In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA ...from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.
Interactions between the tumor necrosis factor (TNF) ligand superfamily and TNF receptor superfamily play critical roles in B-cell development and maturation. A proliferation-inducing ligand (APRIL), ...a member of the TNF ligand superfamily, is secreted from myeloid cells and known to induce the differentiation of memory B cells to plasmacytes.
We sought to elucidate the role of APRIL in B-cell differentiation and immunoglobulin production through the analysis of complete APRIL deficiency in human.
We performed whole exome sequencing in a patient with adult common variable immunodeficiency. His parents were in a consanguineous marriage. TNFSF13 mRNA and protein expression were analyzed in the primary cells and plasma from the patient and in cDNA-transfected cells and supernatants of the cultures in vitro. Immunologic analysis was performed by using flow cytometry and next-generation sequencing. Monocyte-derived dendritic cells differentiated from induced pluripotent stem cells (iPSC-moDCs) were cocultured with memory B cells from healthy controls to examine in vitro plasmacyte differentiation.
We identified a homozygous frameshift mutation in TNFSF13, the gene encoding APRIL, in the patient. APRIL mRNA and protein were completely absent in the monocytes and iPSC-moDCs of the patient. In contrast to the results of previous animal model studies, the patient showed hypogammaglobulinemia with a markedly reduced level of plasmacytes in peripheral blood and a clearly increased level of circulating marginal zone B cells. Although iPSC-moDC–induced in vitro plasmacyte differentiation was reduced in the patient, recombinant APRIL supplementation corrected this abnormality.
The first APRIL deficiency in an adult patient with common variable immunodeficiency revealed the role of APRIL in lifelong maintenance of plasmacytes and immunoglobulin production in humans.
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CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in ...increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti-CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3-kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.
Certain autoimmune diseases result in abnormal bone homeostasis, but association of immunodeficiency with bone is poorly understood. Osteoclasts, which derive from bone marrow cells, are under the ...control of the immune system. Differentiation of osteoclasts is mainly regulated by signaling pathways activated by RANK and immune receptors linked to ITAM-harboring adaptors. However, it is unclear how the two signals merge to cooperate in osteoclast differentiation. Here we report that mice lacking the tyrosine kinases Btk and Tec show severe osteopetrosis caused by a defect in bone resorption. RANK and ITAM signaling results in formation of a Btk(Tec)/BLNK(SLP-76)-containing complex and PLCγ-mediated activation of an essential calcium signal. Furthermore, Tec kinase inhibition reduces osteoclastic bone resorption in models of osteoporosis and inflammation-induced bone destruction. Thus, this study reveals the importance of the osteoclastogenic signaling complex composed of tyrosine kinases, which may provide the molecular basis for a new therapeutic strategy.
Haematopoietic stem cell transplantation (HSCT) is performed for treatment of a broad spectrum of illnesses. Reconstitution of an intact immune system is crucial after transplantation to avoid ...infectious complications, and above all, the establishment of T cell receptor (TCR) diversity is the most important goal in the procedure. Until recently, little has been known of the mechanism of T cell reconstitution in the very early period after HSCT. In this study, we analysed TCR repertoires sequentially in four patients with severe combined immunodeficiency (SCID) before and after HSCT. In all patients, the TCR repertoires were extremely abnormal before HSCT, whereas after transplantation there was progressive improvement in TCR diversity, based on analysis of the TCR Vβ repertoire and CDR3 size distributions. Somewhat unexpectedly, there was a significant but transient expansion of TCR diversity 1 month after transplantation in all cases. Clonotypic analysis of TCRs performed in one case showed that many T cell clones shared identical CDR3 sequences at 1 month and that the shared fraction decreased progressively. These results indicate that early expansion of TCR diversity may reflect transient expansion of pre-existing mature T cells from the donor blood, independent of de novo T cell maturation through the thymus.
Purpose
Owing to recent technological advancements, using next-generation sequencing (NGS) and the accumulation of clinical experiences worldwide, more than 420 genes associated with inborn errors of ...immunity (IEI) have been identified, which exhibit large genotypic and phenotypic variations. Consequently, NGS-based comprehensive genetic analysis, including whole-exome sequencing (WES), have become more valuable in the clinical setting and have contributed to earlier diagnosis, improved treatment, and prognosis. However, these approaches have the following disadvantages that need to be considered: a relatively low diagnostic rate, high cost, difficulties in the interpretation of each variant, and the risk of incidental findings. Thus, the objective of this study is to review our WES results of a large number of patients with IEI and to elucidate patient characteristics, which are related to the positive WES result.
Methods
We performed WES for 136 IEI patients with negative conventional screening results for candidate genes and classified these variants depending on validity of their pathogenicity.
Results
We identified disease-causing pathogenic mutations in 36 (26.5%) of the patients which were found in known IEI-causing genes. Although the overall diagnostic rate was not high and was not apparently correlated with the clinical subcategories and severity, we revealed that earlier onset with longer duration of diseases were associated with positive WES results, especially in pediatric cases.
Conclusions
Most of the disease-causing germline mutations were located in the known IEI genes which could be predicted using patients’ clinical characteristics. These results may be useful when considering appropriate genetic approaches in the clinical setting.
Fanconi anemia is a rare recessive disease characterized by multiple congenital abnormalities, progressive bone marrow failure, and a predisposition to malignancies. It results from mutations in one ...of the 22 known
genes. The number of Japanese Fanconi anemia patients with a defined genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated
genes in Japan and clarify the genotype-phenotype correlations. We studied 117 Japanese patients and successfully subtyped 97% of the cases.
and
pathogenic variants accounted for the disease in 58% and 25% of Fanconi anemia patients, respectively. We identified one
and two
hot spot mutations, which are found at low percentages (0.04-0.1%) in the whole-genome reference panel of 3,554 Japanese individuals (Tohoku Medical Megabank).
was the third most common complementation group and only one
case was identified in our series. Based on the data from the Tohoku Medical Megabank, we estimate that approximately 2.6% of Japanese are carriers of disease-causing
gene variants, excluding missense mutations. This is the largest series of subtyped Japanese Fanconi anemia patients to date and the results will be useful for future clinical management.
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