Sven Kracker

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage

Answers from Exomes Exome sequencing, which targets only the protein-coding regions of the genome, has the potential to identify the underlying genetic causes of rare inherited diseases. Angulo et al. (p. 866, published online 17 October; see Perspective by Conley and Fruman) performed exome sequencing of individuals from seven unrelated families with severe, recurrent respiratory infections. The patients carried the same mutation in the gene coding for the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). The mutation caused aberrant activation of this kinase, which plays a key role in immune cell signaling. Drugs inhibiting PI3Kδ are already in clinical trials for other disorders. Gene sequencing of unrelated patients with recurrent airway infections identifies a common underlying mutation. [Also see Perspective by Conley and Fruman] Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

A human immunodeficiency caused by mutations in the PIK3R1 gene

Recently, patient mutations that activate PI3K signaling have been linked to a primary antibody deficiency. Here, we used whole-exome sequencing and characterized the molecular defects in 4 patients from 3 unrelated families diagnosed with hypogammaglobulinemia and recurrent infections. We identified 2 different heterozygous splice site mutations that affect the same splice site in PIK3R1, which encodes the p85α subunit of PI3K. The resulting deletion of exon 10 produced a shortened p85α protein that lacks part of the PI3K p110-binding domain. The hypothetical loss of p85α-mediated inhibition of p110 activity was supported by elevated phosphorylation of the known downstream signaling kinase AKT in patient T cell blasts. Analysis of patient blood revealed that naive T and memory B cell counts were low, and T cell blasts displayed enhanced activation-induced cell death, which was corrected by addition of the PI3Kδ inhibitor IC87114. Furthermore, B lymphocytes proliferated weakly in response to activation via the B cell receptor and TLR9, indicating a B cell defect. The phenotype exhibited by patients carrying the PIK3R1 splice site mutation is similar to that of patients carrying gain-of-function mutations in PIK3CD. Our results suggest that PI3K activity is tightly regulated in T and B lymphocytes and that various defects in the PI3K-triggered pathway can cause primary immunodeficiencies.

Primary antibody deficiencies

Primary antibody deficiencies (PADs) are the most common inherited immunodeficiencies in humans. The use of novel approaches, such as whole-exome sequencing and mouse genetic engineering, has helped to identify new genes that are involved in the pathogenesis of PADs and has enabled the characterization of the molecular pathways that are involved in B cell development and function. Here, we review the different PADs in terms of their known or putative mechanisms, which can be B cell intrinsic, B cell extrinsic or not defined so far. We also describe the clinical manifestations (including susceptibility to infections, autoimmunity and cancer) that have been associated with the various PADs.

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study

Background: Activated phosphoinositide 3‐kinase &dgr; syndrome (APDS) is a recently described combined immunodeficiency resulting from gain‐of‐function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3‐kinase &dgr; (PI3K&dgr;). Objective: We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. Methods: We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. Results: Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3K&dgr; in the central nervous system; consistent with this, PI3K&dgr; is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. Conclusion: APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3K&dgr; inhibitors offer new prospects for APDS treatment.

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

To the Editor Activated 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, ​,1B1B). Table 1A Table 1B Lymphocyte populations Patient 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. Figure 1 B-cell lymphomas. Patient 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. These 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.

DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions.

Genetic inactivation of the genes encoding several low-fidelity DNA polymerases indicates that DNA polymerase ζ inserts tandem double-base substitutions in the immunoglobulin variable region in mouse B cells.

Impaired induction of DNA lesions during immunoglobulin class-switch recombination in humans influences end-joining repair

Ig class-switch recombination (CSR) is a region-specific process that exchanges the constant Ig heavy-chain region and thus modifies an antibodys effector function. DNA lesions in switch (S) regions are induced by activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase 2 (UNG2), subsequently processed to DNA breaks, and resolved by either the classical nonhomologous end-joining pathway or the alternative end-joining pathway (XRCC4/DNA ligase 4– and/or Ku70/Ku80-independent and prone to increased microhomology usage). We examined whether the induction of DNA lesions influences DNA end-joining during CSR by analyzing Sμ–Sα recombination junctions in various human Ig CSR defects of DNA lesion induction. We observed a progressive trend toward the usage of microhomology in Sμ–Sα recombination junctions from AID-heterozygous to AID–autosomal dominant to UNG2-deficient B lymphocytes. We thus hypothesize that impaired induction of DNA lesions in S regions during CSR leads to unusual end-processing of the DNA breaks, resulting in microhomology-mediated end-joining, which could be an indication for preferential processing by alternative end-joining rather than by classical nonhomologous end-joining.

Mild B-cell lymphocytosis in patients with a CARD11 C49Y mutation

of the KU Leuven. X.B. is a senior clinical investigator of the Research Foundation Flanders. R.R. received a fellowship from the French Ministry of Research. E.M. is supported by a fellowship of the Cancer League (France). S.L. is a senior scientist from the French National Center for Scientific Research. Disclosure of potential conflict of interest: H. Schaballie has received research support from FWO Research Foundation Flanders. A. Fischer has been supported by a senior European Research Council (ERC) grant ‘‘PIDIMMUN’’ (grant no. 249816). I. Meyts has received research support from the Jeffrey Modell Foundation and has received lecture fees from Gilead Sciences. The rest of the authors declare that they have no relevant conflicts of interest.

Potential roles of activation-induced cytidine deaminase in promotion or prevention of autoimmunity in humans

Autoimmune manifestations are paradoxical and frequent complications of primary immunodeficiencies, including T and/or B cell defects. Among pure B cell defects, the Activation-induced cytidine Deaminase (AID)-deficiency, characterized by a complete lack of immunoglobulin class switch recombination and somatic hypermutation, is especially complicated by autoimmune disorders. We summarized in this review the different autoimmune and inflammatory manifestations present in 13 patients out of a cohort of 45 patients. Moreover, we also review the impact of AID mutations on B-cell tolerance and discuss hypotheses that may explain why central and peripheral B-cell tolerance was abnormal in the absence of functional AID. Hence, AID plays an essential role in controlling autoreactive B cells in humans and prevents the development of autoimmune syndromes.

Decreased somatic hypermutation induces an impaired peripheral B cell tolerance checkpoint

Patients with mutations in AICDA, which encodes activation-induced cytidine deaminase (AID), display an impaired peripheral B cell tolerance. AID mediates class-switch recombination (CSR) and somatic hypermutation (SHM) in B cells, but the mechanism by which AID prevents the accumulation of autoreactive B cells in blood is unclear. Here, we analyzed B cell tolerance in AID-deficient patients, patients with autosomal dominant AID mutations (AD-AID), asymptomatic AICDA heterozygotes (AID+/-), and patients with uracil N-glycosylase (UNG) deficiency, which impairs CSR but not SHM. The low frequency of autoreactive mature naive B cells in UNG-deficient patients resembled that of healthy subjects, revealing that impaired CSR does not interfere with the peripheral B cell tolerance checkpoint. In contrast, we observed decreased frequencies of SHM in memory B cells from AD-AID patients and AID+/- subjects, who were unable to prevent the accumulation of autoreactive mature naive B cells. In addition, the individuals with AICDA mutations, but not UNG-deficient patients, displayed Tregs with defective suppressive capacity that correlated with increases in circulating T follicular helper cells and enhanced cytokine production. We conclude that SHM, but not CSR, regulates peripheral B cell tolerance through the production of mutated antibodies that clear antigens and prevent sustained interleukin secretions that interfere with Treg function.