Karin Chen

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

Phevor Combines Multiple Biomedical Ontologies for Accurate Identification of Disease-Causing Alleles in Single Individuals and Small Nuclear Families

Phevor integrates phenotype, gene function, and disease information with personal genomic data for improved power to identify disease-causing alleles. Phevor works by combining knowledge resident in multiple biomedical ontologies with the outputs of variant-prioritization tools. It does so by using an algorithm that propagates information across and between ontologies. This process enables Phevor to accurately reprioritize potentially damaging alleles identified by variant-prioritization tools in light of gene function, disease, and phenotype knowledge. Phevor is especially useful for single-exome and family-trio-based diagnostic analyses, the most commonly occurring clinical scenarios and ones for which existing personal genome diagnostic tools are most inaccurate and underpowered. Here, we present a series of benchmark analyses illustrating Phevors performance characteristics. Also presented are three recent Utah Genome Project case studies in which Phevor was used to identify disease-causing alleles. Collectively, these results show that Phevor improves diagnostic accuracy not only for individuals presenting with established disease phenotypes but also for those with previously undescribed and atypical disease presentations. Importantly, Phevor is not limited to known diseases or known disease-causing alleles. As we demonstrate, Phevor can also use latent information in ontologies to discover genes and disease-causing alleles not previously associated with disease.

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Autoimmunity due to RAG deficiency and estimated disease incidence in RAG1/2 mutations

To the Editor: A growing body of literature demonstrates that recombinase activating gene (RAG1/2) deficiency is seen in patients with autoimmune features and variable immunodeficiency, thus expanding the phenotype beyond T-/B-/NK+ severe combined immunodeficiency (SCID) and Omenn syndrome (OS) (1, 2). Based on data from SCID newborn screening programs in the United States and Puerto Rico, the estimated incidence of SCID is approximately 1:50,000 (3). Recent data from California’s SCID newborn screening identified RAG1/2 mutations in 28.6% of SCID/OS cases, for an incidence of about 1:250,000(4). However, this may not include individuals with less severe phenotypes caused by RAG1/2 defects. We present a family affected by compound heterozygous RAG1 mutations which resulted in a combined immunodeficiency phenotype with autoimmune cytopenias. We performed a population genetic analysis to estimate the incidence of disease caused by RAG1/2 homozygous or compound heterozygous mutations and found that the contribution of immune dysregulatory disease due to RAG1/2 mutations present in the general population may be much higher than previously estimated. Patient 1 (P1) is the eldest of five siblings born to non-consanguineous Caucasian parents (Figure 1, II.1). Clinical and laboratory data are summarized in Table 1 and Table E1 (online repository). Briefly, P1 developed early-onset autoimmune hemolytic anemia (AIHA), recurrent viral and bacterial infections, and nephrotic syndrome. Patient 2 (P2) is the full sibling of P1 (Figure 1, II.4), born full term with an eczematous rash. She developed recurrent bacterial and viral upper respiratory infections, early-onset AIHA, and neutropenia with antineutrophil antibodies. The autoimmune cytopenias did not respond to anti-CD20 treatment (rituximab). Both siblings had significantly reduced naive CD4+ T cell counts. Notable family history includes the siblings’ healthy mother who had false positive Rapid Plasma Reagin (RPR) antibodies and several maternal family members with multiple sclerosis. Figure 1 A. Sanger sequencing chromatogram of parents of the affected children confirms RAG1 mutation carrier status. Arrow indicates mutation position. B. Pedigree of family with RAG1 deficiency. Patient 1(II.1) and patient 2 (II.4) were affected with autoimmune ... Table 1 Clinical features and key laboratory data P1 underwent a 10/10 HLA-matched sibling bone marrow transplant at 8 years following conditioning with busulfan, fludarabine and anti-thymocyte globulin. Graft-versus-host disease (GVHD) prophylaxis included cyclosporine and methotrexate. One year post-transplant, P1 has excellent donor chimerism. Immune globulin replacement was discontinued 16 months post-transplant. P2 received a 10/10 matched unrelated marrow hematopoietic stem cell transplant (HSCT) at 29 months following similar conditioning. She had delayed engraftment of neutrophils with persistence of anti-neutrophil antibodies. Complications have included grade 3 gastrointestinal GVHD 6 months post-transplant, pancytopenia, and concern for graft failure. To identify an underlying genetic defect, P2 was screened for known mutations associated with SCID. No disease-causing mutations were identified in JAK3, DCLRE1C (ARTEMIS), IL7R, or RAG2. She harbored compound heterozygous mutations in exon 2 of RAG1, a genotype that was also confirmed for P1 (Figure 1). The c.1420C>T (p.Arg474Cys) mutation has previously been reported in cases of primary immunodeficiency, including SCID, Omenn syndrome and CD4+ T cell lymphopenia (Table E2, online repository). The c.2949delA (p.Lys983AsnfsX9) mutation is novel. Sanger sequencing of the remaining family members identified each parent as a RAG1 mutation carrier (Figure 1). The c.2949delA mutation is located at the carboxy-terminus of RAG1, at a RAG2 interaction site within the catalytic core (5, 6). A homozygous mutation at nearby amino acid position Q981P has been described by de Villartay et al.(7) with clinical features very similar to P1 and P2. Serum samples from healthy and affected family members were probed against an autoantibody protein microarray. IgG/IgM autoantibodies were increased in P1 and P2, and surprisingly, also in their mother (Figure E1 and Tables E3–E6, online repository). Furthermore, serum was tested for anticytokine antibodies that have been reported among patients with thymoma or autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED)(8, 9). Interestingly, our two affected patients also produce anti-interferon-α (IFNα) autoantibodies that were able to neutralize IFNα-induction of STAT1 phosphorylation in normal peripheral blood mononuclear cells, and may comprise a phenotypic signature for certain RAG-deficient patients (Figure E2, online repository). We assessed the European population carrier rate of RAG1/2 variants that have been identified in primary immunodeficiency diseases (PIDs; for methods, see Online Repository Text). Of more than 100 disease-causing RAG1 variants in the Human Gene Mutation Database, only 2 were present in 379 individuals of European ancestry in the 1000 Genomes database (Table E7, online repository). Seven and three unrelated individuals were heterozygous for the c.1346G>A and c.3016A>G disease-associated RAG1 variants, respectively. Therefore, the predicted population frequency based on Hardy-Weinberg equilibrium is 1:5746 individuals of European descent who are homozygous or compound heterozygous, much higher than what is expected for RAG1/2 SCID or OS cases. The single RAG2 disease-causing variant present in 1000 Genomes is in linkage disequilibrium with the c.3016A>G variant. Thus, the RAG1/2 carrier rate does not change. Our population genetic estimate indicates that RAG1 mutations likely contribute to many more undiagnosed cases of combined immunodeficiency, autoimmune cytopenias, and/or organ-specific autoimmune disease, although it is possible there is incomplete penetrance of these genotypes. California has a diverse and large Hispanic demographic and there may be differences when comparing estimates to a predominantly European population. Our estimate does not account for very rare RAG1 mutations not present in the 1000 Genomes database. Because limited data are available for functional activity in RAG1 variants identified in patients with PIDs, their disease-causing role cannot be easily established. In this case report, compound heterozygous RAG1 mutations resulted in a combined immunodeficiency phenotype with autoimmune cytopenias and multiple autoantibodies, including anti-IFNα antibodies as have been reported in APECED or thymoma patients. The mechanism for generation of these autoantibodies is unclear, but may depend on an underdeveloped thymus with decreased AIRE expression. The mother has a history of false-positive RPR and demonstrated presence of elevated levels of autoantibodies that was not seen in the c.1420C>T male carriers. Our findings suggest that the c.2949delA variant, even in heterozygous state, may contribute to generation of autoantibodies in RAG1-related disease. In patients with autoimmune cytopenias, lymphopenia and moderate-to-severe infections, screening for RAG1/2 mutations may well be worthwhile to identify molecular pathogenesis and treatment options. In our cohort, the presence of mild lymphopenia, autoimmune cytopenias, and a skewed naive:memory T cell ratio led to the diagnosis of RAG1 deficiency and subsequent HSCT. Earlier identification of a monogenic immune disorder and prompt HSCT during infancy might have spared progressive autoimmune complications. These findings provide additional data that RAG has important implications in both immunodeficiency and autoimmune diseases.

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation

Background: X‐linked hyper‐IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients. Objectives: We sought to collect data on the clinical presentation, treatment, and follow‐up of a large sample of patients with XHIGM to (1) compare long‐term overall survival and general well‐being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT. Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan‐Meier method compared with log‐rank tests and modeled by using proportional hazards regression. Results: Twenty‐eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow‐up and vital status information. Sixty‐seven (38%) patients received HCT. The average follow‐up time was 8.5 ± 7.2 years (range, 0.1‐36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987‐1995; the hazard ratio was significantly less than 1 for diagnosis years 1995‐1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2‐10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft‐versus‐host disease, and most deaths occurred within 1 year of transplantation. Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964‐2013). However, survivors treated with HCT experienced somewhat greater well‐being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

A sticky story for signal transducer and activator of transcription 3 in platelets.

The signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic protein that, on appropriate signaling, translocates to the nucleus and binds DNA response elements of target genes.1 As a result, STAT3 mediates the transcription of key mediators involved in mitogenesis, cell survival, apoptosis, cell cycle regulation, angiogenesis, and metastasis development.1,2 STAT3 also regulates the transcription of several genes in megakaryocytes that are required for the formation of platelets (Figure).3 Figure Schematic representation of traditional and nontraditional roles of signal transducer and activator of transcription 3 (STAT3) in megakaryocytes and platelets, respectively, as described by Zhou et al5 and reviewed here. As megakaryocytes form platelets, they transfer STAT3 to proplatelet tips. Consequently, STAT3 is found in platelets that circulate in the bloodstream (Figure). The presence of STAT3 in platelets raises the question of whether it regulates functional responses in platelets or is simply a vestigial remnant of megakaryocytes. An argument for the “leftover without function” hypothesis is the anucleate status of platelets: simply stated, with no nucleus and no nuclear DNA there is no place for STAT3 to stick in platelets. The problem with this argument is that “simple” is no longer a common word used to describe platelets. Moreover, why would platelets expend energy to carry a protein that they do not need, especially since previous studies have shown that STAT3 undergoes signal-dependent phosphorylation in these anucleate cytoplasts?4 Well, any doubt regarding why STAT3 is present in platelets has been cleared up. Using a combination of pharmacological and genetic based tools, Zhou et al5 demonstrate that STAT3 affects how platelets stick to one another and extracellular matrices. In addition, the authors put forth a new role for interleukin 6 (IL-6) and its soluble receptor in enhancing platelet aggregation. A major strength of the group’s findings is the plethora of evidence presented to make the story stick from men to mice and then back to men. First, they used 2 different types of STAT3 inhibitors to block collagen- and collagen-related peptide-dependent aggregation, as well as the formation of thrombi to a collagen substrate under flow conditions in human platelets. Neutralization of STAT3 also reduced collagen-dependent induction of P-selectin surface expression. STAT3 inhibitors, however, did not block ATP release nor did they dampen aggregation induced by ADP or a thrombin receptor activating peptide. Second, platelets from mice deficient in STAT3 aggregated poorly, had a low level of P-selectin surface expression and calcium influx in response to collagen, and formed smaller thrombi when exposed to a collagen matrix under arterial flow. The same platelets reacted normally to ADP and thrombin receptor activating peptide. Additional studies led to studies implying that glycoprotein VI platelet (GPVI) is the primary collagen receptor on platelets linked to the STAT3 signaling pathway. Finally, Zhou et al5 provided the first evidence that the IL-6 signaling complex can influence platelet function. They found that platelets constitutively express glycoprotein 130, which is capable of interacting with exogenous IL-6 and the soluble IL-6 receptor (IL-6R). Together, but not individually, these IL-6 family members induce STAT3 phosphorylation and enhance collagen-dependent platelet aggregation. A transcription-independent role for STAT3 builds on the growing appreciation that previously characterized transcription factors have diverse, noncanonical functions in platelets.6 In activated platelets, the nuclear factor-κB family member B cell lymphoma 3 interacts with Fyn-related tyrosine kinases to contract fibrin-rich clots.7 Nuclear factor-κB itself also has roles in limiting platelet activation,8 and nuclear factor-κB inhibitors attenuate the formation of lipodia in adherent platelets.9 Much like STAT3, peroxisome proliferator-activated receptor-γ regulates collagen-dependent platelet aggregation that is driven by GPVI.10 Ligand-dependent binding of retinoid function and X receptor also controls GTP-binding protein Gq thereby aggregation responses in platelets.11 Cumulatively, these studies point to the sundry function of proteins that were originally thought to have a sole role in transcription. One of the most intriguing findings of the work of Zhou et al5 is the identification of an IL-6 signaling pathway that links inflammation to thrombosis. In response to inflammatory cues, IL-6 is synthesized and released by various types of nucleated cells. IL-6 exerts its activities through 2 molecules, the IL-6R (also known as IL-6Rα) and glycoprotein 130 (also referred to as IL-6Rβ).12 The IL-6R is either membrane bound or soluble. As its name implies, soluble IL-6R is released into the extracellular milieu where it binds IL-6 and then forms a complex with membranous glycoprotein 130. This unique receptor signaling system, termed “IL-6 trans-signaling,”13 induces cellular activation including STAT3-dependent transcriptional responses. Until now there has been no evidence that IL-6 trans-signaling occurs in platelets. Zhou et al5 demonstrate that, in combination with the soluble IL-6R, IL-6 binds membrane-expressed glycoprotein 130 and primes platelets for collagen-induced cellular activation. This suggests that heightened IL-6 trans-signaling in response to inflammation may enhance thrombus formation in a variety of human diseases, such as rheumatoid arthritis, lupus, and sepsis. Conversely, deficiencies in IL-6 production, which have been reported to occur in common variable immune deficiency,14 may lead to dampened thrombus formation and increased bruising and bleeding that is commonly observed in patients with this syndrome. Selective inhibition of IL-6 trans-signaling has recently received considerable attention for the treatment of cancer, and an IL-6R blocking antibody (tocilizumab) was recently approved for Castelman disease and rheumatoid arthritis (reviewed in References 15 and 16). A STAT3 decoy inhibitor is currently being tested in patients with head and neck cancer, and there is emerging evidence that STAT3 inhibitors may prove useful in treating disorders of cardiac-related inflammation.15,17 Thus, it will be important to consider off-target inhibition of platelet activity, which may be good or bad, when patients are treated with IL-6 trans-signaling and STAT3 inhibitors. Indeed, the authors speculate that inhibition of STAT3 may improve inflammation-induced platelet hyper-reactivity and improve the efficacy of aspirin in patients with coronary artery disease. The interplay of IL-6 trans-signaling and STAT3 with GPVI will also have to be pondered as the efficacy of GPVI receptor antagonists are screened in the clinic.18 Thrombosis is often linked to inflammation, but the reverse has received little attention until Zhou et al5 unmasked the STAT3 signaling pathway in anucleate platelets.5 Their results challenge existing paradigms and, in doing so, reveal that we should never underestimate the resolve of platelets to use previously described nuclear-based systems in alternative ways. Identification of a 3-way bridge among IL-6 trans-signaling, STAT3, and GPVI that courses to aggregation adds to the fascinating biology of platelets (Figure). It also creates a sticky story for STAT3 in platelets, and potentially the cytoplasm of nucleated cells, with clinical implications for human disease.

Profound T-cell lymphopenia associated with prenatal exposure to purine antagonists detected by TREC newborn screening

Recognition of non-SCID and secondary causes of T cell lymphopenia detected by TREC newborn screening is important in directing subsequent care and identifying those who would not benefit from more invasive interventions. Here, we report two infants with low TRECs and severe, but self-resolving, T cell lymphopenia identified by SCID NBS that were caused by in utero exposure to purine antimetabolites.

Chronic norovirus infection in primary immune deficiency disorders: an international case series

OBJECTIVE Predictive factors associated with clinical outcomes of chronic norovirus infection (CNI) in primary immunodeficiency diseases (PIDD) are lacking. METHOD We sought to characterize CNI using a multi-institutional cohort of patients with PIDD and CNI using the Clinical Immunology Societys CIS-PIDD Listserv e-mail group. RESULTS Thirty-four subjects (21 males and 13 females) were reported from centers across North America, Europe, and Asia. All subjects were receiving high doses (median IgG dose: 1200 mg/kg/month) of supplemental immunoglobulin therapy. Fifty-three percent had a complete absence of B cells (median B-cell count 0; range 0-139 cells/μL). Common Variable Immune Deficiency (CVID) subjects manifested a unique phenotype with B-cell lymphopenia, non O+ blood type, and villous atrophy (logistic regression model, P = 0.01). Five subjects died, all of whom had no evidence of villous atrophy. CONCLUSION While Norovirus (NoV) is thought to replicate in B cells, in this PIDD cohort of CNI, B-cell lymphopenia was common, indicating that the presence of B lymphocytes is not essential for CNI.