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Autosomal recessive osteopetrosis (ARO) is a heterogeneous disorder, characterized by defective osteoclastic resorption of bone that results in increased bone density. We have studied nine individuals with an intermediate form of ARO, from the county of Västerbotten in Northern Sweden. All afflicted individuals had an onset in early infancy with optic atrophy, and in four patients anemia was present at diagnosis. Tonsillar herniation, foramen magnum stenosis, and severe osteomyelitis of the jaw were common clinical features. Whole exome sequencing, verified by Sanger sequencing, identified a splice site mutation c.212 + 1 G > T in the SNX10 gene encoding sorting nexin 10. Sequence analysis of the SNX10 transcript in patients revealed activation of a cryptic splice site in intron 4 resulting in a frame shift and a premature stop (p.S66Nfs * 15). Haplotype analysis showed that all cases originated from a single mutational event, and the age of the mutation was estimated to be approximately 950 years. Functional analysis of osteoclast progenitors isolated from peripheral blood of patients revealed that stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) resulted in a robust formation of large, multinucleated osteoclasts which generated sealing zones; however these osteoclasts exhibited defective ruffled borders and were unable to resorb bone in vitro.

Osteopetrosis is a genetic condition of increased bone mass, which is caused by defects in osteoclast formation and function. Both autosomal recessive and autosomal dominant forms exist, but this Review focuses on autosomal recessive osteopetrosis (ARO), also known as malignant infantile osteopetrosis. The genetic basis of this disease is now largely uncovered: mutations in TCIRG1, CLCN7, OSTM1, SNX10 and PLEKHM1 lead to osteoclast-rich ARO (in which osteoclasts are abundant but have severely impaired resorptive function), whereas mutations in TNFSF11 and TNFRSF11A lead to osteoclast-poor ARO. In osteoclast-rich ARO, impaired endosomal and lysosomal vesicle trafficking results in defective osteoclast ruffled-border formation and, hence, the inability to resorb bone and mineralized cartilage. ARO presents soon after birth and can be fatal if left untreated. However, the disease is heterogeneous in clinical presentation and often misdiagnosed. This article describes the genetics of ARO and discusses the diagnostic role of next-generation sequencing methods. The management of affected patients, including guidelines for the indication of haematopoietic stem cell transplantation (which can provide a cure for many types of ARO), are outlined. Finally, novel treatments, including preclinical data on in utero stem cell treatment, RANKL replacement therapy and denosumab therapy for hypercalcaemia are also discussed.

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis., (Copyright © 2012 American Society for Bone and Mineral Research.)

Functional, biochemical and genetic studies have over the past decade identified many causative genes in the osteoclast diseases osteopetrosis and Paget's disease of bone. Here, we outline all osteoclast diseases and their genetic associations and then focus specifically on those diseases caused by mutations in the critical osteoclast molecule Receptor Activator of Nuclear factor Kappa B (RANK). Both loss and gain-of-function mutations have been found in humans leading to osteopetrosis and high bone turnover phenotypes, respectively. Osteopetrosis-associated RANK mutations are widely distributed over the RANK molecule. It is likely that some negatively affect ligand binding, whereas others preclude appropriate association of RANK with downstream signalling molecules. In the Paget-like disorders, familial expansile osteolysis, early onset Paget's disease and expansile skeletal hyperphosphatasia, heterozygous insertion mutations are found in the RANK signal peptide. These prevent signal peptide cleavage, trapping the protein translated from the mutated allele in the endoplasmic reticulum. Whole animal studies replicate the hyperactive osteoclast phenotype associated with these disorders and present only with heterozygous expression of the mutation, suggesting an as yet unexplained effect of the mutant allele on normal RANK function. We discuss the cell biological studies and animal models that help us to understand the nature of these different RANK defects and describe how careful dissection of these conditions can help understand critical pathways in osteoclast development and function. We highlight areas that require further study, particularly in light of the pharmacological interest in targeting the RANK signalling pathway to treat diseases caused by excessive bone resorption.

The multinucleated osteoclast has a unique function: degradation of mineralized tissues. It is generally taken that all osteoclasts are alike, independent of the skeletal site where they exert their activity. Recent data, however, question this view as they show that osteoclasts at different bony sites appear to differ, for example in the machinery responsible for resorption. Support for the notion that there may be heterogeneity in osteoclasts is obtained from studies in which osteoclast activity is inhibited and from observations in osteopetrosis and inflammatory bone conditions. In this review we discuss the available evidence and propose the existence of bone-site-specific osteoclast heterogeneity.

Autosomal-Recessive Osteopetrosis (ARO) comprises a heterogeneous group of bone diseases for which mutations in five genes are known as causative. Most ARO are classified as osteoclast-rich, but recently a subset of osteoclast-poor ARO has been recognized as due to a defect in TNFSF11 (also called RANKL or TRANCE, coding for the RANKL protein), a master gene driving osteoclast differentiation along the RANKL-RANK axis. RANKL and RANK (coded for by the TNFRSF11A gene) also play a role in the immune system, which raises the possibility that defects in this pathway might cause osteopetrosis with immunodeficiency. From a large series of ARO patients we selected a Turkish consanguineous family with two siblings affected by ARO and hypogammaglobulinemia with no defects in known osteopetrosis genes. Sequencing of genes involved in the RANKL downstream pathway identified a homozygous mutation in the TNFRSF11A gene in both siblings. Their monocytes failed to differentiate in vitro into osteoclasts upon exposure to M-CSF and RANKL, in keeping with an osteoclast-intrinsic defect. Immunological analysis showed that their hypogammaglobulinemia was associated with impairment in immunoglobulin-secreting B cells. Investigation of other patients revealed a defect in both TNFRSF11A alleles in six additional, unrelated families. Our results indicate that TNFRSF11A mutations can cause a clinical condition in which severe ARO is associated with an immunoglobulin-production defect.

Unlabelled: We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk., Introduction: The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis., Materials and Methods: We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out., Results: Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling., Conclusions: We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.

Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor-KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Tooth eruption depends on the presence of osteoclasts to create an eruption pathway through the alveolar bone. In diseases where osteoclast formation, or function is reduced, such as the various types of osteopetrosis, tooth eruption is affected. Diseases in which osteoclast formation or activity is increased, such as familiar expansile osteolysis and Paget's disease, are associated with dental abnormalities such as root resorption and premature tooth loss. Less is known about the origin of the dental problems in these conditions as there are no rodent models of these diseases as yet. In this short review, the genes currently known to be mutated in human osteoclast diseases will be reviewed and, where known, the effect of osteoclast dysfunction on dental development described. It will focus on human conditions and only mention rodent disease where no clear data in the human are available.

The genetic defects that cause human infantile malignant osteopetrosis, a disease with recessive inheritance characterized by lack of bone resorption and the presence of large numbers of inactive osteoclasts, are only partially known. Studies of osteoclasts in vitro may help to identify or exclude candidate genes in this disorder. Here, we established co-cultures of peripheral blood mononuclear cells with mouse fetal bone rudiments to generate osteoclasts from three infants with malignant osteopetrosis. Osteoclasts generated in vitro displayed the same inability to form ruffled borders and resorb bone as seen in bone biopsies. This culture model may contribute to understanding the pathogenesis of this disease.

Objective: To determine the variability and the natural course of children suffering from autosomal recessive osteopetrosis to allow optimal counseling and decision making with respect to therapeutic intervention., Design: Retrospective and longitudinal evaluation of clinical symptoms and natural course with emphasis on survival and sensoneurologic and hematologic findings., Setting: Two large referral-based pediatric bone marrow transplantation units in Europe., Patients: Thirty-three patients with autosomal recessive osteopetrosis admitted to units in Paris and Leiden between 1972 and 1988 were analyzed until last follow-up or the time at which bone marrow transplantation was performed. The great number of patients and unprecedented amount of data make this report one of the single largest clinical studies on autosomal recessive osteopetrosis., Main Results: Ocular involvement occurring at a median age of 2 months was the symptom at initial examination in half of the patients. A striking variability between patients was found. Retinal degeneration was present in three patients and associated generalized neurodegeneration was present in two. The probability of survival until the age of 6 years was about 30% for the group as a whole. The cumulative risk of developing visual or hematologic impairment in the first year of life was about 75% and leveled off afterward. Patients with early hematologic impairment, ie, before 3 months of age, especially when combined with early visual impairment, had a very poor prognosis regarding life expectancy., Conclusions: Autosomal recessive osteopetrosis seems to be a variable disorder with a poor prognosis, especially in children with early visual and hematologic impairment. Allogenic bone marrow transplantation remains the only curative approach.

Trabecular bone biopsies obtained from six patients with malignant osteopetrosis, one patient with benign osteopetrosis, and two controls were examined by light and electron microscopy. Osteopetrotic osteoclasts showed little to no signs of active involvement in bone resorption. Ruffled borders and clear zones were absent in most cells. In all patients there were large numbers of osteoclasts. Numbers of osteoblasts, bone lining cells, and bone marrow stromal cells were extremely low in all patients with malignant osteopetrosis. In six of the patients a mineralized layer of amorphous organic material lacking collagen fibrils was seen covering large areas of the bone or cartilage matrix. We suggest that this layer represents a pathological calcification on which subsequently organic material has accumulated. The abnormalities in osteopetrotic bone are discussed in the light of the pathogenesis of this disease.

Cell surface structures of isolated osteopetrotic (mi/mi) and normal murine (+/+ and +/mi) and human osteoclasts were examined in a microscope combining light and scanning electron microscopy (LM/SEM). Tartrate-resistant acid phosphatase (TrAP) was used as an histochemical osteoclast marker. In osteopetrotic bone, as in normal murine bone, TrAP activity was exclusively seen in osteoclasts and preosteoclasts and was therefore judged a suitable marker for identification of isolated osteoclasts. A method was developed for preparation of LM/SEM specimens from osteoclast-enriched cell suspensions. In the LM/SEM isolated osteoclasts were easily recognized in the LM mode by TrAP contents. In specimens prepared from murine cells, but not human cells, LM identification of osteoclasts by TrAP was essential. This was in particular true for small, mononuclear, mi/mi osteoclasts. All osteoclasts examined had a villous appearance and were well spread over the glass substrate. There were no differences in cell surface morphology and in adherence to glass between osteopetrotic and normal osteoclasts.