First international symposium on osteopetrosis: Biology and therapy: October 23–24, 2003

Welcome

Steven Hausman, Deputy Director,

National Institute of Arthritis and Musculoskeletal and Skin Diseases

Moderators

Gregory Mundy, The University of Texas Health Science Center at San Antonio, Texas Michael Whyte, Washington University in St Louis, Missouri

Discussion

Jakub Tolar, University of Minnesota

Break 9:45–10:00 a.m.

Discussion

Su Yang, Medical University of South Carolina

Luncheon break 11: 45–1:00 p.m.

Moderators

Paul Orchard, University of Minnesota, Minneapolis, Minnesota

L. Lyndon Key, Medical University of South Carolina, Charleston, South Carolina

Discussion

Kimberly Kasow, St. Jude Children's Research Hospital

Break 2:45–3:00 p.m.

Discussion

Mary Eapen, Medical College of Wisconsin

Closing and Adjournment 4:45 p.m.

Summary of Plenary Session: Dr Michael Whyte:

Osteopetrosis (OP) refers to a heterogeneous group of heritable skeletal disorders characterized by defective osteoclast-mediated bone resorption. One century ago, in 1904, Albers-Schönberg described the radiographic findings of a young man with increased bone density. Since then, wide variation in the clinical severity of OP has been well documented. There are several proposed classifications. The autosomal recessive form, also called “malignant,” is associated with the most severe phenotype leading to reduced medullary space insufficient to support hematopoiesis. Resulting extramedullary hematopoiesis often leads to massive hepatosplenomegaly apparent in infancy. In most children who are severely affected, cranial nerve dysfunction occurs from failed enlargement of bony foramina as the child grows. Significant visual deficits often become evident by several months of age. Unless successfully treated, thrombocytopenia, anemia, and infections commonly lead to death within the first decade of life. A less aggressive course has been described for other presumed autosomal recessive forms. Autosomal dominant OP (ADO) has been divided by some investigators into at least two forms—based primarily on radiographic findings. ADO, type 1 is characterized by pronounced sclerosis of the cranial vault with uniformly increased density of the spine. In contrast, ADO, type 2 features sclerosis of the skull predominately at the base and end-plate thickening in the vertebrae and iliac wings of the pelvis producing a “bone within bone” (endobone) appearance. In these less severe forms of OP, normal life expectancy is anticipated, although frequent fractures may occur in ADO-2 due to the increase in fragility of bone; in addition, osteomyelitis and cranial nerve dysfunction may occur. Aberrant presence of creatine kinase brain isoenzyme (BB-CK) in the circulation seems to be a useful serum marker for genuine forms of OP.

OP classification is requiring re-evaluation as our understanding of the molecular basis of increase bone density is rapidly becoming understood—providing additional insight regarding the pathophysiology and genotypic variation of these conditions. The majority of OP patients have defects in one of three genes involved in the osteoclast acidification pathway. The first relates to the production of protons (H⁺) from water and CO₂ mediated by carbonic anhydrase II. Transport of protons into the resorption lacunae is normally facilitated by an osteoclast-specific H⁺ pump. Defects in the gene encoding the a3 subunit of this pump (TCIRG1) affects approximately half of all children with severe, autosomal recessive OP. Additionally, molecular defects in chloride channel 7 (CLCN7) have been described especially in ADO-2—likely reflecting a dominant-negative effect. In some patients, two defective CLCN7 alleles result in severe, autosomal recessive OP. While the above genotypes are associated with defects in osteoclast function, molecular evidence now indicates that ADO-1 is caused by gain-of-function mutations in the LRP5 gene driving osteoblasts through activation of Wnt signaling.

Another important focus for the future is correlation of genotype and phenotype with prognosis and response to treatment. This effort will be essential to understand how responses to various therapies may differ among groups of OP patients. To date, little information is available regarding this issue. Continuing assessment of the clinical manifestations of OP, molecular defects, and therapeutic interventions and outcomes will produce better evaluation and treatment.

Summary of the Scientific Sessions: Jakub Tolar, Su Yang, Kimberly Kasow, Mary Eapen:

The Bone Resorbing Unit and Osteopetrosis

Gregory R Mundy, MD

Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

The osteoclast is a social cell. It works in conjunction with accessory cells to form a bone-resorbing unit and cause resorption of bone. These cell-cell interactions have been characterized during the past decade, and it is now recognized that the RANKL/RANK/OPG system is responsible in most, if not all, cases for regulating osteoclast formation, differentiation, and activity. Osteopetrosis represents malfunction of this bone-resorbing unit. Many types of osteopetrosis have now been identified. These can be classified into two major groups: those representing abnormalities of the differentiated cells where osteoclasts are present but nonfunctional and those in which there is absence of differentiated osteoclasts. In most cases, osteopetrosis is caused by absence of important intracellular molecules necessary for osteoclast formation or function. In several instances of osteopetrosis, there is absence of extracellular factors that control regulation of osteoclast activity, such as monocyte-macrophage colony stimulating factor and RANKL. There are a number of unanswered questions in this arena. These include the following. (1) Does the RANKL/OPG system mediate osteoclastic bone resorption? (2) In osteopetrosis, why does excess bone formation occur? (3) What does this tell us about the coupling of bone formation to bone resorption? Perhaps the most practical useful offshoot of studies of osteopetrosis is identification of molecular targets for drug discovery. One example among others is the discovery that cathepsin K is required for normal osteoclastic bone resorption, and based on this observation, inhibitors of cathepsin K activity are being developed as drugs for preventing bone resorption and stabilizing bone mass.

Molecular Events Regulating Differentiation and Function of Osteoclasts

F Patrick Ross, PhD

Department of Pathology, Washington University School of Medicine, St Louis, Missouri, USA

Our understanding of osteoclast biology has increased greatly over the last 5 years for several reasons. First, cloning of RANKL, the key osteoclastogenic cytokine, facilitated execution of many biochemical and cell biological studies. Second, advances in genetically based information arising from genome sequencing targeted gene deletions or positional cloning, many of the latter involving the rare disease osteopetrosis, facilitated identification of key molecules regulating the osteoclast. Thus, it is now possible to provide a model that describes many of the important steps involved in osteoclastogenesis and bone resorption. Early stage osteoclast differentiation involves proliferation and differentiation of myeloid precursors, a process dependent of the lineage marker PU.1. As precursors differentiate, they express c-Fms, the receptor for M-CSF, the cytokine regulating their survival and proliferation. Activation of c-Fms results in RANK expression, rendering the cells responsive to RANKL. In short, M-CSF and RANKL are the two cytokines necessary and sufficient for osteoclastogenesis. Studies of signaling downstream of c-Fms and RANK identified a number of molecules required for osteoclast formation and/or function. These include the adaptor proteins TRAF6 and c-src, the transcription factors MITF, c-Fos, its associating molecules c-Jun/Jun D, their downstream targets Fra 1 and NFAT1c (NFAT2), and two members of the NFκB family, p50 plus p52. Thus, absence of genes coding for the cytokines M-CSF or RANKL, their respective receptors c-Fms or RANK, or the transcription factors, PU.1, MITF, c-Fos, Fra1, NFAT1c, or p50 plus p52 lead to severe osteopetrosis. Later steps in bone resorption involve adhesion of osteoclasts to matrix through the integrin α_vβ₃ and formation of a ruffled border, the unique organelle of the osteoclast. Inorganic matrix is dissolved by polarized extrusion of hydrochloric acid into the resorptive space, an event that requires the charge-coupled activity of a vacuolar ATPase complex and a chloride channel. Degradation of organic matrix requires secretion of the acidic protease cathepsin K. Attesting to their unique roles, specific inhibition of the vacuolar ATPase or targeted deletion and/or natural mutations in the major acid-generating enzyme, carbonic anhydrase II, the chloride channel, or cathepsin K, suppress bone resorption. Finally, several recent examples of gain-of-function deletions/mutations have been characterized. These include the decoy RANKL receptor OPG and the lipid phosphatase SHIP, because their genetic deletion leads to unbridled bone resorption, with resulting severe osteoporosis. Finally, clustered point mutations in RANK or its adaptor molecule p62, seen in subsets of pagetic patients, lead to enhanced bone resorption.

Osteoclast Recruitment and Homing

Miep H Helfrich, PhD

Bone Group, Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, Scotland

Models of osteopetrosis in rodents have been instrumental in establishing the hemopoietic origin of the osteoclast. Parabiosis experiments and later bone marrow transplantation showed convincingly that, for most mutations, the osteopetrotic defect could be corrected by replacing the source of osteoclasts by that of a non-osteopetrotic animal. Osteoclast precursors develop initially along the monocyte/macrophage lineage until they become finally committed to osteoclast development after expressing the receptor RANK and binding RANKL. In early mammalian development, osteoclast precursors are found in all tissues where hemopoiesis occurs, in particular in liver and spleen, and they are recruited to sites of developing bone. Once bone marrow spaces are established, osteoclast precursors are mainly present in bone marrow itself and in the peripheral circulation, and they continue to be recruited from there to sites of osteoclast formation and remodeling in bone itself. This process is poorly understood and is likely to be regulated in part by endothelial cells. Embryonic mouse long bones are a good model for studying osteoclast recruitment during bone formation both in vivo and in explant cultures in vitro. During early development, proliferating osteoclast precursors appear in the periosteum of developing long bones, where they reach a postmitotic stage before invading the bone. It is not yet clear what specifically attracts osteoclast precursors to these sites, but the presence of mineralized cartilage appears obligatory. The subsequent invasion of primitive long bones by osteoclast precursors has been shown to depend critically on the activity of matrix metalloproteinase-9 and vascular endothelial growth factor. When in explant cultures, the periosteum is removed to delete the endogenous source of osteoclast precursors, and it becomes possible to use the long bone as “bait” to recruit osteoclast precursors from other tissue sources. Cocultures of so called “stripped” mouse long bones with various mouse tissues, including cross-over cocultures with tissues of osteopetrotic animals, have been instrumental in our understanding of sources of osteoclast precursors and the cellular origin of osteopetrotic defects. In addition, the coculture of mouse long bones with human sources of osteoclast precursors has been accomplished. It was shown that osteoclasts with normal ruffled borders and resorptive capacity are generated from healthy human osteoclast precursors. In contrast, precursors from children with recessive malignant osteopetrosis were recruited as normal and fused into multinuclear cells, but neither developed ruffled borders nor resorbed calcified cartilage or bone, closely mimicking the in vivo situation in these young patients. This coculture model therefore seems useful in understanding the cellular consequences of mutations causing human osteopetrosis in the context of a developing bone.

Murine Models of Osteopetrosis: Lessons for Human Disease

Jean Vacher, DSc

Clinical Research Institute of Montreal, Montreal, Quebec, Canada

Infantile malignant recessive osteopetrosis is the most severe form of the disease. The animal model that most closely resembles this disorder is the mouse grey-lethal (gl) mutant. In addition to osteopetrosis, the gl mutant displays a coat color defect. In this model, osteopetrosis results from inactive osteoclasts with an underdeveloped ruffled border and consequent defective bone resorption. To isolate and characterize the gl gene, we have undertaken a positional cloning approach. Following our genetic localization of the gl locus, detailed physical and transcriptional maps were defined based on establishment of yeast (YAC) and bacterial artificial chromosome (BAC) contigs. Sequence analyses defined 15 genes and expressed tag sequences (ESTs) in this interval. Functional rescue of the gl osteopetrotic defect using BAC transgenic mice reduced the gl candidate region to a single 180-kb genomic clone. Expression analyses revealed that one gene in this BAC was undetectable in homozygous gl/gl animals. Consistent with complete loss of expression, identification of the gl locus was further confirmed through characterization of a genomic rearrangement that corresponds to a deletion of the promotor region, the first exon, and part of first intron. In wildtype animals, the gl gene encodes a unique ∼3-kb transcript that is most prevalent in brain, spleen, and kidney, as well as in osteoclast-like cells (OCLs) and melanocytes. In situ hybridization analysis revealed early embryonic gl expression in hematopoietic, skeletal, and brain tissues and postnatally in gut, kidney, skin, and mature osteoclasts. The novel 338 aa Gl protein is cytosolic and includes a potential signal-peptide and a single trans-membrane domain, with no homology to any known murine protein. The Gl protein is conserved during evolution and seems restricted to multicellular organisms, because no yeast homologous gene has been found. We have also cloned the human Gl gene, which encodes a protein with 83% homology to the murine protein. Of major significance, our screening of osteopetrotic patients for Gl mutation identified the first mutation in the human Gl gene associated with development of very severe form of recessive osteopetrosis. Thus, our studies have identified a novel gene essential for proper osteoclast maturation and bone resorption in mouse and human. Functional characterization of the protein will potentially define the molecular signaling pathway(s) associated with osteoclast activation requiring Gl function.

Development of a System to Differentiate Osteopetrotic Osteoclasts In Vitro

Harry C Blair, MD

Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

We studied osteoclast development in vitro using CD14-selected cells from blood of normal and osteopetrotic subjects. RANKL and CSF-1 were used to induce differentiation; cathepsin K, TRACP, acid secretion, and lacunar bone resorption were studied. Mononuclear cells were selected from 15 ml of peripheral blood using magnetic beads (Miltenyi Biotec, Auburn, CA, USA). Flow cytometry showed that cells expressing CD14 and the integrin subunits CD11b and CD11c were enriched from ∼8% of total peripheral blood mononuclear cells to ∼85% by CD14 selection. Mononuclear cells uniformly expressed α-naphthyl acetate. Cells were cultured 6–10 days in IMDM with 30% serum and a cytokine cocktail. 15 ng/ml of human IL1, IL6, SCF, 10⁻⁹ M 1,25 dihydroxyvitamin D. and 10⁻⁸ M dexamethasone before being differentiated into osteoclasts. This allowed monocytes to proliferate, because starting cells were limited. This had variable efficacy; labeling studies in control (adult) cell cultures showed division in much less than one-half of cells, although some infant cell cultures had increases on the order of 2- to 3-fold. Postgrowth analysis by flow cytometry showed ∼95% of cells were CD11b-CD11c+ CD14+. This antigen profile is typical for pre-osteoclastic cells, although mature osteoclasts lose these antigens and express instead the vitronectin receptor α_vβ₃ integrin. Nonmonocytic cells were lymphocytes (∼3% by antigen labeling) and unidentified cell fragments. The results from studying several controls and osteopetrotic patients with defects in TCIRG1, CLCN7, or neither gene included discovery of a new form of osteopetrosis with defective attachment and podosome formation in subjects with normal TCIRG1 and CLCN7. The phenotype of TCIRG1 mutants was in accord with the expected effect on acid secretion, but CLCN7 cells did not show defective acid secretion; instead they showed abnormal lacunar resorption with retained demineralized matrix. This system can be used to study osteoclast function in any subject where ∼15 ml of peripheral blood is available.

Determinations of Osteoclast Proton Pump and Chloride Channel Function

Paul H Schlesinger, MD, PhD

Department of Cell Biology, Washington University School of Medicine, St Louis, Missouri, USA

Appropriate regulation of osteoclast number and function is essential to healthy bone, and consequently, osteopetrosis results from reduced osteoclast function. The dissolution of bone mineral requires one to two protons per calcium released at pH 7, and a reduction of pH dramatically enhances the rate of bone mineral solution. The pH of the bone resorption compartment has been estimated to be acidic by direct measurement with electrodes as well as fluorescent pH indicators. Furthermore, the resorption of bone can be inhibited by weak bases that will be concentrated in acid compartments and increase their pH. At the molecular level a vacuolar-type proton translocating ATPase has been identified as essential for acid secretion. Because of the electrogenic nature of this pump, we proposed the existence of a short circuit (parallel) current pathway in the osteoclast ruffled border membrane. Without a neutral membrane potential, very powerful driving forces would oppose the required continuous proton entry into the resorption compartment and promote leak of calcium into the cytosol. Using avian osteoclasts that could be obtained in sufficient quantity and purity, the mechanism of resorption compartment acidification has been studied. Analogy with mammalian intracellular vesicles suggested that a chloride channel in parallel with the proton pump could provide charge neutralization over a period of prolonged bone resorption. Ionic and pH balance for the resorbing osteoclast is provided by carbon dioxide hydration by carbonic anhydrase and chloride-bicarbonate exchange on the nonadherent surfaces of the osteoclast. Under these conditions, chloride can be shown to accurately control the pH of ruffled membrane vesicles. By use of these substrates, carbon dioxide, water, and chloride, the osteoclast can dissolve a large bone mass and produce the pits that are visible microscopically. The molecular identity of the chloride channel has been complicated. In avian cells, where we could study transport directly, we isolated a protein of 62 kDa that was immunologically cross-reactive with the CLIC family of proteins. In mammals, the CLC family has clearly been shown to be important in osteoclast function. In either case, the requirement for chloride transport for bone resorption is obvious. This makes defects in chloride transport an important consideration in osteopetrosis.

Superoxide Production in Osteopetrotic Patient Cells

Su Yang, PhD

General Clinical Research Center, Medical University of South Carolina, Charleston, South Carolina, USA

Severe “malignant” osteopetrosis is a metabolic bone disease characterized by defective osteoclastic function and decreased bone resorption. In osteopetrosis, dysfunction of osteoclasts is often accompanied by defects in other types of cells. It has been reported that neutrophils, leukocytes, and macrophages from osteopetrotic patients have reduced superoxide production. Such a reduction contributes to the inability of the immune system to protect against and eradicate infections. In this study, lymphocytes from osteopetrotic patients were immortalized by EBV transformation. Superoxide production from EBV-transformed cells of osteopetrotic patients was found to be one-third that of control cells. Further study revealed that the expression of the p47 subunit of NADPH oxidase was reduced in patient cells, and the total amount of p47 phosphorylation and translocation was decreased in patient samples. To confirm that reduced p47 could account for the decreased superoxide production in patient cells, p47 antisense oligonucleotide was introduced into normal cells. The antisense treatment reduced p47 protein expression and resulted in a subsequent decrease of superoxide generation. These results showed that the defect in p47 expression probably resulted in reduced superoxide production, which may translate into an increased propensity for infection in osteopetrotic patients. In addition to lymphocytes, superoxide production by osteoclasts from osteopetrotic patients was only 30% of that from normal osteoclasts, which is consistent with the finding in lymphocytes. However, when patient osteoclasts were stimulated with IFN-γ, superoxide generation was significantly increased up to 3.5 times higher, which reached 90% of that from normal osteoclasts treated with IFN-γ. These findings suggest that IFN-γ treated osteoclasts are more metabolically active. IFN-γ stimulation results in a greater response of superoxide generation in patient osteoclasts (350% increase) than in normal osteoclasts (15% increase). Because osteoclasts from osteopetrotic patients produce a lower basal level of superoxide, patient cells are more sensitive to IFN-γ stimulation, thus resulting in a greater increase of superoxide production. Increased superoxide amounts in patient osteoclasts are within the range of the normal osteoclast production level. Although IFN-γ has been shown to inhibit bone resorption in normal osteoclasts, IFN-γ may activate patient osteoclasts by stimulating superoxide production.

Update on the Molecular Genetics of Human Osteopetrosis

Wim Van Hul, PhD

Department of Medical Genetics, University of Antwerp, Antwerp, Belgium

The human osteopetroses belong to the group of sclerosing bone dysplasias. The latter comprise in total about 40 different clinical entities with an increased BMD. The osteopetroses are clinically and genetically very heterogeneous but are grouped because all of them are suggested to be caused by impaired bone resorption. These conditions have long time been poorly understood at the molecular level. However, primarily within the last 5 years, major breakthroughs have been made in the understanding of the pathogenic mechanisms underlying most forms of osteopetrosis. Already 20 years ago, it was shown that osteopetrosis with renal tubular acidosis and cerebral calcifications is caused by a deficiency of carbonic anhydrase II (CA II). More recently, evidence was obtained for genetic heterogeneity within the most severe form, also called the malignant or infantile form. After the report of mutations in the ATP6i gene encoding a subunit of the vacuolar H(+)-ATPase proton pump, in some cases mutations were found in the gene encoding chloride channel ClCN7. This year, one patient with the malignant form of osteopetrosis was reported with a mutation in a previously unknown gene called gl since a partial deletion of this gene underlies the grey-lethal (gl) osteopetrotic mouse model. Besides the ATP6i, ClCN7, and gl genes, at least one gene remains to be identified for this type of osteopetrosis, because some patients don't have a mutation in any of these three genes. Interestingly, the proteins encoded by the CAII, ATP6i, and ClCN7 genes all participate in the osteoclastic production of protons and their transport to the extracellular compartment between the bone and the osteoclast. The function of the GL protein is currently unknown. The autosomal dominant form of osteopetrosis has been subdivided in two subtypes with clear radiological and clinical differences. We were able to show that missense mutations in the ClCN7 gene are causing autosomal dominant osteopetrosis type II, most likely because of a dominant negative effect. Very recently, we reported mutations in the LRP5 gene in patients diagnosed with autosomal dominant osteopetrosis type I. Remarkably, so far mutations in this gene were reported in conditions with an increased BMD caused by increased bone formation rather than decreased bone resorption. Further analysis should indicate whether this implies that autosomal dominant osteopetrosis type I should be removed from the group of osteopetroses. In conclusion, positional cloning efforts on the different forms of osteopetrosis have increased the understanding of the underlying pathogenic mechanisms and might lead to novel ways to treat or prevent these conditions.

A Phase III Randomized, Controlled Open-Label Trial of Interferon-γ1b in Patients With Severe Congenital (Malignant) Osteopetrosis

L Lyndon Key, Jr. MD

Medical University of South Carolina, Charleston, South Carolina, USA

To determine if interferon-γ1b (1.5 μg/kg/dose SC, three times per week; Actimmune; Intermune Pharmaceuticals) plus calcitriol (1 μg/kg/day orally) or calcitriol alone is effective in delaying the time to treatment failure, 16 patients with congenital osteopetrosis were recruited from all over the world to participate in the study. They were randomized in a 2:1 ratio on interferon-γ1b plus calcitriol and calcitriol alone, respectively. The drug was administered to the patients three times per week subcutaneously. The patients receiving interferon-γ1b plus calcitriol had their first evidence of failure at a mean of 452 days compared with those on calcitriol alone, who failed at 129.8 days (p = 0.0161). Only 10% of the patients treated with interferon-γ1b experienced a serious infection compared with 67% of the patients on calcitriol alone. In limited data, biopsies obtained in patients treated with interferon-γ1b had a 50% reduction in bone mass, whereas no reduction was seen in one patient treated with calcitriol alone. Optic nerve and auditory canal size increased in subjects treated with interferon-γ1b, but not in those treated with calcitriol only. We conclude that interferon-γ1b is an effective treatment for osteopetrosis, stimulating immune function and osteoclastic activity, slowing the progression of the skeletal abnormalities, and reducing infections.

Pulmonary Arterial Hypertension Complicating Stem Cell Transplantation for Malignant Infantile Osteopetrosis

Colin Steward, BM, BCh, PhD, FRCP

Royal Hospital for Children, Bristol, United Kingdom

The results of stem cell transplantation for malignant infantile osteopetrosis (MIOP) have always been comparatively poor for a benign disease, with a significant proportion of children dying from pulmonary complications. For example, the latest EBMT survey of SCT for MIOP (Driessen et al. 2003) cites a 5-year disease-free survival of only 73% following matched sibling SCT and <45% with other donor types. Eleven of 61 deaths (18%) from this series of 122 transplanted patients were caused by idiopathic pneumonia/pneumonitis. It now seems likely that the development of acute severe pulmonary arterial hypertension (PAH) underlies at least some of these deaths. In conjunction with colleagues from Paris and Ulm, I have identified 9 children from a total of 30 (30%) transplanted at our three centers between 1996 and 2002 who developed acute severe PAH at days −2 to +89 after allogeneic SCT for MIOP (Steward 2004). No child had evidence of PAH before transplant. Typical presentations were with acute dyspnea, hypoxia, and brady/tachycardia, usually in the absence of fever, crepitations, or other evidence of infection. Six patients required assisted ventilation, and five died. There was clinical or pathological evidence of veno-occlusive disease (VOD) in three children, but the absence of VOD in the remaining six suggests that a separate disease process was responsible for PAH. Responses to NO, defibrotide, nicardipine, and steroids in varying combinations were disappointing. Three children showed sustained improvement after administration of epoprostenol (prostacyclin) in conjunction with NO and/or defibrotide and remain well and free of PAH 27, 33, and 34 months after transplant. PAH must therefore be excluded in any child who becomes acutely breathless after SCT for osteopetrosis.

Acquired Osteopetrosis

Deborah Wenkert, MD

Shriners Hospitals for Children, St Louis, Missouri, USA

Acquired forms of osteopetrosis (OP) caused by infection have been reported primarily in animals. Retroviruses were proposed as etiologic agents in the 1980s and 1990s for avian, feline, and murine OP models. However, these animals feature increased osteoblastic (as opposed to decreased osteoclastic) function, making the pathogenesis of their skeletal disease different from patients with true OP. Nevertheless, viral-like inclusions and viral antigens have been described in the osteoclasts of patients with “benign” OP. Furthermore, evidence of retroviral infection in patients was reported in 1990 when reverse transcriptase was detected in mononuclear blood cells from a 27-year-old patient with OP. Because three OP-causing genes have since been identified in humans, mutational analysis of these same patients would be interesting. “Transient osteopetrosis in infancy” suggests that exposure to some factor in utero can cause a reversible form of OP. The first preliminary evidence that there can be a pharmacologic form of OP followed in utero exposure to acetazolamide (a carbonic anhydrase inhibitor). Recently, we reported a case of bisphosphonate-induced osteopetrosis in a child. Pharmacologic OP should manifest differently from congenital OP. In the patient with pamidronate-induced OP, the typical clinical manifestations (except for fracture) of severe, lifelong disease were absent (cranial nerve palsy, hematopoietic compromise from myelophthises, short stature, ankylosed teeth, etc.). Genetically understood forms of OP in humans thus far reflect defective osteoclasts unable to produce and secrete HCl for bone resorption. Patients with malignant disease (homozygous CLCN7 or proton pump deficiency) typically have bone marrow spaces crowded with many ineffective osteoclasts. Bisphosphonates interfere in the recruitment, differentiation, and function of osteoclasts and may cause osteoclast apoptosis. Therefore, increased numbers of osteoclasts within bone are not expected in bisphosphonate-induced OP and were not seen in our patient. This, in addition to normal osteoclast activity before his bisphosphonate exposure, may account for the clinical differences mentioned above. Currently, several pharmaceuticals under development to treat osteoporosis or autoimmune diseases are expected to inhibit osteoclast generation, recruitment, function, or longevity. The bisphosphonates, in particular, are especially long-acting. Thus, it is not surprising that the first detailed report of drug-induced osteopetrosis occurred from a bisphosphonate. However, as more effective and longer-lasting inhibitors of osteoclasts are synthesized, clinicians must be aware of their “osteopetrotic” potential when given to children.

Neuroradiologic Findings in Human Osteopetrosis

Joel K Curé, MD

Department of Radiology, University of Alabama Medical Center, Birmingham, Alabama, USA

This study reviews the cranial neuroimaging findings in osteopetrosis described in the literature as well as the results of two neuroimaging studies conducted at our institution in patients with osteopetrosis. Stenosis of the petrous carotid and cervical vertebral arteries and jugular bulbs has been described in the literature and attributed to bony overgrowth surrounding the involved vascular canals and foramina. Cranial CT studies of infants with the autosomal recessive form of OP showed skull thickening and sclerosis, frontal bossing, a small nasoethmoid complex and posterior nasal stenosis, minimal to absent paranasal sinus and mastoid pneumatization, small orbital volume with proptosis and hypertelorism, small optic canals, and a persistent fetal appearance of the temporal bones. The latter includes small middle ear cavities, trumpet-shaped internal auditory canals, and enlarged subarcuate canaliculi. Other authors have observed stenotic internal auditory and facial nerve canals (but no stenoses of the external auditory canals). Sclerosis and deformity of the ossicles was also reported in most of these patients. A small sella turcica has been noted in some patients. Malformed teeth and mandibular hypoplasia have been described. “Endobones” within the mandible and bones of the skull base, and sclerosis along sutures and synchondroses are characteristic. Nonosseous manifestations included prominent subarachnoid spaces, ventriculomegaly, (due either to atrophy or communicating hydrocephalus) and optic nerve atrophy. We studied the cranial MR images of 47 patients with osteopetrosis (OP). Thirty-four patients had autosomal recessive (malignant) osteopetrosis (AROP), 7 had intermediate osteopetrosis (IOP), 3 had autosomal dominant osteopetrosis type I (ADOP I), and 3 had autosomal dominant osteopetrosis type II (ADOP II). Abnormalities were tabulated and compared among the OP variants. All patients had calvarial thickening and sclerosis. Ventriculomegaly, cerebellar tonsillar ectopia, proptosis, and jugular bulb stenosis occurred in most patients with AROP and ADOP I. Optic nerve sheath dilatation occurred in many of the patients with AROP and all of the patients with ADOP I. Acquired cephaloceles were observed only in patients with AROP and ADOP I. Optic canal stenosis and optic nerve atrophy were observed in a majority of patients with AROP, IOP, and ADOP II. Middle ear fluid was observed in over one-half of patients with AROP and IOP. Features seen exclusively or nearly exclusively in AROP included intracranial internal carotid and cervical vertebral artery stenosis and intracranial extramedullary hematopiesis. In a second study, we examined the petrous carotid canals (PCC) in 20 patients with AROP and compared the mean and minimum PCC diameters of the patients with those measured in 52 control subjects. PCC diameters were also correlated with MR angiograms. This study showed a statistically significant difference in PCC diameters between the study and control groups (smaller in the study patients). A strongly positive correlation between PCC diameter and age was noted in controls, but only a weakly positive correlation in study subjects. One or both internal carotid arteries were stenotic on MRA in all study patients. MRA stenosis grade correlated positively with age but not PCC diameter.

Developmental Spectrum of Children With Congenital Malignant Osteopetrosis

Jane M Charles, MD

Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina, USA

Literature describing the developmental outcome of osteopetrosis has been minimal and has painted a uniformly dismal outcome. This study reports outcomes of an ongoing comprehensive follow-up of children with osteopetrosis using neurodevelopmental tests administered during each patient's inpatient admission. Test selection was based on age and visual acuity. Twenty-five children with osteopetrosis (age, 2 weeks–11 years) followed by the Division of Pediatric Endocrinology were admitted for a routine protocol with a multidisciplinary team every 6–12 months. Eight children have complete visual impairment (VI) because of optic atrophy, 12 have partial visual impairment, and 5 have no optic atrophy. All have varying degrees of sequelae that accompany osteopetrosis. For all children with total or near total VI, the Maxfield-Buchholz Scale of Social Maturity for Preschool Blind Children was administered, yielding a Social Quotient. Children less than 36 months with normal vision or mild loss were administered the CAT/CLAMS. Children over age 3 received the Slosson Intelligence Test. Gross motor (GM) skills were evaluated by standard motor milestone checklists. For children ages 2 weeks–18 months, language quotients ranged from 30 to 100, and GM quotients ranged from 10 to 100. For children 19–36 months, language quotients ranged from 63 to 100, GM quotients ranged from 21 to 50, and social quotients ranged from 76 to 112. For children 3–6 years of age, language quotients ranged from 105 to 110, GM quotients 10 to 100, and social quotients from 28 to 105. For children 7 years and older, language quotients ranged from 50 to 102, GM showed that all were ambulatory, and social quotient on one patient was 73. For those with complete VI, language quotients ranged from moderate delay to average range, GM ranged from profound delay to average range, and adaptive skills ranged from severe MR to high average range. For those with partial VI, language skills ranged from mild delay to average range, GM ranged from moderate delay to average range, and social skills range from moderate delay to average range. For those with no optic atrophy, all but one had language skills in average range, and GM ranged from mild delay to average range. Contrary to conclusions in the literature, a wide range of cognitive and adaptive abilities was seen in these 25 children with osteopetrosis. Despite significant GM delays in early life, the child can be expected to be ambulatory by school age. Language and adaptive skills can vary from the MR range to high average range. Health professionals who care for children with osteopetrosis can assure parents that a wide range of developmental outcomes are seen with this disease.

Results of Bone Marrow Transplantation for Recessive Osteopetrosis

Paul Orchard, MD

Department of Pediatrics/BMT, University of Minnesota, Minneapolis, Minnesota, USA

Allogeneic hematopoietic cell transplantation has been used for several decades as an important treatment modality for patients with autosomal recessive osteopetrosis. In a recent analysis combining data from the International Bone Marrow Transplant Registry (IBMTR) and the National Marrow Donor Program (NMDP), 124 patients with osteopetrosis that were recipients of allogeneic transplants were evaluable for analysis. Transplants used matched related donors (MRD; n = 49), alternative related donors (RD; n = 33), or unrelated sources of stem cells, including bone marrow, peripheral blood, and cord blood (URD; n = 42). The median age at transplant was 8 months, and median follow-up was 49 months. The median time to transplantation after establishing the diagnosis was 4 months. The most used preparative regimen was busulfan and cyclophosphamide (n = 92; 74%), whereas 25 patients (20%) were treated with irradiation and cyclophosphamide. Patients were analyzed for neutrophil and platelet recovery, incidence of graft-versus-host disease, and overall survival. The proportion of patients achieving a neutrophil count ≥500 cells/μl by day 60 was 79%, 61%, and 83% in the MRD, RD, and URD groups, respectively. Platelet recovery (>50,000 cells/μl) at 1 year after transplant was 68%, 50%, and 47% in the three groups. Late graft failure was reported in 11 patients. Peritranplant mortality was high throughout the groups, with 33%, 42%, and 31% of patients transplanted dying by day 100 in the MRD, RD, and URD groups, respectively. The primary causes of death for the 68 patients with sufficient information included graft failure (n = 19, 28% of deaths) and pulmonary-related complications, including interstitial pneumonitis, pulmonary hemorrhage, and ARDS (n = 23, 34%). Other primary causes of death include infection (n = 9, 13%) and GvHD (n = 6, 9%). Overall survival at 5 years for the MRD population was 57% (CI 41–69%), which was significantly different than the results in recipients of URD transplants (24%, CI 10–43%; p = 0.01); the results with alternative donor transplants (38%, CI 21–54) were not significantly worse than that observed in the MRD recipients (p = 1.0). T-cell depletion was performed in patients within all groups, but was less commonly used in the MRD recipients (2 of 49 transplants) in comparison with RD (14 of 33) and URD recipients (7 of 42). In comparisons of rates of engraftment and survival in recipients of TCD grafts with those receiving unmanipulated grafts, recipients of TCD transplants had a worse outcome (p = 0.01). In summary, allogeneic hematopoietic cell transplantation remains an important therapeutic option for patients with severe osteopetrosis. There is a significant advantage to having a matched related donor compared with an unrelated donor. Graft failure remains an obstacle to be overcome, as well as the high incidence of pulmonary complications and early death.

ENT Issues in Severe Osteopetrosis

James D Sidman, MD

Children's Hospital of Minneapolis and Departments of Otolaryngology and Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

Head and neck manifestations of osteopetrosis are usually severe and unremitting. This discussion is focused on clinical otolaryngological expressions of osteopetrosis and the practical problems encountered. These manifestations include infiltration of the temporal bone with resultant hearing loss or deafness and encroachment on the optic nerve at multiple locations along its extra-dural course with resultant visual loss and blindness. Loss of cranial nerve function may also be secondary to disruption of the blood supply by osteopetrosis bone lesions. Other complications of osteopetrosis include infiltration of the paranasal sinuses and nose with associated airway obstruction leading to severe sleep apnea and persistent sinus infections. Chronic infections of the upper and lower jaws with loss of teeth and chronic draining fistulas to the skin of the face and neck were also observed. Treatment options include surgical approaches to the skull base to decompress cranial nerves including the optic, acoustic, and trigeminal nerves. Bony decompression for acute intervention and prophylaxis will be discussed, as well as long-term treatment of chronic oral and neck infections with both surgery and medication. Management of spontaneous facial fractures and fractures of the skull base are additional problems encountered in the patient population. This presentation will focus on clinical examples of these expressions of osteopetrosis and the treatment options used in practice. There is not enough literature on this subject to adapt a “best practice” paradigm, but the author's approach will be described in detail.

Gene Therapy for Osteopetrosis

Sherry L Abboud, MD

Department of Pathology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

Osteopetrosis is a heterogenous group of inherited disorders characterized by excessive accumulation of bone. Studies in humans and rodents indicate that osteopetrosis may be caused by decreased osteoclast function and/or failure of osteoclast formation. The development of gene-targeted strategies to correct these defects will be essential for preventing the progression or ameliorating established osteopetrosis in patients with this disorder. Our laboratory has focused on the osteopetrotic op/op mouse model, which is deficient in both the soluble (s) and membrane bound (m) forms of CSF-1. Absence of stromal-derived CSF-1 decreases osteoclasts and leads to osteopetrosis as well as failure of tooth eruption. To determine whether sCSF-1 or mCSF-1 gene replacement reverses the osteopetrotic defect, we took advantage of the osteocalcin (OC) promoter to selectively express each form of CSF-1 in the bone of op/op mice. Transgenic mice harboring either the human sCSF-1 or mCSF-1 cDNA under the control of the OC promoter were generated and crossbred with heterozygous op/wt mice to establish op/op mutants expressing each transgene. In both sCSF-1– and mCSF-1–expressing op/op mice, high levels of CSF-1 protein were detected in bone, tooth eruption was restored, and skeletal growth was comparable with wt littermates by 14 weeks of age. These findings indicate that each form of CSF-1 is sufficient to drive osteoclast development and that the osteocalcin promoter provides an efficient method for delivering exogenous genes to bone. Results from these studies also lead to the novel observation that CSF-1 is required for normal primary tooth formation. Absence of CSF-1 in op/op mice lead to an enamel/dentin dysplasia, which was partially corrected in mCSF-1 op/op mice and almost normalized in sCSF-1 op/op mice. In humans, severe osteopetrosis has been linked to defective osteoclast resorption, primarily caused by mutations in the ATP6i (TCIRG1) gene encoding the α-3 subunit of the vacuolar proton pump required for extracellular acidification, with fewer cases caused by mutations in the CLCN7 chloride channel, cathepsin K, and gl genes. Allogeneic bone marrow transplantation has provided the only cure, and other options such as transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) may be beneficial. Improved methods for gene transfer, design of viral vectors, and drug selection systems have led to efficient and stable gene expression in HSCs. The availability of murine models that mimic the human counterpart of osteopetrosis, including oc/oc mutants and null mutants for ATP6i, ClC-7, cathepsin K, and gl, provide useful tools for exploring the efficacy of gene replacement therapy in vivo. Our studies in op/op mice and other models may provide novel therapeutic strategies for enhancing osteoclast-mediated bone resorption and have clinical application for correcting skeletal remodeling and dental defects in osteopetrotic disorders.