This study examines the association between bone mineral density (BMD) and a start codon polymorphism (SCP) at the translation initiation site of the vitamin D receptor (VDR) gene. The thymine/cytosine (T/C) polymorphism in the first of two start (ATG) codons can be detected by a restriction fragment length polymorphism (RFLP) using the endonuclease FokI, which recognizes ATG as part of its restriction site. F indicates absence of the first ATG and a VDR that is shorter by three amino acids. The FokI genotype was determined in 154 premenopausal American women (72 black and 82 white) who were 20 -40 years old. BMD of the total body, femoral neck, and lumbar spine were measured by dual-energy X-ray absorptiometry. The distribution of the SCP genotypes differed significantly by race ( p < 0.001): 4% of blacks versus 18% of whites were ff homozygous and 65% of blacks versus 37% of whites were FF homozygous. There was no statistically significant interaction between race and SCP genotype in analyses of BMD at any skeletal site. In the group as a whole, the ff women had femoral neck BMD that was 7.4% lower than that of the FF women. The ff white women had total body BMD values that were 4.3% lower and femoral neck values that were 12.1% lower than FF white women. Total body and femoral neck BMD did not differ significantly by genotype in black women, and spine BMD did not differ by genotype in either race. Addition of the SCP genotype to analysis of covariance models comparing BMD of the black and white women reduced estimated differences in femoral neck BMD between the two groups by about 35%. In conclusion, the SCP polymorphism, detected with the endonuclease FokI, appears to influence peak bone density, particularly at the femoral neck. Racial differences in its distribution may explain some of the racial difference in femoral neck BMD.

The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where "f" indicates the presence of the restriction site and the first ATG, while "F" indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [ ; however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH) 2 D 3 doseresponse (0.1-10 nM) curves revealed that the ED 50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF, Ff, and ff genotypes had similar sensitivity to 1,25(OH) 2 D 3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in

We examined the association of bone mineral density (BMD) with a polymorphism in the gene encoding the vitamin D receptor (VDR) that causes a change in the predicted protein sequence. The polymorphism results from a C-to-T transition and creates an initiation codon (ATG) three codons proximal to a downstream start site. The polymorphism can be defined by a restriction fragment length polymorphism (RFLP) using the restriction endonuclease FokI. The presence of a FokI site, designated f, allows protein translation to initiate from the first ATG. The allele lacking the site (designated F), initiates from a second ATG site. Thus, translation products from these alleles are predicted to differ by three amino acids with the f variant elongated. In a group of 100 postmenopausal Mexican-American Caucasian women, subjects with the ff genotype (15% of the study population) had a 12.8% lower BMD at the lumbar spine than FF subjects (37% of the population) (p = 0.01). Heterozygote (Ff) subjects (48% of the population) had an intermediate BMD. This association between BMD and genotype was not apparent at the femoral neck or forearm. Over a 2-year follow-up period, a decrease in BMD at the femoral neck was greater in ff compared with FF subjects (-4.7% vs. -0.5%, p = 0.005). This trend was not apparent at the lumbar spine or forearm. There were no differences between genotype groups in measurements of 25-hydroxyvitamin D (25(OH)D), calcitriol, parathyroid hormone (PTH), osteocalcin, or urinary pyridinolines. We conclude that the FokI polymorphism of the VDR gene correlates significantly with decreased BMD at the lumbar spine and with an increased rate of bone loss at the hip in ff subjects. We emphasize that these initial data should be interpreted with caution but that the utility of this polymorphism as a genetic marker to determine BMD and osteoporosis risk warrants further study in larger populations with subjects of diverse ethnic backgrounds.

No abstract

Previous studies have demonstrated an association between bone mineral density (BMD) and a start codon polymorphism (SCP) of the vitamin D receptor (VDR) gene in pre-and postmenopausal Caucasian and Japanese women. The SCP can be determined by a restriction fragment length polymorphism defined by the FokI restriction endonuclease. VDR alleles containing the FokI site are denoted by f and alleles lacking the site by F. In this study, the association between BMD and the SCP was examined in a group of 174 premenopausal French women who previously had been studied for a relationship between BMD and the VDR BsmI polymorphism. The SCP genotypes of the French women were FF 40%, Ff 44%, and ff 16% and they were independent of the BsmI genotype. BMD was measured by dual-energy X-ray absorptiometry at the lumbar spine, proximal femur, forearm, and total body. In contrast to previous reports, there was no association of BMD with SCP genotype in this group of Caucasian women at any site. We also measured several biochemical indices of calcium homeostasis and bone turnover. We found no statistically significant associations between SCP genotype and calcium, parathyroid hormone, or vitamin D levels. There was a 33.5% higher level of the skeletal resorption marker N-telopeptides of type I collagen in the women with the ff genotype when compared with women with the FF genotype ( p ‫؍‬ 0.004). Other bone turnover markers failed to show an association with SCP genotype. In summary, the SCP genotype may not be associated with reduced BMD in all geographical or ethnic populations. (J Bone Miner Res 1998;13:31-35)

A 49-year-old man with classic manifestations of Cushing's syndrome had undetectable levels of ACTH, lack of suppression of hypercortisolism with dexamethasone in doses of 2, 8, or 16 mg per day, bilaterally enlarged adrenal glands on MRI, and bilateral adrenal uptake of iodocholesterol. Preoperative treatment with ketoconazole lowered blood pressure and serum cortisol and produced symptoms of steroid withdrawal. Bilateral adrenalectomy revealed massively enlarged adrenal glands (left: 199 g, right: 93 g). Sequencing of the gene encoding the stimulatory G protein, GS alpha, did not show either of two activating mutations previously reported in patients with McCune-Albright syndrome or acromegaly. Twenty-three previous cases of Cushing's syndrome due to ACTH-independent massive bilateral adrenal disease (AIMBAD) have been reported. AIMBAD may cause confusion in the differential diagnosis of Cushing's syndrome as endocrine testing suggests a unilateral, ACTH-independent process while adrenal imaging demonstrates bilateral abnormalities. Bilateral adrenalectomy is curative and appears to carry little risk of Nelson's syndrome. The pathogenesis of AIMBAD appears to be heterogeneous, as recent reports have demonstrated GIP-mediated hypercortisolism and familial AIMBAD. Transition from Cushing's disease to ACTH-independence is not supported by the available data. Future cases of AIMBAD should be investigated carefully to further elucidate the pathogenesis of this disorder.

alpha-Glucosidase (EC 3.2.1.20) was purified to homogeneity from logarithmically growing cells of Saccharomyces carlsbergensis. The purification involved the following steps: (a) ammonium sulfate fractionation; (b) Sephadex G-100 chromatography; (c) DEAE-cellulose chromatography; and (d) hydroxylapatite chromatography. This procedure gave a preparation judged to be greater than 98% pure by Na-DodSO4-polyacrylamide gel electrophoresis. The enzyme was shown to be a monomer of 63 000 daltons by gel filtration on Sephacryl S-200 under native conditions and by polyacrylamide gel electrophoresis under denaturing conditions. The Km values of the enzyme for the substrates maltose and p-nitrophenyl alpha-D-glucoside were found to be 1.66 X 10(-2) and 3.1 X 10(-4) M, respectively. The corresponding Vmax value for maltose was 44.8 X 10(-6) mol min(-1) mg(-1) and that for p-nitrophenyl alpha-D-glucoside was 134 X 10(-6) mol min-1 mg-1. The pH optimum for the purified enzyme was found to be between pH 6.7 and 6.8. The enzyme has an absolute anomeric specificity for alpha-glycosidic linkages and appears to recognize a glucosyl residue in alpha linkage on the nonreducing end of its substrate. For the strain used in this study, which carries the MAL 6 locus, only a single form of the enzyme was detected.

Common polymorphisms in the vitamin D receptor (VDR) gene have been shown to correlate with bone mineral density (BMD). However, attempts to replicate the original findings in other populations have yielded variable results. These disparities may reflect ethnic or environmental differences in the expression of the VDR effect upon BMD. We examined a relatively ethnically homogeneous group of 103 healthy postmenopausal Caucasian women of Mexican descent living in Northern California. We determined the VDR genotype and measured the BMD at the lumbar spine and femoral neck by dual-energy X-ray absorptiometry, as well as several biochemical indices of mineral metabolism. The prevalence of the BB genotype, associated in previous studies with the lowest BMD, was 8% and highly linked to the tt genotype. Absolute and age-adjusted BMD at both hip and spine showed a trend toward lower BMD in the BB, AA, and tt genotypes, but this trend did not achieve statistical significance. There were no consistent intergroup differences in change in BMD over 2 years of follow-up, nor in mean serum concentrations of 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, osteocalcin, or total urinary pyridinolines. Intact parathyroid hormone concentrations were significantly higher in subjects with the AA genotype, with a trend toward higher values in those with the BB and tt genotypes as well. Our data suggest that there may be a decrease in BMD associated with the B, A, and t alleles, but the intergroup difference in BMD is 0.2-0.5 standard deviations (SD) at the lumbar spine and 0.3 SD at the femoral neck, decreases that are smaller than previously reported. Given the relatively low prevalence of the BB/tt genotype in Mexican-American Caucasians, a larger sample would be required to detect a significant association between VDR alleles and differences in BMD of the magnitude suggested by our data. We conclude that a genotype effect of this magnitude, if present, would be clinically relevant, but the impact on BMD is too small to detect with statistical significance in a study of this size. (J Bone Miner Res 1997;12:234-240)