X-linked hyper-IgM Research Articles

Analysis of CD40 ligand gene mutations in patients with primary biliary cirrhosis

An elevated immunoglobulin (Ig)M concentration in serum is a common and distinctive feature of primary biliary cirrhosis (PBC). Little is known, however, about the mechanism of hyper-IgM in PBC. CD40 ligand (CD40L) has a crucial role in immunoglobulin class switching in B cells. Mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome. To identify mutations in the gene for CD40L in PBC patients, we analyzed CD40L gene mutations, using reverse transcription (RT)-PCR single-strand conformation polymorphism (SSCP) analysis. No mutations were detected in cDNA from any of 24 PBC patients by the RT-PCR-SSCP technique. These data suggest that other, unidentified mechanisms are involved in hyper-IgM in PBC patients.

Leukocyte transfusion-associated granulocyte responses in a patient with X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) is a severe congenital immunodeficiency caused by mutations in CD154 (CD40 ligand, gp39), the T cell ligand for CD40 on B cells. Chronic or cyclic neutropenia is a frequent complicating feature that heightens susceptibility to severe infections. We describe a patient with a variant of XHIM who produced elevated levels of serum IgA as well as IgM and suffered from chronic severe neutropenia. Eight of ten leukocyte transfusions with cells from a maternal aunt, performed because of mucosal infections, resulted in similar episodes of endogenous granulocyte production. Transfection studies with the mutant CD154 protein indicate that the protein is expressed at the cell surface and forms an aberrant trimer that does not interact with CD40. The data suggest that allogeneic cells from the patient's aunt, probably activated T cells bearing functional CD154, may interact with CD40+ recipient cells to produce maturation of myeloid precursors in the bone marrow.

Development of a Rapid Whole Blood Flow Cytometry Procedure for the Diagnosis of X-Linked Hyper-IgM Syndrome Patients and Carriers

The CD40 ligand expressed on activated T cells plays a pivotal role in B cell proliferation and differentiation. Mutations in the CD40 ligand gene, which alter its expression on the surface of activated T cells, are associated with the X-linked form of Hyper-IgM syndrome (XHIM). A rapid and simple, three-color whole blood flow cytometry procedure was developed for maximal expression and detection of the CD40L on the surface ofin vitroactivated CD4+ T cells. Approximately 90% ofin vitroactivated CD4+ T cells obtained from healthy controls expressed the CD40L compared to only 5% ofin vitroactivated CD4+ T cells obtained from the XHIM patients. The CD40L was expressed on approximately 50% of thein vitroactivated CD4+ T cells obtained from the mothers of XHIM patients, consistent with a diagnosis of their carrier status. This is the first report of a whole blood procedure adapted for routine clinical use which is able to detect abnormal CD40L expression in XHIM patients and carriers.

Immune function.

To summarize, within the adaptive immune network, the CD4 T cell provides regulatory effects that play a central role in controlling the majority of immune functions and memory cells that enhance host protection. Many of the regulatory effects are mediated through the release of cytokines, a group of hormones that also promotes inflammation and replenishes hematopoietic elements. The CD8 T cell functions as a cytotoxic cell that is a crucial part of the host defense against intracellular pathogens such as viruses. The other major arm of the lymphoid network consists of B cells whose major function is the production of immunoglobulins that are crucial in host protection against encapsulated bacteria and other microbes. The role of the NK cell is less clear, but it appears to participate in host defenses against selected viruses and malignant cells. It also may be important in rejection reactions that are activated following tissue transplantation. The various lymphocyte types within the adaptive immune system are interdependent, as evidenced by the X-linked hyper-IgM syndrome in which a T-cell deficiency (CD40 ligand) manifests clinically as an antibody production problem.

Clinical spectrum of X-linked hyper-IgM syndrome

We report the clinical and immunologic features and outcome in 56 patients with X-linked hyper-IgM syndrome, a disorder caused by mutations in the CD40 ligand gene. Upper and lower respiratory tract infections (the latter frequently caused by Pneumocystis carinii ), chronic diarrhea, and liver involvement (both often associated with Cryptosporidium infection) were common. Many patients had chronic neutropenia associated with oral and rectal ulcers. The marked prevalence of infections caused by intracellular pathogens suggests some degree of impairment of cell-mediated immunity. Although lymphocyte counts and in vitro proliferation to mitogens were normal, a defective in vitro proliferative response to antigens was observed in some patients, and additional defects of cell-mediated immunity may be presumed on the basis of current knowledge of CD40-ligand function. All patients received regular infusions of immunoglobulins. Four patients underwent liver transplantation because of sclerosing cholangitis, which relapsed in three. Three patients underwent bone marrow transplantation. Thirteen patients (23%) died of infection and/or liver disease. X-linked hyper-IgM syndrome, once considered a clinical variant of hypogammaglobulinemia, is a severe immunodeficiency with significant cellular involvement and a high mortality rate. (J Pediatr 1997;131:47-54)

Defective antigen-induced lymphocyte proliferation in the X-linked hyper-IgM syndrome

We examined T-cell proliferation in five patients with X-linked hyper-IgM syndrome (XHIM), using a panel of antigens and lectins. All patients had impaired antigen-induced proliferation, whereas their lectin responses were normal. Thus, in addition to severely depressed antibody responses, patients with XHIM have a defect in antigen-specific T-cell proliferation, which may explain their susceptibility to pathogens such as Pneumocystis carinii. (J Pediatr 1997;131:147-50)

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus.

Molecular Analysis of the X-Linked Hyper IgM Syndrome: Heterogeneity of Clinical Expression and Effect of CD40 Ligand Deficiency on Immune Function.♦ 45

X-linked hyper IgM (HIGMX-1) is a rare inherited immune deficiency disease resulting from a defect in the gene for CD40 ligand (CD40L). Patients exhibit an heterogenous phenotype with normal or elevated IgM, recurrent bacterial infections with or without opportunistic infections, autoimmune disease, or lymphoproliferative disease. Data from CD40 or CD40L gene-targetted mice suggests that, in addition to the defect in humoral immunity, CD40L/CD40 interaction is necessary for T cell priming. Unlike gene-targetted mice, HIGMX-1 patients express mutated mRNA, and heterogeneity of the disease may partly result from differences in the expressed protein. We have investigated two patients with HIGMX-1 who have a deletion of the third exon of CD40L. Analysis of the CD40L genomic DNA showed that the deletion in the third exon resulted from different genomic mutations. The first patient, an 8 year old boy of Chinese origin, had a deletion of the first nucleotide in the donor site of exon 3 while the second patient, a 23 year old boy of Italian origin, had a point mutation (a->g) at the -2 position in the acceptor site of exon 3. The patients' clinical disease differs in levels of IgM and IgA and in the development of opportunistic infections. The differences in clinical phenotype may be related to differences in environmental exposure, or may, in part result from differences in the effect of the differing genomic mutations on mRNA splicing and skipping of the third exon. Next, we examined the effect of the CD40L deficiency on T cell priming in the second patient. In contrast to CD40L-deficient mice, this patient demonstrated reactivity against recall antigens by T cell proliferation in-vitro and by positive delayed-type hypersensitivity skin testing in-vivo. Also, the patient's circulating T cells contained a primed population of CD4+ T cells as determined by CD45RO expression; and in his mother, an obligate carrier of HIGMX-1, only 47% of the CD4+/CD45RO+ T cells expressed CD40L upon stimulation compared to 80% of control CD4+/CD45RO+ T cells indicating that normal CD40L did not provide an advantage for T cell priming. These results suggest that at least some patients with HIGMX-1 have no defect in T cell priming.

Registries of immunodeficiency patients and mutations.

Immunodeficiencies form a distinct group of human hereditary diseases with several rare disorders. During recent years, information has been collected concerning immunodeficiency patients and mutations causing disorders. The large European (ESID) registry contains clinical data for some 7,000 patients. At present, international mutation databases have information for > 1,000 immunodeficiency patients, including X-linked chronic granulomatous disease (XCGD), Wiskott-Aldrich syndrome (WAS), and X-linked thrombocytopenia (XLT), X-linked hyper-IgM syndrome (XHIM), X-linked agammaglobulinemia (XLA), and X-linked severe combined immunodeficiency (XSCID). The databases are available on Internet. The mutation spectra of patients in these registries were compared. Mutational hotspots were found in CpG dinucleotides with a preference for selected flanking bases.

CD40/CD40 Ligand Interactions in Normal, Reactive and Malignant Lympho-Hematopoietic Tissues

CD40 is a 48 Kd integral membrane protein expressed by cells of B cells origin, dentritic cells, monocytes, epithelial cells, endothelial cells and tumor cells including carcinomas, B cell lymphomas/leukemias and Hodgkin and Reed-Sternberg (H-RS) cells of Hodgkin's disease (HD). CD40 has been clustered as a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily with the corresponding counterstructure, the CD40 ligand (L) being mainly expressed by activated CD4+ T cells, but also some activated CD8+ T cells, basophils, eosinophils, mast cells and stromal cells. CD40L shares significant amino acid homology with TNF particularly in its extracellular domain (“TNF homology region”) and is therefore viewed as a member of the TNF ligand superfamily. Binding of CD40L+ T cells to CD40+ B cells is thought to play a major role in T cell-dependent B cell activation, B cell proliferation, Ig isotype switching, memory B cell formation and rescue of B cells from apoptotic death in germinal centers. Mutations of the CD40L gene have been associated with the X-linked hyper-IgM immunodeficiency syndrome, pointing to the critical role of the CD40/CD40L interaction in the T cell-B cell interplay. Accordingly, expression of CD40 by human lympho-hematopoietic tumors has been shown in most of the B cell neoplasias, H-RS cells of HD and some carcinomas. In contrast, CD40L+ tumor cells are almost invariably restricted to CD4+/CD8- T cell lymphomas. Overall, functional CD40/CD40L interactions appear to be critical for cellular activation signals during immune responses and neoplastic tumor cell growth. The understanding of the biology of CD40L has improved our diagnostic and therapeutic repertoire in the management of several human diseases, including CD40+ tumors.

CD40lbase: a database of CD40L gene mutations causing X-linked hyper-IgM syndrome.

X-linked hyper-IgM syndrome (X-HIM) is an immunodeficiency caused by mutations in the gene encoding the CD40 ligand (CD40L). A database (CD40Lbase) of CD40L mutations has now been established, and the resultant information, together with other mutations reported elsewhere in the literature, is presented here.

CD40Lbase: a database of CD40L gene mutations causing X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (X-HIM) is an immunodeficiency caused by mutations in the gene encoding the CD40 ligand (CD40L). A database (CD40Lbase) of CD40L mutations has now been established, and the resultant information, together with other mutations reported elsewhere in the literature, is presented here.

Small cell undifferentiated carcinoma of the colon associated with hepatocellular carcinoma in an immunodeficient patient

Small cell undifferentiated carcinoma (oat cell carcinoma) is a malignant epithelial neoplasm with neuroendocrine features. It can appear as a primary tumor in many organs besides the lung, including the colon. We report a case of primary small cell undifferentiated carcinoma of the left colon with omental metastases in a 23-year-old man with a history of X-linked hyper-IgM syndrome. The patient had a simultaneous primary hepatocellular carcinoma. A literature review of this rare colonic malignancy is presented together with a discussion of the possible relationship of this tumor with hepatic malignancy and immunodeficiency.

Somatic mutations in human Ig variable genes correlate with a partially functional CD40-ligand in the X-linked hyper-IgM syndrome.

X-linked hyper-IgM (HIGM-1) syndrome is a rare disorder resulting from mutations in the CD40-ligand (CD40L) gene. This defect is associated with normal or elevated serum levels of IgM, and with low to undetectable levels of serum IgG, IgA, and IgE. We analyzed the somatic mutation status in Ig V genes from three unrelated HIGM-1 patients by reverse-transcription PCR and sequence analysis. Two patients (B.S. and P.S.) expressed unmutated VH6 genes. In contrast, one patient (A.T.) was found to express mutated VH6 genes. Whether the presence of somatic mutations in this patient was related to a functional CD40L was assessed by deriving T cell clones from his peripheral blood cells. Upon activation, these T cell clones expressed weakly and transiently surface CD40L, and were able to induce limited isotype switch of normal native B cells, indicating residual CD40L function. Altogether, our results 1) confirm the central role played by CD40L in the generation of somatic mutation (patients B.S. and P.S.), 2) provide an unusual illustration of the relative dissociation between somatic mutation and isotype switching (patient A.T.), and 3) demonstrate a further complexity of the X-linked HIGM syndrome that may occur despite a partially functional CD40L.

Classification of mutations in the human CD40 ligand, gp39, that are associated with X‐linked hyper IgM syndrome

The interaction between the T cell activation antigen gp39 and CD40, its receptor CD40 on B cells, plays a critical role in the regulation of humoral immune responses. Using a detailed three-dimensional model of the gp39 extracellular region, we have analyzed 20 mutations in gp39 that were, with one exception, isolated from patients with X-linked hyper IgM (XHIM) syndrome. On the basis of this analysis, the mutations were classified according to their predicted locations and effects. Twelve mutations are thought to compromise the gp39 structure by affecting interactions in hydrophobic core regions or at monomer interfaces, whereas seven others map closely to gp39 residues important for interaction with CD40. The latter mutations may thus, directly or indirectly, interfere with CD40 binding. One naturally occurring mutant whose carrier displays normal immune responses maps to a solvent-exposed position in a loop region of the molecule.

Genomic structure and PCR-SSCP analysis of the human CD40 ligand gene: Its application to prenatal screening for X-linked hyper-IgM syndrome

To develop a general method for analysis of the mutation and prenatal diagnosis of X-linked hyper-IgM syndrome (XHM), the human CD40 ligand (hCD40L) gene was cloned and sequenced with special reference to the 5' and 3' flanking regions and exon/intron boundaries. The hCD40L gene consists of five exons and four introns, as already reported by others. Two major transcription initiation sites were identified at 67 bp and 64 bp upstream from the ATG initiation codon. The hCD40L mRNA transcripts terminated at 321 bp, 327 bp and 987 bp downstream from the TGA stop codon. Based on the intronic sequences, oligonucleotide primers were designed for amplifying the coding region of each exon separately. Polymerase chain reaction--single-strand conformational polymorphism (PCR-SSCP) analysis was successfully applied to screening for the defective hCD40L gene in a family with XHM. The nonsense mutation, Trp140 (TGG)-->stop (TAG) in exon 5, was found in the mother and an affected child. We also performed prenatal diagnosis by PCR-SSCP during the first trimester of pregnancy in this family.

A single strand conformation polymorphism study of CD40 ligand. Efficient mutation analysis and carrier detection for X-linked hyper IgM syndrome.

Mutations in the gene for CD40 ligand are responsible for the X-linked form of hyper IgM syndrome. However, no clinical or laboratory findings that reliably distinguish X-linked disease from other forms of hyper IgM syndrome have been reported, nor are there tests available that can be used to confidently provide carrier detection. To identify efficiently mutations in the gene for CD40 ligand, eight pairs of PCR primers that could be used to screen genomic DNA by single strand conformation polymorphism (SSCP) were designed. 11 different mutations were found in DNA from all 13 patients whose activated T cells failed to bind a recombinant CD40 construct. The exact nature of four of these mutations, a deletion and three splice defects, could not be determined by cDNA sequencing. In addition, SSCP analysis permitted rapid carrier detection in two families in whom the source of the mutation was most likely a male with gonadal chimerism who passed the disorder on to some but not all of his daughters. These studies document the utility of SSCP analysis for both mutation detection and carrier detection in X-linked hyper IgM syndrome.

The roles of CD40 and CD23 in IgE regulation.

IgE production is strictly regulated by a number of cytokines and surface molecules. Th2 helper cell-derived IL-4 and IL-13 positively regulate differentiation of B cells to produce IgE while IFN-γ is known to be a negative regulator1. IL-4 can induce IgE production in human B cells in the presence of T cells although IL-4 alone is not sufficient. Recently, crosslinkage of CD40 by anti-CD40 or CD40 ligand (CD40L) has been shown to induce IgE secretion of B cells even in the absence of T cells2,3. These findings suggest that CD40 may transduce an essential signal to B cells in physical T and B cell interaction. Recently, mutations have been demonstrated in the CD40L gene of patients with X-linked hyper IgM syndrome4,5,6, indicating that the CD40-CD40L system play an important role in in vivo humoral immune responses.

A novel splice-site mutation in the CD40L gene in a patient with X-linked hyper-IgM syndrome.

A novel splice-site mutation in the CD40L gene in a patient with X-linked hyper-IgM syndrome.

CD40—CD40 ligand (CD40L) interactions and X-linked hyperIgM syndrome (HIGMX-1)

Interactions between the B cell surface antigen CD40 and its ligand (CD40L) expressed on activated T cells play a critical role in isotype switching. This is illustrated by failure of isotype switching in patients with X-linked hyperIgM syndrome in whom the CD40L gene is mutated and by failure of isotype switching of CD40-deficient mice in response to T-cell-dependent antigens. We review these findings and discuss the signaling mechanisms of CD40 and the developmental control and transcriptional regulation of CD40L expression.