| Background and ObjectiveOsteoporosis is a systemic skeletal disease characterized by low bone mineral density (BMD) and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (WHO). Nowadays, the incidence of osteoporosis is increasing yearly with the aging society. BMD is the best criterion for osteoporosis diagnosis, while bone turnover markers (BTM) reflect whole body rates of bone resorption and bone formation, and provide a dynamic assessment of the skeleton. In our opinion, bone turnover is active at early postmenopausal period, and then decreasing with age. But nowadays anti-resorption drugs are often effective in the treatment of postmenopausal osteoporosis. The problem is why the anti-resorption drugs are useful when the bone resorption is decreasing. And vitamin D plays an important role in maintaining calcium homeostasis and bone metabolism, which has close association with osteoporosis. Our study is to investigate the status of vitamin D and BTM in a large sample of postmenopausal women, so that to analyze the relationship among BTM, vitamin D, BMD and vertebral fractures.Osteoporosis is a common disease with a strong genetic component. Genetic factors contribute to osteoporosis by influencing not only bone mineral density but also bone turnover, and fractures. GWAS have found out many new single-nucleotide polymorphism (SNP) which had significant association with BMD in Caucasians. This study aims to verify the potential associations between candidate loci and BMD, BTM in Chinese postmenopausal women, these SNPs were reported by one recent GWAS in Iceland people, including rs1038304, rs6929137, rs4870044, rs1999805.Methods1. Collection of clinical samples and data 2070 cases of postmenopausal women in Beijing were selected through random cluster sampling method. Clinical data was collected, including general information, medical history, familial history, menstrual history. At last 1753 cases were into the study. All study subjects were required to have DXA lumbar X-ray. And whole blood samples and serum samples were required in order to detect vitamin D and BTM, includingβCTX and PINP.2. Selection of SNPSelection criteria of SNPs was based on 2 aspects following:①select genetic tag SNP of China Beijing Han provided by Hapmap.②The positive SNP site from abroad study.4 SNPs were selected:rs6929137 (G/A), rs1999805 (T/C) rs4870044 (G/A), rs1038304 (A/G)3. SNP genotypingAll the 4 SNPs were genotyped by Applied Biosystems 7900HT Fast Real-Time PCR System, which were operated by TaqMan probe technology.4. Test of bone turnover markers and vitamin DSerum concentrations of P1NP,β-CTX,25OHD3 were determined by a fully automated Roche electrochemiluminescence system((E170, Roche Diagnostics). The principle was electrochemiluminescence immunoassay (ECLI).5. Test of other biochemical markersSerum concentration of ALT, ALP, calcium and phosphorus were test by automated Siemens system.Results1. General data1753 cases were at last added to the database for statistical analysis. All of them were postmenopausal women, and the mean age was 65.3 years.2. Characteristics of BTMThe level of P1NP in the total study group was 58.39±29.32 (10.5-429.7) ng/ml, andβCTX was 0.45±0.21 (0.04-2.14)ng/ml. Both P1NP andβCTX were higher at early postmenopausal period, then both serum BTM decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Both BTM showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal, so as in vertebral site. Negative correlation were found betweenβCTX and femoral neck BMD, similar situation were found betweenβCTX and lumbar BMD, so as P1NP. 3. Characteristics of vitamin DThe mean level of 25 (OH) D3 was 13.15±5.39 (4-35.11) ng/ml. The prevelance of 25 (OH) D3<20 and≥30ng/ml was 89.9%,0.55% respectively. Negative correlation was found between vitamin D level and age.25 (OH) D3 showed significant differences among groups of femoral neck osteoporosis, osteopenia and normal. Both BTM had negative correlation with the level of 25 (OH) D3.4. Association analysis between SNPs and genotypesSubjects with osteoporosis and osteopenia were defined as cases and the subjects with normal BMD as controls. Subjects with vertebral fractures were defined as cases, and the subjects without were defined as controls. Subjects with higher bone turnover markers (above the 50th percentile) were defined as cases and others were defined as controls. Hardy-Weinberg equilibrium was calculated using the chi-square test. All SNPs were in Hardy-Weinberg equilibrium either in the cases or control groups.Compared osteoporosis/osteopenia group with control group, no association was found in allele frequencies between the case-control analyses. Compared vertebral fracture group with control group, no association was found in allele frequencies. Compared higher bone turnover group with control group, no association was found in allele frequencies.There was significant difference in P1NP level among genotypes of rs1038304. Subjects with genotype AA had higher P1 NP level than who with genotype AG or GG (P=0.015). Compared osteoporosis/osteopenia groups with control group, the P values of genotype frequencies of each SNP were all over 0.05, which had no statistical significance. Compared vertebral fracture group with control group, no association were found in genotypes either. There was no difference of BMD and bone turnover markers within genotypes.In dominance model, significant difference was found in P1NP level between the case and control group of rs1038304. In dominance and recessive models, there was no obvious difference of the genotype frequency of all above SNPs between the case and control group. Data analysis results calculated by Haploview 4.1 software show that rs1038304 and rs6929137 were in the same LD block, haplotype AG, GA and GG can be constructed. There was no significant difference in haplotypes frequency between case and control groups.Matching the age between vertebral osteoporosis group and normal group, the height, weight and average BMD were significantly lower than the control group. No difference was found in allele frequencies and genotypes between the case and control group, and no association was found between genotypes and BMD. No asscioation was found between genotypes and bone turnover markers.ConclusionIn conclusion, we found age-related changes in bone turnover markers in a large cross-sectional study of Chinese postmenopausal women from 49 to 108 years of age. Both P1NP andβCTX were higher at early menopausal period, and then both decreased up till the age of 70-79 years, and showed a trend of increasing older than 80 years. Increases in the levels of biochemical markers were correlated with low BMD. Most subjects were suffering vitamin D insufficiency, negative correlation was found between vitamin D level and bone turnover markers. So, monitoring vitamin D and bone turnover marker may be better for evaluating the comprehensive health condition of bone, which could be better for prevention and treatment of steoporosis,Genotypes of rs1038304 in chromosome 6q25.1 region had significant association with P1NP level. So, rs1038304 may have some association with bone formation, further studies are needed. SNPs (rs1999805, rs4870044, rs1038304, rs6929137) may have no association with BMD and vertebral fracture 'in Chinese postmenopausal women.
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