Buried in the Middle but Guilty: Intronic Mutations in the TCIRG1 Gene Cause Human Autosomal Recessive Osteopetrosis.

Autosomal recessive osteopetrosis (ARO) is a rare genetic bone disease with genotypic and phenotypic heterogeneity, sometimes translating into delayed diagnosis and treatment. In particular, cases of intermediate severity often constitute a diagnostic challenge and represent good candidates for exome sequencing. Here, we describe the tortuous path to identification of the molecular defect in two siblings, in which osteopetrosis diagnosed in early childhood followed a milder course, allowing them to reach the adult age in relatively good conditions with no specific therapy. No clearly pathogenic mutation was identified either with standard amplification and resequencing protocols or with exome sequencing analysis. While evaluating the possible impact of a 3'UTR variant on the TCIRG1 expression, we found a novel single nucleotide change buried in the middle of intron 15 of the TCIRG1 gene, about 150 nucleotides away from the closest canonical splice site. By sequencing a number of independent cDNA clones covering exons 14 to 17, we demonstrated that this mutation reduced splicing efficiency but did not completely abrogate the production of the normal transcript. Prompted by this finding, we sequenced the same genomic region in 33 patients from our unresolved ARO cohort and found three additional novel single nucleotide changes in a similar location and with a predicted disruptive effect on splicing, further confirmed in one of them at the transcript level. Overall, we identified an intronic region in TCIRG1 that seems to be particularly prone to splicing mutations, allowing the production of a small amount of protein sufficient to reduce the severity of the phenotype usually associated with TCIRG1 defects. On this basis, we would recommend including TCIRG1 not only in the molecular work-up of severe infantile osteopetrosis but also in intermediate cases and carefully evaluating the possible effects of intronic changes.

As little as needed: the extraordinary case of a mild recessive osteopetrosis owing to a novel splicing hypomorphic mutation in the TCIRG1 gene.

Mutations in the TCIRG1 gene, coding for a subunit of the osteoclast proton pump, are responsible for more than 50% of cases of human malignant autosomal recessive osteopetrosis (ARO), a rare inherited bone disease with increased bone density owing to a failure in bone resorption. A wide variety of mutations has been described, including missense, nonsense, small deletions/insertions, splice-site mutations, and large genomic deletions, all leading to a similar severe presentation. So far, to the best of our knowledge, no report of a mild phenotype owing to recessive TCIRG1 mutations is present neither in our series of more than 100 TCIRG1-dependent ARO patients nor in the literature. Here we describe an 8-year-old patient referred to us with a clinical diagnosis of ARO, based on radiological findings; of note, no neurological or hematological defects were present in this girl. Surprisingly, we identified a novel nucleotide change in intron 15 of the TCIRG1 gene at the homozygous state, leading to the production of multiple aberrant transcripts, but also, more importantly, of a limited amount of the normal transcript. Our results show that a low level of normal TCIRG1 protein can dampen the clinical presentation of TCIRG1-dependent ARO. On this basis, a small amount of protein might be sufficient to rescue, at least partially, the severe ARO phenotype, and this is particularly important when gene therapy approaches are considered. In addition, we would also recommend that the TCIRG1 gene be included in the molecular diagnosis of mild forms of human ARO.

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions.

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.

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.

Homozygous stop mutation in the SNX10 gene in a consanguineous Iraqi boy with osteopetrosis and corpus callosum hypoplasia.

Recently a mutation in the SNX10 gene that belongs to the sorting nexin family was identified as a cause of a new subset of human autosomal recessive osteopetrosis. Here, we identified a novel homozygous mutation (c.46C > T, p.Arg16X) in SNX10, in an Iraqi boy from a consanguineous family with a history of infantile osteopetrosis. The proband exhibited macrocephaly, prominent forehead, proptosis of the eyes, strabismus, splenomegaly and joint hyperlaxity. Bone X-rays showed increased bone density, metaphyseal under-modelling, transverse alternating bands of greater and lesser density in tubular bones, anteriorly notched vertebral bodies and bone-in-bone appearance. Brain atrophy, external hydrocephalus and thin corpus callosum were noted at the brain MRI and CT scan. Blood test results revealed the presence of anaemia and leukopenia. Our findings confirm the role of SNX10 in autosomal recessive osteopetrosis and help to better define the core set of manifestations associated with this new pathological entity.

Association of severe autosomal recessive osteopetrosis and structural brain abnormalities: a case report and review of the literature.

We describe a fetus with severe osteopetrosis diagnosed on post-mortem radiographs following termination of pregnancy at 29 weeks for major brain malformations detected on ultrasound. SNP microarray confirmed loss of heterozygosity in 5% of the genome, consistent with parental consanguinity. Sequencing of the genes known to cause severe recessive osteopetrosis, TCIRG1, CLCN7, OSTM1 and SNX10, was negative. Brain malformations are not typically considered part of the phenotypic spectrum of osteopetrosis. We review the literature, and propose that this may represent a novel autosomal recessive variant of osteopetrosis.

A homozygous contiguous gene deletion in chromosome 16p13.3 leads to autosomal recessive osteopetrosis in a Jordanian patient.

Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Mutations in the CLCN7 gene are responsible not only for a substantial portion of ARO patients but also for other forms of osteopetrosis characterized by different severity and inheritance. The lack of a clear genotype/phenotype correlation makes genetic counseling a tricky issue for CLCN7-dependent osteopetrosis. Here, we characterize the first homozygous interstitial deletion in 16p13.3, detected by array comparative genomic hybridization in an ARO patient of Jordanian origin. The deletion involved other genes besides CLCN7, while the proband displayed a classic ARO phenotype; however, her early death did not allow more extensive clinical investigations. The identification of this novel genomic deletion involving a large part of the CLCN7 gene is of clinical relevance, especially in prenatal diagnosis, and suggests the possibility that this kind of mutation has been underestimated so far. These data highlight the need for alternative approaches to genetic analysis also in other ARO-causative genes.

Osteopetrosis rescue upon RANKL administration to Rankl(-/-) mice: a new therapy for human RANKL-dependent ARO.

In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.

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.

A new familial sclerosing bone dysplasia.

Osteoscleroses are a heterogeneous group of bone remodeling disorders characterized by an increase in bone density. Here we report on a consanguineous Lebanese family in which two sisters, aged 39 and 36 years, exhibit a severe genu varum, a square-face appearance, high forehead, slight proptosis of the eyes, symmetric enlargement of the jaw, protruding chin, and short stature. Bone X-rays showed the presence of hyperostosis of the cranial base and vault with increased density of the orbits, hyperostosis of the bones, thickening of the cortices, diaphyseal modeling defects, cortical thickening of the medullary cavity, mild enlargement of the medullary cavity of the short long bones, short femoral necks, increased width of the ribs, and narrow interpedicular distances of the lower lumbar spine. Osteodensitometry showed values 200% to 300% above values for age. A cervical MRI revealed the presence of a diffuse osteosclerosis with calcification of the posterior vertebral ligament and a narrow canal between C2 and T2. Blood test results were unremarkable. Serum osteocalcin levels were in the normal range, whereas high values of serum C-telopeptide were noted. A bone biopsy showed only the presence of compact bone and did not allow for histomorphometric analysis. Molecular studies excluded genes known to be involved in sclerosing bone dysplasias as the cause of this condition. In vitro analysis of osteoclast function indicated that contrary to most cases of autosomal recessive osteopetrosis, osteoclasts both formed and resorbed but exhibited a small decrease in resorptive activity compared with osteoclasts generated from normal control individuals. Differential diagnoses are discussed, and the possibility that this may be a novel clinical entity is raised.

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.

Osteopetrosis with micro-lacunar resorption because of defective integrin organization.

In vitro differentiated monocytes were used to characterize the cellular defect in a type of osteopetrosis with minimally functional osteoclasts, in which defects associated with common causes of osteopetrosis were excluded by gene sequencing. Monocytes from the blood of a 28-year-old patient were differentiated in media with RANKL and CSF-1. Cell fusion, acid compartments within cells, and tartrate resistant acid phosphatase (TRAP) activity were normal. However, the osteoclasts made abnormally small pits on the dentine. Phalloidin labeling showed that the cell attachments lacked the peripheral ring structure that supports lacunar resorption. Instead, the osteoclasts had clusters of podosomes near the center of cell attachments. Antibody to the alphavbeta3 integrin pair or to the C-terminal of beta3 did not label podosomes, but antibody to alphav labeled them. Western blots using antibody to the N-terminal of beta3 showed a protein of reduced size. Integrins beta1 and beta5 were upregulated, but, in contrast to observations in beta3 defects, alpha2 had not increased. The rho-GTP exchange protein Vav3, a key attachment organizing protein, did not localize normally with peripheral attachment structures. Vav3 forms of 70 kD and 90 kD were identified on western blots. However, the proteins beta3 integrin, Vav3, Plekhm1, and Src, implicated in attachment defects, had normal exon sequences. In this new type of osteopetrosis, the integrin-organizing complex is dysfunctional, and at least two attachment proteins may be partially degraded.

Characterization of a novel Alu-Alu recombination-mediated genomic deletion in the TCIRG1 gene in five osteopetrotic patients.

Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant-like TCIRG1-dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu-mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.

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.

Prognostic potential of precise molecular diagnosis of Autosomal Recessive Osteopetrosis with respect to the outcome of bone marrow transplantation.

Hematopoietic stem cell transplantation (HSCT) is often the only practical approach to fatal genetic defects. One of the first pathologies which HSCT was applied to was Autosomal Recessive Osteopetrosis (ARO), a rare genetic bone disease in which a deficit in bone resorption by osteoclasts leads to increased bone density and secondary defects. The disease is often lethal early in life unless treated with HSCT. In utero transplantation (IUT) of the oc/oc mouse, reproducing the clinical features of a subset of ARO, has demonstrated that the quality of life and the survival of transplanted animals are greatly improved, suggesting that a similar protocol could be applied to humans. However, recently the dissection of the molecular bases of the disease has shown that ARO is genetically heterogeneous and has revealed the presence of subsets of patients which do not benefit from HSCT. This observation highlights the importance of molecular diagnosing ARO to identify and establish the proper therapies for a better prognosis. In particular, on the basis of experimental results in murine models, efforts should be undertaken to develop approaches such as IUT and new pharmacological strategies.

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.

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.

Molecular study of six families originating from the Middle-East and presenting with autosomal recessive osteopetrosis.

Autosomal recessive osteopetrosis is a severe hereditary bone disease whose cellular basis is in the osteoclast, but with heterogeneous molecular defects. We hereby report the clinical and the molecular study of seven patients affected by the recessive form of osteopetrosis (ARO) from six families originating from the Middle-East: four from Lebanon and two from Syria. Parental consanguinity was found in five families. The mean age of diagnosis was 3 months. Failure to thrive, prominent forehead, exophthalmia, optic atrophy, hepatosplenomegaly, neurological manifestations, anaemia, thrombocytopenia, hypocalcaemia, elevated hepatic enzymes and acid phosphatase, and an early fatal outcome were common. Macrocephaly, strabismus, and brain malformations were relatively less common. Mutations were identified in two genes: TCIRG1 and OSTM1. Phenotype-genotype correlation is discussed.