Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.

The DNA f lanking the 5 sequence of the mouse 1␣-hydroxylase gene has been cloned and sequenced. A TATA box has been located at ؊30 bp and aCCAAT box has been located at ؊79 bp. The gene's promoter activity has been demonstrated by using a luciferase reporter gene construct transfected into a modified pig kidney cell line, AOK-B50. Parathyroid hormone stimulates this promoter-directed synthesis of luciferase by 17-fold, whereas forskolin stimulates it by 3-fold. The action of parathyroid hormone is concentration-dependent. 1,25-Dihydroxyvitamin D 3 does not suppress basal promoter activity and marginally suppresses parathyroid hormone-driven luciferase reporter activity. The promoter has three potential cAMP-responsive element sites, and two perfect and one imperfect AP-1 sites, while no DR-3 was detected. These results indicate that parathyroid hormone stimulates 25-hydroxyvitamin D 3 -1␣-hydroxylase by acting on the promoter of the 1␣-hydroxylase gene.Vitamin D is a major actor in calcium homeostasis of higher animals (1). To carry out these functions, vitamin D must be metabolized to its biologically active hormonal form, 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ). This is a two-step process requiring 25-hydroxylation in the liver and 1␣-hydroxylation in the kidney. The resulting hormone, 1,25-(OH) 2 D 3 , then binds to a nuclear vitamin D receptor (VDR) in target tissues, and the liganded receptor acts as a transcription factor to modulate the expression of specific genes encoding proteins that bring about the actions of vitamin D (2).The importance of the 1␣-hydroxylase enzyme is emphasized by the occurrence of a genetic disorder of vitamin D metabolism, vitamin D dependency rickets type I (VDDRI) (3). This disorder is characterized by very low serum 1,25-(OH) 2 D 3 levels despite normal vitamin D intakes (4) and is thought to be the result of a defect in the 1␣-hydroxylase gene (5). This link has recently been reinforced with the identification of the gene for human 1␣-hydroxylase (6). The gene was mapped to chromosomal region 12q13.1-q13.3, which contains the VDDRI disease locus.The 1␣-hydroxylation step is the most tightly regulated step in vitamin D metabolism. Several physiological factors interact to regulate 1␣-hydroxylase activity that, in turn, determines serum and tissue levels of 1,25-(OH) 2 D 3 . These regulators include parathyroid hormone (PTH) and hypophosphatemia, which stimulate 1␣-hydroxylase activity, and 1,25-(OH) 2 D 3 , which suppresses it (7-9).

Familial hypophosphatemic rickets is a rare disease, which is mostly transmitted as an X-linked dominant trait, and mutations on the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) gene are responsible for the disease in most familial cases. In this study we analyzed PHEX in a large cohort of 118 pedigrees representing 56 familial cases and 62 sporadic cases. The high-resolution melting curves technique was tested as a screening method, along with classical sequencing. PHEX mutations have been found in 87% of familial cases but also in 72% of sporadic cases. Missense mutations were found in 16 probands, two of which being associated with other PHEX mutations resulting into truncated proteins. By plotting missense mutations described so far on a 3D model of PHEX we observed that these mutations focus on two regions located in the inner part of the PHEX protein. Family members of 13 sporadic cases were analyzed and a PHEX mutation was detected in one of the apparently healthy mother. These results highlight the major role of PHEX in X-linked dominant hypophosphatemic rickets, and give new clues regarding the genetic analysis of the disease. A screening of the different family members should be mandatory when a PHEX mutation is assessed in a sporadic case and the search for another PHEX mutation should be systematically proceed when facing a missense mutation.