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.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

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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.

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.

Among 94 osteopetrotic patients presenting with a severe clinical picture and diagnosed early in life, 12 bore mutations in the ClCN7 gene, but only 7 of them had the expected two recessive mutations. The remaining five patients seem to be heterozygous for a ClCN7 mutation, and significant variations were observed in the clinical manifestations of their disease, even within the same family.Introduction: Human osteopetroses are a heterogeneous group of diseases that include both infantile severe, autosomal recessive (ARO) and adult autosomal dominant (ADO) forms. Two genes, Atp6a3 (TCIRG1) and ClCN7, have been shown to be associated with human ARO, the latter of which is also thought to be responsible for ADO-II. However, patients with an intermediate phenotype have been described: the genetic basis of these observances is unknown. Materials and Methods:In this study, we report the clinical and molecular analysis of 94 patients in which a diagnosis of severe osteopetrosis was made within the first 2 years of age. Both TCIRG1 and CLCN7 genes were sequenced in all patients and the molecular findings were correlated to clinical parameters. Results and Conclusions:In 56 of 94 patients with a classical picture of ARO, TCIRG1-dependent recessive mutations were found. In contrast, ClCN7 mutations were found in 12 cases (13%) of severe osteopetrosis, but only 7 of them had two recessive mutations identified: in 6 of these 7 cases, central nervous system manifestations were noted, and these patients had a poor prognosis. The remaining five cases were heterozygous for a ClCN7 mutation,

Human malignant infantile osteopetrosis (arOP; MIM 259700) is a genetically heterogeneous autosomal recessive disorder of bone metabolism, which, if untreated, has a fatal outcome. Our group, as well as others, have recently identified mutations in the ATP6i (TCIRG1) gene, encoding the a3 subunit of the vacuolar proton pump, which mediates the acidification of the bone/osteoclast interface, are responsible for a subset of this condition. By sequencing the ATP6i gene in arOP patients from 44 unrelated families with a worldwide distribution we have now established that ATP6i mutations are responsible for approximately 50% of patients affected by this disease. The vast majority of these mutations (40 out of 42 alleles, including seven deletions, two insertions, 10 nonsense substitutions and 21 mutations in splice sites) are predicted to cause severe abnormalities in the protein product and are likely to represent null alleles. In addition, we have also analysed nine unrelated arOP patients from Costa Rica, where this disease is apparently much more frequent than elsewhere. All nine Costa Rican patients bore either or both of two missense mutations (G405R and R444L) in amino acid residues which are evolutionarily conserved from yeast to humans. The identification of ATP6i gene mutations in two families allowed us for the first time to perform prenatal diagnosis: both fetuses were predicted not to be affected and two healthy babies were born. This study contributes to the determination of genetic heterogeneity of arOP and allows further delineation of the other genetic defects causing this severe condition.

Human recessive osteopetrosis (ARO) represents a group of diseases in which, due to a defect in osteoclasts, bone resorption is prevented. The deficit could arise either from failure in osteoclast differentiation or from inability to perform resorption by mature, multinucleated, but nonfunctional cells. Historically, osteopetrosis due to both these mechanisms was found in spontaneous and artificially created mouse mutants, but the first five genes identified in human ARO (CA-II, TCIRG1, ClCN7, OSTM1, and PLEKHM1) were all involved in the effector function of mature osteoclasts, being linked to acidification of the cell/bone interface or to intracellular processing of the resorbed material. Differentiation defects in human ARO have only recently been described, following the identification of mutations in both RANKL and RANK, which define a new form of osteoclast-poor ARO, as expected from biochemical, cellular, and animal studies. The molecular dissection of ARO has prognostic and therapeutic implications. RANKL-dependent patients, in particular, represent an interesting subset which could benefit from mesenchymal cell transplant and/or administration of soluble RANKL cytokine.