Osteopetrosis
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《新英格兰医药杂志》
Bone is a dynamic tissue in which osteoblasts synthesize bone matrix while osteoclasts resorb bone. Therefore, bone density is dependent on the relative function of these two types of cells. Osteoclasts are multinucleated cells of hematopoietic lineage that are critical for bone remodeling; osteoblasts, in contrast, are of mesenchymal origin.1 Osteoblasts synthesize bone matrix and in so doing lay down a microenvironment that supports osteoclast growth, maturation, and function. They also secrete macrophage colony-stimulating factor (M-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), interleukin-1, and interleukin-6,2 all of which influence the activities of osteoclasts. Direct interactions between osteoblasts or marrow stromal cells and osteoclast precursors are essential for the differentiation of osteoclasts.3,4
Osteopetrosis is a heterogeneous group of heritable conditions in which there is a defect in bone resorption by osteoclasts. A century ago, Albers-Sch?nberg described the radiographic findings in a patient with increased bone density.5 Since then, various types of osteopetrosis have been described. The disease is associated with an increased skeletal mass due to abnormally dense bone. Generalized osteosclerosis is apparent radiographically, often with a "bone within a bone" appearance6,7; transverse radiolucent bands may be observed, and it may be difficult to discern the marrow cavity. The decrease in osteoclast activity also affects the shape and structure of bone by altering its capacity to remodel during growth. In severely affected patients, the medullary cavity is filled with endochondral new bone, with little space remaining for hematopoietic cells. This abnormality contributes to the brittleness of bone in osteopetrosis. The abnormal skeletal radiographs and microscopical appearance of bone can be reversed by hematopoietic stem-cell transplantation (Figure 1).
Figure 1. Appearance of Osteopetrotic Bone.
The characteristic changes of increased bone density with no recognizable bone marrow cavity are apparent in a radiograph of the forearm of an infant with autosomal recessive osteopetrosis (Panel A). Two years after hematopoietic-cell transplantation, normalization of the bone is observed (Panel B). The microscopical appearance of the bone from the same patient before transplantation reveals interweaving bony trabeculae with plates of hyaline cartilage, a minimal medullary cavity, and a markedly decreased number of hematopoietic stem cells (Panel C, hematoxylin and eosin). By 100 days after allogeneic transplantation, islands of hematopoiesis are observed (Panel D, hematoxylin and eosin).
Before the changes in genes that affect the function of osteoclasts were identified, osteopetrosis could be categorized only on the basis of the clinical aspects of the three primary types: infantile, or "malignant," osteopetrosis, inherited in an autosomal recessive inheritance pattern; "intermediate" autosomal recessive osteopetrosis; and autosomal dominant osteopetrosis.6 The incidence of autosomal recessive osteopetrosis is approximately 1 in 300,000 births but is almost 10 times as high in Costa Rica.8 In severe forms of osteopetrosis, the insufficient bone marrow cavity cannot support adequate hematopoiesis. The result is extramedullary hematopoiesis, which causes hepatosplenomegaly. Children who are severely affected can have cranial-nerve dysfunction, and visual deficits are often evident at birth or within the first several months of life.7,9 Thrombocytopenia, anemia, and infectious complications commonly cause death within the first decade of life. In less severe forms of osteopetrosis, patients have a normal life expectancy, but the brittle bone frequently fractures, particularly in autosomal dominant osteopetrosis.10
The origin of osteoclasts from hematopoietic precursors was first suggested by landmark studies in which osteopetrosis in mice was corrected by parabiotic union with normal animals.11,12 Subsequently, the transplantation of splenocytes from unaffected littermates was shown to cure the bony manifestations of the disease; moreover, osteopetrosis developed in unaffected animals after transplantation of splenocytes from osteopetrotic animals.13,14 These experiments in mice prompted treatment of an infant who had severe osteopetrosis with allogeneic bone marrow transplantation, which corrected the bony and hematologic manifestations in the child.15
Osteoclasts and Bone Resorption
The concept that osteoclast precursors are of monocyte lineage was suggested by the capacity of granulocyte–macrophage precursors to differentiate into osteoclasts in vitro16 and by reports that cultures of peripheral-blood monocytes can differentiate into osteoclasts.17 After such differentiation, osteoclasts become adherent to bone. The attachment is facilitated by footlike podosomes containing filamentous actin and the v3 integrin. These molecules associate with the matrix proteins osteopontin and vitronectin on the bone surface,18,19 forming a tight seal of attachment that is termed the clear zone because it is rich in actin filaments but devoid of organelles.20
Polarization of the osteoclast forms two functionally distinct domains, the resorptive surface and the basolateral membrane (Figure 2). The resorption, or Howship's, lacuna is defined by the sealing zone of osteoclasts on bone. An area of complex folding of the osteoclast membrane, termed the "ruffled border," is the resorptive surface of the cell.21 Resorption occurs through acidification of the bony surface, which initiates dissolution of the mineral matrix and secretion of enzymes that digest the organic component of bone.22 In the cytoplasm of the osteoclast, carbonic anhydrase II forms carbonic acid (H2CO3) from carbon dioxide (CO2) and water; the H2CO3 dissociates to bicarbonate (HCO2–) and a proton (H+).23 The protons are transported through the ruffled border into the resorption lacuna by a vacuolar proton pump (H+–ATPase), generating a pH of 4 to 5 in the extracellular space adjacent to bone.24 The electroneutrality of the ruffled membrane is preserved by a chloride-channel charge coupled to the H+–ATPase.25 Acidification of this extracellular environment initiates the degradation of the mineral component of bone, which is composed primarily of hydroxyapatite (Ca3(PO4)2)3? Ca(OH)2). In the presence of protons, hydroxyapatite is degraded to calcium (Ca2+), soluble inorganic phosphate (HPO42–), and water.26
Figure 2. Osteoclast Physiology.
The attachment of the osteoclast to bone is facilitated by podosomes containing filamentous actin and the v3 integrin. To achieve acidification of the resorption lacunae and begin the process of bone demineralization, carbonic anhydrase II (CAII) generates a proton and bicarbonate from carbon dioxide and water. The proton is actively transported across the membrane of the ruffled border through the action of the osteoclast-specific vacuolar-type H+–ATPase "proton pump." A chloride channel coupled to the proton pump facilitates balancing the charge of ions across the membrane. Finally, excess bicarbonate is removed through the basolateral membrane by passive exchange with chloride. The organic matrix of the bone is removed through enzymatic activity, with cathepsin K playing a large role in this process.
The other functional domain of the osteoclast is the basolateral membrane, which is important in exocytosis, a process in which a vesicle releases its contents when it fuses with the cell membrane.27 The organic portions of bone are digested by cathepsin K, a cysteine protease.26 Matrix metalloproteinase activity also contributes to resorption, but the enzyme of principal importance for this activity is unknown.28 The degradation products and debris are transported within vesicles through the cytoplasm and released at the basolateral membrane.29 These vesicles contain the enzyme tartrate-resistant acid phosphatase (TRAP), which generates reactive oxygen species capable of destroying collagen.30 In addition, excess bicarbonate produced in the process of acidification is removed by a chloride–bicarbonate exchanger in the basolateral membrane, thereby maintaining acid–base balance within the cell.31
Insights into the Biology of Osteoclasts
It is clear that numerous kinds of functional defects of osteoclasts have the potential to impair bone resorption. Despite recent advances, however, our understanding of the genetic basis of the disease is incomplete. Clues to genes that may prove to be clinically important have been found through investigations of osteopetrosis in laboratory mice (Table 1).14,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 It is useful to characterize abnormalities of osteoclasts as defects that are intrinsic or extrinsic to the osteoclast. This division is particularly important in evaluating allogeneic hematopoietic stem-cell transplantation as therapy for osteopetrosis, because replacement of the hematopoietic compartment should improve intrinsic osteoclast defects, whereas extrinsic abnormalities may not be corrected. The types of osteoclast defects in laboratory mice can be broadly classified into four categories, as described below.
Table 1. Murine Models of Osteopetrosis.
Early Differentiation Defects: PU.1 and M-CSF
M-CSF is a critical cytokine in the differentiation of osteoclast precursors (Figure 3).60 The osteopetrotic op/op mouse has an insertional mutation in the coding region of the mcsf gene that causes a lack of osteoclasts and circulating monocytes.33,34 Since stromal cells and osteoblasts are the primary source of M-CSF,61 this defect is extrinsic to the osteoclast. Osteopetrosis is also a feature in animals that do not express the receptor for M-CSF.37
Figure 3. Molecules Affecting Osteoclast Differentiation and Function.
The earliest-acting molecule shown to be important in the production of osteoclasts is the transcription factor PU.1, since defects in PU.1 affect the differentiation of other hematopoietic cells, including myeloid cells and B cells. Monocytes are not observed with either PU.1 defects or deficiencies in the production of macrophage colony-stimulating factor (M-CSF). Although the microphthalmia transcription factor (Mitf) has been shown to interact with PU.1, the osteoclast defects observed in Mitf-deficient mice occur later in differentiation, as osteoclasts are observed but are abnormal in appearance. The binding of receptor activator of nuclear factor-B (RANK) ligand (RANKL) produced by osteoblasts to RANK on osteoclast precursors induces osteoclast differentiation; in animals with defects in these genes, no osteoclasts are observed. Signaling following the interaction between RANK and RANKL is mediated by several molecules, including tumor necrosis factor receptor–associated factor 6 (TRAF-6), nuclear factor-B (NF-B), and c-fos. In animals deficient in c-src or 3 and in the grey-lethal (gl/gl) mouse, osteoclasts are normal in number but are unable to produce normal-appearing ruffled borders. In disorders of acidification — including defects in carbonic anhydrase II (CAII), the osteoclast-specific proton pump (encoded by TCIRG1, also termed ATP6i), and the gene encoding a chloride channel (CLCN7) — there are no primary defects in differentiation.
The transcription factor PU.1 is functionally associated with the M-CSF pathway. It binds to the promoter region of the gene for the M-CSF receptor, thereby regulating its synthesis.62 In the absence of PU.1 activity, mice are devoid of osteoclasts, similar to the op/op mice.38,39 However, in contrast to the op/op mice, in PU.1–/– mice (knockout mice in which both copies of the gene are disrupted) the defect is intrinsic to the osteoclast. Bone marrow transplantation restores osteoclasts and reverses the osteopetrosis in PU.1–/– mice.39 Another molecular defect associated with the M-CSF–PU.1 pathway occurs in the microphthalmic (mi/mi) mouse.40 Mutations within the gene encoding the microphthalmia basic helix–loop–helix zipper transcription factor (Mitf ) result in osteopetrosis.41
Receptor Activator of Nuclear Factor-B and Related Proteins
Osteoprotegerin inhibits the differentiation of osteoclast precursors by interacting with a factor termed RANKL (receptor activator of nuclear factor-B ligand, also called osteoprotegerin ligand).63 RANKL is a member of the tumor necrosis factor family and activates its receptor activator of nuclear factor-B (RANK) on osteoclasts and their precursors. Factors that regulate remodeling of bone, such as parathyroid hormone and tumor necrosis factor, do so by regulating expression of osteoprotegerin and RANKL.64 The RANKL–RANK interaction constitutes a signaling pathway that is important in stimulating the formation of osteoclasts and enhancing their function.65,66 RANKL is principally a membrane-bound osteoblast and stromal-cell protein.63 Skeletal mass may be determined by the relative concentrations of RANKL and osteoprotegerin67; this ratio has been shown to be abnormal in the hypercalcemia of cancer with osteolytic bone metastases.
In transgenic animals engineered to express osteoprotegerin under control of the apolipoprotein E gene promoter, osteopetrosis develops because the production of osteoclasts is inhibited by interactions between osteoprotegerin and RANKL.36 Similarly, in RANK or RANKL knockout animals, osteoclasts cannot be produced, and they also have osteopetrosis.35,42,43 In contrast, in osteoprotegerin knockout mice, the production and function of osteoclasts are unregulated and the result is osteoporosis.68 RANK signaling occurs through several intermediates, including c-fos, NF-B pathways, and tumor necrosis factor receptor–associated factor 6 (TRAF-6) (Figure 4).69 Osteopetrosis occurs in knockout animals with deletions in c-fos,44 both NF-B subunits,45 or TRAF-6.46,47
Figure 4. Roles of RANK, RANKL, and Osteoprotegerin in Osteoclast Differentiation and Function.
Receptor activator of nuclear factor-B (RANK) is activated by the cytokine RANK ligand (RANKL), which is produced by other cells, primarily osteoblasts. The expression of RANKL is enhanced by interleukin-1, interleukin-6, and interleukin-11. Osteoprotegerin (OPG) is also produced by osteoclasts and functions as a decoy molecule to regulate osteoclast differentiation and activation by blocking the interaction of RANK and RANKL. Signaling occurs following the association of RANK and RANKL, primarily through tumor necrosis factor receptor–associated factor 6 (TRAF-6), which influences the differentiation of osteoclasts through several pathways, including nuclear factor B (NF-B). In addition, TRAF-6 exerts effects on mitogen-activated protein (MAP) kinases. Targets of MAP kinases include AP-1 transcription factor, which includes c-fos and c-jun. These pathways induce the differentiation of osteoclasts. RANK signaling is also thought to exert anti-apoptotic effects mediated by phosphatidylinositol 3-kinase through c-src.
Functional Osteoclast Defects Leading to Murine Osteopetrosis
Osteopetrosis in mice with osteoclasts has also been described. Mice with deletions of the tyrosine kinase c-src gene have osteopetrosis with a normal number of osteoclasts but abnormal ruffled borders48,49,70; a similar phenotype is observed in the grey-lethal ( gl/gl) mouse.56,57 Animals deficient in the enzyme cathepsin K, important in the degradation of the organic matrix of bone, or TRAP, which appears to have a role in intracellular transport, have nonfunctional osteoclasts.58,59 Animals that have been engineered to be deficient in the 3 integrin also have been shown to have osteopetrosis.51 Osteoclasts derived from these animals have been shown to have an arrest in differentiation, do not spread normally on bone, and have an abnormal ruffled border.71
Four Genotypes Affecting Acidification
Acidification of lysosomes, endosomes, and other organelles is accomplished by a number of cellular proton pumps.72 Within osteoclasts, the ability to generate an acidic environment in the resorption lacuna is critical for bone resorption. In mice, the gene encoding the 3 subunit of an ATP-dependent osteoclast-specific vacuolar proton pump subunit is encoded by the Atp6i gene.73 Targeted disruption of this gene causes osteopetrosis,53 and the naturally occurring osteosclerotic (oc/oc) mouse has been shown to have a 1.6-kb deletion in the Atp6i gene as well.54 The osteopetrosis in the oc/oc mouse is not cured by hematopoietic stem-cell transplantation,52 which is unexpected because the defect is intrinsic to the osteoclast; perhaps in these animals engraftment is impaired. Bone from oc/oc mice undergoes remodeling following implantation in unaffected animals, confirming that bone resorption takes place in the presence of the appropriate cells derived from unaffected animals.74 Related to the acidification pathway is a chloride channel that is charge-coupled to the osteoclast proton pump to achieve electroneutrality.75 Mice that are deficient in expression of the ClC-7 chloride channel have osteopetrosis despite the presence of osteoclasts in the bone cavity.55
Defects of Human Osteoclasts Associated with Osteopetrosis
Most murine models of osteopetrosis are the result of defects in osteoclast differentiation, but comparable defects have not been documented in human osteopetrosis. Following the report of M-CSF deficiency in the op/op mouse, for example, 13 patients with autosomal recessive osteopetrosis were evaluated, but none could be identified with a deficiency in the presence or activity of M-CSF.76 However, the pathways associated with acidification of the resorption lacunae are important in human osteopetrosis (Table 2).
Table 2. Human Osteopetrosis Genotypes.
Carbonic Anhydrase II Deficiency
The first physiological defect described in human osteopetrosis was an autosomal recessive condition with a lack of carbonic anhydrase II activity.23 Patients with this disorder have renal tubular acidosis and may have cerebral calcification. Molecular defects in the carbonic anhydrase II (CAII) gene appear relatively specific to particular geographic regions.79,80,81 Other clinical manifestations of carbonic anhydrase II deficiency are an increased frequency of fractures, short stature, dental abnormalities, cranial-nerve compression, and developmental delay.81 Bone marrow transplantation can correct the hematologic and bony manifestations of carbonic anhydrase II deficiency and arrest the formation of additional cerebral calcifications; renal tubular acidosis, however, is unchanged following transplantation.82 Only a minority of patients with osteopetrosis have a defect in the CAII gene.
Osteoclast Proton Pump Deficiency
After the description of the murine defect in the osteoclast-specific proton pump subunit,53 several groups performed mutational analysis of the human A3 subunit of the osteoclast H+–ATPase proton pump (TCIRG1, also termed ATP6i and OC116), which showed that 50 to 60 percent of children with severe osteopetrosis have mutations in this gene.83,84,85 Most of these homozygous and compound heterozygous mutations were predicted to disrupt the encoded protein. In Costa Rica, where the incidence of autosomal recessive osteopetrosis is approximately 10 times the expected incidence7,9 there is evidence of a founder effect. Two missense mutations account for all of the defects in nine unrelated families.85 Osteoclasts in patients with H+–ATPase proton pump defects are normal-appearing but dysfunctional.86
Defects in the Chloride Channel
Studies have evaluated abnormalities in the chloride channel coupled to the osteoclast H+–ATPase as a cause of osteopetrosis. Kornak et al. described a child with osteopetrosis who had a defect in the CLCN7 chloride-channel gene.55 Mutations in the CLCN7 gene also occur in autosomal dominant osteopetrosis,87 as well as the autosomal recessive "intermediate" form of the disease.88 Defects within the CLCN7 gene appear less frequently as a cause of osteopetrosis than do mutations in the TCIRG1 gene.
The most extensive review of genetic abnormalities in 94 children with severe osteopetrosis reported mutations in the TCIRG1 gene (i.e., the proton-pump gene) in 56 patients (60 percent) and mutations in the CLCN7 gene in 12 patients (13 percent).84 Surprisingly, five children, all of whom had an intermediate to severe phenotype, were heterozygous for mutations in the CLCN7 gene. The variable phenotype in patients with defects in the CLCN7 gene could be a result of modifier genes or of the type of mutation.78 Because the chloride channel exists as a multimer (likely a dimer) of CLCN7 proteins within the cell membrane,89 it is possible that a heterozygous missense mutation could result in a dominant negative effect, in which the abnormal gene product from one allele interferes with the activity of the chloride-channel multimer despite the presence of normal protein expressed from the other allele.78 Many, if not all, cases of what had previously been described as type II autosomal dominant osteopetrosis are now thought to be related to abnormalities in the CLCN7 gene (Table 2).78,84,87,88 This type of autosomal dominant osteopetrosis is associated with characteristic radiographic changes in the vertebrae and pelvis and an increased rate of fractures.90,91,92
Other Genotypes Associated with Clinical Increases in Bone Density
Two severe cases of osteopetrosis with the human equivalent of the grey-lethal mutation in mice have been reported.57,93 The frequency of this mutation appears quite low. Mutations in the cathepsin K gene have been described in pyknodysostosis, which is characterized by delayed closing of cranial sutures, short stature, and skeletal abnormalities.94,95 However, this disorder is generally not recognized as a form of osteopetrosis.
A mutation in the gene that encodes the low-density lipoprotein receptor–related protein 5 (LRP5) has recently been associated with increased bone density.77 The LRP5 gene encodes a protein produced by osteoblasts that serves to increase bone mass; defects in both alleles of LRP5 cause an autosomal recessive disorder called the osteoporosis–pseudoglioma syndrome, in which the susceptibility to fractures is due to a very low bone mass.96 In contrast, there is an autosomal dominant condition in which a gain-of-function mutation in the N-terminal of one allele of the LRP5 gene results in enhanced osteoblast activity and increased bone mass.77,97 It is therefore believed that mutations within the LPR5 gene are primarily responsible for type I autosomal dominant osteopetrosis,77 in which bone density is increased, especially in the cranial vault, with a relatively low risk of fractures.91,92
Since we now have the capacity to characterize these disorders of bone on a molecular and cellular basis, we propose to limit conditions classified as osteopetrosis to defects in osteoclast function. Because the biology of LPR5 mutations alter osteoblast function,97 we propose to exclude this defect from the disorders characterized as osteopetrosis.
"Acquired" Osteopetrosis
Whyte et al. described a case of abnormal bone modeling and increased bone density with histologic features of osteopetrosis associated with extended bisphosphonate therapy in a 12-year-old boy.98 This case suggests that agents that inhibit the recruitment and function of osteoclasts, when given over an extended period of time, may cause a clinical picture comparable to heritable osteopetrosis.
Summary
The three mutations that have been linked to osteopetrosis cause defects in the acidification of bone. The most common of these, found in 50 to 60 percent of patients, results in defects in the A3 subunit of the osteoclast vacuolar H+–ATPase proton pump. The second most clinically significant mutation affects CLCN7, a gene encoding an osteoclast-specific chloride channel. These mutations occur in 10 to 15 percent of patients with severe autosomal recessive osteopetrosis and have been implicated in "intermediate" and autosomal dominant osteopetrosis as well. Carbonic anhydrase II dysfunction is a feature of autosomal recessive osteopetrosis but accounts for a small proportion of patients with osteopetrosis. Several patients have been reported with the equivalent of the murine grey-lethal mutation, but this mutation also occurs in few children with osteopetrosis. It should be noted that a substantial percentage of patients with osteopetrosis have no identifiable gene defect.
Before these molecular abnormalities were found, clinical descriptions were the exclusive means of characterizing osteopetrosis. Classifications based on molecular events and associated physiology will undoubtedly be more precise. It is logical to expect that there is a role for hematopoietic stem-cell transplantation in patients with intrinsic osteoclast defects and severe osteopetrosis. Further investigations will facilitate correlation of the various genotypes with the clinical presentation, anticipated complications, prognosis, and expected response to treatment. This information will provide a basis for making more informed decisions regarding the care of patients with osteopetrosis.
Supported by the Children's Cancer Research Fund and the Minnesota Medical Foundation.
Source Information
From the Program in Blood and Marrow Transplantation, Department of Pediatrics (J.T., P.J.O.) and the Institute of Human Genetics (P.J.O.), University of Minnesota, Minneapolis; and the Department of Pathology and Immunology, Washington University, St. Louis (S.L.T.).
Address reprint requests to Dr. Orchard at 660D CCRB, MMC 366, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, or at orcha001@umn.edu.
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Rajapurohitam V, Chalhoub N, Benachenhou N, Neff L, Baron R, Vacher J. The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function. Bone 2001;28:513-523.
Chalhoub N, Benachenhou N, Rajapurohitam V, et al. Grey-lethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human. Nat Med 2003;9:399-406.
Saftig P, Hunziker E, Wehmeyer O, et al. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc Natl Acad Sci U S A 1998;95:13453-13458.
Hollberg K, Hultenby K, Hayman A, Cox T, Andersson G. Osteoclasts from mice deficient in tartrate-resistant acid phosphatase have altered ruffled borders and disturbed intracellular vesicular transport. Exp Cell Res 2002;279:227-238.
Roth P, Stanley ER. The biology of CSF-1 and its receptor. Curr Top Microbiol Immunol 1992;181:141-167.
Felix R, Halasy-Nagy J, Wetterwald A, Cecchini MG, Fleisch H, Hofstetter W. Synthesis of membrane- and matrix-bound colony-stimulating factor-1 by cultured osteoblasts. J Cell Physiol 1996;166:311-322.
Zhang DE, Hetherington CJ, Chen HM, Tenen DG. The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor. Mol Cell Biol 1994;14:373-381.
Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-176.
Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet 2003;4:638-649.
Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597-3602.
Burgess TL, Qian Y, Kaufman S, et al. The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts. J Cell Biol 1999;145:527-538.
Hofbauer LC, Neubauer A, Heufelder AE. Receptor activator of nuclear factor-kappaB ligand and osteoprotegerin: potential implications for the pathogenesis and treatment of malignant bone diseases. Cancer 2001;92:460-470.
Bucay N, Sarosi I, Dunstan CR, et al. Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 1998;12:1260-1268.
Lee ZH, Kim HH. Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Biophys Res Commun 2003;305:211-214.
Boyce BF, Yoneda T, Lowe C, Soriano P, Mundy GR. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J Clin Invest 1992;90:1622-1627.
Faccio R, Takeshita S, Zallone A, Ross FP, Teitelbaum SL. c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation. J Clin Invest 2003;111:749-758.
Rousselle AV, Heymann D. Osteoclastic acidification pathways during bone resorption. Bone 2002;30:533-540.
Deng W, Stashenko P, Chen W, Liang Y, Shimizu K, Li YP. Characterization of mouse Atp6i gene, the gene promoter, and the gene expression. J Bone Miner Res 2001;16:1136-1146.
Schmitz PE, Marks SC Jr, Wright P, Seifert MF, McGuire JL. Bone particles from osteopetrotic mice not cured by bone marrow transplants are resorbed in normal littermates. Bone 1986;7:473-478.
Blair HC, Teitelbaum SL, Tan HL, Koziol CM, Schlesinger PH. Passive chloride permeability charge coupled to H(+)-ATPase of avian osteoclast ruffled membrane. Am J Physiol 1991;260:C1315-C1324.
Orchard PJ, Dahl N, Aukerman SL, Blazar BR, Key LL Jr. Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis. Exp Hematol 1992;20:103-105.
Van Wesenbeeck L, Cleiren E, Gram J, et al. Six novel missense mutations in the LDL receptor-related protein 5 (LRP5) gene in different conditions with an increased bone density. Am J Hum Genet 2003;72:763-771.
Waguespack SG, Koller DL, White KE, et al. Chloride channel 7 (ClCN7) gene mutations and autosomal dominant osteopetrosis, type II. J Bone Miner Res 2003;18:1513-1518.
Fathallah DM, Bejaoui M, Sly WS, Lakhoua R, Dellagi K. A unique mutation underlying carbonic anhydrase II deficiency syndrome in patients of Arab descent. Hum Genet 1994;94:581-582.
Hu PY, Ernst AR, Sly WS, Venta PJ, Skaggs LA, Tashian RE. Carbonic anhydrase II deficiency: single-base deletion in exon 7 is the predominant mutation in Caribbean Hispanic patients. Am J Hum Genet 1994;54:602-608.
Whyte MP. Carbonic anhydrase II deficiency. Clin Orthop 1993;294:52-63.
McMahon C, Will A, Hu P, Shah GN, Sly WS, Smith OP. Bone marrow transplantation corrects osteopetrosis in the carbonic anhydrase II deficiency syndrome. Blood 2001;97:1947-1950.
Frattini A, Orchard PJ, Sobacchi C, et al. Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 2000;25:343-346.
Frattini A, Pangrazio A, Susani L, et al. Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 2003;18:1740-1747.
Sobacchi C, Frattini A, Orchard P, et al. The mutational spectrum of human malignant autosomal recessive osteopetrosis. Hum Mol Genet 2001;10:1767-1773.
Taranta A, Migliaccio S, Recchia I, et al. Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis. Am J Pathol 2003;162:57-68.
Cleiren E, Benichou O, Van Hul E, et al. Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene. Hum Mol Genet 2001;10:2861-2867.
Campos-Xavier AB, Saraiva JM, Ribeiro LM, Munnich A, Cormier-Daire V. Chloride channel 7 (CLCN7) gene mutations in intermediate autosomal recessive osteopetrosis. Hum Genet 2003;112:186-189.
Jentsch TJ, Stein V, Weinreich F, Zdebik AA. Molecular structure and physiological function of chloride channels. Physiol Rev 2002;82:503-568.
Benichou OD, Laredo JD, de Vernejoul MC. Type II autosomal dominant osteopetrosis (Albers-Schonberg disease): clinical and radiological manifestations in 42 patients. Bone 2000;26:87-93.
el-Tawil T, Stoker DJ. Benign osteopetrosis: a review of 42 cases showing two different patterns. Skeletal Radiol 1993;22:587-593.
Bollerslev J, Mosekilde L. Autosomal dominant osteopetrosis. Clin Orthop 1993;294:45-51.
Van Hul E, Gram J, Bollerslev J, et al. Localization of the gene causing autosomal dominant osteopetrosis type I to chromosome 11q12-13. J Bone Miner Res 2002;17:1111-1117.
Motyckova G, Fisher DE. Pycnodysostosis: role and regulation of cathepsin K in osteoclast function and human disease. Curr Mol Med 2002;2:407-421.
Motyckova G, Weilbaecher KN, Horstmann M, Rieman DJ, Fisher DZ, Fisher DE. Linking osteopetrosis and pycnodysostosis: regulation of cathepsin K expression by the microphthalmia transcription factor family. Proc Natl Acad Sci U S A 2001;98:5798-5803.
Gong Y, Slee RB, Fukai N, et al. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development. Cell 2001;107:513-523.
Boyden LM, Mao J, Belsky J, et al. High bone density due to a mutation in LDL-receptor-related protein 5. N Engl J Med 2002;346:1513-1521.
Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S. Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457-463.(Jakub Tolar, M.D., Ph.D.,)
Osteopetrosis is a heterogeneous group of heritable conditions in which there is a defect in bone resorption by osteoclasts. A century ago, Albers-Sch?nberg described the radiographic findings in a patient with increased bone density.5 Since then, various types of osteopetrosis have been described. The disease is associated with an increased skeletal mass due to abnormally dense bone. Generalized osteosclerosis is apparent radiographically, often with a "bone within a bone" appearance6,7; transverse radiolucent bands may be observed, and it may be difficult to discern the marrow cavity. The decrease in osteoclast activity also affects the shape and structure of bone by altering its capacity to remodel during growth. In severely affected patients, the medullary cavity is filled with endochondral new bone, with little space remaining for hematopoietic cells. This abnormality contributes to the brittleness of bone in osteopetrosis. The abnormal skeletal radiographs and microscopical appearance of bone can be reversed by hematopoietic stem-cell transplantation (Figure 1).
Figure 1. Appearance of Osteopetrotic Bone.
The characteristic changes of increased bone density with no recognizable bone marrow cavity are apparent in a radiograph of the forearm of an infant with autosomal recessive osteopetrosis (Panel A). Two years after hematopoietic-cell transplantation, normalization of the bone is observed (Panel B). The microscopical appearance of the bone from the same patient before transplantation reveals interweaving bony trabeculae with plates of hyaline cartilage, a minimal medullary cavity, and a markedly decreased number of hematopoietic stem cells (Panel C, hematoxylin and eosin). By 100 days after allogeneic transplantation, islands of hematopoiesis are observed (Panel D, hematoxylin and eosin).
Before the changes in genes that affect the function of osteoclasts were identified, osteopetrosis could be categorized only on the basis of the clinical aspects of the three primary types: infantile, or "malignant," osteopetrosis, inherited in an autosomal recessive inheritance pattern; "intermediate" autosomal recessive osteopetrosis; and autosomal dominant osteopetrosis.6 The incidence of autosomal recessive osteopetrosis is approximately 1 in 300,000 births but is almost 10 times as high in Costa Rica.8 In severe forms of osteopetrosis, the insufficient bone marrow cavity cannot support adequate hematopoiesis. The result is extramedullary hematopoiesis, which causes hepatosplenomegaly. Children who are severely affected can have cranial-nerve dysfunction, and visual deficits are often evident at birth or within the first several months of life.7,9 Thrombocytopenia, anemia, and infectious complications commonly cause death within the first decade of life. In less severe forms of osteopetrosis, patients have a normal life expectancy, but the brittle bone frequently fractures, particularly in autosomal dominant osteopetrosis.10
The origin of osteoclasts from hematopoietic precursors was first suggested by landmark studies in which osteopetrosis in mice was corrected by parabiotic union with normal animals.11,12 Subsequently, the transplantation of splenocytes from unaffected littermates was shown to cure the bony manifestations of the disease; moreover, osteopetrosis developed in unaffected animals after transplantation of splenocytes from osteopetrotic animals.13,14 These experiments in mice prompted treatment of an infant who had severe osteopetrosis with allogeneic bone marrow transplantation, which corrected the bony and hematologic manifestations in the child.15
Osteoclasts and Bone Resorption
The concept that osteoclast precursors are of monocyte lineage was suggested by the capacity of granulocyte–macrophage precursors to differentiate into osteoclasts in vitro16 and by reports that cultures of peripheral-blood monocytes can differentiate into osteoclasts.17 After such differentiation, osteoclasts become adherent to bone. The attachment is facilitated by footlike podosomes containing filamentous actin and the v3 integrin. These molecules associate with the matrix proteins osteopontin and vitronectin on the bone surface,18,19 forming a tight seal of attachment that is termed the clear zone because it is rich in actin filaments but devoid of organelles.20
Polarization of the osteoclast forms two functionally distinct domains, the resorptive surface and the basolateral membrane (Figure 2). The resorption, or Howship's, lacuna is defined by the sealing zone of osteoclasts on bone. An area of complex folding of the osteoclast membrane, termed the "ruffled border," is the resorptive surface of the cell.21 Resorption occurs through acidification of the bony surface, which initiates dissolution of the mineral matrix and secretion of enzymes that digest the organic component of bone.22 In the cytoplasm of the osteoclast, carbonic anhydrase II forms carbonic acid (H2CO3) from carbon dioxide (CO2) and water; the H2CO3 dissociates to bicarbonate (HCO2–) and a proton (H+).23 The protons are transported through the ruffled border into the resorption lacuna by a vacuolar proton pump (H+–ATPase), generating a pH of 4 to 5 in the extracellular space adjacent to bone.24 The electroneutrality of the ruffled membrane is preserved by a chloride-channel charge coupled to the H+–ATPase.25 Acidification of this extracellular environment initiates the degradation of the mineral component of bone, which is composed primarily of hydroxyapatite (Ca3(PO4)2)3? Ca(OH)2). In the presence of protons, hydroxyapatite is degraded to calcium (Ca2+), soluble inorganic phosphate (HPO42–), and water.26
Figure 2. Osteoclast Physiology.
The attachment of the osteoclast to bone is facilitated by podosomes containing filamentous actin and the v3 integrin. To achieve acidification of the resorption lacunae and begin the process of bone demineralization, carbonic anhydrase II (CAII) generates a proton and bicarbonate from carbon dioxide and water. The proton is actively transported across the membrane of the ruffled border through the action of the osteoclast-specific vacuolar-type H+–ATPase "proton pump." A chloride channel coupled to the proton pump facilitates balancing the charge of ions across the membrane. Finally, excess bicarbonate is removed through the basolateral membrane by passive exchange with chloride. The organic matrix of the bone is removed through enzymatic activity, with cathepsin K playing a large role in this process.
The other functional domain of the osteoclast is the basolateral membrane, which is important in exocytosis, a process in which a vesicle releases its contents when it fuses with the cell membrane.27 The organic portions of bone are digested by cathepsin K, a cysteine protease.26 Matrix metalloproteinase activity also contributes to resorption, but the enzyme of principal importance for this activity is unknown.28 The degradation products and debris are transported within vesicles through the cytoplasm and released at the basolateral membrane.29 These vesicles contain the enzyme tartrate-resistant acid phosphatase (TRAP), which generates reactive oxygen species capable of destroying collagen.30 In addition, excess bicarbonate produced in the process of acidification is removed by a chloride–bicarbonate exchanger in the basolateral membrane, thereby maintaining acid–base balance within the cell.31
Insights into the Biology of Osteoclasts
It is clear that numerous kinds of functional defects of osteoclasts have the potential to impair bone resorption. Despite recent advances, however, our understanding of the genetic basis of the disease is incomplete. Clues to genes that may prove to be clinically important have been found through investigations of osteopetrosis in laboratory mice (Table 1).14,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59 It is useful to characterize abnormalities of osteoclasts as defects that are intrinsic or extrinsic to the osteoclast. This division is particularly important in evaluating allogeneic hematopoietic stem-cell transplantation as therapy for osteopetrosis, because replacement of the hematopoietic compartment should improve intrinsic osteoclast defects, whereas extrinsic abnormalities may not be corrected. The types of osteoclast defects in laboratory mice can be broadly classified into four categories, as described below.
Table 1. Murine Models of Osteopetrosis.
Early Differentiation Defects: PU.1 and M-CSF
M-CSF is a critical cytokine in the differentiation of osteoclast precursors (Figure 3).60 The osteopetrotic op/op mouse has an insertional mutation in the coding region of the mcsf gene that causes a lack of osteoclasts and circulating monocytes.33,34 Since stromal cells and osteoblasts are the primary source of M-CSF,61 this defect is extrinsic to the osteoclast. Osteopetrosis is also a feature in animals that do not express the receptor for M-CSF.37
Figure 3. Molecules Affecting Osteoclast Differentiation and Function.
The earliest-acting molecule shown to be important in the production of osteoclasts is the transcription factor PU.1, since defects in PU.1 affect the differentiation of other hematopoietic cells, including myeloid cells and B cells. Monocytes are not observed with either PU.1 defects or deficiencies in the production of macrophage colony-stimulating factor (M-CSF). Although the microphthalmia transcription factor (Mitf) has been shown to interact with PU.1, the osteoclast defects observed in Mitf-deficient mice occur later in differentiation, as osteoclasts are observed but are abnormal in appearance. The binding of receptor activator of nuclear factor-B (RANK) ligand (RANKL) produced by osteoblasts to RANK on osteoclast precursors induces osteoclast differentiation; in animals with defects in these genes, no osteoclasts are observed. Signaling following the interaction between RANK and RANKL is mediated by several molecules, including tumor necrosis factor receptor–associated factor 6 (TRAF-6), nuclear factor-B (NF-B), and c-fos. In animals deficient in c-src or 3 and in the grey-lethal (gl/gl) mouse, osteoclasts are normal in number but are unable to produce normal-appearing ruffled borders. In disorders of acidification — including defects in carbonic anhydrase II (CAII), the osteoclast-specific proton pump (encoded by TCIRG1, also termed ATP6i), and the gene encoding a chloride channel (CLCN7) — there are no primary defects in differentiation.
The transcription factor PU.1 is functionally associated with the M-CSF pathway. It binds to the promoter region of the gene for the M-CSF receptor, thereby regulating its synthesis.62 In the absence of PU.1 activity, mice are devoid of osteoclasts, similar to the op/op mice.38,39 However, in contrast to the op/op mice, in PU.1–/– mice (knockout mice in which both copies of the gene are disrupted) the defect is intrinsic to the osteoclast. Bone marrow transplantation restores osteoclasts and reverses the osteopetrosis in PU.1–/– mice.39 Another molecular defect associated with the M-CSF–PU.1 pathway occurs in the microphthalmic (mi/mi) mouse.40 Mutations within the gene encoding the microphthalmia basic helix–loop–helix zipper transcription factor (Mitf ) result in osteopetrosis.41
Receptor Activator of Nuclear Factor-B and Related Proteins
Osteoprotegerin inhibits the differentiation of osteoclast precursors by interacting with a factor termed RANKL (receptor activator of nuclear factor-B ligand, also called osteoprotegerin ligand).63 RANKL is a member of the tumor necrosis factor family and activates its receptor activator of nuclear factor-B (RANK) on osteoclasts and their precursors. Factors that regulate remodeling of bone, such as parathyroid hormone and tumor necrosis factor, do so by regulating expression of osteoprotegerin and RANKL.64 The RANKL–RANK interaction constitutes a signaling pathway that is important in stimulating the formation of osteoclasts and enhancing their function.65,66 RANKL is principally a membrane-bound osteoblast and stromal-cell protein.63 Skeletal mass may be determined by the relative concentrations of RANKL and osteoprotegerin67; this ratio has been shown to be abnormal in the hypercalcemia of cancer with osteolytic bone metastases.
In transgenic animals engineered to express osteoprotegerin under control of the apolipoprotein E gene promoter, osteopetrosis develops because the production of osteoclasts is inhibited by interactions between osteoprotegerin and RANKL.36 Similarly, in RANK or RANKL knockout animals, osteoclasts cannot be produced, and they also have osteopetrosis.35,42,43 In contrast, in osteoprotegerin knockout mice, the production and function of osteoclasts are unregulated and the result is osteoporosis.68 RANK signaling occurs through several intermediates, including c-fos, NF-B pathways, and tumor necrosis factor receptor–associated factor 6 (TRAF-6) (Figure 4).69 Osteopetrosis occurs in knockout animals with deletions in c-fos,44 both NF-B subunits,45 or TRAF-6.46,47
Figure 4. Roles of RANK, RANKL, and Osteoprotegerin in Osteoclast Differentiation and Function.
Receptor activator of nuclear factor-B (RANK) is activated by the cytokine RANK ligand (RANKL), which is produced by other cells, primarily osteoblasts. The expression of RANKL is enhanced by interleukin-1, interleukin-6, and interleukin-11. Osteoprotegerin (OPG) is also produced by osteoclasts and functions as a decoy molecule to regulate osteoclast differentiation and activation by blocking the interaction of RANK and RANKL. Signaling occurs following the association of RANK and RANKL, primarily through tumor necrosis factor receptor–associated factor 6 (TRAF-6), which influences the differentiation of osteoclasts through several pathways, including nuclear factor B (NF-B). In addition, TRAF-6 exerts effects on mitogen-activated protein (MAP) kinases. Targets of MAP kinases include AP-1 transcription factor, which includes c-fos and c-jun. These pathways induce the differentiation of osteoclasts. RANK signaling is also thought to exert anti-apoptotic effects mediated by phosphatidylinositol 3-kinase through c-src.
Functional Osteoclast Defects Leading to Murine Osteopetrosis
Osteopetrosis in mice with osteoclasts has also been described. Mice with deletions of the tyrosine kinase c-src gene have osteopetrosis with a normal number of osteoclasts but abnormal ruffled borders48,49,70; a similar phenotype is observed in the grey-lethal ( gl/gl) mouse.56,57 Animals deficient in the enzyme cathepsin K, important in the degradation of the organic matrix of bone, or TRAP, which appears to have a role in intracellular transport, have nonfunctional osteoclasts.58,59 Animals that have been engineered to be deficient in the 3 integrin also have been shown to have osteopetrosis.51 Osteoclasts derived from these animals have been shown to have an arrest in differentiation, do not spread normally on bone, and have an abnormal ruffled border.71
Four Genotypes Affecting Acidification
Acidification of lysosomes, endosomes, and other organelles is accomplished by a number of cellular proton pumps.72 Within osteoclasts, the ability to generate an acidic environment in the resorption lacuna is critical for bone resorption. In mice, the gene encoding the 3 subunit of an ATP-dependent osteoclast-specific vacuolar proton pump subunit is encoded by the Atp6i gene.73 Targeted disruption of this gene causes osteopetrosis,53 and the naturally occurring osteosclerotic (oc/oc) mouse has been shown to have a 1.6-kb deletion in the Atp6i gene as well.54 The osteopetrosis in the oc/oc mouse is not cured by hematopoietic stem-cell transplantation,52 which is unexpected because the defect is intrinsic to the osteoclast; perhaps in these animals engraftment is impaired. Bone from oc/oc mice undergoes remodeling following implantation in unaffected animals, confirming that bone resorption takes place in the presence of the appropriate cells derived from unaffected animals.74 Related to the acidification pathway is a chloride channel that is charge-coupled to the osteoclast proton pump to achieve electroneutrality.75 Mice that are deficient in expression of the ClC-7 chloride channel have osteopetrosis despite the presence of osteoclasts in the bone cavity.55
Defects of Human Osteoclasts Associated with Osteopetrosis
Most murine models of osteopetrosis are the result of defects in osteoclast differentiation, but comparable defects have not been documented in human osteopetrosis. Following the report of M-CSF deficiency in the op/op mouse, for example, 13 patients with autosomal recessive osteopetrosis were evaluated, but none could be identified with a deficiency in the presence or activity of M-CSF.76 However, the pathways associated with acidification of the resorption lacunae are important in human osteopetrosis (Table 2).
Table 2. Human Osteopetrosis Genotypes.
Carbonic Anhydrase II Deficiency
The first physiological defect described in human osteopetrosis was an autosomal recessive condition with a lack of carbonic anhydrase II activity.23 Patients with this disorder have renal tubular acidosis and may have cerebral calcification. Molecular defects in the carbonic anhydrase II (CAII) gene appear relatively specific to particular geographic regions.79,80,81 Other clinical manifestations of carbonic anhydrase II deficiency are an increased frequency of fractures, short stature, dental abnormalities, cranial-nerve compression, and developmental delay.81 Bone marrow transplantation can correct the hematologic and bony manifestations of carbonic anhydrase II deficiency and arrest the formation of additional cerebral calcifications; renal tubular acidosis, however, is unchanged following transplantation.82 Only a minority of patients with osteopetrosis have a defect in the CAII gene.
Osteoclast Proton Pump Deficiency
After the description of the murine defect in the osteoclast-specific proton pump subunit,53 several groups performed mutational analysis of the human A3 subunit of the osteoclast H+–ATPase proton pump (TCIRG1, also termed ATP6i and OC116), which showed that 50 to 60 percent of children with severe osteopetrosis have mutations in this gene.83,84,85 Most of these homozygous and compound heterozygous mutations were predicted to disrupt the encoded protein. In Costa Rica, where the incidence of autosomal recessive osteopetrosis is approximately 10 times the expected incidence7,9 there is evidence of a founder effect. Two missense mutations account for all of the defects in nine unrelated families.85 Osteoclasts in patients with H+–ATPase proton pump defects are normal-appearing but dysfunctional.86
Defects in the Chloride Channel
Studies have evaluated abnormalities in the chloride channel coupled to the osteoclast H+–ATPase as a cause of osteopetrosis. Kornak et al. described a child with osteopetrosis who had a defect in the CLCN7 chloride-channel gene.55 Mutations in the CLCN7 gene also occur in autosomal dominant osteopetrosis,87 as well as the autosomal recessive "intermediate" form of the disease.88 Defects within the CLCN7 gene appear less frequently as a cause of osteopetrosis than do mutations in the TCIRG1 gene.
The most extensive review of genetic abnormalities in 94 children with severe osteopetrosis reported mutations in the TCIRG1 gene (i.e., the proton-pump gene) in 56 patients (60 percent) and mutations in the CLCN7 gene in 12 patients (13 percent).84 Surprisingly, five children, all of whom had an intermediate to severe phenotype, were heterozygous for mutations in the CLCN7 gene. The variable phenotype in patients with defects in the CLCN7 gene could be a result of modifier genes or of the type of mutation.78 Because the chloride channel exists as a multimer (likely a dimer) of CLCN7 proteins within the cell membrane,89 it is possible that a heterozygous missense mutation could result in a dominant negative effect, in which the abnormal gene product from one allele interferes with the activity of the chloride-channel multimer despite the presence of normal protein expressed from the other allele.78 Many, if not all, cases of what had previously been described as type II autosomal dominant osteopetrosis are now thought to be related to abnormalities in the CLCN7 gene (Table 2).78,84,87,88 This type of autosomal dominant osteopetrosis is associated with characteristic radiographic changes in the vertebrae and pelvis and an increased rate of fractures.90,91,92
Other Genotypes Associated with Clinical Increases in Bone Density
Two severe cases of osteopetrosis with the human equivalent of the grey-lethal mutation in mice have been reported.57,93 The frequency of this mutation appears quite low. Mutations in the cathepsin K gene have been described in pyknodysostosis, which is characterized by delayed closing of cranial sutures, short stature, and skeletal abnormalities.94,95 However, this disorder is generally not recognized as a form of osteopetrosis.
A mutation in the gene that encodes the low-density lipoprotein receptor–related protein 5 (LRP5) has recently been associated with increased bone density.77 The LRP5 gene encodes a protein produced by osteoblasts that serves to increase bone mass; defects in both alleles of LRP5 cause an autosomal recessive disorder called the osteoporosis–pseudoglioma syndrome, in which the susceptibility to fractures is due to a very low bone mass.96 In contrast, there is an autosomal dominant condition in which a gain-of-function mutation in the N-terminal of one allele of the LRP5 gene results in enhanced osteoblast activity and increased bone mass.77,97 It is therefore believed that mutations within the LPR5 gene are primarily responsible for type I autosomal dominant osteopetrosis,77 in which bone density is increased, especially in the cranial vault, with a relatively low risk of fractures.91,92
Since we now have the capacity to characterize these disorders of bone on a molecular and cellular basis, we propose to limit conditions classified as osteopetrosis to defects in osteoclast function. Because the biology of LPR5 mutations alter osteoblast function,97 we propose to exclude this defect from the disorders characterized as osteopetrosis.
"Acquired" Osteopetrosis
Whyte et al. described a case of abnormal bone modeling and increased bone density with histologic features of osteopetrosis associated with extended bisphosphonate therapy in a 12-year-old boy.98 This case suggests that agents that inhibit the recruitment and function of osteoclasts, when given over an extended period of time, may cause a clinical picture comparable to heritable osteopetrosis.
Summary
The three mutations that have been linked to osteopetrosis cause defects in the acidification of bone. The most common of these, found in 50 to 60 percent of patients, results in defects in the A3 subunit of the osteoclast vacuolar H+–ATPase proton pump. The second most clinically significant mutation affects CLCN7, a gene encoding an osteoclast-specific chloride channel. These mutations occur in 10 to 15 percent of patients with severe autosomal recessive osteopetrosis and have been implicated in "intermediate" and autosomal dominant osteopetrosis as well. Carbonic anhydrase II dysfunction is a feature of autosomal recessive osteopetrosis but accounts for a small proportion of patients with osteopetrosis. Several patients have been reported with the equivalent of the murine grey-lethal mutation, but this mutation also occurs in few children with osteopetrosis. It should be noted that a substantial percentage of patients with osteopetrosis have no identifiable gene defect.
Before these molecular abnormalities were found, clinical descriptions were the exclusive means of characterizing osteopetrosis. Classifications based on molecular events and associated physiology will undoubtedly be more precise. It is logical to expect that there is a role for hematopoietic stem-cell transplantation in patients with intrinsic osteoclast defects and severe osteopetrosis. Further investigations will facilitate correlation of the various genotypes with the clinical presentation, anticipated complications, prognosis, and expected response to treatment. This information will provide a basis for making more informed decisions regarding the care of patients with osteopetrosis.
Supported by the Children's Cancer Research Fund and the Minnesota Medical Foundation.
Source Information
From the Program in Blood and Marrow Transplantation, Department of Pediatrics (J.T., P.J.O.) and the Institute of Human Genetics (P.J.O.), University of Minnesota, Minneapolis; and the Department of Pathology and Immunology, Washington University, St. Louis (S.L.T.).
Address reprint requests to Dr. Orchard at 660D CCRB, MMC 366, University of Minnesota, 420 Delaware St. SE, Minneapolis, MN 55455, or at orcha001@umn.edu.
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