Study On Female Osteoporosis And Polymorphisms Of Related Genes

PartⅠINVESTIGATION OF BONE MINERAL DENSITY OF FEMALE ADULTS IN CHONGQING AREAObjective To investigate bone mineral density (BMD) changing pattern at different skeletal sites of female adults in Chongqing area,study BMD change with different influential factors,evaluate the value of different sites in the diagnosis of primary osteoporosis, and provide scientific basis for diagnosis and prevention of osteoporosis (OP). Methods BMD of the proximal femur and lumbar spine (anteroposterior position) in 308 female adults was measured by NORLAND XR-46 dual-energy X-ray absorptiometer.BMD of proximal femur and lumbar spine with age-related changes was fitted in the whole healthy women population by eight kinds of regression models. Best model equations of fitting were found and the reference databases of BMD in women were established.Results (1) The peak bone mass (PBM) of the proximal femur occurred in the age group 40～44, while that of the lumbar spine (L2-4) in 30～34.BMD would gradually decrease along with the age increasing when it achieved PBM. BMD significantly decreased in the 50～65 age groups, and BMD change of Wards triangle area showed 5 years earlier than that of others and obviously decreased. (2) Age and body weight could influence BMD values of proximal femur and L2-4. Body weight only had larger effect on trochanter, but smallest effect on Wards triangle. (3) A cubic regression model was the better model to establish a normal BMD reference curve. R square of cubic regression curve was maximum value. R square in femoral neck was larger than that of other sites. (4) BMD of each site correlated with those of other (P<0.01).There was no significant difference in the incidence rate of OP in different sites (P>0.05). Conclusions BMD of female adults in Chongqing area changed along with increasing age. The PBM age of proximal femur was older than that of L2-4. BMD significantly decreased in the perimenopausal period and early period after menopause. Age and body weight had certain effect on BMD. To keep proper body weight may be in favor of preventing OP. The women reference databases of BMD in Chongqing area were established by using cubic regression model. The BMD of different sites should be measured according to different age groups in the diagnosis of OP.PartⅡSTUDY ON SOME RELATED GENES OF OSTEOPOROSIS IN POSTMENOPAUSAL WOMEN1 STUDY ON POLYMORPHISMS OF CYP19 GENEObjective To investigate the association of the single nucleotide polymorphism (SNP) site Val80 and a TTTA short tandem repeat (STR) in cytochrome P450 family 19 (CYP19) gene with bone mineral density (BMD) and postmenopausal osteoporosis (PMO). Methods (1) Section 1: 200 postmenopausal women in Chongqing were randomly enrolled in the study. Section 2 ( case-control study): involving 78 femoral neck PMO patients vs. 122 controls and 108 L2-4 PMO patients vs. 92 controls. (2) Genotypes for the Val80 polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotypes for the (TTTA)n polymorphism were determined by gene scan and DNA sequence methods. (3) BMD for the proximal femur and L2-4 was measured by NORLAND XR-46 dual-energy X-ray absorptiometry. Results Section 1: (1) The (TTTA)n <10 and (TTTA)n≥10 alleles were designated S and L, respectively. The frequencies of genotypes in 200 postmenopausal women were as follows: GG (19.5%), GA (44.5%), AA (36.0%) for Val80 polymorphism; And SS (24.5%), SL (54.0%), LL (21.5%) for (TTTA)n polymorphism. The genotype distribution was met with Hardy-Weinberg equilibrium (P>0.05). (2) After adjusting for age, menopausal period, height, and weight, no significant difference in BMD was seen in different Val80 genotypes in postmenopausal women (P > 0.05); and BMD was higher in postmenopausal women with LL genotype than those with SS genotype or the combined SS and SL genotype for (TTTA)n polymorphism at L2-4 (P<0.05) ,but no significant difference in BMD at other sites was seen in different genotypes (P>0.05). Section 2 (case-control study): (1) The genotype distribution in each group was met with Hardy-Weinberg equilibrium at each skeletal site (P>0.05). (2) There were no significant differences in the genotype and allele distributions of Val80 polymorphism between PMO group and control group at the femoral neck and L2-4 sites (P>0.05). At the femoral neck site, there were no significant differences in the genotype and allele distributions of (TTTA)n polymorphism between PMO group and control group (P>0.05). But the SS genotype and the S allele frequencies of PMO group were significantly higher than those of control group at the L2-4 (P<0.05). (3) After adjustments for age, years since menopause, menopausal age, and body mass index, Logistic regression analyses revealed only the (TTTA)n polymorphism remained significantly associated with PMO at the L2-4 (P<0.05). The subjects with the combined SS and SL genotype had higher risk of PMO compared with those with the LL genotype (adjusted OR2.246, 95% CI 1.070～4.715 , P=0.032). Conclusions The (TTTA)n polymorphism in the forth intron of CYP19 gene may contribute to the genetic regulation of BMD in postmenopausal women and be independently associated with PMO at the L2-4. The presence of the S allele may dominantly increase the risk of PMO at the L2-4. The results also showed Val80 polymorphism in the third exon of CYP19 gene may not be associated with BMD and PMO.2 STUDY ON POLYMORPHISMS OF ESTROGEN RECEPTORΒGENEObjective To investigate the association of the single nucleotide polymorphism (SNP) site G1082A and a cytosine adenine (CA) short tandem repeat (STR) in estrogen receptorβ(ESR2) gene with bone mineral density (BMD) and postmenopausal osteoporosis (PMO). Methods Study subjects were same as PartⅡ.1. Genotypes for the G1082A polymorphism were determined by PCR-RFLP. Genotypes for the (CA)n polymorphism were determined by gene scan and DNA sequence methods. Results Section 1: (1) Nine different allelic variants, containing 18, 19, 20, 21, 22, 23, 24, 25, and 26 CA repeats, were detected. There was a bimodal distribution of alleles, with two major peaks at 19 and 24 repeats and a very low distribution of the 21-repeat allele and 26-repeat allele. 21 genotypes were present in the subjects. The (CA)n <22 and (CA)n≥22 alleles were designated S and L, respectively. The frequencies of genotypes in 200 postmenopausal women were as follows: SS (25.0%), SL (46.0%), LL (29.0%) for (CA)n polymorphism; and rr (43.0%), Rr (42.0%), RR (15.0%) for G1082A polymorphism. The genotype distribution was met with Hardy-Weinberg equilibrium (P > 0.05). (2) After adjusting for age, menopausal period, height and weight, no significant difference in BMD was seen in different G1082A genotypes in postmenopausal women (P>0.05); and BMD was higher in subjects with SS genotype than with SL/LL/SL+LL genotypes for (CA)n polymorphism at femoral neck (P<0.05). No significant difference in BMD at other sites was seen in different genotypes (P>0.05). Section 2 (case-control study): (1) The genotype distribution in each group was met with Hardy-Weinberg equilibrium at each skeletal site (P>0.05). (2) There were no significant differences in the genotype and allele distributions of G1082A polymorphism between PMO group and control group at the femoral neck and L2-4 site (P>0.05). However, LL genotype and L allele frequencies of PMO group were significantly higher than those of control group at both the two sites (P<0.01). (3) After adjustments for age, years since menopause, menopausal age, and body mass index, Logistic regression analysis showed that the subjects with the combined SL and LL genotype for (CA)n polymorphism had increased risk of PMO compared with those with the SS genotype both at the femoral neck (adjusted OR 4.923, 95% CI 1.986～12.203 , P=0.001) and the L2-4 (adjusted OR 2.267, 95% CI 1.121～4.586, P=0.023). Conclusions The study revealed that the CA repeat number range, alleles,and distributions had race and regional difference. The (CA)n polymorphism in the fifth intron of the ESR2 gene may contribute to the genetic regulation of BMD in postmenopausal women, SS genotype may have some beneficial effect on BMD. The (CA)n polymorphism may be independently associated with PMO at the femoral neck and L2-4 site. The presence of the L allele may dominantly increase the risk of PMO at the two sites. The distribution of G1082A genotypes had race diversity. The results also showed the G1082A polymorphism in the fifth exon of the ESR2 gene may not be significantly associated with BMD and PMO.3 STUDY ON AN ANDROGEN RECEPTOR CAG REPEAT POLYMORPHISMObjective To study the association of a CAG repeat polymorphism of the androgen receptor (AR) gene with bone mineral density (BMD) and postmenopausal osteoporosis (PMO). Methods Study subjects were same as PartⅡ.1. Genotypes for the (CAG) n polymorphism were determined by gene scan and DNA sequence methods. Results Section 1: (1) 11 different allelic variants of CAG repeats and 16 genotypes were detected. The (CAG)n < 25 and (CAG)n≥25 alleles were designated S and L, respectively. The frequencies of genotypes in 200 postmenopausal women were as follows: SS (23.5%), SL (45.0%), LL (31.5%). The genotype distribution was met with Hardy-Weinberg equilibrium (P>0.05). (2) After adjusting for age, menopausal period, height, and weight, we observed that no significant difference in BMD was seen in different genotypes (P>0.05). Section 2 (case-control study): (1) The genotype distribution in each group was met with Hardy-Weinberg equilibrium at each skeletal site (P>0.05). (2) There were no significant differences in the genotype and allele distributions of (CAG)n polymorphism between PMO group and control group at the femoral neck and L2-4 sites (P>0.05). (3) After adjustments for age, years since menopause, menopausal age, and body mass index, Logistic regression analyses showed the subjects with the combined SL and LL genotype had no increased risk of PMO compared with those with the SS genotype both at the femoral neck and the L2-4 site. Conclusions The (CAG)n polymorphism in the first exon of AR gene may not contribute to the genetic regulation of BMD in postmenopausal women and be associated with PMO.4 MICROSATELLITE DNA POLYMORPHISMS OF CYP19, ESR2, AND AR GENE IN CHONGQING POSTMENOPAUSAL WOMEN (HAN POPULATION)Objective To study microsatellite DNA polymorphisms of the CYP19 gene , ESR2 gene, and AR gene in Chongqing postmenopausal women (Han population) and to evaluate its value in population genetics analysis. Methods Alleles and genotypes for microsatellite DNA polymorphism were determined by gene scan and DNA sequence methods in 200 unrelated postmenopausal women (Han population) in Chongqing region, The three DNA loci were (TTTA)n polymorphism of the forth intron in CYP19 gene, (CA)n polymorphism of the fifth intron in ESR2 gene, and (CAG)n polymorphism of the first exon in AR gene. Results The (TTTA)n polymorphism of CYP19 gene, (CA)n polymorphism of ESR2 gene, and (CAG)n polymorphism of AR gene were detected 5, 9, and 11 alleles, respectively. The sizes were 220～243 bp, 170～186 bp, 305～341 bp. The observed heterozygosity was 0.8250, 0.8550, and 0.8650, respectively. The expected heterozygosity was 0.7607, 0.8492, and 0.8677, respectively. The polymorphism information content (PIC) was 0.7195, 0.8303, and 0.8512, respectively. All the three microsatellite DNA were in the Hardy-Weinberg equilibrium (P>0.05). Conclusions Highly genetic polymorphisms of CYP19 gene (TTTA)n, ESR2 gene (CA)n, and AR gene (CAG)n loci were observed in Chongqing postmenopausal women (Han population). The three microsatellite DNA are ideal genetic markers, which could be used to population association analysis.PartⅢSTUDY ON SNPs AND HAPLOTYPES OF OPG GENE IN POSTMENOPAUSAL WOMEN1 STUDY ON SNPs OF OPG GENEObjective To investigate the association of the single nucleotide polymorphism (SNP) sites A163G, T950C, and G1181C in osteoprotegerin (OPG) gene with bone mineral density (BMD) and postmenopausal osteoporosis (PMO). Methods Study subjects were same as PartⅡ.1. Genotypes for the A163G, T950C, and G1181C polymorphism were determined by PCR-RFLP. Results Section 1: (1) The frequencies of genotypes in 200 postmenopausal women were as follows: AA (13.0%), AG (42.0%), GG (45.0%) for A163G polymorphism; TT (35.5%), TC (44.0%), CC (20.5%) for T950C polymorphism. And GG (48.0%), GC (40.5%), CC (11.5%) for G1181C polymorphism. The genotype distribution of each polymorphism site was met with Hardy-Weinberg equilibrium (P>0.05). (2) After adjusting for age, menopausal period, height, and weight, BMD was lower in postmenopausal women with AG/GG/AG+GG genotype than those with AA genotype for A163G polymorphism at femoral neck, Wards triangle, and L2-4 (P<0.05) ,but no significant difference in BMD at the trochanter; and no significant difference in BMD was seen in different T950C & G1181C genotypes in postmenopausal women (P>0.05). Section 2 (case-control study): (1) The genotype distribution in each group was met with Hardy-Weinberg equilibrium at each skeletal site (P > 0.05). (2) The GG genotype frequencies of PMO group were significantly higher than those of control group both at the femoral neck and the L2-4 (P<0.05) . The G allele frequency of PMO group were significantly higher than those of control group at the femoral neck (P<0.05), but not at the L2-4 (P>0.05). There were no significant differences in the genotype and allele distributions of T950C & G1181C polymorphism between PMO group and control group at the two sites (P>0.05). (3) After adjustments for age, years since menopause, menopausal age, and body mass index, Logistic regression analyses revealed the A163G and T950C polymorphism remained significantly associated with PMO at the femoral neck (P < 0.05). Conclusions A163G, T950C, and G1181C genotype distributions of OPG gene had evident race and regional diversity. A163G polymorphism in the promoter region of the OPG gene may contribute to the genetic regulation of BMD in postmenopausal women and be independently associated with PMO at the femoral neck. The presence of the G allele may dominantly increase the risk of PMO at the femoral neck. T950C polymorphism in the promoter region of OPG gene may not be associated with BMD. The influence of T950C on PMO at femoral neck may be further explored. The results also showed G1181C polymorphism in the first exon of OPG gene may not be associated with BMD and PMO.2 ASSOCIATIONS BETWEEN HAPLOTYPES OF OPG GENE AND POSTMENOPAUSAL OSTEOPOROSISObjective To investigate the relationship between haplotypes of the osteoprotegerin (OPG) gene and postmenopausal osteoporosis (PMO). Methods A case-control study, involving 78 femoral neck PMO patients vs. 122 controls and 108 L2-4 PMO patients vs. 92 controls. Arlequin 3.11 software was used to analysis linkage disequilibrium pattern and to contribute haplotype. Online SHEsis software was used to compute linkage disequilibrium coefficient (D′, r2). Results (1) Linkage disequilibrium was found between A163G and T950C or G1181C. T950C and G1181C were in strong linkage disequilibrium. (2) All haplotype groups containing A163G showed significant association with PMO at femoral neck site (P<0.05). Among these a two-SNP (A163G-T950C) haplotype group revealed the most highly significant association (P=0.000137). Among all haplotypes containing A163G SNP, the frequency of the G-T-G haplotype was much more in PMO group than that in control group at femoral neck (P=9.55e-005), whereas the frequency of A-C-G haplotype was significant lower in PMO group than that in control group (P=0.015). So the G-T-G haplotype may be risk factors while the A-C-G haplotype may be protective factors for femoral neck PMO. (3) All haplotype groups containing A163G and T950C showed significant association with PMO at L2-4 site (P<0.05). Among these a three-SNP (A163G-T950C-G1181C) haplotype group revealed the most highly significant association (P=0.015). Among all haplotypes containing A163G-T950C, the frequency of the A-C-G haplotype was much more in PMO group than that in control group at L2-4 (P=0.041), whereas the frequency of A-T haplotype was significant lower in PMO group than that in control group (P=0.006). The A-C-G haplotype may be risk factors while the A-T haplotype may be protective factors for L2-4 PMO. Conclusions A163G polymorphism in the promoter region of the OPG gene may be associated with PMO at femoral neck. The G-T-G haplotype may confer susceptibility to PMO and the A-C-G may be probably a protecting haplotype of PMO.The influence of A163G and T950C on PMO at L2-4 may be further explored.PARTⅣGENE-GENE INTERACTIONS AND GENE-ENVIRONMENT INTERACTIONS WITH POSTMENOPAUSAL OSTEOPOROSISObjective To determine whether interactions among candidate genes and a variety of environment factors are associated with postmenopausal osteoporosis (PMO). Methods We collected clinical data and characterized eight genetic variants and nine environmental factors of 78 femoral neck PMO patients vs. 122 controls and 108 L2-4 PMO patients vs. 92 controls. Multifactor dimensionality reduction (MDR) was used to detect gene-gene or gene-environment interactions. Results (1) The optimal model was an interaction between CYP19 gene (TTTA)n polymorphism and ESR2 gene (CA)n polymorphism of PMO at femoral neck site. However, the gene-gene interactions were not detected of PMO at L2-4 site (P>0.05). (2) A optimal model of gene-environment interactions among ESR2 gene G1082A polymorphism, (CA)n polymorphism and BMI of PMO at femoral neck site was detected which was able to correctly tested disease status with 75.79% (P=0.0107) accuracy. We also detected an interaction between height and BMI of PMO at L2-4 site that tested disease status with 63.41% (P=0.0107) accuracy. Conclusions These findings demonstrate the utility of novel computational approaches for the detection of disease susceptibility genes. The interaction among ESR2 gene G1082A, (CA)n polymorphism and BMI may be associated with PMO at the femoral neck site. The effect of ESR2 gene (CA)n on femoral neck PMO may be more important. Nevertheless, the environment factors just like height and BMI may be chief predisposing factors of PMO at L2-4 site. Low height and low BMI may have synergistic effect on the development of PMO. The contribution of low BMI may be more predominant than that of low height.