Massimiliano Vitali

Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.

Bruton tyrosine kinase mediates TLR9-dependent human dendritic cell activation

BACKGROUND Bruton tyrosine kinase (BTK) plays an essential role in various biologic functions of different cell types. Mutations in BTK lead to X-linked agammaglobulinemia (XLA) in humans. BTK was recently linked to the innate immune system, in particular, the Toll-like receptor (TLR) pathway; however, the TLR9 pathway has never been studied in dendritic cells (DCs) of patients with XLA. OBJECTIVE The aim of this study was to investigate the role of BTK in human DC activation upon TLR9 stimulation. METHODS DCs of patients with XLA and healthy donors were stimulated via TLR4/9 and evaluated for cell activation and cytokine production. RESULTS We showed that BTK plays an essential role in DC responses to unmethylated CpG oligodeoxynucleotide: although responses to lipopolysaccaride/TLR4 induce normal DC activation in terms of upregulation of specific markers (CD86, CD83, CD80, HLA-DR), the CpG/TLR9 pathway is completely impaired in patients with XLA. Furthermore, cytokine production upon TLR9 activation in patients with XLA is radically impaired in terms of IL-6, IL-12, TNF-α, and IL-10 production. Interestingly, BTK mediated STAT1/3 upregulation in a TLR9-dependent manner. The important role of BTK in human DC activation was confirmed after incubation of healthy DCs with ibrutinib, the specific BTK inhibitor, which resulted in impairment of TLR9 responses as seen in patients with XLA. CONCLUSION Analysis of these data suggests that BTK regulates human DC responses upon TLR9 engagement in terms of activation, cytokine production, and STAT1/3 upregulation. These findings may be of important significance for better understanding and managing different clinical conditions, such as agammaglobulinemia and lymphoid malignancies.

Inhibition of heme synthesis alters Amyloid Precursor Protein processing

Decay of mitochondria, energy failure and increased oxidative stress are features commonly detected in brains from Alzheimer’s disease (AD) patients. Recent findings indicate that neuronal heme deficiency may contribute to the appearance of those cytopathologies and potentially alter the course of AD. We repressed heme synthesis in cells by inhibiting ferrochelatase enzyme with small interfering RNA and N-methylprotoporphyrin IX. The treatments induced a severe perturbation of mitochondria and energy production, with decrease of the subunit II of Cytochrome c Oxidase, alteration of the membrane potential and a 50% reduction of intracellular ATP. The state and processing of the Amyloid Precursor Protein (APP) was also affected, with the appearance of APP aggregates and a significant decrease (30–40%) of sAPPα secretion, associated with perturbation of ADAM10 and TACE, enzymes involved in the α-secretase cleavage. The production of sAPPβ was increased, without augment of Amyloid β generation. Our findings strengthen the hypothesis that a reduced availability of heme may play a role in AD pathogenesis.

A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by low immunoglobulin serum levels and increased susceptibility to infections. Underlying genetic causes are only known in less than 15% of patients and encompass mutations in the genes encoding for ICOS, TACI, BAFF-R, CD19, CD20, CD81 and MSH5. TACI is the most frequently mutated gene among CVID patients. We report on two pediatric Italian male siblings with hypogammaglobulinemia and recurrent respiratory and gastrointestinal infections in association with a novel compound heterozygous TACI mutation. Both patients carry the I87N/C104R mutation that has not been reported yet. This results in aberrant TACI expression and abrogates APRIL binding on EBV B cells. This study identifies a novel combined mutation in TNFRSF13B increasing the spectrum of TACI mutations associated with CVID.

Autosomal Recessive Agammaglobulinemia: A Novel Non-sense Mutation in CD79a

This study describes the fifth case worldwide of autosomal recessive agammaglobulinemia due to a novel non-sense mutation in CD79a gene with a severe unusual onset due to an invasive central nervous system infection.

Cohort of Iranian Patients with Congenital Agammaglobulinemia: Mutation Analysis and Novel Gene Defects

ABSTRACT Objectives: Impairment in early B-cell development can cause a predominantly antibody deficiency with severe depletion of peripheral B-cells. Mutations in the gene encoding for Bruton’s-tyrosine-kinase (BTK) and the components of the pre-B-cell receptor complex or downstream signaling molecules have been related to this defect in patients with agammaglobulinemia. Methods: Iranian patients with congenital agammaglobulinemia were included and the correlation between disease-causing mutations and parameters such as clinical and immunologic phenotypes were evaluated in available patients. Results: Out of 87 patients, a molecular investigation was performed on 51 patients leading to identification of 39 cases with BTK (1 novel mutation), 5 cases of µ-heavy chain (3 novel mutations) and 1 case of Igα-deficiencies. Conclusion: Although there is no comprehensive correlation between type of responsible BTK mutation and severity of clinical phenotype, our data suggest that BTK-deficient and autosomal recessive agammaglobulinemia patients differ significantly regarding clinical/immunologic characteristics.

The Genetic Heterogeneity of Common Variable Immunodeficiency (CVID): An Update

B cells are generated in the bone marrow and then enter the periphery, where the maturation process takes place leading to the formation of an effective humoral immune response. Defects in this highly regulated process in the periphery have been considered to be responsible for the pathogenesis of Common Variable Immunodeficiency (CVID) for more than 6 decades. CVID is traditionally characterized by low immunoglobulin serum levels and defective antibody response in the presence of normal peripheral B cell numbers. The clinical spectrum of CVID is highly variable, including recurrent infections, autoimmune complications and increased susceptibility to cancer and lymphomas. However, only in the last decade, the genetic defects underlying this maturational B cell defect have been partially elucidated in a small percentage of affected patients. This review will focus on the current state of art regarding the known genetic alterations associated with the pathogenesis of CVID.

Polymorphisms in the LOC387715/ARMS2 Putative Gene and the Risk for Alzheimer’s Disease

Background: Age-related macular degeneration (ARMD) and Alzheimer’s disease (AD) are neurodegenerative disorders that share a high prevalence among elderly people, the extracellular deposition of β-amyloid and the involvement of genetic factors in their aetiology. Genetic linkage with the chromosome regions 10q26 and 10q24–25 have been shown for ARMD and AD, respectively. The rs10490924 polymorphism, the major determinant of the 10q26 association with ARMD, determines the A69S substitution in the LOC387715/ARMS2 gene. Little information is available about the expression of the gene in humans. Methods: We analysed the expression of the gene by RT-PCR in the brain and we looked for nucleotide variations in the gene sequence by DHPLC. Results: We found specific gene transcripts in the hippocampus, cortex and cerebellum. The genetic analysis identified two other common variations, which determine the R3H change (rs10490923) and a premature stop codon (rs2736911), respectively. The analysis of their distribution in 213 AD patients and 149 controls revealed a trend for a reduced frequency of the variant allele of rs2736911 in AD patients (p = 0.038), with an odds ratio of 0.631. Conclusion: The LOC387715/ARMS2 gene is expressed in the human brain, and it may concur to the individual risk for AD.

BAFF-R mutations in Good's syndrome.

Goods syndrome (GS) is a rare disorder characterized by thymoma and immunodeficiency. Typically the immunological presentation of affected patients includes low B cells, hypogammaglobulinemia and altered T cell function in the presence of normal or elevated T cell counts [1]. Opportunistic infections may accompany the clinical history. The pathogenesis of this disorder is still unknown and no definite genetic studies have been reported so far. Recently in this journal, mutations in TACI were identified in two affected patients [2,3]. The rationale for analyzing TACI in patients with GS was provided by the fact that TACI is mutated in patients affected with Common Variable Immunodeficiency (CVID) [4] and that the immunological phenotype of GS may resemble Common Variable Immunodeficiency (CVID), although with certain differences: for example, GS, unlike CVID, is characterized by B cell deficiency. TACI, together with BAFF-R and BCMA, are members of the Tumor Necrosis Factor Receptor (TNFR) superfamily, and are expressed on B cells. Their binding to specific ligands (all three bind to BAFF while only TACI and BCMA bind to APRIL) is important for B cell maturation and homeostasis. BAFF-R in particular, is essential for B cell development both in mice and humans [4,5]. The animal knockout model for BAFF-R results in peripheral B cell deficiency [4]. Humans affected with BAFF-R deficiency and hypogammaglobulinemia show low peripheral B cell percentages as well (1.1%–3.2%) [5]. Human TACI mutations on the other hand are not typically associated with reduced peripheral B cells [4].

B Cell Responses to CpG Correlate with CXCL16 Expression Levels in Common Variable Immunodeficiency

Broad Toll-like receptor 9 (TLR9) signalling defects after CpG in vitro stimulation have been described in common variable immunodeficiency (CVID). CXCL16, a surface receptor, was recently shown to influence cell responses to CpG. We evaluated the expression and function of CXCL16 on B cells from healthy controls and CVID patients. We report that CXCL16 is normally expressed on B cells throughout peripheral maturation. Decreased B cell expression of CXCL16 was observed in a subgroup of CVID patients that correlated with defective in vitro responses to CpG (such as upregulation of CD69, CD86, AICDA, IL-6, and TLR9). Our data suggest that expression levels of a surface receptor, namely, CXCL16, correlate with B cell responses mediated by TLR9 in common variable immunodeficiency.