Correction to: Comprehensive genetic analyses using targeted next-generation sequencing and genotype-phenotype correlations in 53 Japanese patients with osteogenesis imperfecta.

The original article has been corrected.

Comprehensive genetic analyses using targeted next-generation sequencing and genotype-phenotype correlations in 53 Japanese patients with osteogenesis imperfecta.

To elucidate mutation spectrum and genotype-phenotype correlations in Japanese patients with OI, we conducted comprehensive genetic analyses using NGS, as this had not been analyzed comprehensively in this patient population. Most mutations were located on COL1A1 and COL1A2. Glycine substitutions in COL1A1 resulted in the severe phenotype. INTRODUCTION: Most cases of osteogenesis imperfecta (OI) are caused by mutations in COL1A1 or COL1A2, which encode α chains of type I collagen. However, mutations in at least 16 other genes also cause OI. The mutation spectrum in Japanese patients with OI has not been comprehensively analyzed, as it is difficult to identify using classical Sanger sequencing. In this study, we aimed to reveal the mutation spectrum and genotype-phenotype correlations in Japanese patients with OI using next-generation sequencing (NGS). METHODS: We designed a capture panel for sequencing 15 candidate OI genes and 19 candidate genes that are associated with bone fragility or Wnt signaling. Using NGS, we examined 53 Japanese patients with OI from unrelated families. RESULTS: Pathogenic mutations were detected in 43 out of 53 individuals. All mutations were heterozygous. Among the 43 individuals, 40 variants were identified including 15 novel mutations. We found these mutations in COL1A1 (n = 30, 69.8%), COL1A2 (n = 12, 27.9%), and IFITM5 (n = 1, 2.3%). Patients with glycine substitution on COL1A1 had a higher frequency of fractures and were more severely short-statured. Although no significant genotype-phenotype correlation was observed for bone mineral density, the trabecular bone score was significantly lower in patients with glycine substitutions. CONCLUSION: We identified pathogenic mutations in 81% of our Japanese patients with OI. Most mutations were located on COL1A1 and COL1A2. This study revealed that glycine substitutions on COL1A1 resulted in the severe phenotype among Japanese patients with OI.

Bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lack expression of osteoclast-reactive vacuolar proton pump.

Craniometaphyseal dysplasia (CMD) is a rare craniotubular bone dysplasia transmitted in autosomal dominant or recessive form. This disease is characterized by cranial bone hyperostosis and deformity of the metaphyses of the long bones. Using osteoclast-like cells formed from patient bone marrow cells, we investigated the pathophysiology of CMD in a 3-yr-old patient. Untreated bone marrow cells from the patient differentiated into osteoclast-like cells in vitro. These cells were shown to have vitronectin beta-receptors using a specific monoclonal antibody, i.e., 23C6 (CD51), which reacts with osteoclasts in human bone biopsy samples. However, the number of these osteoclast-like cells formed from the patient's bone marrow was only 40% of the normal controls. 1,25-dihydroxyvitamin-D3, bovine 1-34 parathyroid hormone, recombinant human interleukin-1 beta, recombinant human interleukin-6, or recombinant human macrophage colony-stimulating factor significantly increased, while salmon calcitonin significantly inhibited, the number of osteoclast-like cells. However, these cells could not resorb sperm whale dentin slices and lacked the osteoclast-reactive vacuolar proton pump as evidenced by a monoclonal antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression of the osteoclast-reactive vacuolar proton pump; and (b) a protooncogene c-src was not associated with the pathogenesis of the present CMD patient.

Increased IL-6-production by cells isolated from the fibrous bone dysplasia tissues in patients with McCune-Albright syndrome.

McCune-Albright syndrome (MAS) is characterized by café-au-lait spot, multiple endocrine hyperfunction, and polyostotic fibrous dysplasia. A somatic point mutation of Gsalpha protein was reported to decrease GTPase activity, leading to increase in the GSalpha-associated hormone actions via cAMP. IL-6 is known to stimulate osteoclast formation and in the IL-6 promoter, a cAMP responsive element has been identified. In this paper, we investigated the role of IL-6 in the bone lesions of MAS, using the isolated fibrous cells from the polyostotic fibrous dysplasia tissues in bones of the two patients with MAS. Bone biopsy specimen revealed the increased osteoclast in number. In both patients, a GSalpha mutation (Arg201 -> His) was identified in the cultured fibrous cells. Intracellular cAMP content and IL-6 secretion by the patient cells were increased. Rp-8Br-cAMP significantly inhibited IL-6 production in the patient cells, while it had no effect on normal control. The addition of dibutyryl cAMP significantly increased the synthesis of IL-6 in normal control cells. In contrast, no effect of dibutyryl cAMP on IL-6 synthesis was observed in the cells from one of the MAS patients. These data suggest that IL-6 is, at least, one of the downstream effectors of cAMP and that the increased IL-6 synthesis has a pathogenic role in the bone lesions of MAS patients via increasing the number of osteoclasts. These results may provide a new strategy for the therapy of MAS patients.

Receptor activator of nuclear factor kappaB ligand (RANKL) is a key molecule of osteoclast formation for bone metastasis in a newly developed model of human neuroblastoma.

Neuroblastoma originates from neural crest cells and is the most common extracranial solid tumor in childhood. Bone metastasis in neuroblastoma is an unfavorable prognostic factor even with intensive therapy. In the present study, we screened four cell lines of human neuroblastoma (NB-1, NB-16, NB-19, and NH-6) for tumorigenicity and metastatic capacity in nude mice and found that NB-19 cells caused osteolytic lesions after s.c. injection into mice. To detect micrometastases in the host tissue, we performed two kinds of PCR-based metastasis assays: (a) genomic PCR assay using the primers for human genome-specific Alu sequence; and (b) reverse transcription-nested PCR assay that detects the expression of tyrosine hydroxylase, a marker specific for neuroblastoma. The results of these PCR assays revealed the colonization of human neuroblastoma cells in the bone marrow of the mice that had received the s.c. injection of NB-19 cells. Because osteoclastic bone resorption has been reported to play important roles in osteolysis in some cancers such as breast cancer, we next examined the osteoclast (OC)-inducing activity of NB-19 cells using a coculture system in which NB-19 cells were cultured with murine bone marrow cells containing OC precursors and stromal cells. NB-19 cells induced tartrate-resistant acid phosphatase-positive multinucleated OC-like cells without requirement of 1,25-dihydroxyvitamin D3 or other osteoclastogenic stimulators. To investigate the factors involved in the osteoclastogenesis in the coculture of mouse marrow cells and NB-19 cells, we performed reverse transcription-PCR analysis and revealed the increased expression of receptor activator of nuclear factor kappaB ligand (RANKL) in the coculture compared with the culture of bone marrow cells alone. Interleukin-1alpha and cyclooxygenase-2 expression in the murine marrow cells was also increased in the presence of NB-19 cells. To further study the role of RANKL in the OC-like cell formation in the coculture of NB-19 cells and murine marrow cells, an expression vector encoding the active portion of the murine osteoprotegerin, which is the native inhibitor of RANKL action, was constructed and introduced into COS-7 cells. The conditioned media of the COS-7 cells transfected with the osteoprotegerin expression vector effectively blocked OC-like cell formation in the coculture of the bone marrow cells and NB-19 cells. These results suggested that in the bone microenvironment of NB-19-bearing mice, the stimulated expression of RANKL plays an important role in OC formation, leading to osteolytic bone metastasis.

Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells.

Chondromodulin-1 (ChM-1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. To clarify the tissue-specific expression and the role of ChM-1 in pathophysiological conditions, we analyzed the structure of the human ChM-1 gene and its promoter. On the screening of a human genomic cosmid library using the human ChM-1 complimentary DNA (cDNA) as a probe, two clones were obtained that contained ChM-1 cDNA. The restriction enzyme map and nucleotide sequence revealed the human ChM-1 gene consisting of seven exons and exon-intron boundaries. The human ChM-1 gene was assigned to chromosome 13q14-21 by fluorescence in situ hybridization (FISH) using the clone as a probe. A primer extension analysis using total RNA extracted from human cartilage revealed a major transcription start site with the sequence CGCT+1GG. The region approximately 3-kilobase (kb) nucleotides upstream of the translation start site was then sequenced and analyzed in terms of promoter activity. We found that a region 446 base pairs (bp) upstream of the start site had promoter activity in COS7, HeLa, and ATDC5 cells. In structure the promoter is a TATA-less type without a GC-rich region. The transcription factors Sox9, Og12, and Cart-1 did not affect the promoter activity. The transcription factor Ying-Yang1 suppressed the promoter activity but GABP protein did not change the promoter activity. The construct containing -446/+87 fused to the SV40 enhancer and green fluorescent protein (GFP) exhibited expression of GFP corresponding to the differentiation of ATDC5 cells to mature chondrocytes. These results suggest that the element -446/+87 confers the cartilage-specific expression of this gene by some factor(s) other than Sox9, Og12, and Cart-1.

Hydroxylases involved in vitamin D metabolism are differentially expressed in murine embryonic kidney: application of whole mount in situ hybridization.

In this study we examined the expression of 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase) and 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) by RT-PCR and whole mount in situ hybridization using organ culture of kidney taken from mouse embryo. First, the kidneys of mouse embryo at 11.5-17.5 days gestation were cultured in the presence or absence of forskolin and 1,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)]. Forskolin and 1alpha,25-(OH)(2)D(3) induced the expression of 1alpha-hydroxylase and 24-hydroxylase, respectively, in a dose- and time-dependent manner. In the absence of stimulants, the expression of 1alpha-hydroxylase and 24-hydroxylase was detected from days 13.5-17.5 gestation. The expression of vitamin D receptor and megalin was detected from days 13.5 and 11.5, respectively. Next, signals for the expression of either 1alpha-hydroxylase or 24-hydroxylase were detected by whole mount in situ hybridization in kidney explants taken from embryo at 15.5 days gestation after the appropriate stimulation. However, the localization of signals differed between the two enzymes; 1alpha-hydroxylase messenger RNA was expressed in the inner area of the kidney explants, whereas 24-hydroxylase messenger RNA was expressed in the surface area. The expression of both hydroxylases was restricted to the epithelium of developing renal tubules. The pattern of megalin expression was similar to that of 1alpha-hydroxylase expression. To confirm the difference in distribution of 1alpha-hydroxylase and 24-hydroxylase transcripts, the explants were hybridized with probes for both 1alpha-hydroxylase and 24-hydroxylase using double labeling techniques after simultaneous stimulation with forskolin and 1alpha,25-(OH)(2)D(3), resulting in the detection at different locations of positive signals for the two enzymes. These results suggest that the expression of 1alpha-hydroxylase is induced in a distinct epithelium of renal tubules from that of 24-hydroxylase even at the early stage of kidney development before glomerulogenesis.

Hypercalcemia associated with infantile fibrosarcoma producing parathyroid hormone-related protein.

We describe a 7-month-old boy who manifested severe hypercalcemia associated with mesenchymal neoplasm. A huge hypervascular tumor on the neck had been detected in prenatal ultrasonography. Surgical removal of the entire tumor at birth was not indicated, because the tumor was diagnosed as hemangioma. Chemotherapy and radiotherapy were attempted, but there was no effect on tumor growth. When the infant was 6 months old, the serum calcium level increased rapidly, associated with the expansion of the tumor. Hypophosphatemia due to phosphaturia was also observed. Serum PTH was undetectable, whereas the serum concentration of carboxyl-terminal (C-terminal) fragments of PTH-related protein (PTH-rP) was markedly elevated. Northern blot analysis and immunostaining demonstrated the expression of PTH-rP in the tumor. The tumor was transplantable to nude mice and caused elevation of circulating PTH-rP in the animals. Histological examination of the patient's bone revealed an increased number of osteoclasts. These findings were consistent with humoral hypercalcemia of malignancy caused by the excess production of PTH-rP. The tumor was identified histologically as infantile fibrosarcoma, which has not been reported as a cause of humoral hypercalcemia of malignancy to date. The expression of PTH/PTH-rP receptor messenger ribonucleic acid was detected in the tumor by the RT-PCR, suggesting that PTH-rP may have exerted its effect in the tumor in an autocrine/paracrine manner. In addition to the systemic effect of PTH-rP manifested as hypercalcemia, the PTH-rP secreted from the neoplasm could have been a local factor involved in the growth of the tumor.

Asp361Val Mutant of alkaline phosphatase found in patients with dominantly inherited hypophosphatasia inhibits the activity of the wild-type enzyme.

Hypophosphatasia is characterized by the hypomineralization of bone associated with the mutation of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Although the disease is usually autosomal recessive, an autosomal dominant form is also recognized. Approximately 50 mutations have been found in the TNSALP gene in patients with hypophosphatasia. However, the mutations identified to date do not seem to account for the dominantly inherited form of the disease. We have examined a German family in which the father and all 4 children were affected with hypophosphatasia, whereas the mother was healthy. The affected members of this family showed premature loss of deciduous teeth at or shortly before 2 yr of age and low levels of serum ALP with elevated levels of urinary phosphoethanolamine. DNA analysis by direct sequencing revealed a heterozygous missense mutation that caused the conversion of amino acid Asp to Val at position 361 (D361V) in the patients. Another substitution was detected in exon 12 (Val to Ala conversion at codon 505: V505A) in 1 allele of the mother and 3 children, indicating no association of the substitution with the disease. Reconstruction experiments demonstrated that the D361V mutant protein lost its enzymatic activity and that it inhibited the function of wild-type enzyme when coexpressed in COS-7 cells. On the other hand, the V505A mutant exhibited enzymatic activities equal to those of the wild-type ALP. It is likely that the mutant D361V protein forms dimers with the wild-type protein, and the protein-protein interaction contributes to the dominant effect of the mutant D361V. The mutation that causes D361V is the first one proven to be associated with the dominant form of hypophosphatasia.

Identification of novel missense mutations (Phe310Leu and Gly439Arg) in a neonatal case of hypophosphatasia.

Hypophosphatasia is associated with a defect of the tissue-non-specific alkaline phosphatase gene. We performed a mutational analysis in a surviving patient diagnosed at birth as having hypophosphatasia, on the basis of a low level of serum alkaline phosphatase (ALP) activity and characteristic radiographical findings. She had two sisters, one of whom died of respiratory failure complicated by perinatal hypophosphatasia; the other seemed healthy, with a relatively low activity level of ALP. The patient's parents also had low ALP activity. Sequence analysis of the tissue-nonspecific alkaline phosphatase gene was performed, using genomic DNA and total RNA from the skin fibroblasts of the patient and the peripheral mononuclear cells of her parents. The conversion of Phe to Leu at codon 310 (F310L) and Gly to Arg at 439 (G439R) were identified in the patient. Interestingly, the reconstructive experiments demonstrated that the F310L mutant exhibited an ALP activity level 65% of the normal level, whereas the mutant G439R had no activity. Moreover, the digestion by StuI, after a PCR using complementary DNA extracted from fibroblasts of the patient and lymphocytes of her father, revealed a relatively low messenger RNA level of F310L. These findings suggest that the neonatal case of hypophosphatasia was associated with compound mutations, one of which caused the loss of ALP activity and the other of which caused a slight reduction of the ALP activity, with a relatively low level of messenger RNA.

Analysis of localization of mutated tissue-nonspecific alkaline phosphatase proteins associated with neonatal hypophosphatasia using green fluorescent protein chimeras.

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458-4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

Use of bisphosphonates for the treatment of bone metastasis in experimental animal models.

Therapeutic effectiveness of bisphosphonates (BP) on bone metastases in patients with cancers including those of the breast and prostate has been well documented. However, there are still many important questions that remain unsolved or controversial. To obtain answers for these questions that are not readily addressed in a well-controlled manner in clinical studies, we have developed two animal models of bone metastasis (orthotopic and experimental). Using these models, we studied the effects of BP alone or in combination with anti-cancer agents on the metastasis of breast cancer to bone and visceral organs. In addition, we also determined the effects of BP on osteosclerotic metastases. We found that BP impaired the progression of bone metastases primarily through enhancing apoptosis in osteoclasts and breast cancer cells colonized in bone. In some situations, however, BP alone increased metastases in visceral organs including liver and adrenal glands. However, combination of BP with anti-cancer agents enhanced the suppression of tumour in both bone and visceral organs, leading to prolonged survival of tumour-bearing animals. Of potential importance, preventative administration of BP inhibited the development of eventual osteosclerotic bone metastases. These results suggest that BP exhibits diverse beneficial effects on osteolytic and osteoblastic bone metastasis and non-bone organ metastasis in breast cancer when administered appropriately. They also suggest that the animal models of bone metastasis described here allow us to produce clinically- relevant information that is useful for the design of optimal regimens of BP for the treatment of breast cancer patients with bone and visceral metastases.

Analysis of the rat interpeduncular subnuclei by immunocytochemical double-staining for enkephalin and substance P, with some reference to the coexistence of both peptides.

The rat interpeduncular nucleus (IPN) was immunocytochemically double-stained for enkephalin (ENK) and substance P (SP) on the same sections. On the basis of both peptidergic distribution patterns and topographic relationship, the IPN was divided into nine subnuclei and one cap: the rostral subnucleus (IP-R), the central subnucleus (IP-C), the rostral-lateral subnucleus (IP-RL), the main lateral subnucleus (IP-L), the caudal-lateral subnucleus (IP-CL), the dorsal-lateral subnucleus (IP-DL), the dorsal-medial subnucleus (IP-DM), the apical subnucleus (IP-A), the intermediate subnucleus (IP-I), and the dorsal cap (IP-Cap). As the descriptions of the IP-RL, IP-L, and IP-CL were inconsistent with previous reports, they were reevaluated; the IP-RL was proposed as the region situated in the lateral portion at rostral levels and characterized by the lack of ENK and SP immunoreactive structures, the IP-L as the region situated throughout the rostrocaudal extent in the lateral portion of the IPN and containing the highest density of SP immunoreactive fibers but no ENK immunoreactive fibers, and the IP-CL as the region situated just laterocaudal to the IP-L in the caudal pole of the IPN and containing ENK immunoreactive cells and fibers but no SP immunoreactive structures. Our results also showed that some cells in the IP-R have both ENK and SP immunoreactivity. This coexistence was observed in some small spherical cells of the IP-R, but rarely in larger oval-shaped cells, which occasionally showed only ENK immunoreactivity. In addition, paired ENK immunoreactive fiber bundles entering the IP-R were found to run just rostral to the paired SP immunoreactive columns, both of which composed parts of the interpedunculotegmental tract. A three-dimensional model representing the subnuclear organization of the IPN was proposed on the basis of the present results.

Growth and dissemination of a newly-established murine B-cell lymphoma cell line is inhibited by multimeric YIGSR peptide.

B-cell lymphoma frequently shows simultaneous dissemination to multiple organs. It also occasionally involves bone and causes osteolytic lesions. To study the mechanisms responsible for this capacity of lymphoma cells to grow in different tissue microenvironments and search for effective therapeutic interventions for this hematological malignancy, we established a new murine B-cell lymphoma cell line named MH-95. The tumor disseminated to multiple organs including the lung, liver, kidney, spleen and lymph nodes within 2 weeks after subcutaneous inoculation in nude mice. In addition, the tumor also grew in bone and caused osteoclastic osteolytic lesions. Thus, this tumor model mimics the behavior in many ways of B-cell lymphoma in humans. We studied the role of laminin, a major component of the basement membrane, in this model, since although it has been implicated in solid tumor metastasis, little is known about the involvement of laminin in the growth of B-cell lymphoma in bone and other organs. Immunohistochemical examination showed strong laminin expression in the stroma of the primary subcutaneous tumor and tumors in the bone and other organs. Systemic administration of the antagonistic laminin peptide YIGSR decreased primary tumor growth and tumor cell deposit in the bone, liver and kidney. In addition, the peptide also decreased apparent neovascularization in the tumor, suggesting that the peptide suppressed angiogenesis presumably due to inhibition of laminin binding to its receptors. These results demonstrate that the MH-95 B-cell lymphoma cells express laminin and suggest that laminin plays a critical role in the growth and simultaneous dissemination of tumor cells to multiple organs, similar to what has been described in solid tumors. The results also suggest that suppression of angiogenesis through interfering with laminin actions may be a useful adjuvant therapy for B-cell lymphoma.

Novel mutations in the a3 subunit of vacuolar H(+)-adenosine triphosphatase in a Japanese patient with infantile malignant osteopetrosis.

A case of infantile malignant osteopetrosis is described. The patient died from respiratory hemorrhage at 7 months of age despite treatment that included high doses of active vitamin D and administration of interferon-gamma. A postmortem examination revealed the presence of many osteoclasts in the bone, which lacked ruffled borders. This observation was consistent with the histology of bone reported in Atp6i-knockout mice, which lack the gene encoding the a3 subunit of vacuolar-type H(+)-adenosine triphosphatase (ATPase). Sequence analysis of the TCIRG1 gene encoding the a3 subunit revealed two novel mutations: a deletion/insertion mutation in exon 9 and a T-to-C transition at the splice donor site of intron 19. The former mutation caused a frame shift and premature stop codon. The latter was associated with abnormal splicing, which was confirmed by sequencing the products amplified by reverse transcription-polymerase chain reaction (RT-PCR), using total RNA from the liver specimen as template. Although several mutations in the TCIRG1 gene in infantile malignant osteopetrosis have been reported in other populations, this is the first case of a Japanese patient with a mutation identified in this gene. These results support the important role of the subunit in the function of the proton pump.

Conflicting actions of parathyroid hormone-related protein and serum calcium as regulators of 25-hydroxyvitamin D(3)-1 alpha-hydroxylase expression in a nude rat model of humoral hypercalcemia of malignancy.

In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.

[A case report of glomerulocystic kidney disease with hypothyroidism in a new born infant].

We report a rare case of glomerulocystic kidney disease (GCKD) with congenital hypothyroidism. A gigantic abdominal mass was noted at birth. There was no family history of renal cystic disease. Ultrasonography revealed diffuse granular cysts in the markedly enlarged kidneys. Blood examination showed moderate renal failure and hypothyroidism. Bilateral nephrectomy was conducted at 47 days of age to relieve respiratory failure and severe abdominal distention caused by the growing cystic kidneys. Histological findings of the kidney showed numerous glomerular cysts without renal dysplasia. There were no other malformations. These findings were compatible with GCKD.

Global Consensus Recommendations on Prevention and Management of Nutritional Rickets

"BACKGROUND: Vitamin D and calcium deficiencies are common worldwide, causing nutritional rickets and osteomalacia, which have a major impact on health, growth, and development of infants, children, and adolescents; the consequences can be lethal or can last into adulthood. The goals of this evidence-based consensus document are to provide health care professionals with guidance for prevention, diagnosis, and management of nutritional rickets and to provide policy makers with a framework to work toward its eradication. EVIDENCE: A systematic literature search examining the definition, diagnosis, treatment, and prevention of nutritional rickets in children was conducted. Evidence-based recommendations were developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system that describes the strength of the recommendation and the quality of supporting evidence. PROCESS: Thirty-three nominated experts in pediatric endocrinology, pediatrics, nutrition, epidemiology, public health, and health economics evaluated the evidence on specific questions within five working groups. The consensus group, representing 11 international scientific organizations, participated in a multiday conference in May 2014 to reach a global evidence-based consensus. RESULTS: This consensus document defines nutritional rickets and its diagnostic criteria and describes the clinical management of rickets and osteomalacia. Risk factors, particularly in mothers and infants, are ranked, and specific prevention recommendations including food fortification and supplementation are offered for both the clinical and public health contexts. CONCLUSION: Rickets, osteomalacia, and vitamin D and calcium deficiencies are preventable global public health problems in infants, children, and adolescents. Implementation of international rickets prevention programs, including supplementation and food fortification, is urgently required."

Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.

BACKGROUND: Bone, which abundantly stores a variety of growth factors, provides a fertile soil for cancer cells to develop metastases by supplying these growth factors as a consequence of osteoclastic bone resorption. Accordingly, suppression of osteoclast activity is a primary approach to inhibit bone metastasis, and bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used for the treatment of bone metastases in cancer patients. To obtain further insights into the therapeutic usefulness of BP, the authors studied the effects of BP on bone and visceral metastases in animal models of metastasis. METHODS: The authors used two animal models of breast carcinoma metastasis that they had developed in their laboratory over the last several years. One model uses female young nude mice in which inoculation of the MDA-MB-231 or MCF-7 human breast carcinoma cells into the left cardiac ventricle selectively develops osteolytic or osteosclerotic bone metastases, respectively. Another model uses syngeneic female mice (Balb/c) in which orthotopic inoculation of the 4T1 murine mammary carcinoma cells develops metastases in bone and visceral organs including lung, liver, and kidney. RESULTS: BP inhibited the development and progression of osteolytic bone metastases of MDA-MB-231 breast carcinoma through increased apoptosis in osteoclasts and breast carcinoma cells colonized in bone. In a preventative administration, however, BP alone increased the metastases to visceral organs with profound inhibition of bone metastases. However, combination of BP with anticancer agents such as uracil and tegafur or doxorubicin suppressed the metastases not only in bone but also visceral organs and prolonged the survival in 4T1 mammary tumor-bearing animals. Of interest, inhibition of early osteolysis by BP inhibited the subsequent development of osteosclerotic bone metastases of MCF-7 breast carcinoma. CONCLUSIONS: These results suggest that BP has beneficial effects on bone metastasis of breast carcinoma and is more effective when combined with anticancer agents. They also suggest that the animal models of bone metastasis described here allow us to design optimized regimen of BP administration for the treatment of breast carcinoma patients with bone and visceral metastases.

Severe hypercalcaemia and respiratory insufficiency associated with infantile hypophosphatasia caused by two novel mutations of the tissue-nonspecific alkaline phosphatase gene.

UNLABELLED: We report the case of a male patient with infantile hypophosphatasia associated with severe hypercalcaemia and mild respiratory insufficiency. At the age of 2 months, severe hypercalcaemia, low levels of serum alkaline phosphatase activity, and elevated urinary excretion of calcium and phosphoethanolamine were noted. Radiological findings showed generalized osteopenia and disturbed and irregular ossification of the metaphyses. Their involvement had spontaneously improved at the age of 6 months. A genetic study revealed that the tissue-nonspecific alkaline phosphatase gene of the patient had two novel mutations, K207E and G409C, derived from the mother and father, respectively. A reconsitution experiment revealed that both mutant gene products had low but significant enzymatic activity. CONCLUSION: The detection of tissue-nonspecific alkaline phosphatase gene mutations and expression studies to determine the enzymatic activity of mutant gene products was useful for assessing the clinical course of this patient with hypophosphatasia.