EOMES is a transcription factor involved in the activation and differentiation of effector CD8 T-lymphocytes and NK-cells (Pearce, 2003). Conditional EOMES-KO mice demonstrated its role in anti-viral immune response (Paley, 2012). We studied a patient with a mutation in EOMES who died from fulminant EBV infection.
Whole exome sequencing, Immunofluorescence, EMSA, Crispr-Cas9
We investigated a patient born from a consanguineous family presenting with fatal T-cell lymphoproliferation associated with hemophagocytic lymphohistiocytosis following primary EBV infection at the age of 4. Histological staining of lymph node sections revealed high proportions of EBV-infected T-cells. By WES, we identified a homozygous missense mutation, p.346G>S, in the highly conserved T-box domain of EOMES. The G346S EOMES protein was expressed similarly as the wild-type. Using EMSA, we also confirmed that the mutated protein binds DNA as the WT. However, we observed by immunofluorescence that the G346S EOMES accumulated in the nucleus forming aggregates, which were not observed with the WT, suggesting a greater binding of the mutant protein to DNA. Having no material from the patient, we developed a knock-in mouse using CRISP-Cas9 technology mimicking the human mutation. Preliminary analysis showed no phenotypic abnormalities in the CD8 and CD4 T-cell populations. However, NK-cell development was abnormal with an accumulation of mature NK-cells. In vivo studies of the anti-viral response with LCMV are ongoing.
We identified a missense mutation in EOMES in patient with a fatal EBV infection that may explain this severe immunodeficiency. Additional studies are needed to formally establish the deleterious nature of this mutation.
The molecular cause of severe congenital neutropenia (SCN) is unknown in 30-50% of patients. SEC61A1 encodes the α subunit of the heterotrimeric Sec61 complex, which governs ER signal peptide-dependent protein transport and passive calcium leakage. Recently autosomal dominant mutations in SEC61A1 were reported to be pathogenic in CVID and glomerulocystic kidney disease with an anecdotical association with neutropenia (p.V67G ) in a single family. However the full spectrum of clinical manifestation in SEC61A1 mutations is yet to be explored. We investigated a SCN patient, only child of healthy non-consanguineous Belgian parents.
WES findings were validated and reported mutations compared in parallel by western blotting, Ca+2 flux assays, differentiation of transduced HL-60 cells using 1% DMSO, in vitro differentiation of primary CD34 cells, qPCR for unfolded protein response genes and 10X Genomics single-cell RNA-sequencing on whole bone marrow.
We identified a de novo private missense mutation in SEC61A1 (c.A275G;p.Q92R; CADD 24.5; msc 6.099) in a patient with SCN. The mutation results in diminished expression in PBMC and fibroblasts and an increase in calcium leakage. In vitro differentiation of CD34+ cells recapitulated the patient’s clinical arrest in granulopoeisis. HL-60 cells stably transduced with p.Q92R-Sec61α1 and p.V67G-Sec61α1 showed reduced differentiation to CD11b+CD16+ cells with decreased CD16 expression compared to both WT-Sec61α1 and p.V85D-Sec61α1, mutation associated with CVID. Analysis of CD34 cells on day 16 of differentiation revealed upregulation of CHOP and BiP indicative of increased unfolded protein response.
Specific autosomal dominant mutations in SEC61A1 cause SCN through upregulation of the unfolded protein response.
AK2 encodes the phosphotransferase adenylate kinase 2 (AK2). Human mutations in AK2 cause reticular dysgenesis, a severe combined immunodeficiency with agranulocytosis, lymphopenia, fatal infections, and sensorineural deafness. We investigated the mechanisms underlying recurrent sino-pulmonary infections and hypogammaglobulinemia in 15 patients, ranging from 3 to 34 years of age, from nine kindreds.
Patient cells were studied with next-generation DNA sequencing, tandem mass spectrometry, and assays of lymphocyte and mitochondrial function.
We identified two different homozygous mutations in AK2. AK2G100S and AK2A182D permit residual protein expression, enzymatic activity, and normal numbers of neutrophils and lymphocytes. All but one patient have intact hearing. The patients’ B cells had severely impaired cell cycling and proliferation, reduced survival, and minimal in vitro immunoglobulin secretion. After activation, the patients’ B cells exhibit defective mitochondrial respiration and impaired clearance of dysfunctional mitochondria. The patients’ T cells demonstrated reduced diversity in Vβ usage in CD4+ and CD8+ T cells and impaired proliferation to antigens. However, in contrast to severely defective B cells function, the patients’ T cell proliferation to phytohemagglutinin was partially impaired in only 3 patients and intact in the remaining patients studied. These findings are consistent with the patients’ limited susceptibility to opportunistic infections and suggest a less strict cellular dependence of T cell function on AK2 activity.
These AK2 hypomorphic mutations demonstrate the importance of mitochondrial function for B cell activation and antibody production.
CD48 is expressed on nearly all cells of haematopoietic origin. CD48 interacts with the 2B4 receptor on NK cells, γδ T lymphocytes and activated cytotoxic T lymphocytes. In humans, 2B4 acts as an activating receptor. Phenotypes of an inherited defect in CD48 have never been described in humans. In a patient suffering from recurrent episodes of fever, rash, bi-cytopenia, hyperferritinaemia and IL-6 hypercytokinaemia, we found a heterozygous mutation in CD48. We sought to assess the consequences of the mutation on CD48 expression, cytotoxic effectiveness and systemic inflammation.
We performed whole exome sequencing on DNA samples of the patient, his brother and parents. RNA and protein expression were measured; flow cytometry and NK-cell killing assays of patient’s cells and killing assays of a transfected cell line were conducted. Signs for systemic inflammation were assessed in lymphocytic choriomeningitis virus (LCMV)-infected CD48-mutant mice.
The patient’s heterozygous mutation led to reduced cell surface expression of CD48, thereby reducing susceptibility to NK-cell mediated killing. A murine model was used to assess the effect of germline CD48 mutation in immune regulation. Viral infection induced hyperferritinaemia and IL-6 hypercytokinaemia in CD48-deficient mice, similar to the pattern observed in the patient during flares.
Our results show that reduced CD48-expression may lead to immune dysregulation. One possible explanation for this immune dysregulation is that the decreased CD48 expression on target cells enables their escape from killing, thereby interfering with the immune-regulatory function of cytotoxic lymphocytes.
HEM1 is a hematopoietic cell-specific essential subunit of the WAVE regulatory complex (WRC) that activates ARP2/3-mediated actin branching and lamellipodia formation. To date no human disease has been linked to mutations in HEM1 (also known as NCKAP1L) and the significance of HEM1 for immunity is unknown.
We performed exome sequencing of Patient1 (P1) to identify the underlying genetic defect. We used patient derived material and characterized a Hem1-/- mouse model for further molecular and cellular analysis.
P1 was diagnosed with SLE due to skin rashes, oral ulcers, photosensitivity, joint pain and positive serum levels of anti-dsDNA antibodies at early age. P2 presented with recurrent upper respiratory tract infections. At a cellular level, HEM1-deficiency led to destabilization of the WRC, reduced filamentous actin and failure to assemble lamellipodia. We show that Hem1-/- mice display systemic autoimmunity and splenomegaly, phenocopying the human disease. Human and murine Hem1-deficient B cells show increased activated and immature/transitional cell populations. Single-cell sequencing of Hem1-/- cells identified dysregulated BAFFR levels suggesting aberrant B cell development resulting in increased B-cell apoptosis and biased differentiation towards autoreactive plasmablasts.
We identified HEM1 deficiency underlying an inborn error of immunity with systemic autoimmunity in the patients. HEM1 deficiency in humans and mice uncovers a pivotal role of the WRC in B-cell differentiation and immune homeostasis.
Severe combined immunodeficiencies (SCIDs) form a heterogeneous group of life-threatening genetic disorders defined by the absence of autologous T cells and an intrinsic or extrinsic defect in the B-cell compartment. We studied three newborn patients with clinical and biological features of SCID associated with bone-marrow hypoplasia and unknown molecular diagnosis. Given the severity of the clinical presentation, the patients had to undergo allogenic hematopoietic stem cell transplantation in the first weeks of life.
Whole-genome sequencing (WES), biochemical analyses and in vitro cultures were performed in order to characterize the molecular defect.
In the three patients we identified the same heterozygous, dominant missense mutation in the RAC2 gene . The mutation, located in the GDP/GTP binding pocket of the RAC2 GTPase protein (p.G12R), is associated with a gain of function. The results of in vitro differentiation assays highlighted that RAC2 signalling pathway has a key function in regulating the survival and differentiation of hematopoietic stem/progenitor cells toward lymphoid and myeloid lineages.
The present study is the first to report an autosomal dominant form of SCID and suggest that RAC2 gene sequencing should be considered for patients with SCID clinical presentation.
Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative mutations in STAT3. The patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, an allergic triad (elevated serum IgE, eosinophilia, and eczema) and extra-hematopoietic manifestations, mostly affecting the skeleton.
We studied 7 patients from 3 unrelated kindred with AD-HIES
We found two heterozygous mutations in IL6ST (I719fs and T761fs), one of which (I719fs) is shared by two unrelated kindreds including one in which it occurred de novo. The two mutant alleles encode truncated GP130 proteins that retain the transmembrane domain, but lack both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the two mutant proteins are loss-of-function for cellular responses to IL-6, IL-11, LIF, and OSM, and hypomorphic for IL-27. They also accumulate at the cell surface and exert a potent dominant-negative effect for cellular responses to IL-6, IL-11, IL-27, LIF and OSM. The patients’ PBMCs and fibroblasts respond poorly to IL-6, IL-11, and IL-27. Like STAT3-mutated patients, IL6ST-mutated patients have infectious and allergic phenotypes seen in complete IL-6R deficiency, and skeletal anomalies seen in complete IL-11R deficiency.
Dominant-negative STAT3 and IL6ST mutations underlie clinical phenocopies by impairing IL-6 and IL-11 responsive pathways.
PU.1, encoded by the gene SPI1, is a pioneer transcription factor that determines hematopoietic cell fate. Although PU.1 deficiency is embryonically lethal in mice and SPI1 mutations are common in human myeloid leukemias, a primary PU.1-mediated disease has not been previously described. We have identified four unrelated agammaglobulinemic subjects each harboring different, novel, non-synonymous heterozygous SPI1mutations. We sought to determine if and how these mutations caused disease.
PU.MA subjects and their family members were identfied and genotyped via whole exome sequencing. Peripheral blood and bone marrow samples were analyzed by flow cytometry, RNA-sequencing and immunohistochemical stains. Mutant PU.1 proteins were identified with immunoblots and interactions with other transcriptional partners explored with co-immunoprecipitation. Nuclear translocation of mutant PU.1 into HEK293 nuclei was measured via con-focal microscopy. Mutant PU.1 binding to PU.1-box nucleotide sequences was assessed with electrophoretic mobility shift assays. Custom PU.1-reporter lines measured the transcriptional effects of PU.1. mutants.
PU.MA subjects lacked blood IgM-expressing B cells. Bone marrow analyses demonstrated pro-B cell arrest. Two mutant SPI1variants encoded proteins with single amino acid substitutions in the ETS DNA-binding domain. A frame-shift variant produced a truncated PU.1 protein and a stop-gain variant failed to be expressed. Expressed mutant proteins homodimerized and IRF4 heterodimerized normally, entered the nucleus efficiently but did not bind the PU.1 consensus nucleotide sequence. Mutant PU.1 failed to drive transcription in reporter lines but did not interfere with wild type PU.1’s ability to do so.
We identified and molecularly describe a new autosomal dominant form of agammaglobulinemia caused by developmental arrest of PU.1 mutated pro-B cells.