| Objective:Leucine-rich repeat containing G protein-coupled Receptor 4(LGR4)was found to play an important role in body weight control through regulating the white-to-brown fat transition.A genome-wide association study founded that a rare nonsense mutation in the LGR4 gene was strongly associated with low BMD and with osteoporotic fractures.Our objective was to determine whether polymorphisms in the LGR4 gene were related to variations in peak bone mineral density(BMD)and obesity phenotypes in Chinese nuclear families with female offspring.Methods:22 SNPs located in the LGR4 gene were genotyped in a total of 1296 subjects from 390 Chinese nuclear families comprising 780 parents and 478 female children and 38 male children.The dual-energy X-ray absorptiometry was used to measure BMD of the lumbar spine,femoral neck and total hip,as well as total fat mass(TFM),total lean mass(TLM)and trunk fat mass.Meanwhile,weight and height were measured.Body mass index(BMI)was calculated as weight in kilograms divided by the square of height in meters.The percentage of lean mass(PLM)and the percentage of fat mass(PFM)was the ratio of lean mass to body weight and fat mass to body weight respectively.The selection of evaluated tagSNPs depended on the dbSNP database and HapMap database.Twenty-one tagSNPs(rs11030016,rs7936621,rs1083518,rs4542364,rs2447995,rs4128868,rs4923447,rs12787344,rs1102998,rs2219783,rs1531557,rs6484295,rs11030014,rs4923445,rs12796247,rs4074516,rs16917037,rs4514364,rs7927234,rs10835171 and rs10835173)and the 376C>T site were selected.Genotyping was identified by the 3 730XL genetic analyze sequenator using imLDR multiplex kit.The significance level of linkage disequilibrium(LD)between the LGR4 gene markers was assessed on the basis of observed haplotype and allelic frequencies by using Haploview version 4.2.We then obtained haplotypes from the population genotype data by using the PHASE program(version 2.1).The association between SNPs and haplotypes of LGR4 and peak BMD,TFM,TLM,percentage fat mass(PFM),percentage lean mass(PLM),trunk fat mass and body mass index was measured using quantitative transmission disequilibrium test(QTDT)and ANCOVA.Results:1.C376T was excluded from further analysis when only one genotype(GG)was found after genotypic analysis.The minor allele frequency of rs 11029986 in this study was lower than 0.05.Therefore,we also excluded this SNP from the subsequent statistical analyses.For ANCOVA tests,rs7936621 were associated with femoral neck BMD and total hip BMD(P=0.023,0.020).Using QTDT for within-family association analyses,significant associations were found between rs7936621 and femoral neck BMD,total hip BMD,lumbar spine BMD(P=0.031,0.020,0.017 respectively),After 1000 permutations significant associations were also found between rs7936621 and femoral neck BMD,total hip BMD,lumbar spine(P=0.038,0.029,0.031 respectively).We conducted haplotype analyses using genotype data from all SNPs and obtained five blocks.rs7936621 is in block 3.For the haplotypes,we found significant association between GAG in block 2 and total hip BMD,lumbar spine BMD(P=0.048,0.047),between TACTTC and CGCCTC in block 5 and lumbar spine BMD(P=0.047,0.033)by using ANCOVA.For the within family association of haplotypes,significant association were detected between AGCGT in block 3 and total hip BMD(P=0.032),between TGCTCC in block 5 and femoral neck BMD and total hip BMD(P=0.015,0.041),between TACTTC in block 5 and lumbar spine BMD(P=0.040)by using QTCT.Furthermore,after 1000 permutations significant within family associations were kept between AGCGT in block 3 and total hip BMD(P=0.043),between TGCTCC in block 5 and femoral neck BMD(P=0.025),between TACTTC in block 5 and femoral neck BMD and lumbar spine BMD(P=0.024 and 0.037 respectively).2.Using ANCOVA tests,significant association was found between rs2219783 and PFM and PLM(P=0.022,0.018),between rs7927234 and TFM,PFM and PLM(P=0.013,0.046 and 0.042 respectively),between rs4923445 and BMI(P=0.012).Performing QTDT for within-family association analyses,significant associations were found between trunk fat mass and rs2447995,rs 10835173,rs7936621,rs4128868 and rs1103001(P=6.00E-29,P=6.00E-29,P=8.00E-29,P=7.00E-29,P=1.00E-28 respectively).Using QTDT for within-family association after 1000 permutations,significant associations were found between rs10835171 and trunk fat mass(P=0.045),rs6484295 and BMI(P=0.043).By using Haploview we calculated 5 blocks.rs10835171 and rs6484295 were in block 2.For ANCOVA tests,significant association were found between GAA in block 2 and TFM,BMI and trunk fat mass(P=0.033,0.014,0.012 respectively),between TTG in block 4 and TFM,TLM,BMI,and trunk fat mass(P=0.017,0.029,0.016,0.011 respectively).Using QTDT for within-family association analyses,significant association were detected between trunk fat mass and CC,CT and TC in block 1(P=1.00E-28,7.00E-29 and 5.00E-29 respectively),between GAA in block 2 and trunk fat mass and BMI(P=0.043 and 0.031 respectively),between GACCC in block 3 and trunk fat mass(P=0.049),between TTA in block 4 and TFM(P=0.033).Furthermore,after 1000 permutations significant associations were kept between GAA in block 2 and trunk fat mass and BMI(P=0.027 and 0.019 respectively).Conclusion:1.Our study found that rs7936621 of LGR4 was associated with the peek BMD of the lumbar spine,femoral neck and total hip,rs793 6621 was located in block 3 and AGCGT in block 3 was related to total hip BMD.We concluded rs7936621 was the quantitative trait loci underlying peak BMD.2.Our study observed that rs10835171 was associated with trunk fat mass and rs6484295 was correlated with BMI,rs10835171 and rs6484295 were in block 2 and GAA in block 2 was also related to trunk fat mass.We concluded rs10835171 and rs6484295 were the quantitative trait loci underlying the phenotype of obesity.3.The LGR4 gene might be a new candidate for osteoporosis and obesity in Chinese Han ethnic females... |
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