Alessandra Pangrazio

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.

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.

Infantile malignant, autosomal recessive osteopetrosis: The rich and the poor

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.

Mutations in OSTM1 (Grey Lethal) Define a Particularly Severe Form of Autosomal Recessive Osteopetrosis With Neural Involvement

We report three novel osteopetrosis patients with OSTM1 mutations and review two that have been previously described. Our analysis suggests that OSTM1 defines a new subset of patients with severe central nervous system involvement. This defect is also present in the gl mouse, which could represent a good model to study the role of the gene in the pathogenesis of this disease.

Molecular and Clinical Heterogeneity in CLCN7-dependent Osteopetrosis: Report of 20 Novel Mutations

The “Osteopetroses” are genetic diseases whose clinical picture is caused by a defect in bone resorption by osteoclasts. Three main forms can be distinguished on the basis of severity, age of onset and means of inheritance: the dominant benign, the intermediate and the recessive severe form. While several genes have been involved in the pathogenesis of the different types of osteopetroses, the CLCN7 gene has drawn the attention of many researchers, as mutations within this gene are associated with very different phenotypes. We report here the characterization of 25 unpublished patients which has resulted in the identification of 20 novel mutations, including 11 missense mutations, 6 causing premature termination, 1 small deletion and 2 putative splice site defects. Careful analysis of clinical and molecular data led us to several conclusions. First, intermediate osteopetrosis is not homogeneous, since it can comprise both severe dominant forms with an early onset and recessive ones without central nervous system involvement. Second, the appropriateness of haematopoietic stem cell transplantation in CLCN7‐dependent ARO patients has to be carefully evaluated and exhaustive CNS examination is strongly suggested, as transplantation can almost completely cure the disease in situations where no primary neurological symptoms are present. Finally, the analysis of this largest cohort of CLCN7‐dependent ARO patients together with some ADO II families allowed us to draw preliminary genotype‐phenotype correlations suggesting that haploinsufficiency is not the mechanism causing ADO II. The availability of biochemical assays to characterize ClC‐7 function will help to confirm this hypothesis.

SNX10 mutations define a subgroup of human autosomal recessive osteopetrosis with variable clinical severity.

Human Autosomal Recessive Osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. In 2000, we found that mutations in the TCIRG1 gene encoding for a subunit of the proton pump (V‐ATPase) are responsible for more than one‐half of ARO cases. Since then, five additional genes have been demonstrated to be involved in the pathogenesis of the disease, leaving approximately 25% of cases that could not be associated with a genotype. Very recently, a mutation in the sorting nexin 10 (SNX10) gene, whose product is suggested to interact with the proton pump, has been found in 3 consanguineous families of Palestinian origin, thus adding a new candidate gene in patients not previously classified. Here we report the identification of 9 novel mutations in this gene in 14 ARO patients from 12 unrelated families of different geographic origin. Interestingly, we define the molecular defect in three cases of “Västerbottenian osteopetrosis,” named for the Swedish Province where a higher incidence of the disease has been reported. In our cohort of more than 310 patients from all over the world, SNX10‐dependent ARO constitutes 4% of the cases, with a frequency comparable to the receptor activator of NF‐κB ligand (RANKL), receptor activator of NF‐κB (RANK) and osteopetrosis‐associated transmembrane protein 1 (OSTM1)‐dependent subsets. Although the clinical presentation is relatively variable in severity, bone seems to be the only affected tissue and the defect can be almost completely rescued by hematopoietic stem cell transplantation (HSCT). These results confirm the involvement of the SNX10 gene in human ARO and identify a new subset with a relatively favorable prognosis as compared to TCIRG1‐dependent cases. Further analyses will help to better understand the role of SNX10 in osteoclast physiology and verify whether this protein might be considered a new target for selective antiresorptive therapies.

RANK-Dependent Autosomal Recessive Osteopetrosis: Characterization of Five New Cases With Novel Mutations

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.

Identification of the first deletion in the LRP5 gene in a patient with Autosomal Dominant Osteopetrosis type I

In the last decade, the low-density lipoprotein receptor-related protein 5 (LRP5) gene, coding for a coreceptor in the canonical Wnt signalling pathway, has been shown to play an important role in regulating bone mass and to be involved in the pathogenesis of several bone disorders. Here we describe a patient who presented with a clinical picture of Autosomal Dominant Osteopetrosis type I (ADO I), in whom we could identify the first deletion in the LRP5 gene causing increased bone mass. This mutation caused the in-frame deletion of two amino acids in the fourth blade of the first propeller of the protein, namely the highly conserved glycine at position 171 and the following glutamate residue. In vitro studies suggested that the pathogenic effect of this novel mutation could be due to a decreased inhibition of Wnt signalling by the antagonistic proteins sclerostin and Dickkopf-1, encoded respectively by the SOST and DKK1 genes, in the presence of mutated LRP5. Our results highlight an increasing molecular heterogeneity in LRP5-related bone diseases.

RANKL Cytokine: From Pioneer of the Osteoimmunology Era to Cure for a Rare Disease

Since its identification, the RANKL cytokine has been demonstrated to play a crucial role in bone homeostasis and lymphoid tissue organization. Genetic defects impairing its function lead to a peculiar form of autosomal recessive osteopetrosis (ARO), a rare genetic bone disease presenting early in life and characterized by increased bone density due to failure in bone resorption by the osteoclasts. Hematopoietic stem cell transplantation (HSCT) is the only option for the majority of patients affected by this life-threatening disease. However, the RANKL-dependent ARO does not gain any benefit from this approach, because the genetic defect is not intrinsic to the hematopoietic osteoclast lineage but rather to the mesenchymal one. Of note, we recently provided proof of concept of the efficacy of a pharmacological RANKL-based therapy to cure this form of the disease. Here we provide an overview of the diverse roles of RANKL in the bone and immune systems and review the clinical features of RANKL-deficient ARO patients and the results of our preclinical studies. We emphasize that these patients present a continuous worsening of the disease in the absence of a cure and strongly wish that the therapy we propose will be further developed.