The CYP3A4*18 genotype in the cytochrome P450 3A4 gene, a rapid metabolizer of sex steroids, is associated with low bone mineral density.

Osteoporosis is influenced by genetic factors. The interindividual variability in the activity of CYP3A, the metabolic enzyme of sex hormones, may result from genetic polymorphisms. In a study of 2,178 women of ages 40-79 years, the presence of the CYP3A4*18 variant was found to be significantly associated with low bone mass. In vitro functional analyses indicate that CYP3A4*18 is a gain-of-function mutation in sex steroid metabolism, resulting in rapid oxidation of estrogens and testosterone; in vivo pharmacokinetics using midazolam (MDZ) verify the altered activity of the CYP3A4*18, showing lower metabolic turnover in the mutant than in the wild type. Molecular modeling reveals the structural changes in the substrate recognition sites of CYP3A4*18 that can cause changes in enzymatic activity and that potentially account for the difference between the catalytic activities of estrogen and MDZ, depending on the genotype. The results indicate that a genetic variation in the CYP3A4 gene--as a gain-of-function mutation in the metabolism of certain CYP3A substrates, including sex steroids--may predispose individuals to osteoporosis.

Frequencies of CYP3A5 genotypes and haplotypes in a Korean population.

BACKGROUND AND OBJECTIVE: CYP3A, the drug-metabolizing enzyme is an important factor in the pharmacokinetics of many drugs. Polymorphism of the CYP3A5 gene is known to influence the functionality of the CYP3A5 enzymes. The full extent of CYP3A5 genetic polymorphism was analysed in a Korean population. METHODS: Specific polymerase chain reaction-restriction fragment length polymorphism tests for CYP 3AP1 through CYP3A5*7 or direct sequencing were used to identify reported CYP3A5 variant alleles, using 194 unrelated samples. RESULTS AND DISCUSSION: The most frequent single nucleotide polymorphism (SNP) was 6986A>G (CYP3A5*3). The next most frequent SNP was 31611C>T. Haplotype analysis using detected SNPs revealed that the most frequent haplotype was *3A (frequency: 0.724), followed by *1E (frequency: 0.211), *3C (frequency: 0.034) and *1A (frequency: 0.023). We did not find CYP3AP1*3, CYP3A5*6, or *7 in this Korean sample. CONCLUSION: A large proportion of Koreans may have relatively low levels of metabolically active CYP3A5 protein and therefore may be at risk of high levels of drugs metabolized by this enzyme, after administration of conventional doses.

Nonassociation of interleukin-1 receptor antagonist genotypes with bone mineral density, bone turnover status, and estrogen responsiveness in Korean postmenopausal women.

Interleukin-1 receptor antagonist (IL-1ra), a natural inhibitor of interleukin-1 (IL-1), completely inhibits the stimulatory effects of IL-1 on bone resorption. Bioactivity of IL-1 increases in the estrogen-deficient state with an increased IL-1:IL-1ra ratio and decreases after estrogen replacement therapy with a decreased IL-1:IL-1ra ratio. An association was found between an 86 basepair variable number tandem-repeat (VNTR) polymorphism of the IL-1ra gene and an increased production of IL-1ra in a cultured monocyte system. The IL-1ra VNTR polymorphism, therefore, is an attractive candidate gene for osteoporosis susceptibility as well as hormone responsiveness after estrogen replacement. We examined the association of this VNTR polymorphism with bone mass, bone turnover, and the change of bone mineral density (BMD) after 1 year of hormone replacement therapy (HRT). The frequencies of the five alleles were as follows: A1, 90.8% (410 bp, four repeats); A2, 7.2% (240 bp, two repeats); A3, 1.6% (500 bp, five repeats); A4, 0.4% (326 bp, three repeats); and A5, 0% (595 bp, six repeats), in 714 healthy ethnically Korean postmenopausal women, aged 41-74 years (55.2 +/- 6.3 years mean +/- SD). Spine (L2-4) and femoral neck BMD were not significantly different among IL-1ra genotypes, and no significant genotypic differences were found in bone markers. There were no differences in genotypic proportions when we categorized the subjects into a high-loss group and a normal-loss group with regard to levels of bone marker. No significant genotypic differences were found in changes in lumbar and femoral neck BMD and those in bone markers before and after 1 year of HRT in 312 women. Our data suggest that these IL-1ra polymorphisms are not associated with BMD, bone turnover, or the change of BMD after 1 year of HRT in Korean women.

Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells.

Identification of a mutation in the human raloxifene response element of the transforming growth factor-beta 3 gene.

The human transforming growth factor-beta 3 (TGF-beta 3) is an important cytokine to maintain bone mass by inhibiting osteoclast differentiation. Recently raloxifene response element (RRE), a new enhancer with a polypurine sequence for estrogen receptor (ER)-mediated gene activation, was identified on the TGF-beta 3 gene. Functional analysis of the RRE-mediated pathway has shown that this would be an important pathway for bone preserving effect. We found a novel mutation in the RRE sequence by single-strand conformational polymorphism analysis in one of 200 Korean women. Cloning and sequencing revealed a heterozygote in which one allele had an insertion of 20 nucleotides (AGAGAGGGAGAGGGAGA GGG) between nucleotide +71 and +72 and a point mutation at nucleotide +75 (G-A transition), and the other allele had normal sequence. The insertion was a nearly perfect tandem duplication of the wild type DNA sequence. The bone mineral density of the affected woman was not much lower than that of age-matched controls. Transient transfection of the mutant allele showed no significantly different activity compared with that of the wild type allele. These observations suggest that the heterozygote variation of the RRE sequence seems not to be operative in determination of bone mass.

Metabolic characteristics and prevalence of osteoporosis among women in Tae-An area.

Understanding the metabolic changes in women is one of the important ways to prevent and treat osteoporosis. To reveal the metabolic characteristics of 289 healthy women aged between 35-65 yr in Tae-An, Korea we evaluated the association between bone mass assessed by broadband ultrasound attenuation (BUA) using quantitative ultrasound 2 (QUS2) and various parameters such as age, body mass index, serum levels of alkaline phosphatase, calcium, phosphorus, parathyroid hormone, 25(OH)D, and urinary ratios of calcium/creatinine and deoxypyridinoline (Dpyd)/creatinine. Among the subjects, 3.0% were osteoporotic, and 40.9% were osteopenic. When the subjects were classified according to their years since menopause (YSM) and age, the prevalence of osteoporosis increased along with an increase of YSM and age. Bone turnover markers such as serum alkaline phosphatase and fasting urinary Dpyd/creatinine were significantly higher in the group with low bone mass than in the normal group. In summary, this study shows, by use of biochemical markers of bone turnover and QUS2, the prevalence of osteoporosis in women aged between 35-65 in Tae-An was 3.0% and the risk of low bone mass increased with the bone turnover markers.

Expression of the monocyte chemoattractant protein-1 receptor CCR2 is increased in hypercholesterolemia. Differential effects of plasma lipoproteins on monocyte function.

Monocytes are recruited from the circulation into the subendothelial space where they differentiate into mature macrophages and internalize modified lipoproteins to become lipid-laden foam cells. The accumulation of monocytes is mediated by the interaction of locally produced chemoattractant protein-1 (MCP-1) with its receptor CCR2. The objective of the present study is to demonstrate the differential effects of plasma lipoproteins on monocyte CCR2 expression. The CCR2 expression was increased about 2.4-fold in monocytes isolated from hypercholesterolemic patients, compared to monocytes from normal controls. There was a significant correlation between CCR2 expression and plasma low density lipoprotein (LDL). Elevated levels of high density lipoprotein (HDL) blunted and even reverted the effects of LDL on CCR2 expression, both in vivo and in vitro. The causal relationship between plasma lipoproteins and CCR2 expression was further confirmed by modulating the lipoprotein profile. Estrogen supplement therapy decreased plasma LDL cholesterol, increased plasma HDL cholesterol, and reduced CCR2 expression in hypercholesterolemic postmenopausal women, but had no effect on the plasma lipid profile or CCR2 expression in normocholesterolemic subjects. The physiological significance of altered CCR2 expression was tested by chemotaxis assay, and our results demonstrated that treatment of THP-1 monocytes with LDL induced CCR2 expression and substantially enhanced the chemotaxis elicited by MCP-1. Our findings suggest that plasma lipoproteins differentially control monocyte function and that monocytes from hypercholesterolemic subjects are hyperresponsive to chemotactic stimuli. This may increase their accumulation in the vessel wall and accelerate the pathogenic events of atherogenesis.

Lack of an intronic Sp1 binding-site polymorphism at the collagen type I alpha1 gene in healthy Korean women.

Osteoporosis is a disease that is strongly genetically influenced. However, the genes responsible for the disease are poorly defined. Recent data show that a G-T transition polymorphism of the Sp1 binding site at the collagen type I alpha1 gene (Sp1 polymorphism) is associated significantly with bone mineral density (BMD) and osteoporotic fracture in British women. To establish the association between the Sp1 genotypes and BMD in Korean women, we examined 200 healthy postmenopausal women of Korean ethnicity, ranging in age from 44 to 66 years (mean+/-SD: 54.7+/-5.3 years). PCR amplification using the same primers as those used previously, with enzyme digestion, revealed no restriction site in our samples. We also performed a single-strand conformational polymorphism (SSCP) analysis in 100 of the 200 samples and could not find any polymorphic sites in the PCR amplification region. Based on our study, the Sp1 polymorphism at the type I collagen alpha1 gene was not found in Korean women. Therefore, we suggest that the Sp1 polymorphism at the type I collagen alpha1 gene is absent or rare in Korean women. Based on the present findings, this polymorphism does not seem to be responsible for the entire genetic contribution to BMD.

Nonassociation of estrogen receptor genotypes with bone mineral density and estrogen responsiveness to hormone replacement therapy in Korean postmenopausal women.

Hormone replacement therapy (HRT) prevents bone loss in postmenopausal women, but some women are resistant to therapy. A recently reported case of severe estrogen resistance caused by a germline mutation at the estrogen receptor (ER) gene locus suggests the possibility that other variants of the ER gene could be responsible for resistance to HRT and could also be an answer to the heritable components of bone density. Three restriction fragment length polymorphisms (RFLPs) at the ER gene locus, represented as BstUI (or B variant), PvuII, and XbaI, and their relationship to bone mineral density (BMD) and estrogen responsiveness to HRT were examined in 248 healthy postmenopausal women, aged 41-68 yr (mean +/- SD, 52.0 +/- 4.6 yr) in Korea. The BstUI restriction site was not found in Korean women. The distribution of the PvuII and XbaI RFLPs was as follows: PP, 35 (14.1%); Pp, 136 (54.8%); pp, 77 (31.1%) and XX, 18 (7.3%); Xx, 72 (29.0%); and xx, 158 (63.7%), respectively (capital letters signify the absence of and lower case letters signify the presence of the restriction site of each RFLP). There was no significant relation between ER genotypes and z score values of lumbar spine BMD. Also, no significant genotypic differences were found in the change in lumbar spine BMD and those in biochemical markers before and after 1 yr of HRT. These data indicate no significant effects of ER genotypes on BMD and estrogen responsiveness after HRT.