Associations Of Genetic Variants In SEMA3A Pathway Genes And Gut Microbiota With The Risk Of Osteoporosis

Semaphorin3A（SEMA3A）pathway plays an important role in bone metabolism,promoting osteoblast formation and inhibiting osteoclast formation;SEMA3A pathway genes may be potential genetic susceptibility genes to osteoporosis（OP）.Emerging evidence also suggests that gut microbiota could affect bone metabolism,identify disease-related microbial biomarkers may provide new directions for OP treatment in future.Therfore,this study was conducted to explore the associations of genet ic variants in SEMA3A pathway genes,gut microbiota and OP risk through the following three parts.Part I:Association between genetic variants in SEMA3A pathway genes and the risk of osteoporosisObjective:To identify OP-related genetic variants in SEMA3A pathway genes,and further explore the corresponding functional role in the development of OP.Methods:In the present study,a total of 481 OP cases and 778 controls were recruited from two communities in Wuhan,Physical Examination Center and Rehabilitation Center of Union Hospital of Tongji Medical College from 2016 to 2019.The next-generation sequencing technology was applied for targeted region sequencing of SEMA3A,NRP1,PLXNA1,PLXNA2 and PLXNA3;the single nucleotide polymorphism（SNP）identified in the sequencing stage were genotyped using i MLDR technology.Logistic regression and multiple linear regression analyses were performed to explore the associations of common variants with OP and bone mineral density（BMD）.Fisher’s exact test was applied to compare the allele frequency distribution between groups.And simple linear regression was applied to analyze the associations between rare variants and BMD.Bioinformatics tools were used to annotate the function of disease-related variants,and functional experiments were performed to explore the possible mechanism of the variants affecting the development of OP.Results:In the sequencing stage,10 SNPs were found to associate with OP risk.In the replication stage,PLXNA2 rs2274446 was observed to associate with decreased risk of OP(CT/TT vs CC:OR=0.63,95%CI=0.46–0.87,P_FDR=0.027).In the combined stage,NRP1rs2070296(TT vs CC/CT:OR=1.63,95%CI=1.21–2.19,P_FDR=0.007)and rs2274446(TT vs CT vs CC:OR=0.72,95%CI=0.58–0.89,P_FDR=0.016)were found to significantly associate with OP risk;the TT genotype of rs2070296 was associated with decreased BMD at femoral neck(β=-0.03,P_FDR=0.004);the CT/TT genotype of rs2274446 was correlated with increased BMD at femoral neck(β=0.03,P_FDR=0.002);BMD at lumbar spine(β=-0.17,P_FDR=0.032)and femoral neck(β=-0.12,P_FDR=0.040)were decreased significantly with the increase of rs180868035 G allele.NRP1 rs180868035 T>G（p.I140L）was predicted to have damaging effect on protein function by SIFT and Mutation Taster.Through functional experiments,we found that NRP1 I140L significantly decreased the expression of osteoblast-specific genes,inhibited proliferation of osteoblast,while promoted osteoblast apoptosis;moreover,NRP1 I140L significantly increased the expression of osteoclast-specific genes,promoted proliferation of osteoclast,while inhibited osteoclast apoptosis.Conclusions:NRP1 rs2070296 and PLXNA2 rs2274446 were significantly associated with the risk of OP and BMD at femoral neck,and NRP1 rs180868035 was associated BMD at lumbar spine and femoral neck significantly.Functional experiments indicated that nonsynonymous variant rs180868035（p.I140L）might influence bone mass by reducing bone formation and promoting bone resorptionPart II:Association between gut microbiota and prevalent osteoporosis riskObjective:To explore the alternations of gut microbiota in OP cases and identified disease-related bacterial taxa.Methods:A total of 108 elderly were included in the study who were recruited from the Rehabilitation Center,Union Hospital of Tongji Medical College in Wuhan City during2018–2019 and provided stool samples.Absolute quantification 16S r RNA sequencing approach was applied for identification and absolute quantification of microbiota.The Alpha diversity of gut microbiota was assessed with the Observed Species,Chao1,ACE,Shannon,Simpson and Coverage index.Permutational multivariate analysis of variance（PERMANOVA）was performed to evaluate between-group differences of microbial community composition,and linear discriminant analysis effect size（LEf Se）analysis was performed to assess between-group differences of microbiota.Results:In this study,44 OP cases and 64 controls were included.There was no significant between-group differences among the Alpha-diversity indices（P>0.05）.PERMANOVA test revealed different microbial community composition between OP cases and controls（P=0.022）.According to the results of LEf Se,we found that the absolute abundances of the Bacteroidetes phylum,Bacteroidia class and Bacteroidales order were enriched in the OP cases.At the family level,compared with the controls,the OP cases had higer absolute abundance of Bacteroidaceae,Lactobacillaceae and Porphyromonadaceae.At the genus level,the abundance of Bacteroides,Lactobacillus,Clostridium＿Xl Va,Eggerthella,Eisenbergiella and Coprobacillu genera elevated and the Veillonella decreased in the OP cases referring to absolute abundance quantifications.Conclusions:The gut microbota composition of OP cases was significant different from controls,which mainly reflected in the changes of several specific taxon absolute abundance belongs to Bacteroidetes and Firmicutes phyla.Our study suggest that gut microbiota alterations may contribute to the development of OP.Part III:The role of gut microbiota in the association between genetic variants in SEMA3A pathway genes and the risk of osteoporosisObjective:To explore the role of gut microbiota in the association between genetic variants in SEMA3A pathway genes and OP risk.Methods:Based on part I and part II,study subjects with both gut microbiota data and genotyping data were included in the present study.OP-related common variants NRP1rs2070296 and PLXNA2 rs2274446 were included in this study.The Alpha diversity of gut microbioat was assessed with the Observed Species,Chao1,ACE,Shannon,Simpson and Coverage index.PERMANOVA was performed to evaluate between-genotype differences of microbial community composition,and LEf Se was conducted to assess between-genotype differences of microbiota.Mediation analysis was then performed to explore the mediating effect of gut microbiota in the association between SNPs and OP.Logistic regression model was applied to detect gene-gut microbiota interaction.Results:In this study,37 OP cases and 57 controls were included.Individuals with NRP1 rs2070296 TT genotype had lower Chao1 and ACE than those with CC/CT genotype（Chao1:P=0.040;ACE:P=0.039）;compared with PLXNA2 rs2274446 CC genotype carriers,the Alpha diversity indices of Simpson was lower and Shannon was higher in those with CT/TT genotype（Shannon:P=0.038;Simpson:P=0.042）.PERMANOVA test revealed different microbial community composition between individuals with rs2274446CT/TT genotype and CC genotype carriers（P=0.020）.LEf Se analysis showed that individuals with rs2274446 CT/TT genotype had lower absolute abundances of Bacteroidaceae and Bacteroide than those with CC genotype.Comparing with the results of part 2,Bacteroidaceae and Bacteroides were found to significantly associate with both rs2274446 and OP risk.Mediation analysis suggested that Bacteroidaceae and Bacteroides had significant indirect effect on the association of rs2274446 and OP risk.No interactions between rs2070296 or rs2274446 and gut microbiota were found in this study.Conclusions:NRP1 rs2070296 and PLXNA2 rs2274446 were associated with decreased diversity of gut microbiota,indicating that gut microbiota may be influenced by host genetic factor.Bacteroidaceae and Bacteroides presented significant indirect effects on the relation between PLXNA2 rs2274446 and OP,suggesting such association may be partly realized by regulating gut microbiota.