Normalization of C1 Inhibitor in a Patient with Hereditary Angioedema.

Hereditary angioedema is a potentially life-threatening autosomal dominant condition, causing attacks of angioedema due to failure to regulate bradykinin. Nearly all cases of hereditary angioedema are caused by mutations in the gene encoding C1 inhibitor, SERPING1. C1 inhibitor is a multifunctional protein produced in the liver that regulates the kallikrein-kinin system at multiple points. An infant with genetically confirmed hereditary angioedema and low C1 inhibitor levels (but without previous episodes of angioedema) underwent liver transplantation for biliary atresia, an unrelated condition. Liver transplantation led to normalization of the C1 inhibitor level and function. To our knowledge, this represents the first patient to be potentially cured of hereditary angioedema.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Combined multidimensional single-cell protein and RNA profiling dissects the cellular and functional heterogeneity of thymic epithelial cells.

The network of thymic stromal cells provides essential niches with unique molecular cues controlling T cell development and selection. Recent single-cell RNA sequencing studies have uncovered previously unappreciated transcriptional heterogeneity among thymic epithelial cells (TEC). However, there are only very few cell markers that allow a comparable phenotypic identification of TEC. Here, using massively parallel flow cytometry and machine learning, we deconvoluted known TEC phenotypes into novel subpopulations. Using CITEseq, these phenotypes were related to corresponding TEC subtypes defined by the cells' RNA profiles. This approach allowed the phenotypic identification of perinatal cTEC and their physical localisation within the cortical stromal scaffold. In addition, we demonstrate the dynamic change in the frequency of perinatal cTEC in response to developing thymocytes and reveal their exceptional efficiency in positive selection. Collectively, our study identifies markers that allow for an unprecedented dissection of the thymus stromal complexity, as well as physical isolation of TEC populations and assignment of specific functions to individual TEC subtypes.

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK.

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort.

Developmental dynamics of the neural crest-mesenchymal axis in creating the thymic microenvironment.

The thymic stroma is composed of epithelial and nonepithelial cells providing separate microenvironments controlling homing, differentiation, and selection of hematopoietic precursor cells to functional T cells. Here, we explore at single-cell resolution the complex composition and dynamic changes of the nonepithelial stromal compartment across different developmental stages in the human and mouse thymus, and in an experimental model of the DiGeorge syndrome, the most common form of human thymic hypoplasia. The detected gene expression signatures identify previously unknown stromal subtypes and relate their individual molecular profiles to separate differentiation trajectories and functions, revealing an unprecedented heterogeneity of different cell types that emerge at discrete developmental stages and vary in their expression of key regulatory signaling circuits and extracellular matrix components. Together, these findings highlight the dynamic complexity of the nonepithelial thymus stroma and link this to separate instructive roles essential for normal thymus organogenesis and tissue maintenance.

Resolving the polygenic aetiology of a late onset combined immune deficiency caused by NFKB1 haploinsufficiency and modified by PIK3R1 and TNFRSF13B variants.

Genetic variants in PIK3CD, PIK3R1 and NFKB1 cause the primary immune deficiencies, activated PI3Kδ syndrome (APDS) 1, APDS2 and NFκB1 haploinsufficiency, respectively. We have identified a family with known or potentially pathogenic variants NFKB1, TNFRSF13B and PIK3R1. The study's aim was to describe their associated immune and cellular phenotypes and compare with individuals with monogenic disease. NFκB1 pathway function was measured by immunoblotting and PI3Kδ pathway activity by phospho-flow cytometry. p105/p50 expression was absent in two individuals but elevated pS6 only in the index case. Transfection of primary T cells demonstrated increased basal pS6 signalling due to mutant PIK3R1, but not mutant NFKB1 or their wildtype forms. We report on the presence of pathogenic variant NFKB1, with likely modifying variants in TNFRSF13B and PIK3R1 in a family. We describe immune features of both NFκB1 haploinsufficiency and APDS2, and the inhibition of excessive PI3K signalling by rapamycin in vitro.

FOXN1 forms higher-order nuclear condensates displaced by mutations causing immunodeficiency.

The transcription factor FOXN1 is a master regulator of thymic epithelial cell (TEC) development and function. Here, we demonstrate that FOXN1 expression is differentially regulated during organogenesis and participates in multimolecular nuclear condensates essential for the factor's transcriptional activity. FOXN1's C-terminal sequence regulates the diffusion velocity within these aggregates and modulates the binding to proximal gene regulatory regions. These dynamics are altered in a patient with a mutant FOXN1 that is modified in its C-terminal sequence. This mutant is transcriptionally inactive and acts as a dominant negative factor displacing wild-type FOXN1 from condensates and causing athymia and severe lymphopenia in heterozygotes. Expression of the mutated mouse ortholog selectively impairs mouse TEC differentiation, revealing a gene dose dependency for individual TEC subtypes. We have therefore identified the cause for a primary immunodeficiency disease and determined the mechanism by which this FOXN1 gain-of-function mutant mediates its dominant negative effect.

Expanding the Nude SCID/CID Phenotype Associated with FOXN1 Homozygous, Compound Heterozygous, or Heterozygous Mutations.

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

Inborn errors of thymic stromal cell development and function.

As the primary site for T cell development, the thymus is responsible for the production and selection of a functional, yet self-tolerant T cell repertoire. This critically depends on thymic stromal cells, derived from the pharyngeal apparatus during embryogenesis. Thymic epithelial cells, mesenchymal and vascular elements together form the unique and highly specialised microenvironment required to support all aspects of thymopoiesis and T cell central tolerance induction. Although rare, inborn errors of thymic stromal cells constitute a clinically important group of conditions because their immunological consequences, which include autoimmune disease and T cell immunodeficiency, can be life-threatening if unrecognised and untreated. In this review, we describe the molecular and environmental aetiologies of the thymic stromal cell defects known to cause disease in humans, placing particular emphasis on those with a propensity to cause thymic hypoplasia or aplasia and consequently severe congenital immunodeficiency. We discuss the principles underpinning their diagnosis and management, including the use of novel tools to aid in their identification and strategies for curative treatment, principally transplantation of allogeneic thymus tissue.

Histology of Interstitial Lung Disease in Common Variable Immune Deficiency.

Interstitial lung disease (ILD) is an important non-infectious complication in several primary immune deficiencies. In common variable immune deficiency (CVID) it is associated with complex clinical phenotypes and adverse outcomes. The histology of ILD in CVID is heterogeneous and mixed patterns are frequently observed within a single biopsy, including non-necrotising granulomatous inflammation, lymphoid interstitial pneumonitis, lymphoid hyperplasia, follicular bronchiolitis, organizing pneumonia, and interstitial fibrosis; ILD has to be differentiated from lymphoma. The term granulomatous-lymphocytic interstitial lung disease (GLILD), coined to describe the histopathological findings within the lungs of patients with CVID with or without multisystem granulomata, is somewhat controversial as pulmonary granulomata are not always present on histology and the nature of infiltrating lymphocytes is variable. In this mini review we summarize the literature on the histology of CVID-related ILD and discuss some of the factors that may contribute to the inter- and intra- patient variability in the histological patterns reported. Finally, we highlight areas for future development. In particular, there is a need for standardization of histological assessments and reporting, together with a better understanding of the immunopathogenesis of CVID-related ILD to resolve the apparent heterogeneity of ILD in this setting and guide the selection of rational targeted therapies in different patients.

Ageing compromises mouse thymus function and remodels epithelial cell differentiation.

Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus.

Biologically indeterminate yet ordered promiscuous gene expression in single medullary thymic epithelial cells.

To induce central T-cell tolerance, medullary thymic epithelial cells (mTEC) collectively express most protein-coding genes, thereby presenting an extensive library of tissue-restricted antigens (TRAs). To resolve mTEC diversity and whether promiscuous gene expression (PGE) is stochastic or coordinated, we sequenced transcriptomes of 6,894 single mTEC, enriching for 1,795 rare cells expressing either of two TRAs, TSPAN8 or GP2. Transcriptional heterogeneity allowed partitioning of mTEC into 15 reproducible subpopulations representing distinct maturational trajectories, stages and subtypes, including novel mTEC subsets, such as chemokine-expressing and ciliated TEC, which warrant further characterisation. Unexpectedly, 50 modules of genes were robustly defined each showing patterns of co-expression within individual cells, which were mainly not explicable by chromosomal location, biological pathway or tissue specificity. Further, TSPAN8+ and GP2+ mTEC were randomly dispersed within thymic medullary islands. Consequently, these data support observations that PGE exhibits ordered co-expression, although mechanisms underlying this instruction remain biologically indeterminate. Ordered co-expression and random spatial distribution of a diverse range of TRAs likely enhance their presentation and encounter with passing thymocytes, while maintaining mTEC identity.

Sequencing of human genomes with nanopore technology.

Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.

FOXN1 deficient nude severe combined immunodeficiency.

Nude severe combined immunodeficiency is a rare inherited disease caused by autosomal recessive loss-of-function mutations in FOXN1. This gene encodes a transcription factor essential for the development of the thymus, the primary lymphoid organ that supports T-cell development and selection. To date nine cases have been reported presenting with the clinical triad of absent thymus resulting in severe T-cell immunodeficiency, congenital alopecia universalis and nail dystrophy. Diagnosis relies on testing for FOXN1 mutations, which allows genetic counselling and guides therapeutic management. Options for treating the underlying immune deficiency include HLA-matched genoidentical haematopoietic cell transplantation containing mature donor T-cells or thymus tissue transplantation. Experience from other severe combined immune deficiency syndromes suggests that early diagnosis, supportive care and definitive management result in better patient outcomes. Without these the prognosis is poor due to early-onset life threatening infections.

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.

Challenges in the Role of Gammaglobulin Replacement Therapy and Vaccination Strategies for Hematological Malignancy.

Patients with chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are prone to present with antibody production deficits associated with recurrent or severe bacterial infections that might benefit from human immunoglobulin (Ig) (IVIg/SCIg) replacement therapy. However, the original IVIg trial data were done before modern therapies were available, and the current indications do not take into account the shift in the immune situation of current treatment combinations and changes in the spectrum of infections. Besides, patients affected by other B cell malignancies present with similar immunodeficiency and manifestations while they are not covered by the current IVIg indications. A potential beneficial strategy could be to vaccinate patients at monoclonal B lymphocytosis and monoclonal gammopathy of undetermined significance stages (for CLL and MM, respectively) or at B-cell malignancy diagnosis, when better antibody responses are attained. We have to re-emphasize the need for assessing and monitoring specific antibody responses; these are warranted to select adequately those patients for whom early intervention with prophylactic anti-infective therapy and/or IVIg is preferred. This review provides an overview of the current scenario, with a focus on prevention of infection in patients with hematological malignancies and the role of Ig replacement therapy.

Secondary immunodeficiency in lymphoproliferative malignancies.

Secondary immunodeficiencies occur as a consequence of various diseases, including hematological malignancies, and the use of pharmacological therapies, such as immunosuppressive, anti-inflammatory, and biological drugs. Infections are the main cause of morbidity and mortality in multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) patients. Recent advances in treatment have prolonged the duration of remission and the time between relapse phases in MM and CLL patients. However, managing multiple relapses and the use of salvage therapies can lead to cumulative immunosuppression and a higher risk of infections. The pathogenesis of immune deficiency secondary to lymphoproliferative malignancy is multifactorial including disease- and treatment-related factors. Supportive treatment, including early vaccination, anti-infective prophylaxis, and replacement immunoglobulin, plays a key role in preventing infections in MM and CLL. This article provides an overview of the basic immunology necessary to understand the pathogenesis of secondary immunodeficiency and the infectious complications in MM and CLL. We also discuss the evidence supporting the role of prophylactic replacement immunoglobulin treatment in patients with antibody failure secondary to MM and CLL and the indications for its use. Copyright © 2016 John Wiley & Sons, Ltd.

Secondary antibody deficiencies.

PURPOSE OF REVIEW: Antibody deficiency can occur in the context of primary immune deficiency due to inherited genetic defects or secondary to a variety of causes. This review aims to summarize current data concerning the causes of secondary antibody deficiency and where possible evidence regarding the use of prophylactic replacement immunoglobulin. (Figure is included in full-text article.) RECENT FINDINGS: Advances in immune-mediated therapies ranging from monoclonal antibodies to novel B-cell-targeted therapeutics are responsible for an expansion in the possible iatrogenic causes of antibody deficiency. SUMMARY: Causes of secondary antibody deficiency include B-cell lymphoproliferative disease, notably chronic lymphocytic leukaemia and multiple myeloma, protein losing states, disorders of lymphatic circulation, increased immunoglobulin catabolism and a growing number of therapeutic agents. At-risk patients should be closely monitored for the development of hypogammaglobulinaemia, B-cell function should be defined where appropriate with specific antibody responses to immunization antigens and where there is a significant burden of infections patients should be treated with prophylactic antibiotics and/or replacement immunoglobulin.

Identification of a novel mutation in MAGT1 and progressive multifocal leucoencephalopathy in a 58-year-old man with XMEN disease.

XMEN disease (X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus infection and Neoplasia) is a novel primary immune deficiency caused by mutations in MAGT1 and characterised by chronic infection with Epstein-Barr virus (EBV), EBV-driven lymphoma, CD4 T-cell lymphopenia, and dysgammaglobulinemia [1]. Functional studies have demonstrated roles for magnesium as a second messenger in T-cell receptor signalling [1], and for NKG2D expression and consequently NK- and CD8 T-cell cytotoxicity [2]. 7 patients have been described in the literature; the oldest died at 45 years and was diagnosed posthumously [1-3]. We present the case of a 58-year-old Caucasian gentleman with a novel mutation in MAGT1 with the aim of adding to the phenotype of this newly described disease by detailing his clinical course over more than 20 years.

Antibody deficiency secondary to chronic lymphocytic leukemia: Should patients be treated with prophylactic replacement immunoglobulin?

Hypogammaglobulinemia is a common finding in chronic lymphocytic leukemia (CLL). Its incidence increases with disease duration and stage such that it is present in up to 85 % of patients at some point in their disease course. It is therefore important to monitor patients for the development of an antibody deficiency. However, not all patients with antibody deficiency secondary to CLL are symptomatic with bacterial infections. In addition patients are susceptible to viral, fungal and opportunistic infections as a result of iatrogenic immunosuppression and through a variety of disease-related mechanisms, which affect cellular immunity and phagocytes. Published guidelines suggest that patients with a history of recurrent bacterial infections and a documented failure of antibody production should be treated with antibiotic prophylaxis in the first instance, with replacement immunoglobulin reserved for those who continue to suffer with significant bacterial infections. Here we present a review of the existing literature in order to provide a practical approach, based on best available evidence, to the investigation, monitoring and treatment of patients with antibody failure secondary to CLL; and we highlight areas in which further studies are needed.