Research Of The Identification And Functional Investigation Of Susceptible Loci For Sarcopenia

Sarcopenia is an age-related syndrome with decrease of skeletal muscle mass,strength and physical function.It limits exercise capacity in the elderly and inter-links with a series of aging disorders,seriously endangering public health.Lean body mass(LBM),which reflect skeletal muscle mass,is considered as one of diagnostic methods for sarcopenia.Sarcopenia is a highly heritable trait with heritability up to 60%,but its genetic mechanisms are still unknown.Only a few candidate genes were identified through Genome-wide association studies(GWASs)and their functional mechanisms are needed to explore.Purpose:The aim of this study was to identify susceptible loci for sarcopenia through efficient GWAS method and to study the functional mechanisms of the loci.Methods:The study contained two parts.Part I: Based on the database of Genotype And Phenotypes(db GAP),we conducted a GWAS of LBM in Framingham Heart Study(FHS)with 6,586 family participants,and a replication study in 3 independent samples(Women's Health Initiative African-American sub-sample(WHI-AA),N=847;WHI Hispanic sub-sample(WHI-HIS),N=445;Kansas-City Osteoporosis Study(KCOS),N=2,219).Part II: We performed functional annotations and predictions to the identified variants.Dual luciferase reporter gene assays were used to examine the changes of promoter activities.Electrophoretic mobility shift assays(EMSA)and DNA pull-down experiment were performed to explore the interaction between transcription factors and genomic variants.Results:Part I: SNPs rs551145,rs524533,rs571770 and rs545970 at 6p21.1 locus associated with leg LBM were identified at the genome-wide significance level(rs551145 p=3.40x10-9,rs524533 p=9.77x10-9,rs571770 p=9.02x10-9,rs545970 p=7.46x10-9)in the discovery sample and successfully replicated in two replication samples(rs551145 pWHI-AA=4.5x10-3,pKCOS=0.02;rs524533 pWHI-AA=3.2x10-3,pKCOS=0.03;rs571770 pWHI-AA=0.01,pKCOS=0.03;rs545970 pWHI-AA=0.07,pKCOS=0.03).The 4 SNPs have strong leveal of linkage disequilibrium(LD,r2=0.72-1.00)with each other in European.Part II: The functional annotations by Haplo Reg showed the 4 SNPs rs551145,rs524533,rs571770 and rs545970 are located in the inter-genic upstream region between TMEM151 B and NFKBIE gene and are cis-expression quantitative trait loci(cis-e QTL)of NFKBIE gene with promoter or enhancer activities.About 30 transcription factors were predicted combining with SNPs regions by PROMO.The dual luciferase reporter gene assays showed the mutations from major alleles to minor alleles of rs551145(C/T)and rs571770(C/G)resulted in decreased promoter activation.And the mutations between rs545970(C/G)and rs524533(C/T)resulted in increased promoter activation.The results of EMSA further showed that there were combinations between transcription factors and regions of 2 SNPs(rs524533 and rs571770),and the binding abilities were enhanced when the major alleles mutated to minor alleles.However,there was no combination between the other two(rs551145 and rs545970)and transcription factors.DNA pull-down results also showed there were more than one proteins combining with rs524533 variants.Conclusions:This study identified a novel locus 6p21.1 associated with leg lean mass.The mutations from major alleles to minor alleles of the SNPs rs524533 and rs571770 at this locus resulted in enhanced binding of transcription factor complexes to the SNP regions.Our study initially explored the functional mechanisms of the candidate genes discovered by GWAS,that is,the variants may affect the expression of lean body mass by changing the binding abilities between transcription factor complexes and promoter.The results provide a scientific basis for subsequent genetic etiology studies of sarcopenia.