Patients with inherited CARMIL2 or CD28 deficiency have defective T cell CD28 signaling, but their immunological and clinical phenotypes remain largely unknown. We show that only one of three CARMIL2 ...isoforms is produced and functional across leukocyte subsets. Tested mutant CARMIL2 alleles from 89 patients and 52 families impair canonical NF-κB but not AP-1 and NFAT activation in T cells stimulated via CD28. Like CD28-deficient patients, CARMIL2-deficient patients display recalcitrant warts and low blood counts of CD4+ and CD8+ memory T cells and CD4+ TREGs. Unlike CD28-deficient patients, they have low counts of NK cells and memory B cells, and their antibody responses are weak. CARMIL2 deficiency is fully penetrant by the age of 10 yr and is characterized by numerous infections, EBV+ smooth muscle tumors, and mucocutaneous inflammation, including inflammatory bowel disease. Patients with somatic reversions of a mutant allele in CD4+ T cells have milder phenotypes. Our study suggests that CARMIL2 governs immunological pathways beyond CD28.
Abstract
Background
Children with very early onset inflammatory bowel diseases (VEO-IBD) often have a refractory and severe disease course. A significant number of described VEO-IBD-causing monogenic ...disorders can be attributed to defects in immune-related genes. The diagnosis of the underlying primary immunodeficiency (PID) often has critical implications for the treatment of patients with IBD-like phenotypes.
Methods
To identify the molecular etiology in 5 patients from 3 unrelated kindred with IBD-like symptoms, we conducted whole exome sequencing. Immune workup confirmed an underlying PID.
Results
Whole exome sequencing revealed 3 novel CARMIL2 loss-of-function mutations in our patients. Immunophenotyping of peripheral blood mononuclear cells showed reduction of regulatory and effector memory T cells and impaired B cell class switching. The T cell proliferation and activation assays confirmed defective responses to CD28 costimulation, consistent with CARMIL2 deficiency.
Conclusion
Our study highlights that human CARMIL2 deficiency can manifest with IBD-like symptoms. This example illustrates that early diagnosis of underlying PID is crucial for the treatment and prognosis of children with VEO-IBD.
Patients with inherited CARMIL2 deficiency can present with pediatric inflammatory bowel disease. Early diagnosis of the underlying primary immunodeficiency has critical implications for the clinical management of affected children.
Purpose
RAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency (PID) characterized by CD4
+
T cell lymphopenia and Epstein-Barr virus ...(EBV)-associated B cell lymphoma. Our report of three novel patients widens the scope of RASGRP1 deficiency by providing new clinical and immunological insights on autoimmunity, immune cell development, and predisposition to lymphoproliferative disease.
Methods
One patient of Turkish origin (P1) and two Palestinian patients (P2, P3) were evaluated for immunodeficiency. To decipher the molecular cause of disease, whole exome sequencing was conducted. Identified mutations were validated by immunological and biochemical assays.
Results
We report three patients presenting with similar clinical characteristics of immunodeficiency and EBV-associated lymphoproliferative disease. In addition, P2 and P3 exhibited overt autoimmune manifestations. Genetic screening identified two novel loss-of-function mutations in
RASGRP1
. Immunoblotting and active Ras pull-down assays confirmed perturbed ERK1/2 signaling and reduced Ras-GTPase activity in heterologous Jurkat cells with ectopic expression of RASGRP1 mutants. All three patients had CD4
+
T cell lymphopenia. P2 and P3 showed decreased mitogen-induced lymphocyte proliferation, reduced T cell receptor excision circles, abnormal T cell receptor (TCR) Vβ repertoires, and increased frequencies of TCRγδ cells. TCR gamma repertoire diversity was significantly reduced with a remarkable clonal expansion.
Conclusions
RASGRP1 deficiency is associated with life-threatening immune dysregulation, severe autoimmune manifestations, and susceptibility to EBV-induced B cell malignancies. Early diagnosis is critical and hematopoietic stem cell transplantation might be considered as curative treatment.
& I. Somekh and B. Marquardt contributed equally
§ C. Klein and R. Somech contributed equally
Background
RAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary ...immunodeficiency disorder in one patient (Salzer et al, Nat Immunol 2016). Clinically, the disease was characterized by recurrent episodes of pneumonia, bronchiectasis, severe failure to thrive, CD4 T-cell lymphopenia, elevated TCRγδ cell proportions as well as low-grade Epstein-Barr virus (EBV)-associated B cell lymphoma. Immunologically, RASGRP1 deficiency is associated with defective proliferation and activation of T and B cells, respectively. Here, we report 3 novel patients with RASGRP1 deficiency from 2 unrelated consanguineous kindred.
Methods
One patient of Turkish origin (P1) and 2 Palestinian 1st degree cousins (P2, P3) were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material and genetically engineered T-cell lines using CRISPR/Cas9.
Results
Clinical findings for all three patients included failure to thrive, hepatosplenomegaly, lymphadenopathy, and recurrent pulmonary infections resulting in bronchiectasia. Two patients had overt auto-immunity (AIHA and autoimmune thrombocytopenia, respectively). One patient had increased anti-nuclear antibody titers. One patient (P1) developed EBV-associated diffuse large B cell lymphoma. He underwent autologous hematopoietic stem cell transplant due to relapse after combined rituximab and NHL-BFM-90 treatment protocol and is currently in remission at the age of 14 years. P2 had deceased at the age of 3 years due to disseminated intravascular coagulation. P3 developed diffuse large B cell lymphoma at the age of 3 years, treated with R-CHOP protocol and is currently in remission.
Immune workup of P2 and P3 revealed low levels of T-cell receptor excision circles (TREC), an abnormal T-cell receptor (TCR) Vß repertoire and a markedly increased proportion of TCRγδ cells. Phytohemagglutinin (PHA)-induced lymphocyte proliferation and anti-CD3 mitogen proliferation responses were decreased. EBV-viral load in peripheral blood was elevated for both patients, as measured by PCR.
Genetic analysis by whole exome sequencing identified an autosomal recessive homozygous mutations in RASGRP1. P1 had an inversion (c.649_650inv; p.Glu217Ser) and P2/P3 had a deleterious frameshift mutation (c.1111_1114del; p.Asp371Ilefs*7). The variants were confirmed by Sanger sequencing and segregated with the disease phenotype. To confirm the functional relevance of the RASGRP1 mutations, we engineered Jurkat cells via CRISPR/Cas9 editing. RASGRP1 -/- Jurkat cells showed decreased phosphorylation of ERK1/2 and decreased CD69 expression in comparison to wildtype cells. Jurkat cells with RASGRP1 knockin mutants are currently under analysis.
Conclusions
RASGRP1 deficiency is a novel primary immunodeficiency disorder associated with immune dysregulation and susceptibility to EBV-induced B-cell malignancies. Early diagnosis of RASGRP1 deficiency is critical. Even though clinical remission of EBV-lymphoma may be reached using high dose chemotherapy regimens, long term cures may only be reached by allogeneic hematopoietic stem cell transplantation.
No relevant conflicts of interest to declare.
Genetic defects of pediatric-onset inflammatory bowel disease (IBD) provide critical insights into molecular factors controlling intestinal homeostasis. NOX1 has been recently recognized as a major ...source of reactive oxygen species (ROS) in human colonic epithelial cells. Here we assessed the functional consequences of human NOX1 deficiency with respect to wound healing and epithelial migration by studying pediatric IBD patients presenting with a stop-gain mutation in NOX1.
Functional characterization of the NOX1 variant included ROS generation, wound healing, 2-dimensional collective chemotactic migration, single-cell planktonic migration in heterologous cell lines, and RNA scope and immunohistochemistry of paraffin-embedded patient tissue samples.
Using exome sequencing, we identified a stop-gain mutation in NOX1 (c.160C>T, p.54R>*) in patients with pediatric-onset IBD. Our studies confirmed that loss-of-function of NOX1 causes abrogated ROS activity, but they also provided novel mechanistic insights into human NOX1 deficiency. Cells that were NOX1-mutant showed impaired wound healing and attenuated 2-dimensional collective chemotactic migration. High-resolution microscopy of the migrating cell edge revealed a reduced density of filopodial protrusions with altered focal adhesions in NOX1-deficient cells, accompanied by reduced phosphorylation of p190A. Assessment of single-cell planktonic migration toward an epidermal growth factor gradient showed that NOX1 deficiency is associated with altered migration dynamics with loss of directionality and altered cell-cell interactions.
Our studies on pediatric-onset IBD patients with a rare sequence variant in NOX1 highlight that human NOX1 is involved in regulating wound healing by altering epithelial cytoskeletal dynamics at the leading edge and directing cell migration.
Introduction: Acute leukemia is the most common malignancy in children and develops within the bone marrow. Consequently, bone marrow derived T cells of leukemia patients can be defined as tumor ...infiltrating lymphocytes (TILs). Dysfunctional TILs have been described in several other malignancies. However, in pediatric patients the interaction between leukemic blasts and TILs remains largely unknown. In order to understand the impact of leukemic blasts on bone marrow T cells we profiled T cells in the bone marrow of pediatric leukemia patients by surface marker and transcriptome wide analysis.
Methods: First, artificial changes in marker expression due to cryopreservation and thawing were ruled out (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (HD, n=23) were identified in a local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T cells in primary (n=49) and relapse leukemia samples (n=28). As we could not detect surface CTLA4 expression on T cells, CTLA4 was stained intracellularly. Additionally, RNA-Seq on sorted bone marrow derived CD8 T cells (n=48; TCP-ALL: 12, AML: 20, HD: 16) was performed. Analysis of RNA-Seq data was based on Reads Per Kilobase Million (RPKM) normalization and False Discovery Rate (FDR, Benjamini-Hochberg) statistics. 172 differentially expressed genes were found when comparing bone marrow derived CD8 T cells from healthy donors (n=16) and leukemia patients (n=32) using the following criteria: RPKM>2 in both groups, fold change>2 and FDR<0.05).
Results: The frequency of bone marrow T cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p<0.001). This reduction was more pronounced in BCP-ALL than in AML (0.9% vs. 8.4%, p<0.001). LAG3 and CTLA4 protein expression of T cells was increased in leukemia patients vs. healthy controls (LAG3: CD4: 2.6% vs. 0.7%, p<0.001; CD8: 8.6% vs. 2.2%, p<0.001; CTLA4: CD4: 7.3% vs. 3.8%, p=0.001; CD8: 1.2 vs. 0.3%, p<0.001). For CD8 T cells, those findings could be confirmed by RNA-Seq of sorted CD8 T cells (LAG3: 60.4 vs. 23.3 (RPKM), FDR=0.0044; CTLA4: 28.7 vs 4.7 (RPKM), FDR=0.046). Equally, TIM3 on T cells showed higher expression in leukemia patients vs. healthy controls (CD4: 3.7% vs. 1.3%, p=0.002; CD8: 8.5% vs. 3.3%, p<0.001). However, the same analysis of RNA-Seq data on sorted CD8 T cells did not yield a significant difference (18.1 vs. 5.6 (RPKM), FDR=0.29). PD1 was the only surface marker found to be more highly expressed in relapse samples than in primary diagnosis samples than in healthy controls (CD4: 42.3% vs. 28.9% vs. 19.8%, p<0.001; CD8: 45.2% vs. 33.3% vs. 26.5%, p=0.002). For CD8 T cells, RNA-Seq did not recapitulate this finding as no significant difference of PD1 transcript abundancy could be observed between leukemia patients and healthy donors by RNA-Seq (21.4 vs. 16.9 (RPKM), FDR=0.92). Finally, RNA-Seq on sorted CD8 T cells showed a pronounced overexpression of genes that are involved in the cytotoxic granule machinery in leukemia patients indicating an increase of effector phenotype in those cells. Contrarily, genes crucial for T cell function and memory formation were significantly downregulated in CD8 T cells from leukemia patients.
Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls we confirm that bone marrow T cells of leukemia patients show signs of exhaustion compared to healthy individuals. Importantly, PD1 surface expression on T cells was identified as a marker that correlates with disease status (relapse > primary > healthy). A significant increase of exhaustion markers could be demonstrated both on protein and transcriptome level (LAG3, CTLA4) or on protein level only (TIM3, PD1). Moreover, we observed an increase of many elements of the cytotoxic granule machinery which is compatible with a loss of naïve/memory CD8 T cells. Additionally, genes essential for T cell memory formation were found to be downregulated in CD8 T cells from leukemia patients. These findings reflect an insufficient immune surveillance of pediatric leukemia by bone marrow T cells and may provide a rationale for future therapeutic interventions.
No relevant conflicts of interest to declare.
Various autoimmune diseases may be associated with primary immune deficiencies. We reported a case with a loss-of-function mutation in DNASE1L3, a gene described previously in families with systemic ...lupus erythematosus. In addition, the patient showed a novel homozygous missense variant in DOCK8, a gene known to be responsible for the hyper-IgE recurrent infection syndrome (HIES). A 3-year-old girl born to consanguine parents presented with chronic urticarial rash, hemolytic anemia, pulmonary hemorrhage, and hypovolemic shock findings. She had a low hemoglobin level, a positive direct antiglobulin test, antinuclear antibody and anti-double stranded DNA, low C3 and C4, third-degree tricuspid regurgitation, and severe enlargement of the right ventricle on echocardiography, suggesting pulmonary embolism. Despite treatment with intravenous immunoglobulin, pulse metilprednisolone, rituximab, and supportive treatment for shock, the patient died on the seventh day. Whole-exome sequencing indicated a homozygous stop variant c.537G>A (p. Trp179Ter) in DNASE1L3. In addition, a possibly pathogenic homozygous missense variant in the HIES gene DOCK8 was detected. The occurrence of potentially clinically relevant, genetic variants in several genes posed various challenges with respect to diagnosis, treatment, and prognosis.
C-terminal variants in CDC42 encoding cell division control protein 42 homolog underlie neonatal-onset cytopenia, autoinflammation, rash, and hemophagocytic lymphohistiocytosis (NOCARH). Pyrin ...inflammasome hyperactivation has been shown to contribute to disease pathophysiology. However, mortality of NOCARH patients remains high despite inflammasome-focused treatments. Here, we demonstrate in four NOCARH patients from three families that cell-intrinsic activation of type I interferon (IFN) is a previously unrecognized driver of autoinflammation in NOCARH. Our data show that aberrant innate immune activation is caused by sensing of cytosolic nucleic acids released from mitochondria, which exhibit disturbances in integrity and dynamics due to CDC42 dysfunction. In one of our patients, treatment with the Janus kinase inhibitor ruxolitinib led to complete remission, indicating that inhibition of type I IFN signaling may have an important role in the management of autoinflammation in patients with NOCARH.
•C-terminal mutations in CDC42 lead to a previously unrecognized type I interferonopathy in NOCARH syndrome.•Autoinflammation is caused by innate immune activation due to sensing of cytosolic nucleic acids released from mitochondria.•JAK1/2 inhibition with ruxolitinib can control autoinflammatory symptoms in patients with NOCARH syndrome.
Aminoacyl‐tRNA synthetases (ARSs) catalyze the first step of protein biosynthesis (canonical function) and have additional (non‐canonical) functions outside of translation. Bi‐allelic pathogenic ...variants in genes encoding ARSs are associated with various recessive mitochondrial and multisystem disorders. We describe here a multisystem clinical phenotype based on bi‐allelic mutations in the two genes (FARSA, FARSB) encoding distinct subunits for tetrameric cytosolic phenylalanyl‐tRNA synthetase (FARS1). Interstitial lung disease with cholesterol pneumonitis on histology emerged as an early characteristic feature and significantly determined disease burden. Additional clinical characteristics of the patients included neurological findings, liver dysfunction, and connective tissue, muscular and vascular abnormalities. Structural modeling of newly identified missense mutations in the alpha subunit of FARS1, FARSA, showed exclusive mapping to the enzyme's conserved catalytic domain. Patient‐derived mutant cells displayed compromised aminoacylation activity in two cases, while remaining unaffected in another. Collectively, these findings expand current knowledge about the human ARS disease spectrum and support a loss of canonical and non‐canonical function in FARS1‐associated recessive disease.
Bi‐allelic variants in the two genes (FARSA, FARSB) encoding distinct subunits of tetrameric cytosolic phenylalanyl‐tRNA synthetase (FARS1) cause a similar clinical phenotype that affects multiple organ systems.
RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an ...early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations.
Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts.
We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect.
De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome.
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