| Part 1 The association between genetic variants in the R-spondin/Wnt signaling network and osteoporosis riskObjective:This study aimed to explore the relationship between genetic variants of R-spondin/Wnt signaling pathway genes and osteoporosis risk,and to identify the genes and variants associated with osteoporosis.Methods:In this study,middle-aged and elderly people in Wuhan city were selected as the research subjects,and finally 1168 subjects that meet the criteria were included.The next-generation sequencing technology was used for targeted region sequencing in 15 key genes of R-spondin/Wnt signal pathway(RSPO1,RSPO2,RSPO3,RSPO4,LGR4,LGR5,LGR6,LRP5,LRP6,FZD1,FZD4,WNT3A,WNT4,WNT10B,WNT11)in 400menopausal women.Sequence Kernel Association Test(SKAT)and Proxy External Controls Association Test(Prox ECAT)were used for Region of Interest(ROI)and gene levels analyses;Multiple Linear Regression,Logistic Regression and Fisher's exact test were used for Single Nucleotide Variants(SNVs)analyses.For rare variants,the East Asian in Gnom AD database were used as external controls for gene and SNVs levels analyses.SNPscan technology was used to verify SNVs identified in the sequencing stage among 768middle-aged and elderly people.A fracture risk assessment tool was used to predict the ten year probability of osteoporotic and hip fracture.Results:A total of 228 osteoporosis patients and 172 controls were included in the sequencing stage,and the results showed that the ROI composed of low-frequency and rare varients in the R-spondin/Wnt signaling pathway was significantly associated with osteoporosis risk and 10-year probability of hip fracture(P<0.05).At the gene level,the LGR6 gene was significantly associated with lumbar spine T-score(P-FDR=0.045),Z-score(P-FDR=0.030),and osteoporosis risk(P-FDR=0.030);the LRP6 gene was significantly associated with the 10-year probability of osteoporotic fracture(P-FDR=0.003)and hip fracture(P-FDR=5.13×10-6).The Prox ECAT,based on the external controls of Gnom AD database East Asian population,found that the LGR4 gene was significantly associated with osteoporosis risk(Pweighted=1.34×10-4;P-FDRweighted=0.002).At the SNVs level,individuals with rs10920362 CT/TT genotype had a significantly higher risk of osteoporosis than the individuals with CC genotype(OR=2.01,95%CI=1.23-3.29,P=5.3×10-3).In additive model,with the increase of rs11178860 risk allele A,femoral neck BMD(?=-0.02,P=9.6×10-4),T-score(?=-0.18,P=8.6×10-3)and Z-score(?=-0.15,P=0.027)decreased significantly.The CT genotype of rare mutation rs750193863 were significantly correlated with the increase of lumbar spine BMD(?=0.52,P=5.02×10-6),T-score(?=4.31,P=6.39×10-6),and Z-score(?=4.42,P=2.16×10-6).Regarding the East Asian population in the Gnom AD database as external controls,Fisher's exact test showed that the rare non-synonymous variant rs201256460 was significantly associated with osteoporosis risk(OR=3.92,95%CI=1.41-10.96,P=0.023).A total of 356 osteoporosis patients and 412 controls were included in the verification stage,and the associations between rs10920362(OR=2.04,95%CI=1.48-2.81,P-FDR=1.36×10-4)and rs11178860(OR=1.75,95%CI=1.25-2.46,P-FDR=6.33×10-3)with osteoporosis risk were successfully verified.Using the East Asian population in the Gnom AD database as external controls,the association between rs201256460 and osteoporosis was verified(OR=4.40,95%CI=1.98-9.78,P-FDR=0.020).Analyzing the combined data,we found the existed association between rs10920362(OR=1.86,95%CI=1.44-2.41,P-FDR=2.50×10-5)and rs11178860(OR=1.97,95%CI=1.41-2.75,P-FDR=6.95×10-4)with osteoporosis risk.We also found that BMD,T-score and Z-score increased significantly with the increase of rs750193863 allele T(P-FDR<0.05).Conclusion:The R-spondin/Wnt signaling pathway,LGR6 and LGR4 genes were significantly associated with osteoporosis risk,and the LRP6 gene was associated with 10-year probability of fracture.The rare non-synonymous variant rs201256460 in the FZD4gene,the common variant rs10920362 in the LGR6 gene,and the common variant rs11178860 in the LGR5 gene were significantly associated with BMD and osteoporosis risk.Part 2 Association analyses between gut microbiota composition and osteoporosis riskObjective:This study aimed to systematically analyze the association between gut microbiota composition and osteoporosis risk,and to identify the disease-related gut microbiota.Methods:16S r RNA amplicon sequencing technology was used for sequencing of stool samples from 180 middle-aged and elderly people.Alpha diversity analysis,Beta diversity analysis,non-parametric test,Spearman correlation,ridge regression model,KEGG pathway enrichment analysis were used for data analyses,and the data were analyzed at three microbiota levels including phylum,family and genus.Results:A total of 77 osteoporosis patients and 103 controls were included in this study.1699 Operational Taxonomic Units(OTUs)were detected in 180 stool samples.1288OTUs were detected in the case group,and 1556 OTUs were detected in the control group.The number of bacteria kinds in the case group at each classification level was less than that in the case group.The Alpha diversity indices including observed?species,Chao,and ACE in the case group were significantly lower than those in the control group.Beta diversity analyses showed a significant difference of the gut microbiota composition between the cases and controls(F=3.413,P=1.00×10-4).At the phylum level,Firmicutes,Proteobacteria,and Actinobacteria were less abundant in the case group compared with the control group,and they were positively correlated with BMD,T-score,and Z-score.The relative abundance of Bacteroidetes in the case group was higher than that of the control s,and it was negatively related to BMD,T-score,and Z-score.At the family level,Lachnospiraceae,Bifidobacteriaceae,and Lactobacillaceae were less abundant in the case group compared with the control group,and they were positively correlated with BMD,T-score,and Z-score;the relative abundance of Bacteroidaceae in case group was higher than that in the control group,and the BMD,T-score,and Z-score decreased with the increases of Bacteroidaceae abundance.At the genus level,Blautia,Bifidobacterium,and Lactobacillus were less abundant in the case group compared with the control group,and they were positively correlated with the BMD,T-score,and Z-score;Bacteroides and Parabacteroides were more abundant in the case group compared with the control group,and the femur neck BMD and hip T-score decreased with the increase of Bacteroides abundance.85 KEGG metabolic pathways were significantly different between two groups.55(64.7%)pathways were enriched in the case group,and mainly included lipopolysaccharide biosynthesis,lipopolysaccharide biosynthetic protein,sphingolipid metabolism,et al.30(35.3%)pathways were enriched in the control group,and mainly included signal transduction,transport proteins,ribosomal biosynthesis,et al.Conclusion:The species richness of the osteoporosis case group was lower than that of the control group.The relative abundance of the phylum level microbiota(Firmicutes,Proteobacteria and Actinobacteria),the family level microbiota(Lachnospiraceae,Bifidobacteriaceae and Lactobacillaceae),and the genus level microbiota(Blautia,Bifidobacterium,and Lactobacillus)was positively correlated with BMD,T-score and Z-score,and negatively correlated with osteoporosis risk.The relative abundance of Bacteroidetes,Bacteroidaceae,Bacteroides and Parabacteroides w as negatively correlated with BMD,T-score and Z-score,and positively correlated with osteoporosis risk.A significant association was found between the gut microbiota and the osteoporosis risk.Part 3 Exploration of R-spondin/Wnt signaling network gene affecting osteoporosis risk via gut microbiotaObjective:This study aimed to explore the effects of genetic variants in the R-spondin/Wnt signaling network on gut microbiota composition,and to explore whether R-spondin/Wnt signaling network gene polymorphisms affect osteoporosis risk via changing gut microbiota composition.Methods:The high-frequency SNVs rs11178860 and rs10920362 associated with osteoporosis risk,were selected for analyses.We grouped with rs10920362 and rs11178860genotypes to analyze the microbiota differences in phylum,family,and genus levels.Then,we combined with statistical methods such as Spearman correlation analysis and generalized linear model to identify the microbiota associated with rs10920362 or rs11178860.Compared with the microbiota associated with osteoporosis risk,the overlapping microbiota associated with both osteoporosis risk and SNVs rs10920362 or rs11178860 were identified.Results:A total of 53 osteoporosis patients and 60 controls were included in this study.The results found that individuals with rs10920362 CC genotype had more unique OTU kinds(348)than individuals with CT/TT genotype(200);Individuals with rs11178860 GG genotype had less unique OTU kinds(88)than individuals with GA/AA genotype(569).Grouped with rs10920362 genotype,individuals with CT/TT genotype were significantly less abundant of Actinobacteria,Bifidobacteriaceae,and Bifidobacterium than individuals with CC genotype.Grouped with rs11178860 genotype,no microbiota with significant abundance difference was found.The generalized linear model adjusted for factors such as gender,age,body mass index(BMI),smoking,and drinking found that,rs10920362 CT/TT genotype was associated significantly with reduced relative abundance of Actinobacteria(?=-1.47,P=1.47×10-12),Bifidobacteriaceae(?=-1.83,P<0.0001)and Bifidobacterium(?=-1.83,P<0.0001).By comparing with the microbiota associated with osteoporosis risk,we found that Actinobacteria,Bifidobacterium and Bifidobacteriaceae were significantly associated with both osteoporosis risk and rs10920362 polymorphism.Conclusion:This study is the first time to find that the rs10920362 CT/TT genotype was significantly associated with the decreased relative abundance of Actinobacteria,Bifidobacteriaceae and Bifidobacterium.The host gene rs10920362 CT/TT genotype may increase the osteoporosis risk by reducing the relative abundance of Actinobacteria,Bifidobacteriaceae and Bifidobacterium.The exploration of the interaction between host genes and gut microbiota can provide new ideas for the prevention and treatment of primary osteoporosis. |
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