Biomedical subjects

S Giliani

Publications and source records attributed to S Giliani.

At least 19 recordsLinked to original sources

PubMed2001

Mutations of CD40 gene cause an autosomal recessive form of immunodeficiency with hyper IgM.

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4(+) T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for either CD40L or CD40 gene, and in patients with X-linked hyper IgM syndrome, a disease caused by CD40L/TNFSF5 gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis of CD40 genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of the CD40 gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.

CD40 Antigens↗

PubMed2001

Monocytes from Wiskott-Aldrich patients differentiate in functional mature dendritic cells with a defect in CD83 expression.

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by congenital thrombocytopenia and progressive deterioration of the immune function. Dendritic cells (DC) are key effectors in the induction of specific immunity and are highly specialized in antigen uptake and subsequent migration to draining lymph nodes. DC were generated in vitro from circulating monocytes from ten WAS patients characterized by a different disease score. Immature DC showed similar morphology and membrane phenotype, as compared to normal DC. In chemotaxis assay, immature DC had a reduced migration in response to MIP-1alpha/CCL3, but efficiently endocytosed the macromolecules FITC-dextran and FITC-albumin. Upon terminal differentiation with LPS or CD40 ligand, the acquisition of a mature surface phenotype was variably achieved among WAS patients, with increased expression of CD80, CD86 and DC-LAMP. In contrast, the expression of CD83 was usually low. A defective up-regulation of CD83 was also observed in the lymph node from one WAS patient, whose DC stained positively for DC-LAMP. Mature DC from all the patients tested, but one, significantly migrated in vitro in response to MIP-3beta, a finding confirmed in vivo by the detection of HLA-DR/DC LAMP-positive cells in secondary lymphoid organs. When tested in MLR assays, both immature and mature WAS DC induced allogenic T cell proliferation in a manner comparable to control DC. Collectively these results suggest that, although many functional activities of WAS DC are essentially similar to normal DC, subtle and selective alterations of DC differentiation were also observed, with reduced migratory activity of immature DC and defective CD83 expression upon maturation.

Antigen-Presenting Cells↗

PubMed2001

Primary immunodeficiency mutation databases.

Primary immunodeficiencies are intrinsic defects of immune systems. Mutations in a large number of cellular functions can lead to impaired immune responses. More than 80 primary immunodeficiencies are known to date. During the last years genes for several of these disorders have been identified. Here, mutation information for 23 genes affected in 14 immunodefects is presented. The proteins produced are employed in widely diverse functions, such as signal transduction, cell surface receptors, nucleotide metabolism, gene diversification, transcription factors, and phagocytosis. Altogether, the genetic defect of 2,140 families has been determined. Diseases with X-chromosomal origin constitute about 70% of all the cases, presumably due to full penetrance and because the single affected allele causes the phenotype. All types of mutations have been identified; missense mutations are the most common mutation type, and truncation is the most common effect on the protein level. Mutational hotspots in many disorders appear in CPG dinucleotides. The mutation data for the majority of diseases are distributed on the Internet with a special database management system, MUTbase. Despite large numbers of mutations, it has not been possible to make genotype-phenotype correlations for many of the diseases.

Alleles↗

PubMed2000

X-linked lymphoproliferative disease. 2B4 molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill Epstein-Barr virus-infected cells.

2B4 is a surface molecule involved in activation of the natural killer (NK) cell-mediated cytotoxicity. It binds a protein termed Src homology 2 domain-containing protein (SH2D1A) or signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), which in turn has been proposed to function as a regulator of the 2B4-associated signal transduction pathway. In this study, we analyzed patients with X-linked lymphoproliferative disease (XLP), a severe inherited immunodeficiency characterized by critical mutations in the SH2D1A gene and by the inability to control Epstein-Barr virus (EBV) infections. We show that, in these patients, 2B4 not only fails to transduce triggering signals, but also mediates a sharp inhibition of the NK-mediated cytolysis. Other receptors involved in NK cell triggering, including CD16, NKp46, NKp44, and NKp30, displayed a normal functional capability. However, their activating function was inhibited upon engagement of 2B4 molecules. CD48, the natural ligand of 2B4, is highly expressed on the surface of EBV(+) B cell lines. Remarkably, NK cells from XLP patients could not kill EBV(+) B cell lines. This failure was found to be the consequence of inhibitory signals generated by the interaction between 2B4 and CD48, as the antibody-mediated disruption of the 2B4-CD48 interaction restored lysis of EBV(+) target cells lacking human histocompatibility leukocyte antigen (HLA) class I molecules. In the case of autologous or allogeneic (HLA class I(+)) EBV(+) lymphoblastoid cell lines, restoration of lysis was achieved only by the simultaneous disruption of 2B4-CD48 and NK receptor-HLA class I interactions. Molecular analysis revealed that 2B4 molecules isolated from either XLP or normal NK cells were identical. As expected, in XLP-NK cells, 2B4 did not associate with SH2D1A, whereas similar to 2B4 molecules isolated from normal NK cells, it did associate with Src homology 2 domain-containing phosphatase 1.

Antigens, CD↗

PubMed2000

Structural basis for SH2D1A mutations in X-linked lymphoproliferative disease.

X-linked lymphoproliferative disease (XLP) is a rare and severe immune deficiency, characterized by abnormal immune responses to the Epstein-Barr virus. Recently, the gene responsible for XLP, SH2D1A, has been identified and shown to code for a small cytoplasmic protein with an SH2 domain that interacts with SLAM and 2B4, two receptorial molecules involved in signal transduction in T and NK cells, respectively. A variety of SH2D1A gene mutations have been reported thus far in XLP males. Here we describe a single-strand conformation polymorphism assay for mutation analysis in XLP. Four novel patients with SH2D1A mutations are described. These mutants, and the others previously reported in the literature, have been included in a Registry (SH2D1Abase) that is fully accessible on the World Wide Web. A three-dimensional model of the SH2 domain of the SH2D1A protein has been developed, based on homology with other SH2 domains. The structural consequences of disease-causing SH2D1A mutations are discussed.

Amino Acid Sequence↗

PubMed2000

Development of autologous T lymphocytes in two males with X-linked severe combined immune deficiency: molecular and cellular characterization.

We report on two patients affected with severe combined immune deficiency (SCID) with an unusual immunological phenotype and a substantial number of autologous, poorly functioning T cells. Distinct mutations identified at the IL2RG locus in the two patients impaired IL-2-mediated signaling but affected T-cell lymphopoiesis differently, resulting in generation of a polyclonal or oligoclonal T-cell repertoire. These observations add to the growing complexity of the immunological spectrum of SCID in humans and indicate the need for detailed immunological and molecular investigations in atypical cases.

Adolescent↗

PubMed2000

Complete genomic organization of the human JAK3 gene and mutation analysis in severe combined immunodeficiency by single-strand conformation polymorphism.

JAK3 deficiency in humans results in autosomal recessive T-B+ severe combined immunodeficiency disease (SCID), a severe immunodeficiency that can only be cured by bone marrow transplantation. We unraveled the complete organization of the human JAK3 gene, which includes 23 exons. This information allowed us to set up a molecular screening test that enabled us to diagnose JAK3 deficiency in 14 patients from 12 unrelated families with T-B+ SCID. In order to define the mutations, we used a nonradioactive single-strand conformation polymorphism (SSCP)/heteroduplex (HD) assay based on exon-specific polymerase chain reaction (PCR). In this cohort of patients, 15 independent JAK3 gene mutations have been identified, including 7 that have not been described previously. Mutation analysis information was used for genetic counseling and prenatal diagnosis.

Consanguinity↗

PubMed2000

Combined immunodeficiencies due to defects in signal transduction: defects of the gammac-JAK3 signaling pathway as a model.

Combined immune deficiencies comprise a spectrum of genetic disorders characterized by developmental or functional defects of both T and B lymphocytes. Recent progress in cell biology and molecular genetics has unraveled the pathophysiology of most of these defects. In particular, the most common form of severe combined immune deficiency in humans, with lack of circulating T cells, a normal or increased number of B lymphocytes, and an X-linked pattern of inheritance (SCIDXI) has been shown to be due to defects of the IL2RG gene, encoding for the common gamma chain (gammac), shared by several cytokine receptors. Furthermore, defects of the JAK3 gene, encoding for an intracellular tyrosine kinase required for signal transduction through gammac-containing cytokine receptors, have been identified in patients with autosomal recessive T-B+ SCID. Characterization of the functional properties of cytokines that signal through the gammac-JAK3 signaling pathway has been favored by the detailed analysis of SCID patients. Specifically, the key role of IL-7 in promoting T cell development has been substantiated by the identification of rare patients with T-B+ SCID who have a defect in the alpha subunit of the IL-7 receptor (IL7Ralpha). The heterogeneity of genetic defects along the same signaling pathway that may lead to combined immune deficiency is paralleled by the heterogeneity of immunological phenotypes that may associate with defects in the same gene, thus creating a need for detailed immunological and molecular investigations in order to dissect the spectrum of combined immune deficiencies in humans.

Animals↗

PubMed2000

Of genes and phenotypes: the immunological and molecular spectrum of combined immune deficiency. Defects of the gamma(c)-JAK3 signaling pathway as a model.

Cytokines play a major role in lymphoid development. Defects of the common gamma chain (gamma(c)) or of the JAK3 protein in humans have been shown to result in a severe combined immune deficiency (SCID), with a profound defect in T and natural killer (NK)-cell development, whereas B-cell generation is apparently unaffected (T-B+NK-SCID). While extensive molecular and biochemical analysis of these patients has been instrumental in understanding better the biological properties of the gamma(c) and JAK3 protein, an unexpected phenotypic heterogeneity of gamma(c) and JAK3 deficiency has emerged, indicating the need for appropriate and extensive investigations even in patients with atypical presentations. At the same time, characterization of the defects has been instrumental in the development of novel therapeutic approaches, from in utero hematopoietic stem cell transplantation to gene therapy.

Cytokines↗

PubMed2000

Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.

Osteopetrosis includes a group of inherited diseases in which inadequate bone resorption is caused by osteoclast dysfunction. Although molecular defects have been described for many animal models of osteopetrosis, the gene responsible for most cases of the severe human form of the disease (infantile malignant osteopetrosis) is unknown. Infantile malignant autosomal recessive osteopetrosis (MIM 259700) is a severe bone disease with a fatal outcome, generally within the first decade of life. Osteoclasts are present in normal or elevated numbers in individuals affected by autosomal recessive osteopetrosis, suggesting that the defect is not in osteoclast differentiation, but in a gene involved in the functional capacity of mature osteoclasts. Some of the mouse mutants have a decreased number of osteoclasts, which suggests that the defect directly interferes with osteoclast differentiation. In other mutants, it is the function of the osteoclast that seems to be affected, as they show normal or elevated numbers of non-functioning osteoclasts. Here we show that TCIRG1, encoding the osteoclast-specific 116-kD subunit of the vacuolar proton pump, is mutated in five of nine patients with a diagnosis of infantile malignant osteopetrosis. Our data indicate that mutations in TCIRG1 are a frequent cause of autosomal recessive osteopetrosis in humans.

Alternative Splicing↗

PubMed1999

Prenatal molecular diagnosis of Wiskott-Aldrich syndrome by direct mutation analysis.

We have performed prenatal diagnosis for Wiskott Aldrich syndrome (WAS) in two unrelated families by direct gene analysis. Using a combined non-radioactive analysis of single-strand conformational polymorphism (SSCP) and heteroduplex formation (HD), followed by automated sequencing, we studied DNA from chorionic villus sampling (CVS), allowing the diagnosis of one affected and one healthy male at the 12th week of gestation.

Base Sequence↗

PubMed1999

Prenatal diagnosis of JAK3 deficient SCID.

The JAK3 gene, encoding a tyrosine kinase functionally coupled to cytokine receptors which share the common gamma chain, has been identified as the defective gene for autosomal recessive severe combined immunodeficiency (SCID). Thus, specific mutational diagnosis has become possible. We screened all exons with a combined single strand conformational polymorphism and hetero-duplex formation assay followed by sequence analysis to identify specific mutations in two families. This assay was used on chorionic villus sampling derived DNA in two fetuses from two unrelated families, where we found mutations in both parents. We were able to exclude the mutations in both fetuses by the 12th week of gestation. The described method for first-trimester prenatal diagnosis of autosomal recessive T-B+SCID provides a valid tool to aid in genetic counselling and possibly prenatal therapy in this disease.

Alleles↗

PubMed1998

Partial V(D)J recombination activity leads to Omenn syndrome.

Genomic rearrangement of the antigen receptor loci is initiated by the two lymphoid-specific proteins Rag-1 and Rag-2. Null mutations in either of the two proteins abrogate initiation of V(D)J recombination and cause severe combined immunodeficiency with complete absence of mature B and T lymphocytes. We report here that patients with Omenn syndrome, a severe immunodeficiency characterized by the presence of activated, anergic, oligoclonal T cells, hypereosinophilia, and high IgE levels, bear missense mutations in either the Rag-1 or Rag-2 genes that result in partial activity of the two proteins. Two of the amino acid substitutions map within the Rag-1 homeodomain and decrease DNA binding activity, while three others lower the efficiency of Rag-1/Rag-2 interaction. These findings provide evidence to indicate that the immunodeficiency manifested in patients with Omenn syndrome arises from mutations that decrease the efficiency of V(D)J recombination.

Amino Acid Sequence↗

PubMed1998

Defective actin polymerization in EBV-transformed B-cell lines from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by eczema, thrombocytopenia, and immunodeficiency. An allelic variant of the disease is characterized by isolated thrombocytopenia (XLT). The gene responsible for WAS/XLT (WASP) encodes for a 502 amino acid protein (WASP) that is possibly involved in actin binding and cytoskeleton organization. The expression of WASP and the distribution of F-actin and alpha-actinin (which binds to and stabilizes actin filaments) have been analysed in lymphoblastoid cell lines from six patients with WAS and one with XLT. Western blot and immunocytochemistry did not reveal WASP expression in four WAS patients, whereas two WAS patients (with a moderate clinical course) expressed trace amounts of mutant WASP. In contrast, the XLT patient expressed normal amounts of WASP. Furthermore, cell lines from WAS and XLT patients also markedly differed in F-actin polymerization and alpha-actinin distribution. In particular, severe defects of cytoplasmic F-actin expression and of F-actin-positive microvillus formation, and impaired capping of alpha-actinin, were observed in all patients who lacked WASP. As a whole, the degree of impairment of WASP protein expression in WAS/XLT seems to correlate with anomalies of cytoskeletal organization, strongly supporting a role for WASP in the regulation of F-actin polymerization.

Actinin↗

PubMed1998

Mutation analysis by a non-radioactive single-strand conformation polymorphism assay in nine families with X-linked severe combined immunodeficiency (SCIDX1).

X-linked severe combined immunodeficiency (SCIDX1) is an inherited disease characterized by profound abnormalities of cell-mediated and humoral immunity. Patients with SCIDX1 have defects in the common cytokine receptor gamma chain gene (IL2RG) that encodes a shared, essential component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9 and IL-15. We have characterized nine SCIDX1 families by using a DNA-based, non-radioactive screening method and DNA sequencing. Nine different mutations were found, scattered from exon 1 to exon 5 of the IL2RG gene. Two of these mutations have been previously identified in other unrelated patients; the other seven are novel mutations that differ from all of the 95 already reported in the IL2RG mutation data base. In addition to describing novel mutations in the IL2RG gene, this study shows that the knowledge of the genetic defect and the use of an efficient, non-radioactive, and rapid screening approach have important implications for prenatal and postnatal diagnosis, carrier female identification, and possibly prenatal therapy.

DNA Mutational Analysis↗

PubMed1997

Structural and functional basis for JAK3-deficient severe combined immunodeficiency.

Mutations of the Janus family kinase JAK3 have been found to be responsible for autosomal recessive severe combined immunodeficiency (SCID) in humans. We report here the analysis of four new unrelated patients affected by JAK3-deficient SCID. The genetic defects were heterogeneous and included a large intragenic deletion as well as different point mutations, leading to missense substitutions, early stop codons, or splicing defects. We performed a series of studies of the biochemical events induced by cytokines on lymphoblastoid B-cell lines obtained from these patients. Abnormalities in tyrosine phosphorylation of JAK3 in response to interleukin-2 (IL-2) and IL-4 were present in all patients. Accordingly, IL-2-mediated phosphorylation of STAT5 was also absent or barely detectable. On the contrary, in all cases, we could show reduced but clear phosphorylation of STAT6 upon IL-4 stimulation. In one patient carrying a single amino acid change (Glu481Gly) in the JH3 domain of JAK3, we observed partially conserved IL-2 responses resulting in reduced but detectable levels of JAK3 and STAT5 phosphorylation. Interestingly, the patient bearing this mutation developed a substantial number of circulating CD4(+)/CD45RO+ activated T lymphocytes that were functionally impaired. In two cases, patients' cells expressed JAK3 proteins with mutations in the JH2 pseudo-kinase domain. A single cysteine to arginine substitution (Cys759Arg) in this region resulted in high basal levels of constitutive JAK3 tyrosine phosphorylation unresponsive to either downregulation by serum starvation or cytokine-mediated upregulation. The characterization of the genetic defects and biochemical abnormalities in these JAK3-deficient patients will help define the role of JAK3 in the ontogeny of a competent immune system and may lead to a better understanding of the JAK3 functional domains.

B-Lymphocytes↗

PubMed1997

A boy with X-linked hyper-IgM syndrome and natural killer cell deficiency.

We present a boy with hyper-IgM syndrome with a previously not reported mutation in the CD40 ligand gene. He also had a concomitant natural killer (NK) cell deficiency. He had no CD56+ or CD16+ cells and no NK activity as determined in 4 h chromium release cytotoxicity assay. After 5 days in culture with IL-2-containing medium, however, his peripheral blood mononuclear cells lysed both NK-sensitive and NK-resistant targets, showing that he had lymphokine-activated killer cell precursors in the circulation. Due to the associated neutropenia, he was treated with granulocyte colony-stimulating factor (G-CSF) and responded well. In the same period we observed a transient increase in the number of NK cells. Isolated NK cell deficiencies are extremely rare. We suggest that the defect in our patient is part of the hyper-IgM syndrome, probably representing the phenotype of the new mutation described. Thus, it is possible that both the neutropenia and the NK cell deficiency are due to lack of growth-promoting signals normally delivered by the CD40 ligand.

CD40 Antigens↗

PubMed1996

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Antigens, CD34↗