabolism traits,FGF-21 and serotonin.These results suggest that higher diabetic complications in T2DM patients may be partly mediated by gut microbiota dysbiosis,and GCK gene plays important roles in defining the structural of gut microbiota.Our finding will facilitate the early prevention and intervention for diabetic complications in future.Part 2 Clinical characteristics and genetic analysis in Chinese families with familial renal glucosuriaObjectiveFamilial renal glucosuria?FRG?is an inherited renal tubular disorder characterized by persistent isolated glucosuria with normal blood glucose in the absence of any other signs of tubular dysfunction.SLC5A2 gene mutation was the causative of FRG.This study aims to perform clinical characteristics and molecular genetic analysis in families with FRG.MethodsMolecular genetic analysis of SLC5A2 gene by PCR and Sanger sequencing was conducted in two unrelated non-consanguineous Chinese families with isolated glucosuria.Extensive laboratory test and physical examination were performed.In silico algorithms were used to predict the pathogenicity of identified mutation.We also analyzed the conservation of the amino acid residues.ResultsAmissense mutation?c.877A>T,p.Ser293Cys?was detected in proband 1 with mild glucosuria?24h urine glucose:8.06g?and in her father with normal phenotype.In family 2,a previously reported compound heterozygous mutation?c.229G>C,p.Gly77Arg;c.1540C>T,p.Pro514Ser?was identified in the proband?24h urine glucose:more than 10g?,and her healthy parents were heterozygous mutation carriers.Ser293,Gly77 and pro514 residue were found to be highly conserved throughout different mammalian species.ConclusionsSLC5A2 mutation S293C;G77R and P514S was responsible for the onset of FRG.Our study further confirmed the co-dominant inheritance trait with variable penetrance and provided new insight into the clinical and genetic spectrum of FRG.