Kohsuke Imai

Human uracil–DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination

Activation-induced cytidine deaminase (AID) is a master molecule in immunoglobulin (Ig) class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil–DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (immunoglobulin switch or variable regions), followed by uracil removal by UNG.

X-linked thrombocytopenia (XLT) due to WAS mutations: clinical characteristics, long-term outcome, and treatment options

A large proportion of patients with mutations in the Wiskott-Aldrich syndrome (WAS) protein gene exhibit the milder phenotype termed X-linked thrombocytopenia (XLT). Whereas stem cell transplantation at an early age is the treatment of choice for patients with WAS, therapeutic options for patients with XLT are controversial. In a retrospective multicenter study we defined the clinical phenotype of XLT and determined the probability of severe disease-related complications in patients older than 2 years with documented WAS gene mutations and mild-to-moderate eczema or mild, infrequent infections. Enrolled were 173 patients (median age, 11.5 years) from 12 countries spanning 2830 patient-years. Serious bleeding episodes occurred in 13.9%, life-threatening infections in 6.9%, autoimmunity in 12.1%, and malignancy in 5.2% of patients. Overall and event-free survival probabilities were not significantly influenced by the type of mutation or intravenous immunoglobulin or antibiotic prophylaxis. Splenectomy resulted in increased risk of severe infections. This analysis of the clinical outcome and molecular basis of patients with XLT shows excellent long-term survival but also a high probability of severe disease-related complications. These observations will allow better decision making when considering treatment options for individual patients with XLT.

WASP (Wiskott-Aldrich syndrome protein) gene mutations and phenotype.

Purpose of reviewWiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), characterized by chronic microthrombocytopenia with and without immunodeficiency, are caused by mutations of the WAS protein (WASP) gene. WASP has been reported to interact with many cytoplasmic molecules linking cellular signaling to the actin cytoskeleton. In this review we will focus on recent molecular findings that provide a better understanding of the pathogenesis of this complex disease and explore the correlation of genotype and clinical phenotype. Recent findingsRecent investigations have provided evidence that WASP and several related proteins are involved in the reorganization of the actin cytoskeleton by activating Arp2/3-mediated actin polymerization. This function is controlled mainly by a small GTPase Cdc42. Activated GTP-bound Cdc42 dissociates the intramolecular autoinhibitory loop formation of WASP. In addition, WASP is involved in cytoplasmic signaling by its interaction with a variety of adaptor molecules or kinases and serves as a link to actin reorganization, which is important for immunological synapse formation, cell trafficking and motility. Tyrosine or serine phosphorylation of WASP increases the actin polymerization activity of WASP via Arp2/3. Mutation analysis of WAS/XLT patients has provided evidence for a strong correlation between phenotype and genotype. Gene therapy for WASP-deficient human lymphocytes and Wasp-deficient mice was performed successfully. SummaryThe study of WASP and its mutations has led to a better understanding of the pathogenesis of the syndrome (thrombocytopenia, immunodeficiency, atopic dermatitis, autoimmune and malignant diseases) and the mechanisms required for cell mobility, cell-cell interaction and cytoplasmic signaling, as well as thrombopoiesis and maintenance of the number of platelets.

The Block in Immunoglobulin Class Switch Recombination Caused by Activation-Induced Cytidine Deaminase Deficiency Occurs Prior to the Generation of DNA Double Strand Breaks in Switch μ Region

Affinity maturation of the Ab repertoire in germinal centers leads to the selection of high affinity Abs with selected heavy chain constant regions. Ab maturation involves two modifications of the Ig genes, i.e., somatic hypermutation and class switch recombination. The mechanisms of these two processes are not fully understood. As shown by the somatic hypermutation and class switch recombination-deficient phenotype of activation-induced cytidine deaminase (AID)-deficient patients (hyperIgM type 2 syndrome) and mice, both processes require the AID molecule. Somatic DNA modifications require DNA breaks, which, at least for class switch recombination, lead to dsDNA breaks. By using a ligation-mediated PCR, it was found that class switch recombination-induced dsDNA breaks in Sμ switch regions were less frequent in AID-deficient B cells than in AID-proficient B cells, thus indicating that AID acts upstream of DNA break induction.

Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles Quantification Using Neonatal Guthrie Cards

OBJECTIVEnTo assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID).nnnSTUDY DESIGNnReal-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3).nnnRESULTSnTRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study.nnnCONCLUSIONnTRECs quantification can be used as a neonatal mass screening for patients with SCID.

Clinical and genetic characteristics of XIAP deficiency in Japan.

Deficiency of X-linked inhibitor of apoptosis (XIAP) caused by XIAP/BIRC4 gene mutations is an inherited immune defect recognized as X-linked lymphoproliferative syndrome type 2. This disease is mainly observed in patients with hemophagocytic lymphohistiocytosis (HLH) often associated with Epstein–Barr virus infection. We described nine Japanese patients from six unrelated families with XIAP deficiency and studied XIAP protein expression, XIAP gene analysis, invariant natural killer T (iNKT) cell counts, and the cytotoxic activity of CD8+ alloantigen-specific cytotoxic T lymphocytes. Of the nine patients, eight patients presented with symptoms in infancy or early childhood. Five patients presented with recurrent HLH, one of whom had severe HLH and died after cord blood transplantation. One patient presented with colitis, as did another patient’s maternal uncle, who died of colitis at 4xa0years of age prior to diagnosis with XIAP deficiency. Interestingly, a 17-year-old patient was asymptomatic, while his younger brother suffered from recurrent HLH and EBV infection. Seven out of eight patients showed decreased XIAP protein expression. iNKT cells from patients with XIAP deficiency were significantly decreased as compared with age-matched healthy controls. These results in our Japanese cohort are compatible with previous studies, confirming the clinical characteristics of XIAP deficiency.

Occurrence of B-cell lymphomas in patients with activated phosphoinositide 3-kinase δ syndrome.

To the Editor n nActivated Phosphoinositide 3-Kinase δ syndrome (APDS) is a novel autosomal dominant (AD) primary immunodeficiency (PID), caused by a heterozygous gain-of-function mutation in the PIK3CD gene encoding the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ)(1). The c.3061G>A mutation results in a substitution of a glutamic acid by a lysine at position 1021 (E1021K). This new PID is characterized by recurrent respiratory infections, leading to bronchiectasis, progressive lymphopenia, and defective antibody production. Both T and B cell compartments are affected as shown by the propensity of CD4+ and CD8+ T cells to die after in vitro stimulation and their poor capacity for cytokine production, as well as an immunoglobulin (Ig) class switch recombination defect (CSR-D). Most of the cases have an increase of serum IgM levels and a decrease of IgG2 isotype, while total IgG and IgA levels can be either normal or strongly decreased. The clinical presentation is variable, ranging from combined immunodeficiency requiring hematopoietic stem cell transplantation to an isolated primary antibody deficiency which can be well controlled by IgG substitution. In order to identify new APDS patients, we genotyped the PIK3CD gene at position c.3061G as described previously (1) in a cohort of 139 patients with immunological phenotype of Ig CSR-D. We found 8 new APDS patients with the E1021K heterozygous mutation in the PIK3CD gene (“see Tables E1 and E2”) in addition to the 17 described previously (1), bringing the total number of known patients carrying this PIK3CD mutation to 25. We noticed that among these eight new APDS patients two developed B-cell lymphomas, suggesting that a constitutively active PI3Kδ predisposes to malignancies. These two cases are herein reported (Table1A, u200b,1B1B). n n n nTable 1A n n n n n nTable 1B n nLymphocyte populations n n n nPatient 1 has no familial history of PID, but his mother died at 35 years of age of sub-arachnoid haemorrhage. He was referred to our hospital at the age of 2 years with recurrent bronchopulmonary infections, lymphadenopathy, hepato-splenomegaly, liver disease (elevated transaminases and portal septal fibrosis at liver biopsy). He had increased serum IgM levels (4.25g/L), normal IgG (5.7 g/L) and decreased IgA (0.65g/L) levels, compatible with the diagnosis of CSR-D. The CD40L and CD40 defects were excluded and intravenous IgG substitution was initiated. At 8 years of age, he developed a high grade diffuse large B-cell lymphoma (DLBCL, WHO classification) of biliary tract (Figure 1 a-c). In situ hybridization for Epstein Barr virus (EBV) was negative and Bcl-6 was expressed as shown by immunohistochemistry. The patient recovered after nine courses of chemotherapy (UKCCSG 9002 protocol; “see E3”). At 19 years of age, under IgG substitution, he again developed a high grade EBV(-) DLBCL of the colon, which was found to be Bcl-6 negative (Figure 1 d-f). He received CHOP (Cyclophophamide, vincristine, steroids) plus rituximab. He died from large bowel perforation and bleeding 12 days after the third course of chemotherapy. n n n nFigure 1 n nB-cell lymphomas. n n n nPatient 2 belongs to a family in which two siblings were reported as suffering from a CSR-D (data from the affected sister P7 “see Tables E1 and E2”). From the age of 5 months, he suffered recurrent upper (recurrent acute otitis media) and lower respiratory tract infections complicated by bronchiectasis, chronic non-infectious diarrhea with malabsorption syndrom and failure to thrive. Other infections were also noticed, including pericarditis caused by Echo virus infection and recurrent synovitis. The diagnosis of CSR-D was made, according to his familial history and IgG substitution was started. At 6 and 8 years of age, he displayed episodes of massive enlargement of lymph nodes (cervical and mesenteric) with no malignant feature at biopsy. Serum Ig levels revealed an increase of IgM (4.5g/L at 5 years and 13g/L at 11 years) and a decrease of IgG (<1.9g/L) and IgA (0.41 g/L). At 11 years of age, he had a new episode of cervical lymph nodes enlargement which led to the diagnosis of Hodgkin disease, histological type nodular sclerosis, stage III with localization to cervical, mediastinum, retroperitoneum and spleen (EBV status was unknown and could not be studied retrospectively) (Figure 1 g-i). Patient received chemotherapy and radiotherapy with irradiation of regions above and below diaphragma, which induced complete remission. He is now well on IgG substitution and prophylactic antibiotherapy with a follow-up of more than 10 years. n nThese observations extend our previous data reporting one case of marginal zone B-cell lymphoma in an adult APDS patient (1). Moreover, a recent study reports one further APDS patient who developed an EBV+ diffuse B cell lymphoma. Interestingly, authors describe a similar PID phenotype with two other gain of function mutations (E525K and N334K) in PIK3CD gene, including one case of EBV+ nodular sclerosis form of classical Hodgkin lymphoma (E525K) (2). Altogether these observations pinpoint to the fact that PI3Kδ hyperactivation predisposes to multiple types of B-cell lymphomas. Activation of the PI3K pathway is associated with malignant transformations and it has been shown that overexpression of p110δ can transform cells (3). Constitutive PI3K activation has been found in B-cell malignancies, e.g. Burkitt lymphomas (4, 5). Recently, somatic E1021K mutations of p110δ have been detected in diffuse large B-cell lymphomas from two patients (6) similar to our patient #1, which further supports our observation that activation of PI3Kδ signalling contributes to B cell neoplasia. We propose that a combination of defective T cell mediated immune surveillance and uncontrolled lymphoproliferation of B cells predisposes this PID to B cell lymphomagenesis. So far, only the minority of patients carrying PIK3CD mutation (5 out of 39, 13%) had been diagnosed with lymphomas. However, the risk of malignancies is likely to increase with age, modified by additional acquired somatic mutations. Most APDS patients currently receive treatment with antibiotics and IgG replacement. Such treatment reduces infections, but is unlikely to prevent lymphomas. We have found that selective p110δ inhibitors IC87114 and GS-1101 (CAL-101 or Idelalisib) reduce activity of the mutant p110δ in vitro and in cells of APDS patients ex vivo (1). GS-1101 has been in clinical trials for treatment of chronic lymphocytic leukemia (CLL) and early data suggest that the drug is well tolerated for extended periods of exposure (7). Therefore, GS-1101 and other selective p110δ inhibitors may provide a novel specific therapy for APDS patients that prevent lymphoma development. Susceptibility to lymphomas is observed in other well defined PID, such as the AD hyper-IgE syndrome due to heterozygous mutations in STAT3 and in autosomal recessive IL10RA or IL10RB-deficiencies, suggesting a role for the IL-10R/STAT3 pathway in controlling lymphomagenesis (8). Patients with common variable immunodeficiency are also prone to develop lymphomas (9). Physicians should be aware of this complication that strongly worsens the prognosis for PID patients.

Hematopoietic stem cell transplantation for 30 patients with primary immunodeficiency diseases: 20 years experience of a single team.

We retrospectively analyzed our results of 30 patients with three distinctive primary immunodeficiency diseases (PIDs) – severe combined immunodeficiency (SCID, n=11), Wiskott–Aldrich syndrome (WAS, n=11) and X-linked hyper-immunoglobulin M (IgM) syndrome (XHIM, n=8) – who underwent hematopoietic SCT (HSCT) during the past 20 years. Until 1995, all donors were HLA-haploidentical relatives with T-cell depletion (TCD) (n=8). Since 1996, the donors have been HLA-matched related donors (MRD) (n=8), unrelated BM (UR-BM) (n=7) and unrelated cord blood (UR-CB) (n=7). Twenty-seven of 30 patients had various pre-existing infections with or without organ damages before HSCT. Conditioning regimen and GVHD prophylaxis were determined according to disease, donor and pretransplant status. Although one of eight patients transplanted with TCD is alive with full engraftment, the other seven died. On the other hand, 18 of 22 patients transplanted without TCD are alive and well, including six of eight transplanted from MRD, seven of seven from UR-BM and five of seven from UR-CB. All 19 survivors did not require Ig supplementation after HSCT. These results indicate that UR-CBT as well as UR-BMT provides good results for PID comparable to MRD-SCT, and that early diagnosis, HSCT at early stage, careful supportive therapy and monitoring for various pathogens are important for the successful HSCT.

Quantification of κ-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects

In conclusion, the associations among asthma, biofilmforming bacteria, and revision ESS are strong and robust after adjusting for other factors in patients with CRS from a tertiary medical center. Despite its limitations, this study may improve our understanding of refractory CRS pathogenesis, possibly leading to more effective treatment strategies, such as incorporating the treatments of asthma and biofilm infection into conventional CRS therapies. Prospective cohort studies in diverse populations are needed to assess the causality of these associations.

Two Brothers with Ataxia-Telangiectasia-like Disorder with Lung Adenocarcinoma

We report on 2 brothers with ataxia-telangiectasia-like disorder with lung adenocarcinoma. They both had ataxia with cerebellar atrophy and mental retardation. They had the same mutation of the MRE11 gene, which has not been reported previously (c.727T>C and g.24994G>A).