Main results

 

Researchers from this Unit further actively pursued the molecular and cellular basis of the genetic diseases they have so far successfully investigated: primary immunodeficiencies and ARO.

 

1. Omenn syndrome (SCID T+ B-)

Ten years ago, the group identified Rag genes as responsible for Omenn Syndrome (OS), a peculiar severe combined immunodeficiency (SCID) characterized by absence of B cells associated to a normal or even elevated number of activated T cells. In the following years the group has further contributed to the description of the clinical and molecular features of Rag-dependent SCID.

In order to further study OS, in the last few years the group has constructed an experimental model of OS. A point (missense) mutation in the Rag-2 gene, previously detected in an OS patient was engineered in the model by homologous recombination in ES cells and a knock-in model was produced. Very interestingly, this model recapitulates all the symptoms and signs of human OS, allowing the group to investigate several aspects that for ethical and practical reasons cannot be pursued in humans.

 

2.  Molecular basis of Autosomal recessive osteopetrosis (ARO)

In 2000, the group identified the first gene involved in ARO. In 2003 they contributed to the identification of the OSTM1-dependent ARO form (the human counterpart of the gl mutation in experimental model) as well as to the characterization of the intermediate ARO form. In 2007 they contributed, in collaboration with van Hul’s group in Belgium, to the characterization of the PLEKHM1-dependent ARO (the human counterpart of the ia rat mutation). The same year they identified RANKL as the first gene mutated in osteoclast poor ARO, while in 2008 they reported that RANK mutations can also be found in ARO, constituting a second subset of the osteoclast-poor form. They have collected more than 200 infants affected by ARO and provide genetic analysis to Institutions from all the world.

 

Main objectives and research lines

 

1.   Further studies on the molecular genetics of human ARO

About 30% of ARO patients in our series, which is the largest in the world, do not have mutations in the genes (TCIRG1, ClCN7, OSTM1, PLEKHM1, RANKL and RANK) which are known to be responsible for ARO. We plan to analyse more patients and to classify them on the basis of the gene found mutated in each case. We have already selected some patients with an osteoclast-poor form without RANKL and RANK mutations. This observation shows that also the osteoclast-poor ARO subgroup is heterogeneous from a molecular point of view, so in this subset we will investigate other genes involved in the osteoclastogenic process. We will focus first on genes leading to osteopetrosis lacking osteoclasts in model, when knocked-out. Indeed, a number of genes are reported to determine such a phenotype in murine models. Among them, c-src, c-fos, PU.1, Dap12/FcRgamma and Rgs10 have been demonstrated as causing severe osteoclast-poor osteopetrosis in experimental models, while M-CSF, M-CSFR, NF-kB, Dap12 (alone) lead to a milder form of osteopetrosis with absence of osteoclasts, when knocked-out. All these genes can be considered good candidates as responsible for the disease in the subgroup of patients we plan to focus on; some of them have been investigated in the past by our group, however, as it is likely that they account for only 2-3% or even a smaller percentage of ARO patients, they could have gone undetected if the population under investigation had not been selected ad hoc. Therefore, we will sequence these genes, starting from those involved in the RANKL/RANK axis, because of the huge amount of data reported in the last years, strongly demonstrating the pivotal role of this signaling pathway in osteoclast-precursors differentiation and mature osteoclast function (Asagiri et al, 2007).

 

2.   Immunological characterization of patients carrying defects in RANKL and RANK genes

We plan to perform another set of experiments to verify the presence of immunological defects in RANKL and RANK-mutated ARO patients. As reported in literature, rankl ^-/-and rank ^-/- models display osteopetrosis with immunodeficiency due to marked deficiency of B cells in the spleen and complete lack of peripheral lymph-nodes. So, we will collect information on the immune status of newly identified RANKL and RANK-dependent patients from their respective clinicians and, in the case of untransplanted patients, we will further investigate the functionality of their immune system, in terms of percentages of the various cellular subpopulations, proliferation capacity, cytokine and immunoglobulin production. Furthermore, we will analyze regulatory T cell compartment (nTreg) to test if defects in RANKL/RANK pathway can affect mechanisms of peripheral tolerance. We will assay the ability of nTreg cells, defined as CD4+CD25+CD127- obtained by sorting from peripheral blood of RANK and RANKL patients, to suppress the proliferation of effector cells (CD4+CD25- ).

This way, we aim to add new knowledge to the field of the molecular basis of human ARO and, from a more general point of view, to the field of bone homeostasis. In particular, the discovery of a clear immunological defect in RANKL and RANK-mutated ARO patients would add to the recently defined concept of osteoimmunology, describing the strong interplay between bone and the immune system both in physiological and in pathological conditions.

 

3.   In vitro characterization of the cellular defect in ARO patients.

The main aim is to obtain a series of data about the cellular defects in ARO patients, which will be correlated to the molecular defects and will be useful to better understand the whole phenotype. If the mutation lies in a gene which has not yet been involved in the pathogenesis of the disease, the identification of the altered cellular pathway/function will direct the molecular analysis towards the most probable candidate-gene.

For this purpose, we will try to obtain blood samples from all the non-transplanted patients whose DNA has been analysed, in order to isolate peripheral blood mononuclear cells (PBMC) for cellular analysis and in vitro testing. In this respect the collaboration with Prof. Mike Rogers at the University of Aberdeen has been extremely important, because it has allowed the establishment, in our own lab, of a standardized protocol of in vitro differentiation in the presence of recombinant, soluble RANKL and M-CSF. Osteoclasts formed will be analysed through immunostaining and Electron Microscopy (EM), with respect to their morphology and to the presence of structures peculiar to this cellular type (for example, the actin ring, the ruffled border, various kinds of vesicles, others). Moreover their resorptive capability will be evaluated in vitro, when osteoclasts are seeded on dentin discs.

Therefore, the osteoclast differentiation assay will be used in all our patients with unknown mutations whose cells are available. In osteoclast-rich forms, monocytes differentiate to multinucleated osteoclasts unable to resorb bone, while in osteoclast-poor forms a defect in the formation of multinucleated osteoclasts should be apparent. By performing the assay in patients with unknown mutations, we will verify whether a defect in differentiation does exist; if this is the case, we will be able to define the specific step at which the differentiation is blocked and this will help in the identification of the gene/s involved.

 

4.   Preclinical study of RANKL therapy in a model of rankl defect (rankl-/- strain)

We aim to evaluate the long-term effects of RANKL administration in RANKL-dependent osteopetrotic models, in order to assess whether, as predicted by previous studies, the pharmacological