Daniele Moratto

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.

Long-term outcome and lineage-specific chimerism in 194 patients with Wiskott-Aldrich syndrome treated by hematopoietic cell transplantation in the period 1980-2009: an international collaborative study.

In this retrospective collaborative study, we have analyzed long-term outcome and donor cell engraftment in 194 patients with Wiskott-Aldrich syndrome (WAS) who have been treated by hematopoietic cell transplantation (HCT) in the period 1980- 2009. Overall survival was 84.0% and was even higher (89.1% 5-year survival) for those who received HCT since the year 2000, reflecting recent improvement of outcome after transplantation from mismatched family donors and for patients who received HCT from an unrelated donor at older than 5 years. Patients who went to transplantation in better clinical conditions had a lower rate of post-HCT complications. Retrospective analysis of lineage-specific donor cell engraftment showed that stable full donor chimerism was attained by 72.3% of the patients who survived for at least 1 year after HCT. Mixed chimerism was associated with an increased risk of incomplete reconstitution of lymphocyte count and post-HCT autoimmunity, and myeloid donor cell chimerism < 50% was associated with persistent thrombocytopenia. These observations indicate continuous improvement of outcome after HCT for WAS and may have important implications for the development of novel protocols aiming to obtain full correction of the disease and reduce post-HCT complications.

A novel primary human immunodeficiency due to deficiency in the WASP-interacting protein WIP.

A homozygous mutation that gave rise to a stop codon in the WIPF1 gene resulted in WASP protein destabilization and in symptoms resembling those of Wiskott-Aldrich syndrome

Stem cell transplantation for the Wiskott-Aldrich syndrome: a single-center experience confirms efficacy of matched unrelated donor transplantation.

The treatment of Wiskott–Aldrich syndrome (WAS), a once uniformly fatal disorder, has evolved considerably as the use of hematopoietic stem cell transplant has become more widespread. For the majority of patients who lack an human leukocyte antigen-identical sibling, closely matched unrelated donor bone marrow transplant (MUD BMT) at an early age is an excellent option that nevertheless is not uniformly chosen. We retrospectively analyzed our experience with transplantation in 23 patients with WAS from 1990 to 2005 at the University of Brescia, Italy, of whom 16 received MUD BMT. Myeloablative chemotherapy was well tolerated with median neutrophil engraftment at day 18, and no cases of grade III or IV graft-vs-host disease. Overall survival was very good with 78.2% (18/23) of the whole cohort and 81.2% (13/16) of MUD BMT recipients surviving. Among 18 survivors, full donor engraftment was detected in 12 patients, and stable mixed chimerism in all blood lineages in four patients. Deaths were limited to patients who had received mismatched related BMT or who had severe clinical symptomatology at the time of transplantation, further emphasizing the safety and efficacy of MUD BMT when performed early in the clinical course of WAS.

X-chromosome inactivation analysis in a female carrier of FOXP3 mutation

Immune dysregulation, polyendocrinopathy and enteropathy with X‐linked inheritance (IPEX) is a serious disease arising from mutations in FOXP3. This gene codifies for a transcription factor whose dysfunction results in hyperactivation of T cells. It is not clear, however, why an intermediate phenotype is not seen in heterozygous females, who are completely healthy. In order to address this question, we investigated X‐chromosome inactivation in peripheral blood lymphocytes from a heterozygous female with a child affected by IPEX. No preferential inactivation was shown in freshly sorted CD4+, CD8+, CD19+ cells or in IL‐2 cultured CD4 and CD8 T cells, indicating that peripheral blood lymphocytes in these women are randomly selected. Moreover, only one single FOXP3 transcript was expressed by CD4 T cell clones analysed by RT‐PCR, confirming that this gene is subject to X‐ inactivation. We hypothesize that hyper‐activation of T cell in carriers of FOXP3 mutations is regulated by the presence of normal regulatory T cells.

Whole-exome sequencing identifies tetratricopeptide repeat domain 7A (TTC7A) mutations for combined immunodeficiency with intestinal atresias

BACKGROUND Combined immunodeficiency with multiple intestinal atresias (CID-MIA) is a rare hereditary disease characterized by intestinal obstructions and profound immune defects. OBJECTIVE We sought to determine the underlying genetic causes of CID-MIA by analyzing the exomic sequences of 5 patients and their healthy direct relatives from 5 unrelated families. METHODS We performed whole-exome sequencing on 5 patients with CID-MIA and 10 healthy direct family members belonging to 5 unrelated families with CID-MIA. We also performed targeted Sanger sequencing for the candidate gene tetratricopeptide repeat domain 7A (TTC7A) on 3 additional patients with CID-MIA. RESULTS Through analysis and comparison of the exomic sequence of the subjects from these 5 families, we identified biallelic damaging mutations in the TTC7A gene, for a total of 7 distinct mutations. Targeted TTC7A gene sequencing in 3 additional unrelated patients with CID-MIA revealed biallelic deleterious mutations in 2 of them, as well as an aberrant splice product in the third patient. Staining of normal thymus showed that the TTC7A protein is expressed in thymic epithelial cells, as well as in thymocytes. Moreover, severe lymphoid depletion was observed in the thymus and peripheral lymphoid tissues from 2 patients with CID-MIA. CONCLUSIONS We identified deleterious mutations of the TTC7A gene in 8 unrelated patients with CID-MIA and demonstrated that the TTC7A protein is expressed in the thymus. Our results strongly suggest that TTC7A gene defects cause CID-MIA.

Defect of regulatory T cells in patients with Omenn syndrome

BACKGROUND Omenn syndrome (OS) is an autosomal-recessive disorder characterized by severe immunodeficiency and T-cell-mediated autoimmunity. The disease is caused by hypomorphic mutations in recombination-activating genes that hamper the process of Variable (V) Diversity (D) Joining (J) recombination, leading to the generation of autoreactive T cells. We have previously shown that in OS the expression of autoimmune regulator, a key factor governing central tolerance, is markedly reduced. OBJECTIVE Here, we have addressed the role of peripheral tolerance in the disease pathogenesis. METHODS We have analyzed forkhead box protein P3 (FOXP3) expression in peripheral blood T cells of 4 patients with OS and in lymphoid organs of 8 patients with OS and have tested the suppressive activity of sorted CD4(+) CD25(high) peripheral blood T cells in 2 of these patients. RESULTS We have observed that CD4(+)CD25(high)T cells isolated ex vivo from patients with OS failed to suppress proliferation of autologous or allogenic CD4(+) responder T cells. Moreover, despite individual variability in the fraction of circulating FOXP3(+) CD4 cells in patients with OS, the immunohistochemical analysis of FOXP3 expression in lymph nodes and thymus of patients with OS demonstrated a severe reduction of this cell subset compared with control tissues. CONCLUSION Overall, these results suggest a defect of regulatory T cells in OS leading to a breakdown of peripheral tolerance, which may actively concur to the development of autoimmune manifestations in the disease.

Defect of plasmacytoid dendritic cells in warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome patients.

Warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome is a genetic disease that is caused by heterozygous mutations of the CXCR4 gene. These mutations confer an increased leukocyte response to the CXCR4-ligand CXCL12, resulting in abnormal homeostasis of many leukocyte types, including neutrophils and lymphocytes. Analysis of the myeloid and plasmacytoid dendritic cell blood counts in WHIM patients revealed a striking defect in the number of plasmacytoid dendritic cells as well as a partial reduction of the number of myeloid dendritic cells, compared with healthy subjects. Moreover, the production of interferon-α by mononuclear cells in response to herpes simplex infection, or after stimulation with the Toll-like receptor 9 ligand CpG, was undetectable in WHIM patients. Because plasmacytoid dendritic cells play a key role in the defense against viruses and their generation and motility are in part dependent on CXCR4, we hypothesized that the susceptibility of WHIM patients to warts is related to the abnormal homeostasis of plasmacytoid dendritic cells.

Defective migration of monocyte-derived dendritic cells in LAD-1 immunodeficiency

β2 Integrins (CD18) are required for leukocyte migration. In fact, the absence of CD18 results in type‐1 leukocyte adhesion deficiency (LAD‐1). We analyzed the distribution phenotype and function of dendritic cells (DCs) in three LAD‐1 patients with homozygous mutations of CD18. Two of them did not express CD18 (Patients A and C), and the other subject (Patient B) displayed reduced expression of β2 integrins because of a missense mutation. Analysis of DCs derived from Patients A and B showed an abnormal morphology and a severe impairment in transendothelial migration and chemotactic response to CCL19/macrophage inflammatory protein‐3β, suggesting that CD18 is required for migration of monocyte‐derived DCs. Nevertheless, DCs displayed normal macropinocytosis and underwent normal maturation after addition of tumor necrosis factor α. Finally, immunohistochemical analysis of lymph nodes from subjects B and C revealed a significant reduction in the number of factor‐XIIIa+ interstitial DCs in the interfollicular area in both patients, suggesting that CD18 plays a role in the migration of these cells in vivo.

SH2‐domain mutations in STAT3 in hyper‐IgE syndrome patients result in impairment of IL‐10 function

Autosomal‐dominant hyper‐IgE syndrome (AD‐HIES) is a primary immunodeficiency caused by STAT3 mutations. This inherited condition is characterized by eczema, staphylococcal cold abscesses and recurrent pulmonary infections. Given that STAT3 is involved in IL‐10 signaling, we examined the immunoregulatory role of IL‐10 in inflammation by studying the effects of IL‐10 on monocytes, neutrophils and monocyte‐derived DCs from HIES subjects. Analysis of gene expression in PBMCs and neutrophils isolated from HIES patients and stimulated with LPS in the presence of IL‐10 showed reduced expression of IL1RN, which encodes IL‐1 receptor antagonist (IL‐1ra), and SOCS3 mRNA but increased CXCL8 mRNA expression. Moreover, secretion of the anti‐inflammatory protein IL‐1ra was reduced in AD‐HIES patients. DCs from HIES patients secreted higher levels of TNF‐α, IL‐6 and, to a lesser extent, IL‐12 when these cells were cultured in the presence of IL‐10. These results suggest that IL‐10 activity is affected in myeloid cells (e.g. monocytes, DCs) of HIES patients. Impairment of IL‐10 signaling in patients with AD‐HIES might result in an altered balance between pro‐inflammatory and anti‐inflammatory signals and might lead to persistent inflammation and delayed healing after infections.