Silje F. Jørgensen

Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders

Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. Objective: We sought to investigate the ability of whole‐exome screening methods to detect disease‐causing variants in patients with PIDDs. Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole‐exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome‐tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD‐causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. Conclusion: This high‐throughput genomic approach enabled detection of disease‐related variants in unexpected genes; permitted detection of low‐grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

Association of CLEC16A with human common variable immunodeficiency disorder and role in murine B cells.

Common variable immunodeficiency disorder (CVID) is the most common symptomatic primary immunodeficiency in adults, characterized by B cell abnormalities and inadequate antibody response. CVID patients have considerable autoimmune comorbidity and we therefore hypothesized that genetic susceptibility to CVID may overlap with autoimmune disorders. Here, in the largest genetic study performed in CVID to date, we compare 778 CVID cases with 10,999 controls across 123,127 single nucleotide polymorphisms (SNPs) on the Immunochip. We identify the first non-HLA genome-wide significant risk locus at CLEC16A (rs17806056, P=2.0×10−9) and confirm the previously reported human leukocyte antigen (HLA) associations on chromosome 6p21 (rs1049225, P =4.8×10−16). Clec16a knock down (KD) mice showed reduced number of B cells and elevated IgM levels compared to controls, suggesting that CLEC16A may be involved in immune regulatory pathways of relevance to CVID. In conclusion, the CLEC16A associations in CVID represent the first robust evidence of non-HLA associations in this immunodeficiency condition.

Altered gut microbiota profile in common variable immunodeficiency associates with levels of lipopolysaccharide and markers of systemic immune activation

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by low immunoglobulin (Ig)G and IgA, and/or IgM. In addition to bacterial infections, a large subgroup has noninfectious inflammatory and autoimmune complications. We performed 16S ribosomal RNA-based profiling of stool samples in 44 CVID patients, 45 patients with inflammatory bowel disease (disease controls), and 263 healthy controls. We measured plasma lipopolysaccharide (LPS) and markers of immune cell activation (i.e., soluble (s) CD14 and sCD25) in an expanded cohort of 104 patients with CVID and in 30 healthy controls. We found a large shift in the microbiota of CVID patients characterized by a reduced within-individual bacterial diversity (alpha diversity, P<0.001) without obvious associations to antibiotics use. Plasma levels of both LPS (P=0.001) and sCD25 (P<0.0001) were elevated in CVID, correlating negatively with alpha diversity and positively with a dysbiosis index calculated from the taxonomic profile. Low alpha diversity and high dysbiosis index, LPS, and immune markers were most pronounced in the subgroup with inflammatory and autoimmune complications. Low level of IgA was associated with decreased alpha diversity, but not independently from sCD25 and LPS. Our findings suggest a link between immunodeficiency, systemic immune activation, LPS, and altered gut microbiota.

A Cross-Sectional Study of the Prevalence of Gastrointestinal Symptoms and Pathology in Patients With Common Variable Immunodeficiency.

OBJECTIVES:The objective of this study was to study the prevalence of gastrointestinal (GI) symptoms and histopathology in patients with common variable immunodeficiency (CVID) as well as linking the findings to GI infections and markers of systemic immune activation.METHODS:In this cross-sectional study, we addressed GI symptoms in 103 patients and GI histopathological findings in 53 patients who underwent upper and lower endoscopic examination. The most frequent histopathological findings were linked to GI symptoms, B-cell phenotype, and markers of systemic immune activation (soluble (s)CD14, sCD25, and sCD163). Microarray analysis compared “celiac-like disease” in CVID to celiac disease. Screening for selected bacterial and viral infections in fecal samples and gut mucosal biopsies was performed.RESULTS:The main findings of this study were as follows: most common GI symptoms were bloating (34%), pain (30%), and diarrhea (26%). The most frequent histopathological findings were increased intraepithelial lymphocytes in the descending part of the duodenum, i.e., “celiac-like disease” (46% of patients), decreased numbers of plasma cells in GI tract mucosa (62%), and lymphoid hyperplasia (38%), none of which were associated with GI symptoms. Reduced plasma cells in GI mucosa were associated with B-cell phenotypic characteristics of CVID, and increased serum levels of sCD14 (P=0.025), sCD25 (P=0.01), and sCD163 (P=0.04). Microarray analyses distinguished between CVID patients with “celiac-like disease” and celiac disease. Positive tests for bacterial and viral infections were scarce both in fecal samples and gut mucosal biopsies, including PCR test for norovirus in biopsy specimens (0 positive tests).CONCLUSIONS:In conclusion, GI pathology is common in CVID, but does not necessarily cause symptoms. However, reduced plasma cells in GI mucosa were linked to systemic immune activation, “celiac-like disease” in CVID and true celiac disease appear to be different disease entities, as assessed by gene expression, and infections (including norovirus) are rarely a cause of the CVID enteropathy.

IRF4 Is a Critical Gene in Retinoic Acid–Mediated Plasma Cell Formation and Is Deregulated in Common Variable Immunodeficiency–Derived B Cells

In the present study, we aimed at identifying the mechanisms whereby the vitamin A metabolite all-trans retinoic acid (RA) promotes the formation of plasma cells upon stimulation of B cells via the innate immunity receptors TLR9 and RP105. Most often, differentiation of B cells involves the sequential events of class switch recombination and somatic hypermutations characteristic of germinal center reactions, followed by plasma cell formation. By studying the regulatory networks known to drive these reactions, we revealed that RA enhances the expression of the plasma cell–generating transcription factors IFN regulatory factor (IRF)4 and Blimp1, and paradoxically also activation-induced deaminase (AID) involved in somatic hypermutations/class switch recombination, in primary human B cells. IRF4 was identified as a particularly important protein involved in the RA-mediated production of IgG in TLR9/RP105-stimulated B cells. Based on kinetic studies, we present a model suggesting that the initial induction of IRF4 by RA favors AID expression. According to this model, the higher level of IRF4 that eventually arises results in sustained elevated levels of Blimp1. Regarded as a master regulator of plasma cell development, Blimp1 will in turn suppress AID expression and drive the formation of IgG-secreting plasma cells. Notably, we demonstrated IRF4 to be deregulated in B cells from common variable immunodeficiency patients, contributing to the observed aberrant expression of AID in these patients. Taken together, the present study both provides new insight into the mechanisms whereby RA induces differentiation of B cells and identifies IRF4 as a key to understand the defective functions of B cells in common variable immunodeficiency patients.

Liver transplantation in patients with primary antibody deficiency.

In summary, we have engineered an allergen-specific mAb targeting Blo t 5, the major allergen of the HDM species B tropicalis, and showed that this mAb is capable of antagonizing Blo t 5–specific IgE binding in patients’ sera. More critically, we have demonstrated, for the first time, the protective effects of an allergen-specific mAb treatment in a murine model of severe allergic asthma driven by a clinically relevant and dominant allergen instead of the traditional ovalbumin-induced asthma. It is also important to recognize that murine models with asthma may not reflect the spectrum of immunologic and physiologic changes that underlie the pathology of established asthma in humans. However, airway remodeling, an important hallmark of chronic asthma that is often missing in animal models, was prominent in the mouse model used in this study and anti–Blo t 5 mAb administration was capable of ameliorating this phenomenon. Last, we have used chimeric antibodies to highlight the potential of using allergen-specific IgG4 antibodies in inhibiting basophil activation. Although immunogenic murine components limit the prolonged use of chimeric antibodies in clinical settings, full antibody humanization can potentially reduce the likelihood of this problem arising. This potentially mirrors the approach used when anti–TNF-a is used for chronic inflammatory diseases. Taken together, this work has important implications on the current understanding of allergic asthma and may have an impact on future design of targeted therapeutics for an allergic disease lacking an optimal treatment regime. J. H. Sherlynn Chan, BSc(Hons) Yen Leong Chua, MSc Hong Yong Peh, BSc(Hons) Vojislav Jovanovic, PhD Nicholas R. J. Gascoigne, PhD W. S. Fred Wong, PhD Fook Tim Chew, PhD Brendon J. Hanson, PhD David M. Kemeny, PhD, FRCPath Paul A. MacAry, PhD From Immunology Programme, Life Sciences Institute, the Department of Microbiology & Immunology, Yong Loo Lin School ofMedicine, and NUSGraduate School for Integrative Sciences and Engineering (NGS), National University of Singapore, Singapore; the Department of Pharmacology, Yong Loo Lin School of Medicine, National University Health System, Singapore; the Department of Biological Sciences, National University of Singapore, Singapore, and the Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore. E-mail: paul_macary@nuhs.edu.sg. This work was supported by the National Research Foundation (grant no. NRF370062HUJ-NUS). S.J. H.C. was supported by the NUS Graduate School for Integrative Sciences and Engineering (NGS) scholarship. Disclosure of potential conflict of interest: J. H. Sherlynn Chan, V. Jovanovic, and P. A. MacAry receive grant support from the National Research Foundation, Singapore. N. R. J. Gascoigne receives grant support from the National Medical Research Council and Ministry of Education (MOE) and travel support from the Japan Society of Immunology and European Molecular Biology Organization (EMBO). F. T. Chew receives grant support from the Singapore Ministry of Education Academic Research Fund, the Singapore Immunology Network, the National Medical Research Council (Singapore), and the Agency for Science Technology and Research (A*STAR) (Singapore); serves as a consultant for Sime Darby Technology Centre, First Resources Ltd, and Olam International; and is an employee of National University of Singapore. B. J. Hanson is an employee of National University of Singapore Life Sciences Institute. D. M. Kemeny receives grant support from the National Medical Research Council. The rest of the authors declare that they have no relevant conflicts of interest.

Patterns of constitutively phosphorylated kinases in B cells are associated with disease severity in common variable immunodeficiency.

Patients with common variable immunodeficiency (CVID) constitute a clinically and immunologically heterogeneous group characterized by B-cell dysfunction with hypogammaglobulinemia and defective immunoglobulin class switch of unknown etiology. Current classification systems are insufficient to achieve precise disease management. Characterization of signaling pathways essential for B-cell differentiation and class switch could provide new means to stratify patients. We evaluated constitutive and induced signaling by phospho-specific flow cytometry in 26 CVID patients and 18 healthy blood donors. Strong responses were induced both in CVID and healthy donor B cells upon activation. In contrast, constitutive phosphorylation levels of STAT3,-5,-6, Erk, PLC-γ and Syk were significantly increased in CVID B cells only. Hierarchical clustering revealed a subgroup of CVID patients with elevated constitutive phosphorylation of Syk and PLC-γ. All these patients had non-infectious complications, indicating that a distinct phosphorylation pattern of kinases in B cells identifies a clinically important subgroup of CVID patients.

Genetic and transcriptional analysis of inflammatory bowel disease-associated pathways in patients with GUCY2C-linked familial diarrhea

Abstract Objective: Activating mutations in the GUCY2C gene, which encodes the epithelial receptor guanylate cyclase C, cause diarrhea due to increased loss of sodium chloride to the intestinal lumen. Patients with familial GUCY2C diarrhea syndrome (FGDS) are predisposed to inflammatory bowel disease (IBD). We investigated whether genes in the guanylate cyclase C pathway are enriched for association with IBD and reversely whether genetic or transcriptional changes associated with IBD are found in FGDS patients. Methods: (1) A set of 27 genes from the guanylate cyclase C pathway was tested for enrichment of association with IBD by Gene Set Enrichment Analysis, using genome-wide association summary statistics from 12,882 IBD patients and 21,770 controls. (2) We genotyped 163 known IBD risk loci and sequenced NOD2 in 22 patients with FGDS. Eight of them had concomitant Crohns disease. (3) Global gene expression analysis was performed in ileal tissue from patients with FGDS, Crohns disease and healthy individuals. Results: The guanylate cyclase C gene set showed a significant enrichment of association in IBD genome-wide association data. Risk variants in NOD2 were found in 7/8 FGDS patients with concomitant Crohns disease and in 2/14 FDGS patients without Crohns disease. In ileal tissue, downregulation of metallothioneins characterized FGDS patients compared to healthy controls. Conclusions: Our results support a role of guanylate cyclase C signaling and disturbed electrolyte homeostasis in development of IBD. Furthermore, downregulation of metallothioneins in the ileal mucosa of FGDS patients may contribute to IBD development, possibly alongside effects from NOD2 risk variants.

CVID and Celiac Disease

4 Institute of Clinical Medicine, University of Oslo , Oslo , Norway ; 5 Section of Gastroenterology, Department of Transplantation Medicine, Oslo University Hospital Rikshospitalet , Oslo , Norway . Correspondence: Dr Silje F. Jørgensen, MD, Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet , Postbox 4950, Nydalen , Oslo 0424 , Norway . E-mail: s.f.jorgensen@medisin.uio.no