Defective Bone Mineralization Research Articles

Association of COLIA1 Sp1 Alleles with Defective Bone Nodule Formation In Vitro and Abnormal Bone Mineralization In Vivo

Previous work identified a G/T polymorphism affecting a Sp1 binding site in a regulatory region of the COLIA1 gene that predisposes to osteoporotic fractures by affecting bone strength through mechanisms that are partly independent of differences in bone mineral density (BMD). To clarify the mechanisms by which COLIA1 Sp1 alleles influence bone strength we used quantitative backscattered electron imaging (qBEI) to characterize bone mineralization in biopsy samples from subjects of different COLIA1 genotype and studied the ability of osteoblast-like cells cultured from subjects of different genotypes to form mineralized bone nodules. The qBEI analysis showed a significant (P = 0.014) reduction in mineralization in bone biopsies from G/T heterozygotes (n = 6) compared with G/G homozygotes (n = 7) and a significant increase in heterogeneity of mineralization (P = 0.017). The in vitro studies showed that osteoblasts derived from G/T heterozygotes (n = 5) were significantly less able to produce mineralized bone nodules than G/G homozygotes (n = 10) at all time-points examined (P < 0.0001). We conclude that carriage of the COLIA1 Sp1 "T" allele is associated with an impaired ability of osteoblast-like cells to form mineralized bone nodules in vitro and with abnormalities of bone mineralization in vivo. This suggests that the increased bone fragility in carriers of the COLIA1 Sp1 allele may result in part from defects in bone mineralization.

Loss of ZMPSTE24 (FACE-1) causes autosomal recessive restrictive dermopathy and accumulation of Lamin A precursors

Restrictive dermopathy (RD) is characterized by intrauterine growth retardation, tight and rigid skin with prominent superficial vessels, bone mineralization defects, dysplastic clavicles, arthrogryposis and early neonatal death. In two patients affected with RD, we recently reported two different heterozygous splicing mutations in the LMNA gene, leading to the production and accumulation of truncated Prelamin A. In other patients, a single nucleotide insertion was identified in ZMPSTE24. This variation is located in a homopolymeric repeat of thymines and introduces a premature termination codon. ZMPSTE24 encodes an endoprotease essential for the post-translational cleavage of the Lamin A precursor and the production of mature Lamin A. However, the autosomal recessive inheritance of RD suggested that a further molecular defect was present either in the second ZMPSTE24 allele or in another gene involved in Lamin A processing. Here, we report new findings in RD linked to ZMPSTE24 mutations. Ten RD patients were analyzed including seven from a previous series and three novel patients. All were found to be either homozygous or compound heterozygous for ZMPSTE24 mutations. We report three novel 'null' mutations as well as the recurrent thymine insertion. In all cases, we find a complete absence of both ZMPSTE24 and mature Lamin A associated with Prelamin A accumulation. Thus, RD is either a primary or a secondary laminopathy, caused by dominant de novo LMNA mutations or, more frequently, recessive null ZMPSTE24 mutations, most of which lie in a mutation hotspot within exon 9. The accumulation of truncated or normal length Prelamin A is, therefore, a shared pathophysiological feature in recessive and dominant RD. These findings have an important impact on our knowledge of the pathophysiology in Progeria and related disorders and will help direct the development of therapeutic approaches.

Role of Matrix Extracellular Phosphoglycoprotein in the Pathogenesis of X-Linked Hypophosphatemia

X-linked hypophosphatemia (XLH), a disorder characterized by hypophosphatemia, impaired skeletal mineralization, and aberrant regulation of 1, 25(OH)(2)D(3), is caused by inactivating mutations of Phex, which results in the accumulation of putative phosphaturic factors, called phosphatonins. Matrix extracellular phosphoglycoprotein (Mepe) is a proposed candidate for phosphatonin. The authors found that Hyp mice had increased expression of the MEPE and another phosphaturic factor, Fgf23. To establish MEPE's role in the pathogenesis of the XLH, Mepe-deficient mice were back-crossed onto the Hyp mouse homologue of XLH and phenotypes of wild-type, Mepe(-/-), Hyp, and Mepe(-/-)/Hyp mice were examined. Transfer of Mepe deficiency onto the Phex-deficient Hyp mouse background failed to correct hypophosphatemia and aberrant serum 1,25(OH)(2)D(3) levels. Increased Fgf23 levels in Hyp mice were not affected by superimposed Mepe deficiency. In addition, Mepe-deficient Hyp mice retained bone mineralization defects in vivo, characterized by decreased bone mineral density, reduced mineralized trabecular bone volume, lower flexural strength, and histologic evidence of osteomalacia; however, cultures of Hyp-derived bone marrow stromal cells in the absence of Mepe showed improved mineralization and normalization of osteoblast gene expression profiles observed in cells derived from Mepe-null mice. These results demonstrate that MEPE elevation in Hyp mice does not contribute to the hypophosphatemia associated with inactivating Phex mutations and is therefore not phosphatonin.

Histological Evaluation of Biopsies Obtained From Vertebral Compression Fractures: Unsuspected Myeloma and Osteomalacia

A histological evaluation of biopsies obtained from presumed osteoporotic vertebral compression fractures (VCF) to confirm possible osteomalacia after tetracycline labeling. To describe the results of a series of biopsies obtained at the time of vertebral augmentation in presumed osteoporotic VCF, with special reference to the presence of unmineralized bone (osteomalacia) and occult or unconfirmed plasma cell dyscrasia. Vertebral augmentation is now widely performed as a method to treat osteoporotic or osteolytic VCF. However, the influence of underlying pathology on the effect of treatment is unclear. As of October 2003, 178 biopsies were obtained from 142 patients with VCF during 246 kyphoplasty procedures. There were 110 one-level, 28 two-level, and 4 three-level biopsies. Patients included 41 men and 101 women, with an average age of 72 years (range 40-90). The patients consented to this procedure, and 25 received tetracycline (1g/day, in 2 doses separated by 6 days). Vertebral body biopsies were taken using a trephine just before the kyphoplasty procedure. The biopsies were fixed, embedded, and stained with toluidine blue and hematoxylin eosin, and were viewed with transmitted light. Unstained sections were viewed under fluorescent light to detect tetracycline labels. The 178 biopsy levels included: T4 (3), T5 (1), T6 (4), T7 (13), T8 (12), T9 (8), T10 (11), T11 (17), T12 (28), L1 (25), L2 (14), L3 (13), L4 (17), and L5 (12). All specimens showed fragmented bone with variable amounts of unmineralized bone (osteoid), suggesting bone remodeling and/or fracture healing. Woven bone and cartilaginous tissue were often present, representing fracture callus formation. The biopsies obtained from 30 patients (21%), including 4 who received tetracycline, showed significantly increased osteoid, suggesting either increased bone remodeling activity or mineralization defect (osteomalacia). One sample from these 4 patients who received tetracycline showed no tetracycline labels, essentially diagnostic of osteomalacia. The biopsies also provided definitive diagnoses for one case of unsuspected and 3 cases of unconfirmed plasma cell dyscrasia. The majority of biopsies from this series of patients revealed findings consistent with various stages of fracture healing. Osteoid seams were increased in 30 patients, representing either increased bone remodeling or osteomalacia. More cases with tetracycline labeling will help elucidate the true incidence of osteomalacia in this population. As we confirmed 4 cases of plasma cell dyscrasia, we advocate a biopsy during each first-time vertebral augmentation procedure.

Over‐expression of fibroblast growth factor‐2 causes defective bone mineralization and osteopenia in transgenic mice

Over-expression of human FGF-2 cDNA linked to the phosphoglycerate kinase promoter in transgenic (TgFGF2) mice resulted in a dwarf mouse with premature closure of the growth plate and shortening of bone length. This study was designed to further characterize bone structure and remodeling in these mice. Bones of 1-6 month-old wild (NTg) and TgFGF2 mice were studied. FGF-2 protein levels were higher in bones of TgFGF2 mice. Bone mineral density was significantly decreased as early as 1 month in femurs from TgFGF2 mice compared with NTg mice. Micro-CT of trabecular bone of the distal femurs from 6-month-old TgFGF2 mice revealed significant reduction in trabecular bone volume, trabecular number (Tb.N), and increased trabecular separation (Tb.Sp). Osteoblast surface/bone surface, double-labeled surface, mineral apposition rate, and bone formation rates were all significantly reduced in TgFGF2 mice. There were fewer TRAP positive osteoclasts in calvaria from TgFGF2 mice. Quantitative histomorphometry showed that total bone area was similar in both genotypes, however percent osteoclast surface, and osteoclast number/bone surface were significantly reduced in TgFGF2 mice. Increased replication of TgFGF2 calvarial osteoblasts was observed and primary cultures of bone marrow stromal cells from TgFGF2 expressed markers of mature osteoblasts but formed fewer mineralized nodules. The data presented indicate that non-targeted over-expression of FGF-2 protein resulted in decreased endochondral and intramembranous bone formation. These results are consistent with FGF-2 functioning as a negative regulator of postnatal bone growth and remodeling in this animal model.

Assessment of effects of lanthanum carbonate with and without phosphate supplementation on bone mineralization in uremic rats

Previous studies have indicated that impaired bone mineralization in 5/6 th nephrectomized rats given high doses of lanthanum carbonate is due to phosphorus depletion caused by excessive binding to, and reduced absorption of, dietary phosphate. This study aimed to test this hypothesis by: 1) directly comparing the effects of a supratherapeutic dose of lanthanum carbonate or dietary phosphorus restriction on bone mineralization in a rodent model of chronic renal failure (CRF); and 2) investigating whether phosphorus supplementation would prevent the bone mineralization defect associated with lanthanum carbonate treatment. Male Sprague-Dawley rats were subjected to sham surgery or a two-step 5/6th nephrectomy to induce CRF and randomized across five treatment groups: sham, CRF, CRF + dietary phosphorus deficiency, CRF + lanthanum carbonate (1000 mg/kg/ day), and CRF + lanthanum carbonate + parenteral phosphorus repletion. Rats with 5/6th nephrectomy had elevated serum creatinine, blood urea concentration, and urine volume and protein, consistent with impaired renal function, and increased urinary phosphorus and serum parathyroid hormone, consistent with hyperparathyroidism. Lanthanum carbonate and dietary phosphate insufficiency induced parallel changes in serum and urine markers of phosphate homeostasis and increased osteoid formation. These changes induced by lanthanum carbonate were normalized by systemic phosphate supplementation. These findings provide further support for the concept that supratherapeutic doses of lanthanum carbonate induce effects on bone mineralization in uremic rats via an indirect pharmacological mechanism (phosphate depletion) and not via direct bone toxicity.

Bilateral Transverse (Bowdler) Fibular Spurs with Hypophosphatasia in an Adolescent Girl

Hypophosphatasia is a clinically heterogeneous inheritable disorder characterized by defective bone mineralization and the deficiency of serum and tissue liver/bone/kidney alkaline phosphatase activities. Due to the mineralization defect of the bones, various skeletal findings can be radiologically observed in hypophosphatasia. Bowing and Bowdler spurs of long bones are the characteristic findings. The Bowdler spurs reported on in the previous pertinent literature were observed in the perinatal aged patients and these lesions have rarely involved adolescents. We herein report on a 14-year-old girl with fibular Bowdler spurs.

Hypophosphatasie de l'enfant : à propos d'un cas caractérisé par une nouvelle mutation

Hypophosphatasia is characterized by defective bone mineralization associated with impaired activity of the tissue non-specific alkaline phosphatase (TNSALP) due to mutations in the TNSALP gene. We describe a child with a mutation that has not been described up to now. Case report. – A 4-year-old child presented with clinical symptoms of rickets and premature loss of decideous teeth. Reduced serum alkaline phosphatase activity and radiographic features led to the diagnosis of hypophosphatasia, which was confirmed by genetic investigation. The molecular study showed two missense mutations, of which one is a novel mutation. Conclusion. – Hypophosphatasia is suspected in a child with rickets and premature loss of decideous teeth. Such symptoms should prompt the search of a reduced serum alkaline phosphatase activity. The clinical and molecular diagnosis of the disease is important for the genetic counseling but also for a proper determination of prognosis, as it is related to the type of mutation.

Growth and Metabolic Control during Puberty in Girls with X-Linked Hypophosphataemic Rickets

Objective: X-linked hypophosphataemic rickets (XLH) results in defective bone mineralization and impaired growth. Treatment with oral phosphate (Pi) and calcitriol improves but does not normalize growth. This study assessed whether pubertal growth and metabolic control contribute to the height deficit. Methods: Study included patients with XLH who were treated with Pi-calcitriol from diagnosis to adult height; their hospital records, biochemistry and radiographs were reviewed. Results: Six females with XLH were included. Their mean peak height velocity and total height gain during puberty were nearly normal despite deteriorating metabolic control. Conclusions: In treated girls with XLH, the pubertal growth is nearly normal despite suboptimal metabolic control. The major height loss occurs prior to puberty and is not recovered during the pubertal growth spurt.

Génétique de l'hypophosphatasie

Hypophosphatasia is a rare inherited disorder characterized by defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase activity. The symptoms are highly variable in their clinical expression, which ranges from stillbirth without mineralized bone to early loss of teeth without bone symptoms. Currently, there is no treatment for the disease. Recent developments in molecular biology allow to better understand the genetics of the disease, especially its transmission that may be recessive or dominant, to improve genetic counseling and molecular diagnosis, and offer new perspectives of treatment.

Osteomalacia

Osteomalacia is characterized by defective mineralization of bone, leading to an accumulation of unmineralized bone matrix (osteoid). Rickets represents the occurrence of this defect in growing children before the closure of epiphysis. In osteomalacia, there is a reduction in the mineralized bone:matrix ratio, unlike osteoporosis, where this ratio remains normal. There is sufficient bone volume, but the bone has reduced calcification.

The effect of intravenous zoledronic acid on glucocorticoid-induced multiple vertebral fractures in juvenile systemic lupus erythematosus

Glucocorticoids are widely used in the treatment of lupus patients, and adverse effects, which include osteoporosis and associated fractures, are frequent. Treatment of osteoporosis of young patients should be effective and not harmful to bone growth and remodeling. Bisphosphonates are drugs that decrease the incidence of bone fractures, but their use in juvenile patients is still controversial because of their possible side effects on the growing skeleton. However, recently published studies showed that linear growth continued normally after treatment with these drugs, and there was no excessive suppression of bone remodeling or mineralization defects. Zoledronic acid is a new intravenous bisphosphonate that has been approved by the US FDA for use with hypercalcemia of malignancies and might be an effective treatment for postmenopausal osteoporosis. The authors report a case of a young girl with systemic lupus who developed multiple vertebral collapses due to glucocorticoid therapy, and zoledronic acid was used producing significant clinical and densitometric improvement.

Defective bone mineralization and osteopenia in young adult FGFR3-/- mice.

Mutations that cause constitutive activation of fibroblast growth factor receptor 3 (FGFR3) result in skeletal disorders that are characterized by short-limbed dwarfism and premature closure of cranial sutures. In previous work, it was shown that congenital deficiency of FGFR3 led to skeletal overgrowth. Using a combination of imaging, classic histology and molecular cell biology we now show that young adult FGFR3(-/-) mice are osteopenic due to reduced cortical bone thickness and defective trabecular bone mineralization. The reduction in mineralized bone and lack of trabecular connectivity observed by micro-computed tomography were confirmed in histological and histomorphometric analyses, which revealed a significant decrease in calcein labelling of mineralizing surfaces and a significant increase in osteoid in the long bones of 4-month-old FGFR3(-/-) mice. These alterations were associated with increased staining for recognized markers of differentiated osteoblasts and increased numbers of tartrate-resistant acid phsophatase postitive osteoclasts. Primary cultures of adherent bone marrow-derived cells from FGFR3(-/-) mice expressed markers of differentiated osteoblasts but developed fewer mineralized nodules than FGFR3(+/+) cultures of the same age. Our observations reveal a role for FGFR3 in post-natal bone growth and remodelling, which identifies it as a potential therapeutic target for osteopenic disorders and those associated with defective bone mineralization.

Abnormalities in bone mineral density and bone histology in thalassemia.

This study demonstrated that there was extensive iron staining on trabecular surface and marked reduction in trabecular bone volume without significant alteration in bone formation and bone resorption rates as well as significant reduction in bone mineral density in 18 thalassemic patients. Serum IGF-I was reduced and may modulate the reduction of bone mass. Bone histomorphometric studies in thalassemia to show alterations in bone histology and their relationship to biochemical parameters are very limited. Therefore, this study was systematically conducted to determine the alterations in thalassemia patients. Serum biochemical parameters, trans-iliac crest bone biopsy, and determination of bone mineral density of femur and lumbar spine were done in 18 thalassemic patients (10 females and 8 males). Serum osteocalcin, carboxy terminal teleopeptide fragment of type I collagen, and parathyroid hormone levels were within normal limits, but serum 25(OH) vitamin D (19.3 +/- 1.6 ng/ml) and 1,25(OH)2 vitamin D (33.77 +/- 1.51 pg/ml) levels were decreased. Serum insulin-like growth factor I (IGF-I; 145.2 +/- 20 ng/ml) was suppressed, whereas serum ferritin (1366.6 +/- 253.9 ng/ml) was markedly elevated. Reduced bone mineral density was found in all studied areas. Trabecular bone volume was significantly decreased (16.65 +/- 1.12%), whereas bone formation rate, eroded surface, and other bone histomorphometric parameters were within normal limits. The trabecular bone volume varied significantly with bone mineral density of total femur (r = 0.48, p = 0.04). There was an extensive stainable iron surface on the mineral front (9-60%). Significant correlation between serum IGF-I, serum ferritin, stainable iron surface, and bone mineral density, lumbar spine, and total femur were found. Serum IGF-I correlated with trabecular bone volume (r = 0.6, p = 0.03), inversely with both serum ferritin level (r = -0.6, p < 0.01), and inversely with stainable iron surface (r = -0.53, p = 0.02). Multiple regression analysis demonstrated that IGF-I was the only independent variable that determined bone mineral density of lumbar spine and total femur. Low bone mineral density and reduced trabecular bone volume with extensive iron deposition are the predominant findings in thalassemic patients. There was no evidence of increased bone resorption or mineralization defect. A reduction in circulatory IGF-I may modulate the reduction of bone mass.

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene.

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.

Optimizing bone metabolism in osteoporosis: insight into the pharmacologic profile of strontium ranelate.

Strontium ranelate (SR) is currently being developed for the treatment of osteoporosis. Pharmacologic studies in animal models have shown that its efficacy on bone mass is based on its original mode of action on bone formation and bone resorption. In normal mice, SR increased bone formation and vertebral bone mass. In normal rats, SR increased bone mass and the mechanical properties of vertebral, humeral and femoral bones, associated with increased femoral shaft diameter. Vertebral bone mineral density and bone strength were also increased by SR, whereas stiffness was not altered, underlining that the improvement in bone strength occurs without inducing defective bone mineralization. In normal adult monkey alveolar bone, SR decreased bone resorption and increased bone. In ovariectomized (OVX) rats, SR limited the reduction in bone mineral content and the decrease in trabecular bone volume induced by estrogen deficiency, by inhibiting bone resorption while maintaining bone formation. Curative treatment with SR also partially restored bone mass in OVX rats. In the model of hind limb immobilization in rats, SR reduced bone resorption and partially limited long bone loss, as assessed by bone mineral content, bone volume, and histomorphometric and biochemical indices of bone resorption. The unique mode of action of SR on bone formation and resorption is also supported by in vitro studies. In calvaria culture systems and osteoblastic cell cultures, SR enhanced the replication of preosteoblastic cells and consequently increased collagen synthesis. Moreover, SR inhibited the bone-resorbing activity of isolated mouse osteoclasts and devreased osteoclast differentiation markers in chicken bone marrow cultures. Altogether, these pharmacologic results suggest that SR optimizes bone metabolism by decreasing bone resorption and promoting bone formation, which may be of potential value in the treatment of osteoporosis.

Prolonged high-dose phosphate treatment: a risk factor for tertiary hyperparathyroidism in X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets is characterized by renal phosphate wasting, hypophosphatemia and defective bone mineralization. Treatment with oral phosphate (Pi) and calcitriol improves skeletal changes but associates with secondary hyperparathyroidism and nephrocalcinosis. Tertiary hyperparathyroidism is a rare complication of the treatment. The aim of the present study was to identify treatment-related factors that might be associated with the transition of secondary hyperparathyroidism to tertiary hyperparathyroidism in patients with X-linked hypophosphatemic rickets. Thirteen patients with X-linked hypophosphatemic rickets and secondary or tertiary hyperparathyroidism were included in the study. Their hospital records were reviewed and compared for onset, duration and dosage of treatment, and for age of diagnosis and degree of secondary hyperparathyroidism. Two patients developed tertiary hyperparathyroidism and 11 patients secondary hyperparathyroidism during the treatment. Patients with tertiary hyperparathyroidism had, on average, earlier onset and longer duration of treatment, higher dose of Pi and longer duration of treatment with very high Pi doses (> 100 mg/kg/day) compared to the 11 patients with secondary hyperparathyroidism. However, variation of all parameters was great with considerable overlap. Very high S-PTH levels > or = 42 pmol/l were observed in those who later developed tertiary hyperparathyroidism. Prolonged very high dose oral Pi treatment is a major risk factor for the development of tertiary hyperparathyroidism in X-linked hypophosphatemic rickets.

Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression.

Inactivating mutations and/or deletions of PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans and in the murine homolog Hyp. The predominant osteoblastic expression of Phex has implicated a primary metabolic osteoblast defect in the pathophysiology of this disorder. By targeting PHEX expression to osteoblasts in the Hyp genetic background, we aimed to correct the corresponding biochemical and morphological abnormalities and obtain information on their pathogenetic mechanism. When transgene Phex expression, driven by a mouse pro-alpha1(I) collagen gene promoter, was crossed into the Hyp background, it improved the defective mineralization of bone and teeth but failed to correct the hypophosphatemia and altered vitamin D metabolism associated with the disorder. Ex vivo bone marrow cultures confirmed the amelioration in the Hyp-associated matrix mineralization defect after Phex expression. These findings suggest that while the Hyp bone and teeth abnormalities partially correct after PHEX gene transfer, additional factors and/or sites of PHEX expression are likely critical for the elaboration of the appropriate molecular signals that alter renal phosphate handling and vitamin D metabolism in this disorder.

Evidence of a founder effect for the tissue-nonspecific alkaline phosphatase (TNSALP) gene E174K mutation in hypophosphatasia patients.

Hypophosphatasia is a rare inborn error of metabolism characterised by defective bone mineralisation caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The clinical expression of the disease is highly variable, ranging from stillbirth with a poorly mineralised skeleton to pathologic skeletal fractures which develop in late adulthood only. This clinical heterogeneity is due to the strong allelic heterogeneity in the TNSALP gene. We found that mutation E174K is the most frequent in Caucasian patients, and that it was carried by 31% of our patients with mild hypophosphatasia. Because the mutation was found in patients of various geographic origins, we investigated whether it had a unique origin or rather multiple origins due to recurrence of de novo mutations. Three intragenic polymorphisms, S93S, 472+12delG and V505A, were genotyped in patients carrying E174K and in normal unrelated individuals. Our results show that all the E174K mutations are carried by a common ancestral haplotype, also found at low frequency in normal and hypophosphatasia chromosomes. We conclude that the TNSALP gene E174K mutation is the result of a relatively ancient ancestral mutation that occurred on a single chromosome in the north of Western Europe and spread throughout the rest of Europe and into the New World as a result of human migration.

New approach to osteopenia in phenylketonuric patients

Aim: To study bone mineralization in a group of phenylketonuric patients and to search for a possible relationship between bone mineral density, dietary control, serum minerals and nutrition intake. The response to treatment with low‐dose 1.25‐(OH)2 vitamin D in patients with osteopenia was evaluated. Methods: Twenty‐eight phenylketonuric patients (age range: 10–33 y) on dietary treatment were investigated. Bone density at the lumbar spine (Dual Energy X‐ray Absorptiometry), bone formation markers (osteocalcin and bone alkaline phosphatase), serum minerals, index of dietary control and protein, vitamin D and mineral intakes were determined. Results: Of the patients studied, 78.6% had good dietary compliance (462 ± 89 μmol/L). Mean protein, vitamin D and mineral intakes met the recommended dietary allowances (RDAs). Nevertheless, 8 patients had calcium intakes lower than 1000 g/d, and a positive correlation between Z‐score and calcium (r= 0.585; p= 0.002) or phosphorus intake (r= 0.546; p= 0.005) was observed. Osteopenia was detected in 14 patients (50%). Moreover, bone alkaline phosphatase in phenylketonuric patients older than 18 y of age was significantly lower than that in controls (p &gt; 0.0001). No correlation was found between bone mineral density, age, serum minerals, bone formation markers or index of dietary control. Treatment with 0.25 μg/d calcitriol significantly increased bone density in 6 patients. Conclusion: A defect in bone mineralization was detected in 50% of patients in our series. The correct amount of formula intake seems to be necessary for bone mineralization in phenylketonuric patients. Calcitriol can be a useful treatment for these patients, although more studies are needed to confirm these results. Hypercalcaemia and hypercalciuria need to be carefully monitored.