Novel mutations in hyper-IgM syndrome type 2 and X-linked agammaglobulinemia detected in three patients with primary immunodeficiency disease.

BACKGROUND: Ambiguous or atypical phenotypes can make a definite diagnosis of primary immunodeficiency diseases based on biochemical indices alone challenging. Further, mortality in early life because of infections in patients with these conditions supports the use of genetic tests to facilitate rapid and accurate diagnoses. METHODS: Genetic and clinical analyses of three unrelated Chinese children with clinical manifestations of recurrent infections, who were considered to have primary immunodeficiency diseases, were conducted. Patient clinical features and serum immunological indices were recorded. Next-generation sequencing was used to screen for suspected pathogenic variants. Family co-segregation and in silico analysis were conducted to evaluate the pathogenicity of identified variants, following the American College of Medical Genetics and Genomics guidance. RESULTS: All three patients were found to have predominant antibody defects. Sequencing analysis revealed that one had two compound heterozygous variants, c.255C>A and c.295C>T, in the autosomal gene, activation-induced cytidine deaminase (AICDA). The other two patients were each hemizygous for the variants c.1185G>A and c.82C>T in the Bruton's tyrosine kinase (BTK) gene on the X chromosome. In silico analysis revealed that identified substituted amino acids were highly conserved and predicted to cause structural and functional damage to the proteins. CONCLUSION: Four pathogenic variants in AICDA and BTK were confirmed to cause different forms of hyper-IgM syndrome type 2 (HIGM2) and X-linked agammaglobulinemia (XLA); two were novel mutations that have never been reported previously. This is the first report of HIGM2 caused by AICDA deficiency in a patient from the Chinese mainland.

Haematopoietic stem cell transplant for hyper-IgM syndrome due to CD40 defects: a single-centre experience.

Hyper-IgM syndrome due to CD40 deficiency (HIGM3) is a rare disease with only a few reported cases of haematopoietic stem cell transplantation (HSCT). In retrospective study, we reviewed all patients with HIGM3 who underwent HSCT at King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia, between 2008 and 2013. Six patients were identified. Three male and three female patients from three families. The median age of diagnosis was 13 months (range, 1-28 months). All lacked CD40 expression on B cells by flow cytometry. The median time from diagnosis to transplantation was 8.5 months (range, 1-17 months). For all patients, the donors were HLA-identical siblings, with the exception of one patient for whom the donor was a sibling with one antigen mismatch. The conditioning regimen was busulfan and cyclophosphamide in five patients and busulfan, cyclophosphamide and antithymocyte globulin in one patient. For GVHD prophylaxis, cyclosporine and methotrexate was used. All patients engrafted. The survival rate was 100%, with a median follow-up of 54 months (range, 30-116 months). One patient developed acute GVHD. All patients showed complete immune recovery with positive CD40 expression on B cells and discontinued IVIG replacement. Our study shows that HSCT is potentially effective at curing the disease.

A tetrameric form of CD40 ligand with potent biological activities in both mouse and human primary B cells.

CD40 ligand (CD40 L) expressed by activated T cells interacts with CD40 on B cells and triggers B cell survival, proliferation and differentiation. Deficiency in CD40 L or CD40 in humans causes hyper IgM syndrome due to a defect in T-B interaction that is essential for Ig gene class switch recombination (CSR). CD40 L belongs to the tumor necrosis factor family and normally forms a homotrimer on the cell surface, which is important for its biological activity. To generate a multimeric CD40 L that can be used to stimulate both mouse and human B cells, we fused the extracellular domain of mouse CD40 L, which is known to also bind human CD40, with streptavidin (SA) that forms a stable tetramer under physiological conditions. As expected, 293 T cells transiently transfected with an SA-CD40 L expression vector secreted tetrameric SA-CD40 L in the culture supernatant. The secreted SA-CD40 L exhibited > 25-fold stronger activities in inducing the survival, activation and proliferation of both mouse and human primary B cells than did an agonistic anti-mouse or anti-human CD40 antibody. In the presence of IL-4, SA-CD40 L also induced efficient CSR and plasma cell differentiation in both mouse and human B cells. Moreover, administration of SA-CD40 L in mice induced activation and proliferation of spleen B cells in vivo. These results demonstrate that the SA-CD40 L fusion protein generated in the present study recapitulates the function of membrane-bound trimeric CD40 L and has potent biological activities in both mouse and human primary B cells.

The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes.

Activation-induced cytidine deaminase (AID) mediates class switching by binding to a small fraction of single-stranded DNA (ssDNA) to diversify the antibody repertoire. The precise mechanism for highly selective AID targeting in the genome has remained elusive. Here, we report an RNA-binding protein, ROD1 (also known as PTBP3), that is both required and sufficient to define AID-binding sites genome-wide in activated B cells. ROD1 interacts with AID via an ultraconserved loop, which proves to be critical for the recruitment of AID to ssDNA using bi-directionally transcribed nascent RNAs as stepping stones. Strikingly, AID-specific mutations identified in human patients with hyper-IgM syndrome type 2 (HIGM2) completely disrupt the AID interacting surface with ROD1, thereby abolishing the recruitment of AID to immunoglobulin (Ig) loci. Together, our results suggest that bi-directionally transcribed RNA traps the RNA-binding protein ROD1, which serves as a guiding system for AID to load onto specific genomic loci to induce DNA rearrangement during immune responses.

Activation-induced cytidine deaminase targets SUV4-20-mediated histone H4K20 trimethylation to class-switch recombination sites.

Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin (Ig) genes. Association of AID with RNA Pol II and occurrence of epigenetic changes during Ig gene diversification suggest participation of AID in epigenetic regulation. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. Here, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that AID binding to the IgH locus promotes an increase in H4K20me3. In 293F cells, we demonstrate interaction between co-transfected AID and the three SUV4-20 histone H4K20 methyltransferases, and that SUV4-20H1.2, bound to the IgH switch (S) mu site, is replaced by SUV4-20H2 upon AID binding. Analysis of HIGM2 mutants shows that the AID truncated form W68X is impaired to interact with SUV4-20H1.2 and SUV4-20H2 and is unable to bind and target H4K20me3 to the Smu site. We finally show in mouse primary B cells undergoing class-switch recombination (CSR) that AID deficiency associates with decreased H4K20me3 levels at the Smu site. Our results provide a novel link between SUV4-20 enzymes and CSR and offer a new aspect of the interplay between AID and histone modifications in setting the epigenetic status of CSR sites.

Clinical, molecular, and T cell subset analyses in a small cohort of Chinese patients with hyper-IgM syndrome type 1.

Type 1 hyper-IgM syndrome (HIGM1) is a rare primary immunodeficiency disease caused by mutations in the CD40L gene. Patients often present with recurrent infections and autoimmune manifestations. We investigated the clinical and molecular characteristics of HIGM1 in thirteen patients from the Chinese mainland and examined the proportion of CD4(+)CD25(+)FoxP3(+)Treg, Th17, and Th1 cells in the peripheral blood. We identified ten distinct CD40L mutations in eleven patients: one missense mutation, one nonsense mutation, one insertion mutation (in frame), and seven deletions. Six of these mutations were novel. We observed the percentage of Tregs in the peripheral blood of HIGM1 patients decreased markedly compared with that in healthy controls, but no statistically significant differences was found in the percentages of Th17 and Th1. The identified mutations reflect the heterogeneity of the CD40L gene in HIGM1. Precise genetic diagnosis of HIGM1 will enable appropriate therapeutic interventions, reliable detection of carriers, and genetic counseling. Skewed Treg, Th17/Treg, and Th1/Treg profiles may be associated with immune responses to autoimmunity or infection, which requires replication in larger studies.

The loss of Gnai2 and Gnai3 in B cells eliminates B lymphocyte compartments and leads to a hyper-IgM like syndrome.

B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gαi2 and Gαi3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gαi2 and Gαi3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gαi2 and Gαi3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.

Clinical features and genetic analysis of Taiwanese patients with the hyper IgM syndrome phenotype.

OBJECTIVES: Hyper IgM syndrome (HIGM), characterized by recurrent infections, low serum IgG and IgA, normal or elevated IgM, defective class switch recombination and somatic hypermutation, are heterogeneous disorders with at least 6 distinct molecular defects, including the CD40 ligand (CD40L) and the nuclear factor κB essential modulator (NEMO, also known as IKKγ) genes (both X-linked), the CD40, activation-induced cytidine deaminase (AICDA or AID), uracil-DNA glycosylase genes (autosomal recessive) and IκBα (IKBA) (autosomal dominant). Our objective was to determine the molecular basis and clinical features of Taiwanese patients with the HIGM phenotype. METHODS: Clinical manifestations and candidate genes were analyzed in a nationwide population-based study. RESULTS: Among 14 patients (12 unrelated families) since 2003, 10 patents were identified (8 families) with CD40L mutations, including 2 novel deletions of "A" nucleotide (Del 347A and Del 366A), both frameshift and stop at the 127th location; 1 novel AID deletion mutation lack of the 37thAsp and 38th Ser; 1 ataxia-telangiectasia mutation; and 1 deletion of chromosome 1q42. An adult-onset patient with mutant (Thr254Met)CD40L had approximately 30% detectable affinity and therefore less severity. Memory B cells decreased in patients with CD40L and activation-induced cytidine deaminase mutations. Three mortalities encompassed renal cell carcinoma in 1 patient with (Tyr169Asn)CD40L, pneumothorax in 1 with (Tyr140Stop)CD40L and pneumonia after chemotherapy in an ataxia-telangiectasia patient. One patient without detectable genetic defects but normal lymphocyte proliferation resembled the mild form of common variable immune deficiency phenotype. CONCLUSIONS: In contrast to those with AICDA mutation, small chromosome 1 q42 deletion and unknown genetic defect, the majority (10/14; 71.4%) with CD40L mutations except (Thr254Met) and an ataxia-telangiectasia patient had the severe form of HIGM phenotype.