Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1-3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of-and interplay between-novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.

Publisher Correction: Whole-genome sequencing of a sporadic primary immunodeficiency cohort.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Defective Interferon-Gamma Production Is Common in Chronic Pulmonary Aspergillosis.

BACKGROUND: Immune defects in chronic pulmonary aspergillosis (CPA) are poorly characterized. We compared peripheral blood cytokine profiles in patients with CPA versus healthy controls and explored the relationship with disease severity. METHODS: Interferon-gamma (IFNγ), interleukin (IL)-17, tumor necrosis factor-α, IL-6, IL-12, and IL-10 were measured after in vitro stimulation of whole blood with lipopolysaccharide (LPS), phytohemagglutinin, ß-glucan, zymosan (ZYM), IL-12 or IL-18, and combinations. Clinical parameters and mortality were correlated with cytokine production. RESULTS: Cytokine profiles were evaluated in 133 patients (57.1% male, mean age 61 years). In comparison to controls, patients with CPA had significantly reduced production of IFNγ in response to stimulation with ß-glucan + IL-12 (312 vs 988 pg/mL), LPS + IL-12 (252 vs 1033 pg/mL), ZYM + IL-12 (996 vs 2347 pg/mL), and IL-18 + IL-12 (7193 vs 12 330 pg/mL). Age >60 (hazard ratio [HR], 1.71; 95% confidence interval [CI], 1.00-2.91; P = .05) and chronic obstructive pulmonary disease (HR, 1.69; 95% CI, 1.03-2.78; P = .039) were associated with worse survival, whereas high IFNγ production in response to beta-glucan + IL-12 stimulation (HR, 0.48; 95% CI, .25-0.92; P = .026) was associated with reduced mortality. CONCLUSIONS: Patients with CPA show impaired IFNγ production in peripheral blood in response to stimuli. Defective IFNγ production ability correlates with worse outcomes. Immunotherapy with IFNγ could be beneficial for patients showing impaired IFNγ production in CPA.

Prescribing Immunoglobulin Replacement Therapy for Patients with Non-classical and Secondary Antibody Deficiency: an Analysis of the Practice of Clinical Immunologists in the UK and Republic of Ireland.

BACKGROUND: Immunologists are increasingly being asked to assess patients with non-classical and secondary antibody deficiency to determine their potential need for immunoglobulin replacement therapy (IGRT). Immunoglobulin is a limited, expensive resource and no clear guidance exists for this broad patient group. The purpose of this survey is to establish what factors influence the decision to commence IGRT in adult patients, when diagnostic criteria for primary antibody deficiency are not fulfilled. METHODS: Under the auspices of the United Kingdom Primary Immunodeficiency Network (UKPIN), a study group was established which circulated an online questionnaire to the consultant body across the UK and Ireland. Results provided a snapshot of the current clinical practice of 71% of consultant immunologists, from 30 centers. RESULTS: In order of importance, factors which influence the decision to commence IGRT include number of hospital admissions with infection, serum IgG level, bronchiectasis, radiologically proven pneumonia, number of positive sputum cultures, number of antibiotic courses, and results of immunization studies. The commonest test vaccine used was Pneumovax 23 with measurement of serotype-specific responses at 4 weeks, with a threshold of 0.35 µg/ml in 2/3 of serotypes measured. Eighty-six percent of patients are treated with a trial of prophylactic antibiotics prior to consideration of IGRT. Efficacy of IGRT trial is assessed at between 6 and 12 months. CONCLUSIONS: There was consistency in clinical practice using a combination of clinical history, evidence of infections, and vaccination testing for diagnosis. However, there was some variation in the implementation of this practice, particularly in vaccine choice and assessment of response to vaccination.

Liver transplantation in adults with liver disease due to common variable immunodeficiency leads to early recurrent disease and poor outcome.

Common variable immunodeficiency (CVID) is the most common form of primary immunodeficiency characterized by antibody deficiency, recurrent bacterial infections, and autoimmunity. Advanced chronic liver disease occurs in a subset of patients with CVID and manifests with various histological features, such as nodular regenerative hyperplasia, inflammation, fibrosis, and cholangiopathy. We present a case series characterizing the outcomes in adult patients transplanted for primary CVID-related liver disease. We discuss the unique transplantation challenges faced in this primary immunodeficiency group including susceptibility to infections and early disease recurrence. There is a statistically significant decrease in 3-year and 5-year survival after liver transplantation in those with CVID-related liver disease (55% at 3 and 5 years) compared with all-comers (89% at 3 years, 81% at 5 years), prompting a need for discussion of suitability of transplantation in this group of patients as well as methods for reducing posttransplantation risk such as scrupulous search for infectious agents and reduction of immunosuppression. Liver Transplantation 24 171-181 2018 AASLD.

Protein microarrays identify disease-specific anti-cytokine autoantibody profiles in the landscape of immunodeficiency.

BACKGROUND: Anti-cytokine autoantibodies (ACAAs) are pathogenic in a handful of rare immunodeficiencies. However, the prevalence and significance of other ACAAs across immunodeficiencies have not yet been described. OBJECTIVE: We profiled ACAAs in a diverse cohort of serum samples from patients with immunodeficiency and assessed the sensitivity and specificity of protein microarrays for ACAA identification and discovery. METHODS: Highly multiplexed protein microarrays were designed and fabricated. Blinded serum samples from a cohort of 58 immunodeficiency patients and healthy control subjects were used to probe microarrays. Unsupervised hierarchical clustering was used to identify clusters of reactivity, and after unblinding, significance analysis of microarrays was used to identify disease-specific autoantibodies. A bead-based assay was used to validate protein microarray results. Blocking activity of serum containing ACAAs was measured in vitro. RESULTS: Protein microarrays were highly sensitive and specific for the detection of ACAAs in patients with autoimmune polyendocrine syndrome type I and pulmonary alveolar proteinosis, detecting ACAA levels consistent with those reported in the published literature. Protein microarray results were validated by using an independent bead-based assay. To confirm the functional significance of these ACAAs, we tested and confirmed the blocking activity of select ACAAs in vitro. CONCLUSION: Protein microarrays are a powerful tool for ACAA detection and discovery, and they hold promise as a diagnostic for the evaluation and monitoring of clinical immunodeficiency.

Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.

TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell- specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n=39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n=41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P< .001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P< .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD(-)CD27(+) B cells (P= .019), benign lymphoproliferation (P< .001), and autoimmune complications (P= .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation.

Impaired T-cell receptor activation in IL-1 receptor-associated kinase-4-deficient patients.

BACKGROUND: IL-1 receptor-associated kinase 4 (IRAK-4) is an effector of the Toll-like receptor and IL-1 receptor pathways that plays a critical role in innate immune responses. The role of IRAK-4 in adaptive immune functions in human subjects is incompletely understood. OBJECTIVE: We sought to evaluate T-cell function in IRAK-4 deficient patients. METHODS: We compared upregulation of CD25 and CD69 on T cells and production of IL-2, IL-6, and IFN-gamma after stimulation of PBMCs from 4 IRAK-4-deficient patients and healthy control subjects with anti-CD3 and anti-CD28. RESULTS: Upregulation of CD25 and CD69 on T cells and production of IL-6 and IFN-gamma, but not IL-2, was significantly reduced in IRAK-4-deficient patients. CONCLUSIONS: IRAK-4-deficient patients have defects in T-cell activation.

Rituximab Associated Hypogammaglobulinemia in Autoimmune Disease.

Objective: To evaluate the characteristics of patients with autoimmune disease with hypogammaglobulinemia following rituximab (RTX) and describe their long-term outcomes, including those who commenced immunoglobulin replacement therapy. Methods: Patients received RTX for autoimmune disease between 2003 and 2012 with immunoglobulin G (IgG) <7g/L were included in this retrospective series. Hypogammaglobulinemia was classified by nadir IgG subgroups of 5 to <7g/L (mild), 3 to <5g/L (moderate) and <3g/L (severe). Characteristics of patients were compared across subgroups and examined for factors associated with greater likelihood of long term hypogammaglobulinemia or immunoglobulin replacement. Results: 142 patients were included; 101 (71%) had anti-neutrophil cytoplasm antibody (ANCA) associated vasculitis (AAV), 18 (13%) systemic lupus erythematosus (SLE) and 23 (16%) other conditions. Mean follow-up was 97.2 months from first RTX. Hypogammaglobulinemia continued to be identified during long-term follow-up. Median time to IgG <5g/L was 22.5 months. Greater likelihood of moderate hypogammaglobulinemia (IgG <5g/L) and/or use of immunoglobulin replacement therapy at 60 months was observed in patients with prior cyclophosphamide exposure (odds ratio (OR) 3.60 [95% confidence interval (CI) 1.03 - 12.53], glucocorticoid use at 12 months [OR 7.48 (95% CI 1.28 - 43.55], lower nadir IgG within 12 months of RTX commencement [OR 0.68 (95% CI 0.51 - 0.90)] and female sex [OR 8.57 (95% CI 2.07 - 35.43)]. Immunoglobulin replacement was commenced in 29/142 (20%) and associated with reduction in infection rates, but not severe infection rates. Conclusion: Hypogammaglobulinemia continues to occur in long-term follow-up post-RTX. In patients with recurrent infections, immunoglobulin replacement reduced rates of non-severe infections.

Low penetrance, broad resistance, and favorable outcome of interleukin 12 receptor beta1 deficiency: medical and immunological implications.

The clinical phenotype of interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency and the function of human IL-12 in host defense remain largely unknown, due to the small number of patients reported. We now report 41 patients with complete IL-12Rbeta1 deficiency from 17 countries. The only opportunistic infections observed, in 34 patients, were of childhood onset and caused by weakly virulent Salmonella or Mycobacteria (Bacille Calmette-Guérin -BCG- and environmental Mycobacteria). Three patients had clinical tuberculosis, one of whom also had salmonellosis. Unlike salmonellosis, mycobacterial infections did not recur. BCG inoculation and BCG disease were both effective against subsequent environmental mycobacteriosis, but not against salmonellosis. Excluding the probands, seven of the 12 affected siblings have remained free of case-definition opportunistic infection. Finally, only five deaths occurred in childhood, and the remaining 36 patients are alive and well. Thus, a diagnosis of IL-12Rbeta1 deficiency should be considered in children with opportunistic mycobacteriosis or salmonellosis; healthy siblings of probands and selected cases of tuberculosis should also be investigated. The overall prognosis is good due to broad resistance to infection and the low penetrance and favorable outcome of infections. Unexpectedly, human IL-12 is redundant in protective immunity against most microorganisms other than Mycobacteria and Salmonella. Moreover, IL-12 is redundant for primary immunity to Mycobacteria and Salmonella in many individuals and for secondary immunity to Mycobacteria but not to Salmonella in most.

Tuberculous meningitis: protracted course and clinical response to interferon-gamma.

A 12-year-old girl with protracted tuberculous meningitis received standard chemotherapy and dexamethasone and had a progressive cerebrospinal fluid neutrophilia, raised protein and depressed glucose levels. Her temperature was raised for 5 months until a second course of dexamethasone was given. At week 15, multiple tuberculomas and hydrocephalus were detected followed by acute hydrocephalus (week 58), which required a ventricular-peritoneal shunt. Tuberculomas resolved after a second course of dexamethasone but recurred 15 months later. Immunological investigations were normal including integrity of the type 1 cytokine pathway. From month 24, interferon-gamma was given subcutaneously (initially 50 microg/m(2)) and continued for 19 months. Within 2 weeks she responded clinically followed by a reduction in inflammatory signs on magnetic resonance imaging scan (but not in the tuberculomas). At month 44, when chemotherapy was stopped, the cerebrospinal fluid/serum albumin quotient was 57x10(-3) (normal <6.0x10(-3)), which supports continuing major impairment of the blood-brain barrier. Gene expression in peripheral blood mononuclear cells before and during treatment with interferon-gamma, assessed by gene array analysis, showed reduction in a number of cytokine and chemokine genes. The response to interferon-gamma might have been secondary to downregulation of certain cytokine and chemokine genes.

Host genetic factors and mycobacterial infections: lessons from single gene disorders affecting innate and adaptive immunity.

This review summarizes the association of increased susceptibility to mycobacterial disease in patients with genetic defects affecting innate and adaptive immunity. The optimum function of CD4 T-cell and macrophage function is critically important for immunity against mycobacteria. Antibody, complement and neutrophil function is not required for effective anti-mycobacterial immunity.

Failure to clear persistent vaccine-derived neurovirulent poliovirus infection in an immunodeficient man.

BACKGROUND: Individuals who chronically excrete neurovirulent poliovirus of vaccine-origin are of considerable concern to the Global Polio Eradication programme. Chronic infection with such polioviruses is a recognised complication of hypogammaglobulinaemia. METHODS: We did a series of in-vitro and in-vivo therapeutic studies, with a view to clearing persistent neurovirulent poliovirus infection in an individual with common variable immunodeficiency, using oral immunoglobulin, breast milk (as a source of secretory IgA), ribavirin, and the anti-picornaviral agent pleconaril. We undertook viral quantitation, antibody neutralisation and drug susceptibility assays, and viral gene sequencing. FINDINGS: Long-term asymptomatic excretion of vaccine-derived neurovirulent poliovirus 2 was identified in this hypogammaglobulinaemic man, and was estimated to have persisted for up to 22 years. Despite demonstrable in-vitro neutralising activity of immunoglobulin and breast milk, and in-vitro antiviral activity of ribavirin, no treatment was successful at clearing the virus, although in one trial breast milk significantly reduced excretion levels temporarily. During the course of study, the virus developed reduced susceptibility to pleconaril, precluding the in-vivo use of this drug. Sequence analysis revealed the emergence of a methionine to leucine mutation adjacent to the likely binding site of pleconaril in these isolates. INTERPRETATION: Chronic vaccine-associated poliovirus infection in hypogammaglobulinaemia is a difficult condition to treat. It represents a risk to the strategy to discontinue polio vaccination once global eradication has been achieved.

Autoantibodies to interferon-gamma in a patient with selective susceptibility to mycobacterial infection and organ-specific autoimmunity.

We evaluated a patient with disseminated Mycobacterium tuberculosis and Mycobacterium chelonae infection, of which he died. He also developed autoimmune (type I) diabetes and primary hypothyroidism. His serum contained a high titer of immunoglobulin G autoantibody to interferon-gamma (IFN-gamma) capable of blocking in vitro responses to this cytokine by peripheral blood mononuclear cells from normal donors. These results suggest that autoantibodies to IFN-gamma can induce susceptibility to disseminated mycobacterial infection, which may be refractory to chemotherapy.