Whole-Exome Sequencing-Based Approach for Germline Mutations in Patients with Inborn Errors of Immunity.

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.

APRIL-dependent lifelong plasmacyte maintenance and immunoglobulin production in humans.

BACKGROUND: 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. OBJECTIVE: We sought to elucidate the role of APRIL in B-cell differentiation and immunoglobulin production through the analysis of complete APRIL deficiency in human. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Multicolor Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases.

PURPOSE: Primary immunodeficiency diseases (PIDDs) are rare inherited diseases that impair the human immune system. We established a multicolor flow cytometric assay to comprehensively evaluate the immune status and immunological characteristics of patients with PIDDs. METHODS: Fifty-nine normal controls and 75 patients with PIDDs, including X-linked severe combined immunodeficiency (X-SCID), X-linked agammaglobulinemia (XLA), X-linked hyper IgM syndrome (X-HIGM), ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), hyper IgE syndrome (HIES), and chronic mucocutaneous candidiasis disease (CMCD), were enrolled in this study. Immunophenotyes were evaluated by multicolor flow cytometry using seven different panels that allowed the detection of major leukocyte populations in peripheral blood. RESULTS: Multicolor flow cytometry revealed distinct leukocyte populations and immunological features of patients with X-SCID, XLA, X-HIGM, AT, WAS, HIES, and CMCD. CONCLUSIONS: Immunophenotyping by multicolor flow cytometry is useful to evaluate immune status and contributes to the diagnosis and management of patients with PIDDs.

Multilateral Functional Alterations of Human Neutrophils in Sepsis: From the Point of Diagnosis to the Seventh Day.

Neutrophil functional changes caused by sepsis itself and their time-course variation have not been fully elucidated because previous studies targeted patients who had received therapeutic interventions. We explored the multilateral functions of circulating neutrophils in patients with severe sepsis or septic shock who had not yet undergone interventions, and followed their changes. Patients were treated based on the Surviving Sepsis Campaign Guidelines 2012. Neutrophil functions were evaluated on days 0 (before therapeutic intervention), 3, and 7 in 59 septic patients. The clinical severity score (APACHE II and SOFA) and serum pro-/anti-inflammatory cytokine concentrations of the patients were significantly increased on day 0 and normalized on day 3. However, neutrophil priming state, estimated by measuring the fMLP-stimulated reactive oxygen species, was significantly elevated on day 0, further augmented on day 3, and then returned to day 0 levels on day 7 despite general resolution of the inflammatory response. The expression of CXC chemokine receptor 2 and paired immunoglobulin-like receptor α, assessed as surrogate markers of transmigration and adhesion potency, was suppressed most strongly on day 0 and gradually recovered. To conclude, contrary to the patient's clinical course, neutrophil priming state was augmented most strongly at 3 days after diagnosis of sepsis. Impaired transmigration and excessive adhesion potency were observed most prominently at diagnosis. These observations would partially explain the mechanism of development of multiple organ dysfunction of the host who is subjected to a secondary insult, and may provide an important perspective for the implementation of additional immune-modulating therapy in sepsis.