Anna Villa

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis

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.

Partial V(D)J Recombination Activity Leads to Omenn Syndrome

Genomic rearrangement of the antigen receptor loci is initiated by the two lymphoid-specific proteins Rag-1 and Rag-2. Null mutations in either of the two proteins abrogate initiation of V(D)J recombination and cause severe combined immunodeficiency with complete absence of mature B and T lymphocytes. We report here that patients with Omenn syndrome, a severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels, bear missense mutations in either the Rag-1 or Rag-2 genes that result in partial activity of the two proteins. Two of the amino acid substitutions map within the Rag-1 homeodomain and decrease DNA binding activity, while three others lower the efficiency of Rag-1/Rag-2 interaction. These findings provide evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination.

Osteoclast-poor human osteopetrosis due to mutations in the gene encoding RANKL

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.

Grey-lethal mutation induces severe malignant autosomal recessive osteopetrosis in mouse and human.

The spontaneous mouse grey-lethal (gl) mutation is responsible for a coat color defect and for the development of the most severe autosomal recessive form of osteopetrosis. Using a positional cloning approach, we have mapped and isolated the gl locus from a ∼1.5 cM genetic interval. The gl locus was identified in a bacterial artificial chromosome (BAC) contig by functional genetic complementation in transgenic mice. Genomic sequence analysis revealed that the gl mutation is a deletion resulting in complete loss of function. The unique ∼3 kb wild-type transcript is expressed primarily in osteoclasts and melanocytes as well as in brain, kidney, thymus and spleen. The gl gene is predicted to encode a 338–amino acid type I transmembrane protein that localizes to the intracellular compartment. Mutation in the human GL gene leads to severe recessive osteopetrosis. Our studies show that mouse Gl protein function is absolutely required for osteoclast and melanocyte maturation and function.

Human osteoclast-poor osteopetrosis with hypogammaglobulinemia due to TNFRSF11A (RANK) mutations.

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.

Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis.

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.

Osteopetrosis: genetics, treatment and new insights into osteoclast function.

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.

Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency caused by mutations in the gene encoding for WASP, a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WASP result in a wide spectrum of clinical manifestations ranging from the relatively mild X-linked thrombocytopenia to the classic full-blown WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, and high susceptibility to developing tumors and autoimmune manifestations. The life expectancy of patients affected by severe WAS is reduced, unless they are successfully cured by bone marrow transplantation from related identical or matched unrelated donors. Because many patients lack a compatible bone marrow donor, the administration of WAS gene-corrected autologous hematopoietic stem cells could represent an alternative therapeutic approach. In the present review, we focus on recent progress in understanding the molecular and cellular mechanisms contributing to the pathophysiology of WAS. Although molecular and cellular studies have extensively analyzed the mechanisms leading to defects in T, B, and dendritic cells, the basis of autoimmunity and thrombocytopenia still remains poorly understood. A full understanding of these mechanisms is still needed to further implement new therapeutic strategies for this peculiar immunodeficiency.

WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells.

A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4+CD25+FOXP3+ natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS−/− mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS−/− nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS−/− nTreg cells failed to proliferate and to produce transforming growth factor β upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS−/− nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS−/− nTreg cells showed reduced in vitro suppressor activity on both WT and WAS−/− effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4+ effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

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.