PHEX analysis in 118 pedigrees reveals new genetic clues in hypophosphatemic rickets.

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.

Vitamin D receptor genotype in hypophosphatemic rickets as a predictor of growth and response to treatment.

CONTEXT: Treatment of X-linked hypophosphatemic rickets improves bone mineralization and bone deformities, but its effect on skeletal growth is highly variable. OBJECTIVE: Genetic variants in the promoter region of the vitamin D receptor (VDR) gene may explain the response to treatment because this receptor mediates vitamin D action. DESIGN: We studied the VDR promoter haplotype structure in a large cohort of 91 patients with hypophosphatemic rickets including 62 patients receiving 1alpha-hydroxyvitamin D3 derivatives and phosphates from early childhood on. RESULTS: Treatment improved bone deformities and final height, but 39% of treated patients still had short stature at the end of growth (-2 sd score or below). Height was closely associated with VDR promoter Hap1 genotype. Hap1(-) patients (35% of the cohort) had severe growth defects. This disadvantageous association of Hap1(-) status with height was visible before treatment, under treatment, and on to adulthood. Gender and age at initiation of treatment could not account for the Hap1 effect. No association with growth was found with a polymorphism of the PTH receptor gene otherwise found to be associated with adult height. Compared with Hap1(+) patients, those who were Hap1(-) had a higher urinary calcium response to 1alpha-hydroxyvitamin D3 and had significantly lower circulating FGF23 levels (C-terminal assay), taking into account their phosphate and 1alpha-hydroxyvitamin D3 intakes. CONCLUSIONS: The present work identifies the VDR promoter genotype as a key predictor of growth under treatment with 1alpha-hydroxyvitamin D3 derivatives in patients with hypophosphatemic rickets, including those with established PHEX alterations. The VDR promoter genotype appears to provide valuable information for adjusting treatment and for deciding upon the utility of early GH therapy.

Possible involvement of pregnane X receptor-enhanced CYP24 expression in drug-induced osteomalacia.

Vitamin D controls calcium homeostasis and the development and maintenance of bones through vitamin D receptor activation. Prolonged therapy with rifampicin or phenobarbital has been shown to cause vitamin D deficiency or osteomalacia, particularly in patients with marginal vitamin D stores. However, the molecular mechanism of this process is unknown. Here we show that these drugs lead to the upregulation of 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) gene expression through the activation of the nuclear receptor pregnane X receptor (PXR; NR1I2). CYP24 is a mitochondrial enzyme responsible for inactivating vitamin D metabolites. CYP24 mRNA is upregulated in vivo in mice by pregnenolone 16alpha-carbonitrile and dexamethasone, 2 murine PXR agonists, and in vitro in human hepatocytes by rifampicin and hyperforin, 2 human PXR agonists. Moreover, rifampicin increased 24-hydroxylase activity in these cells, while, in vivo in mice, pregnenolone 16alpha-carbonitrile increased the plasma concentration of 24,25-dihydroxyvitamin D(3). Transfection of PXR in human embryonic kidney cells resulted in rifampicin-mediated induction of CYP24 mRNA. Analysis of the human CYP24 promoter showed that PXR transactivates the sequence between -326 and -142. We demonstrated that PXR binds to and transactivates the 2 proximal vitamin D-responsive elements of the human CYP24 promoter. These data suggest that xenobiotics and drugs can modulate CYP24 gene expression and alter vitamin D(3) hormonal activity and calcium homeostasis through the activation of PXR.

Adverse interaction of low-calcium diet and low 25(OH)D levels on lumbar spine mineralization in late-pubertal girls.

No consensus has been reached on the serum 25-hydroxyvitamin D [25(OH)D] levels required to ensure optimal bone health around menarche. We searched for a possible interaction of 25(OH)D levels and calcium intake on lumbar spine mineralization and on biologic features of bone metabolism in healthy late-pubertal girls. Lumbar spine parameters (ie, area, mineral content, and density) and calcium intake were evaluated in 211 healthy white adolescent girls at pubertal stages IV-V (11 to 16.9 years), together with biologic markers of calcium and bone metabolism and with International External Quality Assessment Scheme for Vitamin D Metabolite (DEQAS)-validated serum 25(OH)D levels. A high prevalence of 25(OH)D levels ≤ 30 nmol/L (41%), ≤ 40 nmol/L (61%), and ≤ 50 nmol/L (70%) was found during winter-spring. Parathyroid hormone (PTH) levels were inversely associated with 25(OH)D levels (p = .0021). In contrast, lumbar spine mineral content and density were not associated with 25(OH)D, excepted when calcium intake was below 600 mg/day (p = .0081). Girls with such low calcium intake and 25(OH)D levels of 40 nmol/L or less (9% of the cohort) had a 0.4 to 0.7 SD lower mean areal bone mineral density Z-score than girls with higher calcium intake and/or higher 25(OH)D status. The adverse association between lumbar spine mineralization and combined calcium deficiency-low 25(OH)D levels remained significant in the 91 girls who could be followed over 4 years after their initial evaluation. We conclude that low 25(OH)D levels (≤40 nmol/L) are observed frequently during winter-spring in late-pubertal European girls, which may exacerbate the negative impact of calcium deficiency on lumbar spine mineralization.

Growth, calcium status and vitamin D receptor (VDR) promoter genotype in European children with normal or low calcium intake.

Calcium homeostasis and bone metabolism require the vitamin D receptor (VDR) to function properly, as evidenced in patients and transgenic mice with VDR mutations. We have shown that (A/G) polymorphism at the -1012 locus of the VDR promoter (rs4516035) is frequent in European populations, may influence VDR expression, is associated with height in French adolescent girls, and is associated with their lumbar spine mineral density in case of insufficient milk intake. Here, an association study was performed in a cohort of Moldovan children and adolescents, living at latitude similar to the first cohort but receiving a cereal-based diet with very low milk/dairy product intakes. Children and adolescents in this cohort had similar 25-(OH) D levels, but a short stature and low serum calcium levels, compared to the first cohort. Their height remained associated with the A-1012G VDRp genotype. In addition, their serum calcium levels were associated with VDRp polymorphism, excepted when their 25-(OH) D levels were low (below 33 nmol/L). In conclusion, the -1012 VDRp genotype appears to be associated with height in European children whatever their calcium/dairy product intakes, and may modulate their calcium homeostasis in conditions of low calcium/milk intakes when vitamin D status is sufficient.

Two single-nucleotide polymorphisms in the human vitamin D receptor promoter change protein-DNA complex formation and are associated with height and vitamin D status in adolescent girls.

Numerous association studies have dealt with single-nucleotide polymorphisms (SNPs) in coding and intronic regions of the human vitamin D receptor (hVDR) gene. We have hypothesized that phenotypic traits may also be associated with variations in VDR expression due to the presence of SNPs in promoter regions. In this work, we have studied two SNPs located 1521 bp (G/C) and 1012 bp (A/G) upstream of the transcriptional start site of the main human VDR gene promoter. One base-change in any of the two variant sites led to a dramatic change in protein-DNA complex formation using nuclear extracts from HEK293, Caco-2 and COS-7 cells. Genetic analysis of 185 healthy adolescent girls evidenced two major haplotypes: 1521G/1012A and 1521C/1012G and three main genotypes: homozygous for 1521G/1012A (21.1%), homozygous for 1521C/1012G (17.3%) and heterozygous 1521CG/1012GA (57.3%). On the basis of transfection data, promoter activity was nearly 2-fold higher with the 1521G/1012A haplotype, when compared with the 1521C/1012G haplotype. Clinical and biological association study in the adolescent cohort showed that girls with a CC/GG genotype had (i) lower circulating levels of 25-dihydroxyvitamin D, with no detectable consequence on calcium metabolism, (ii) lower serum IGF-1 levels and (iii) smaller height from 11 years of age up to adult height.