Bone particles from osteopetrotic mice not cured by bone marrow transplants are resorbed in normal littermates

Osteosclerotic (oc/oc) and osteopetrotic (op/op) mice are not cured by bone marrow transplantation from normal littermates. The possibility that this is due to production of poorly resorbable bone was examined by comparing the fate of mutant and normal bone particles implanted subcutaneously in normal hosts. Bone, removed aseptically from calvarial and tibial sites of normal littermates and mutants, was cleaned of adherent soft tissue, ground and sieved to a particle size of 70–300 μm. Aliquots (17–20 mg) of bone from each phenotype of each stock were pelleted and implanted beneath the anterior thoracic skin of normal littermates for two weeks. Particle density in tissue sections was determined as percent of field by a point-counting method. Giant cell response was recorded as number per high-power field. Percent bone present initially was determined in pellets implanted for less than 24 hr. Bone particles were reduced in each pellet with time, about 25% of the original volume being removed in two weeks. No statistically significant differences were noted in the rates of disappearance of mutant and normal bone or in the percentage or number of giant cells in implants of mutant and normal bone in either stock. Furthermore, these values were not different from identical studies in microphthalmic mice, an osteopetrotic stock cured by bone marrow transplantation. These data suggest that the failure of osteopetrotic and osteosclerotic mice to be cured by bone marrow transplants from normal littermates is not due to the presence of unresorbable bone.     

Failure of normal osteoclasts to resorb calcified cartilage from osteosclerotic (oc/oc) mice in vitro

Osteosclerosis is an osteopetrotic mutation in the mouse characterized by reduced bone resorption, numerous small osteoclasts lacking elaborate ruffled borders, and resistance to cure by bone marrow transplants from normal littermates. The failure of osteosclerotic mice to be cured by bone marrow transplants could be due to the production of bone that is not resorbable by normal osteoclasts. We tested this hypothesis using a modification of the metatarsal organ culture system of Burger et al. (1982), in which metatarsals are cultured with various tissues that act as sources of osteoclast precursors. Metatarsals from neonatal mutants were isolated, and live bone rudiments were cultured with small cubes of liver or spleen from normal littermates for 7 days. Controls included normal and mutant metatarsals cultured alone or with spleen or liver from littermates of the same or different genotype. Mutant metatarsals cultured alone or with mutant tissue had small osteoclasts, no marrow spaces, and no evidence of bone resorption. Mutant metatarsals cultured with normal liver or spleen had larger osteoclasts, evidence of resorption of bone but not cartilage, and no marrow spaces because the calcified cartilage cores of metaphyseal trabeculae persisted. Normal metatarsals cultured with normal liver had large osteoclasts, bone resorption, and marrow spaces. By transmission EM, mutant trabeculae contained a layer of amorphous material between the central core of calcified cartilage and the surrounding bone matrix. This material was not present in normal metatarsals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Administration of colony stimulating factor-1 to toothless osteopetrotic rats normalizes osteoblast, but not osteoclast, gene expression

The toothless (tl) osteopetrotic mutation in the rat is characterized by generalized skeletal sclerosis, a severe reduction in the numbers of osteoclasts, monocytes, and macrophages, and absence of tooth eruption. Studies examining gene expression in bone-derived cells of tl rats and their normal littermates have shown that genes related to osteoblast function are aberrantly expressed in tl rats compared to normal littermates. We have previously shown that exogenous administration of colony stimulating factor-1 (CSF-1) to tl rats results in a dramatic reduction of the skeletal sclerosis and significant increases in the number of osteoclasts. Thus, we examined the effects of CSF-1 on osteoblast and osteoclast gene expression in tl rats as demonstrated by Northern blot analysis. While osteoblast-related gene expression as reflected by mRNA levels of alkaline phosphatase, osteocalcin, osteopontin, and type I collagen was normalized, osteoclastrelated gene expression, as reflected by mRNA levels of carbonic anhydrase II and tartrate-resistant adenosine triphosphatase, remained significantly lower in CSF-1-treated tl rats compared to untreated normal littermates. Since previous studies have not demonstrated the CSF-1 receptor on osteoblasts, these results suggest that osteoblast abnormalities in tl rats are an effect of the osteopetrotic condition rather than the cause of the disease.     

Defective bone formation by Hyp mouse bone cells transplanted into normal mice: evidence in favor of an intrinsic osteoblast defect.

The hypophosphatemic (Hyp) mouse is an animal model for human hypophosphatemic vitamin D-resistant rickets. We have reported that bone cells isolated from Hyp mice born to homozygous mutant females produce abnormal bone when transplanted into normal mice. To test whether an environmentally acquired defect of the mutant cells contributed to the impaired bone formation observed in transplants, periostea and osteoblasts from normal and Hyp littermates were transplanted intramuscularly into normal animals. To test more specifically for an hypophosphatemia-induced cell alteration before transplantation, bone cells isolated from phosphate-depleted normal mice were transplanted into normal animals. The bone nodules formed in 2 week transplants were characterized by measuring their osteoid thickness and volume. Impaired bone formation was evidenced in Hyp transplants compared to normal littermate transplants by increased osteoid thickness and volume. In contrast to cells from mutant mice, cells isolated from normal mice with comparable hypophosphatemia produced normal bone. These results indicate that the inability of Hyp osteoblasts to produce normal bone when placed in a normal environment is not the consequence of prior exposure to an altered environmental but likely of an intrinsic cellular abnormality. These observations add further support to the concept that the osteoblast is an important target for the Hyp mutation.     

Auditory ossicle abnormalities and hearing loss in the toothless (osteopetrotic) mutation in the rat and their improvement after treatment with colony-stimulating factor-1.

Osteopetrosis describes a group of skeletal metabolic diseases of heterogeneous etiology and varied severity that produces a generalized accumulation of skeletal mass, the result of reduced bone resorption. Inherited in a variety of species including humans, the most severe forms are lethal. Among common features are progressive blindness and deafness of controversial etiologies for which there are no universally effective treatments. We have studied the auditory responsiveness and auditory ossicle quantitative histomorphology and temporal bone vasculature in the toothless (tl) rat, a lethal osteopetrotic mutation with few osteoclasts, very low bone turnover, and limited angiogenesis in the axial skeleton. Compared with normal littermates, 3-week-old mutants showed significantly reduced auditory responsiveness, a hearing loss due to abnormalities in both form and tissue composition of the stapes, and little capillary sprouting in the vascular bed of the temporal bone. Treatment of mutants with colony-stimulating factor 1 (CSF-1), known to greatly reduce sclerosis in the axial skeleton, significantly improved hearing, stapedial form and tissue composition, and angiogenesis in the temporal bone. In normal rats, the stapes consisted of 89.3% bone, 9.1% mineralized cartilage, and 0.8% porosity. In osteopetrotic rats, the stapes consisted of 48.3% bone, 35.9% mineralized cartilage, and 15.9% porosity, while after CSF-1 treatment, the bone content increased to 55.2%, cartilage was decreased to 21.7%, and porosity increased to 23.0%, respectively. This is the first demonstration of an auditory abnormality in an osteopetrotic animal mutation and shows that the hearing loss in tl rats can be significantly improved following treatment with CSF-1.     

Treatment of congenital osteopetrosis in the rabbit with high-dose 1,25-dihydroxyvitamin D

Osteopetrosis is a congenital metabolic bone disease characterized by skeletal sclerosis resulting from defective osteoclast-mediated bone resorption. Osteopetrosis has been described in several animal species (mouse, rat, and rabbit) and in children. Bone marrow transplantation, originally shown to reverse the skeletal sclerosis in some animal mutations, has been effective in curing osteopetrosis in some children. Unfortunately, not all children with osteopetrosis are candidates for or respond to bone marrow transplantation. Recent studies have shown that several animal mutations and some children inheriting osteopetrosis have significantly elevated serum levels of 1,25-(OH)2D. Based on the possibility that there may be a resistance to 1,25-(OH)2D, high-dose calcitriol therapy has been used to treat some children and stimulated some parameters of resorption. In this study, we have examined the effects of high-dose calcitriol therapy on various serum and skeletal parameters in the osteopetrotic rabbit. Mutant rabbits and normal littermates were given continuous infusions of calcitriol via subcutaneously implanted osmotic minipumps for 2 weeks at a dose of 0.5, 2.5, or 25 micrograms/kg/per day. Untreated mutant rabbits are hypocalcemic and hypophosphatemic in the presence of elevated serum 1,25-(OH)2 levels in comparison with their normal littermates. Calcitriol infusions resulted in dose-dependent increases in circulating 1,25-(OH)2D levels in both normal and mutant rabbits. However, evaluation of other serum parameters and the skeletal response demonstrated significant differences between osteopetrotic and normal rabbits. At the highest dose, normal animals rapidly became hypercalcemic and osteoporotic, accompanied by weight loss and a failure to thrive; mutants remained hypocalcemic and osteopetrotic but did not exhibit the deleterious physical effects seen in treated normal littermates. Although the number of osteoclasts increased in both mutants and normals, osteoclast phenotype in the former remained abnormal. These data indicate that although very high levels of circulating 1,25-(OH)2D were achieved in osteopetrotic mutants, activation of osteoclast-mediated bone resorption with subsequent improvement of skeletal sclerosis was not observed.     

Mineral and matrix alterations in the bones of incisors-absent (ia/ia) osteopetrotic rats

Summary The bones of incisors-absent (ia/ia) osteopetrotic rats differ from those of their normal littermates (ia/+) in histologic and radiographic appearance and in mechanical properties. This study examined how the mineral and matrices of osteopetrotic metaphyses and calvaria differed from normal controls. Bones of 11-day-old and 52-day-old osteopetrotic animals had higher ash (mineral) contents that age-matched controls; osteopetrotic metaphyses had elevated hexosamine contents, indicative of the persistence of cartilage. Calcium acidic phospholipid phosphate complexes, involved in initiation of hydroxyapatite formationin vivo andin vitro, were significantly reduced in content in all osteopetrotic bones. These results suggest that in the osteopetrotic rat, where osteoclast activity is defective, new mineral formation is reduced and replaced by accretion of mineral on existing crystals. Fractionation of osteopetrotic bone particles by density centrifugation demonstrated that the osteopetrotic bone was much more mineralized than that of age-matched controls. X-ray diffraction analysis of the mineralized fractions indicated a lack of growth of mineral crystals during maturation of the osteopetrotic animals. The absence of remodeling was apparent from the elevated Ca:P ratios of the highly mineralized osteopetrotic bone fractions, and from the high hexosamine content of these dense fractions. These observations may explain some of the unusual mechanical properties of osteopetrotic bone.     

The biology of macrophages in osteopetrosis. Structure and function

Osteoclasts from ia rats are present in excessive numbers, contain an excessive amount of the lysosomal enzyme acid phosphatase, and show reduced-bone-resorptive function due to lack of ruffled borders. Given the current theory of a mononuclear phagocytic cell origin for osteoclasts, it was anticipated that the presence or absence of similarities between macrophages of both phenotypes might address the relatedness of these two cells. Thus, peritoneal macrophages from littermate normal and incisors-absent (ia) osteopetrotic rats were examined for differences in structure, acid phosphatase content, and phagocytic activity. The results of this investigation did not reveal any significant differences in these parameters in mutants compared with normal littermates. From these data it is suggested that macrophages are distinct, differentiated cells with functions separate from those of osteoclasts but that they may be related through a common stem cell early in development. Use of more immature cells or monocyte subpopulations may represent better choices for future studies of defects within the mononuclear phagocyte system in the ia mutation.     

Treatment with recombinant human macrophage colony-stimulating factor resorbs blood clot and restores osteoclastogenesis in heterotopic bone induced by partially purified native bone morphogenetic protein in osteopetrotic (op/op) mice

Native bone morphogenetic protein and associated noncollagenous proteins induced the formation of heterotopic bone in the hindquarter muscles of osteopetrotic (op/op) mice and those of their phenotypically normal littermates (+/?). In op/op mice, the heterotopic bone consisted of a disorganized, densely packed mixture of irregular calcified cartilage, osleoid, Chondro-osteoid, and fibrous tissue. Injections of recombinant human macrophage colony-stimulating factor initiated bone resorption that began in the peripheral vascularized regions of the metaphyses and continued in central areas of uncalficified avascular chondro-osteoid. On vascularized surfaces, osteoclasts were stained with tartrate-resistant acid phosphatase. In op/op mice treated with macrophage colony-stimulating factor, the osteoclasts were small, with only two or three nuclei and they did not exhibit tartrate-resistant acid phosphatase staining. In untreated op/op mice, surgical blood clots persisted in the heterotopic sites as late as 3 weeks after the operation, whereas in treated op/op mice, the blood clots were absorbed almost as rapidly as in normal mice. Histologically, recombinant human macrophage colony-stimulating factor restored normal macrophage functions: absorption and organization of blood clot, osteoclastogenesis. synthesis of tartrate-resistant acid phosphatase, bone remodeling, islands of myelopoiesis, and construction of an ossicle complete with a cortex and a medulla filled with functioning hematopoietic bone marrow.     

Effects of Altered Bone Remodeling and Retention of Cement Lines on Bone Quality in Osteopetrotic Aged c-Src-Deficient Mice

Cement lines represent mineralized, extracellular matrix interfacial boundaries at which bone resorption by osteoclasts is followed by bone deposition by osteoblasts. To determine the contribution of cement lines to bone quality, the osteopetrotic c-Src mouse model—where cement lines accumulate and persist as a result of defective osteoclastic resorption—was used to investigate age-related changes in structural and mechanical properties of bone having long-lasting cement lines. Cement lines of osteopetrotic bones in c-Src knockout mice progressively mineralized with age up to the level that the entire matrix of cement lines was lost by EDTA decalcification. While it was anticipated that suppressed and abnormal remodeling, together with the accumulation of cement line interfaces, would lead to defective bone quality with advancing age of the mutant mice, unexpectedly, three-point bending tests of the long bones of 1-year-old c-Src-deficient mice indicated significantly elevated strength relative to age-matched wild-type bones despite the presence of numerous de novo microcracks. Among these microcracks in the c-Src bones, there was no sign of preferential propagation or arrest of microcracks along the cement lines in either fractured or nonfractured bones of old c-Src mice. These data indicate that cement lines are not the site of a potential internal failure of bone strength in aged c-Src osteopetrotic mice and that abundant and long-lasting cement lines in these osteopetrotic bones appear to have no negative impacts on the mechanical properties of this low-turnover bone despite their progressive hypermineralization (and thus potential brittleness) with age.     

Characterization of a novel bipotent hematopoietic progenitor population in normal and osteopetrotic mice.

Several reports indicate that osteoclasts and B-lymphocytes share a common progenitor. This study focuses on the characterization of this bipotent progenitor from the bone marrow of the osteopetrotic oc/oc mouse, where the bipotent progenitor population is amplified, and of normal mice. INTRODUCTION: Osteoclasts have a myelomonocytic origin, but they can also arise in vitro from pro-B-cells, suggesting that a subset of normal pro-B-cells is uncommitted and may reorient into the myeloid lineage representing a B-lymphoid/osteoclastic progenitor. The aim of this study was to characterize this progenitor population. MATERIALS AND METHODS: The osteopetrotic oc/oc mouse was used as a choice model because it displays an increased number of both osteoclasts and pro-B-cells in the bone marrow. Our results have been confirmed in normal littermates. Bone marrow cells from these animals were analyzed by flow cytometry. After sorting, the cells were cultured under different conditions to assess their differentiation capacity. RESULTS: Pro-B-cells from oc/oc and normal mice include an unusual biphenotypic population expressing markers from the B-lymphoid (CD19, CD43, CD5) and the myeloid (F4/80) lineages. This population also expresses progenitor markers (CD34 and Flt3) and is uncommitted. After sorting from the oc/oc bone marrow, this population is able to differentiate in vitro into osteoclast-like cells in the presence of RANKL and macrophage colony-stimulating factor (M-CSF), into dendritic-like cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and TNFalpha, and into immature B-cells when seeded onto ST2 cells in the presence of IL-7. CONCLUSION: Our results show the existence of a novel bipotent biphenotypic hematopoietic progenitor population present in the bone marrow that has retained the capacity to differentiate into myeloid and B-lymphoid cells.     

Calcitonin receptor binding as a marker of osteoclast heterogeneity in osteopetrotic rodents

We have employed a radioautographic technique to examine in vivo receptor binding of calcitonin to osteoclasts in four rodent mutants with osteopetrosis. 125I-Labeled calcitonin was injected intravenously alone or with excess unlabeled calcitonin to osteopetrotic (op/op), osteosclerotic (oc/oc), and microphthalmic (mi/mi) mice and to incisor absent (ia/ia) rats. Similar experiments were performed simultaneously in phenotypically normal littermates. Specific binding of calcitonin to receptors on osteoclasts and osteoclast morphology were then examined by light and electron microscope radioautography. Calcitonin binding was increased in mi/mi mice, where osteoclasts were abundant but reduced in size, and was also increased in op/op mice in association with an undulated and redundant osteoclast cell membrane. Binding of the hormone was markedly diminished on osteoclasts of oc/oc mice and ia/ia rats. Thus, in these rodent models of osteopetrosis all of which manifest reduced skeletal remodeling and share a recessive pattern of inheritance, considerable heterogeneity of osteoclast characteristics was demonstrable. Although calcitonin may play no primary pathogenetic role in most forms of this disease, calcitonin receptor binding is a morphological and functional marker of osteoclasts that can be used in assessing the pathophysiology of disorders of bone remodeling.     

Effect of gene dose and parental origin on bone histomorphometry in X-linked Hyp mice

X-linked hypophosphatemia (XLH) is characterized by rickets and osteomalacia and arises from mutations in the Phex and PHEX genes in mice (Hyp) and humans, respectively. The present study was undertaken to examine the effect of gene dose on the skeletal phenotype using a histomorphometric approach. Metrical traits (vertebral length, growth plate thickness, cancellous osteoid volume per bone volume, and cancellous, endocortical, and periosteal osteoid thickness) were compared in caudal vertebrae of mutant female (Hyp/+, Hyp/Hyp) and male (Hyp/Y) mice and their normal female (+/+) and male (+/Y) littermates. Mutant animals had trait values that differed significantly from those of normal animals. However, with the exception of vertebral length and cancellous osteoid thickness, values were not significantly different between the three mutant genotypes. We also examined the effect of gamete-of-origin on histomorphometric parameters in obligate Hyp/+ females derived from male or female transmitting parents. The metrical trait values in both groups of Hyp/+ mice were similar, with the exception of vertebral length and cancellous osteoid volume per bone volume. In summary, we demonstrate that the amount of osteoid per bone volume is similar in the three mutant genotypes and conclude that the extent and magnitude of the mineralization defect is fully dominant and likely not affected by gene dose. The differences in vertebral length in the mutants suggest that rickets and osteomalacia are not the only causes of decreased vertebral growth in Hyp mice and that Phex protein may influence bone growth and mineralization by distinct pathways.     

Qualitative defects in natural killer cell function in ia osteopetrotic rats.

Recent studies have provided evidence that cells of the immune system and their associated cytokines function in the regulation of bone turnover. The incisors absent (ia) osteopetrotic rat represents a model in which a defect in the immune system and bone resorption can be studied. Osteopetrosis in the ia rat is characterized by a generalized excess accumulation of bone as a result of reduced bone resorption by defective osteoclasts that lack a ruffled border and the ability to exocytose their osteolytic enzymes. Previous attempts to identify associated defects in the ia immune system have proven unsuccessful; ia rats demonstrate normal delayed hypersensitivity, mitogenic activity, and macrophage function. Inasmuch as the skeletal manifestations of the ia mutation may be the result of a defect in exocytosis, related defects may be evident in immune cells utilizing exocytosis of granules or enzymes for their cytolytic function. Natural killer (NK) cells function by such a mechanism. Therefore, these studies were undertaken to evaluate the natural immune system in ia rats. NK activity assessed by 51Cr release assays was significantly reduced in ia animals compared to normal littermates. Mononuclear cells isolated from the peripheral blood of ia rats revealed a significantly greater percentage of large granular lymphocytes than normal littermates. Comparison of NK cell phenotypes using two phenotypic parameters for NK cells (OX8+, OX19- cells and 3.2.3+ cells) revealed that the mononuclear isolates of spleen and peripheral blood of mutant animals had significantly greater percentages of OX8+, OX19- and 3.2.3+ cells than normal controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correction of the mineralization defect in hyp mice treated with protease inhibitors CA074 and pepstatin

Increased expression of several osteoblastic proteases and MEPE (a bone matrix protein) occurs in X-linked hypophosphatemic rickets (hyp). This is associated with an increased release of a protease-resistant MEPE peptide (ASARM peptide), a potent inhibitor of mineralization. Cathepsin B cleaves MEPE releasing ASARM peptide and hyp osteoblast/osteocyte cells hypersecrete cathepsin D, an activator of cathepsin B. Our aims were to determine whether cathepsin inhibitors correct the mineralization defect in vivo and whether hyp-bone ASARM peptide levels are reduced after protease treatment. Normal littermates and hyp mice (n = 6) were injected intraperitoneally once a day for 4 weeks with pepstatin, CAO74 or vehicle. Animals were then sacrificed and bones plus serum removed for comprehensive analysis. All hyp mice groups (treated and untreated) remained hypophosphatemic with serum 1,25 vitamin D3 inappropriately normal. Serum PTH was significantly elevated in all hyp mice groups relative to normal mice (P = 0.0017). Untreated hyp mice had six-fold elevated levels of serum alkaline-phosphatase and two-fold elevated levels of ASARM peptides relative to normal mice (P < 0.001). In contrast, serum alkaline phosphatase and serum ASARM peptides were significantly reduced (normalized) in hyp mice treated with CA074 or pepstatin. Serum FGF23 levels remained high in all hyp animal groups (P < 0.0001). Hyp mice treated with protease inhibitors showed dramatic reductions in unmineralized osteoid (femurs) compared to control hyp mice (Goldner staining). Also, hyp animals treated with protease inhibitors showed marked and significant improvements in growth plate width (42%), osteoid thickness (40%) and cortical area (40%) (P < 0.002). The mineralization apposition rate, bone formation rate and mineralization surface were normalized by protease-treatment. High-resolution pQCT mineral histomorphometry measurements and uCT also confirmed a marked mineralization improvement. Finally, the growth plate and cortical bone of hyp femurs contained a massive accumulation of osteoblast-derived ASARM peptide(s) that was reduced in hyp animals treated with CA074 or pepstatin. This study confirms in vivo administration of cathepsin inhibitors improves bone mineralization in hyp mice. This may be due to a protease inhibitor mediated decrease in proteolytic degradation of the extracellular matrix and a reduced release of ASARM peptides (potent mineralization inhibitors).     

Mineral density and bone strength are dissociated in long bones of rat osteopetrotic mutations.

Bone mineral density (BMD) and mechanical strength generally show strong positive correlations. However, osteopetrosis is a metabolic bone disease with increased skeletal density radiographically and increased risk of fracture. We have evaluated mechanical strength and mineral density in three osteopetrotic mutations in the rat (incisors-absent [ia/ia], osteopetrosis [op/op], and toothless [tl/tl]) to test the hypothesis that reduced bone resorption in one or more of these mutations results in weaker bones in the presence of greater mineral density and skeletal mass. Peripheral quantitative computed tomography (pQCT) was used to analyze BMD and cross-sectional geometry in the tibial diaphysis and metaphysis as well as the femoral diaphysis and femoral neck. The bending breaking force of tibial and femoral midshafts was obtained using the three-point bending test and femoral neck strength was tested by axial loading. Osteopetrotic mutants were significantly smaller than their normal littermates (NLMs) in each stock. The pQCT analysis showed that BMD and bone mineral content (BMC) were higher than or equal to NLMs in all skeletal sites measured in the osteopetrotic mutants. However, the mechanical breaking force was equal to or lower than their NLMs in all sites. The cross-sectional structure of long bone shafts was markedly different in osteopetrotic mutants, having a thin cortex and a medullary area filled with primary trabecular bone. These results indicate that osteopetrotic mutations in the rat increase bone density and decrease bone strength. The tibial diaphysis was significantly weaker in tl/tl and ia/ia mutants and the tibial metaphysis showed the greatest increase in BMD in all mutants. These data are another illustration that an increased BMD does not necessarily lead to stronger bones.     

In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects.

We studied osteoclastic differentiation from normal and osteopetrotic human CD14 cells in vitro. Defects in acid transport, organic matrix removal, and cell fusion with deficient attachment were found. Analysis of genotypes showed that TCIRG1 anomalies correlated with acid transport defects, but surprisingly, organic matrix removal failure correlated with CLCN7 defects; an attachment defect had normal TCIRG1 and CLCN7. INTRODUCTION: Osteopetrotic subjects usually have normal macrophage activity, and despite identification of genetic defects associated with osteopetrosis, the specific developmental and biochemical defects in most cases are unclear. Indeed, patients with identical genotypes often have different clinical courses. We classified defects in osteoclast differentiation in vitro using four osteopetrotic subjects without immune or platelet defects, three of them severe infantile cases, compared with normals. MATERIALS AND METHODS: Osteoclast differentiation used isolated CD14 cells; results were correlated with independent analysis of two key genes, CLCN7 and TCIRG1. CD14 cell attachment and cell surface markers and extent of differentiation in RANKL and colony-stimulating factor (CSF)-1 were studied using acid secretion, bone pitting, enzyme, and attachment proteins assays. RESULTS AND CONCLUSIONS: CD14 cells from all subjects had similar lysosomal and nonspecific esterase activity. With the exception of cells from one osteopetrotic subject, CD14 cells from osteopetrotic and control monocytes attached similarly to bone or tissue culture substrate. Cells from one osteopetrotic subject, with normal CLCN7 and TCIRG1, did not attach to bone, did not multinucleate, and formed no podosomes or actin rings in RANKL and CSF-1. Attachment defects are described in osteopetrosis, most commonly mild osteopetrosis with Glantzman's thrombasthenia. However, this case, with abnormal integrin alphavbeta3 aggregates and no osteoclasts, seems to be unique. Two subjects were compound heterozygotes for TCIRG1 defects; both had CD14 cells that attached to bone but did not acidify attachments; cell fusion and attachment occurred, however, in RANKL and CSF-1. This is consistent with TCIRG1, essential for H+-ATPase assembly at the ruffled border. A compound heterozygote for CLCN7 defects had CD14 cells that fused in vitro, attached to bone, and secreted acid, TRACP, and cathepsin K. However, lacunae were shallow and retained demineralized matrix. This suggests that CLCN7 may not limit H+-ATPase activity as hypothesized, but may be involved in control of organic matrix degradation or removal.     

A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene

X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR1 or ARHR2) are hereditary fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets showing similar clinical features. We here show a patient with hypophosphatemic rickets and widespread ossification of posterior longitudinal ligament (OPLL). The proband is a 62-year-old female. Her parents are first cousins and showed no signs of rickets or osteomalacia. She showed hypophosphatemic rickets with elevated FGF23 level and had been clinically considered to be suffering from XLH. However, direct sequencing of all coding exons and exon–intron junctions of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), FGF23 and dentin matrix protein 1 (DMP1) genes, responsible genes for XLH, ADHR and ARHR1, respectively, showed no mutation. A novel homozygous splice donor site mutation was found at the exon–intron junction of exon 21 of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene responsible for ARHR2 (IVS21 + 1_3(GTA > CACC)). Subsequent analysis of mRNA revealed that this mutation caused skipping of exon 21 which created a premature stop codon in exon 22. These results indicate that genetic analysis is mandatory for the correct diagnosis of hereditary FGF23-related hypophosphatemic rickets. Because Enpp1 knockout mouse is a model of OPLL, this case also suggests that OPLL is associated with ARHR2.