Pyruvate kinase deficiency in 29 Turkish patients with two novel intronic variants.

Pyruvate kinase (PK) is a key enzyme of anaerobic glycolysis. The genetic heterogeneity of PK deficiency (PKD) is high, and over 400 unique variants have been identified. Twenty-nine patients who had been diagnosed as PKD genetically in seven distinct paediatric haematology departments were evaluated. Fifteen of 23 patients (65.2%) had low PK levels. The PK:hexokinase ratio had 100% sensitivity for PKD diagnosis, superior to PK enzyme assay. Two novel intronic variants (c.695-1G>A and c.694+43C>T) have been described. PKD should be suspected in patients with chronic non-spherocytic haemolytic anaemia, even if enzyme levels are falsely normal. Total PKLR gene sequencing is necessary for the characterization of patients with PKD and for genetic counselling.

DIAPH1-Deficiency is Associated with Major T, NK and ILC Defects in Humans.

Loss of function mutations in Diaphanous related formin 1 (DIAPH1) are associated with seizures, cortical blindness, and microcephaly syndrome (SCBMS) and are recently linked to combined immunodeficiency. However, the extent of defects in T and innate lymphoid cells (ILCs) remain unexplored. Herein, we characterized the primary T, natural killer (NK) and helper ILCs of six patients carrying two novel loss of function mutation in DIAPH1 and Jurkat cells after DIAPH1 knockdown. Mutations were identified by whole exome sequencing. T-cell immunophenotyping, proliferation, migration, cytokine signaling, survival, and NK cell cytotoxicity were studied via flow cytometry-based assays, confocal microscopy, and real-time qPCR. CD4+ T cell proteome was analyzed by mass spectrometry. p.R351* and p.R322*variants led to a significant reduction in the DIAPH1 mRNA and protein levels. DIAPH1-deficient T cells showed proliferation, activation, as well as TCR-mediated signaling defects. DIAPH1-deficient PBMCs also displayed impaired transwell migration, defective STAT5 phosphorylation in response to IL-2, IL-7 and IL-15. In vitro generation/expansion of Treg cells from naïve T cells was significantly reduced. shRNA-mediated silencing of DIAPH1 in Jurkat cells reduced DIAPH1 protein level and inhibited T cell proliferation and IL-2/STAT5 axis. Additionally, NK cells from patients had diminished cytotoxic activity, function and IL-2/STAT5 axis. Lastly, DIAPH1-deficient patients' peripheral blood contained dramatically reduced numbers of all helper ILC subsets. DIAPH1 deficiency results in major functional defects in T, NK cells and helper ILCs underlining the critical role of formin DIAPH1 in the biology of those cell subsets.

Thiamine status and genes encoding intestinal thiamine transporters and transcription factors in obese subjects.

BACKGROUND AND AIMS: The inconsistent data on thiamine status in obese subjects necessitates an examination of genes associated with intestinal absorption of thiamine. We aimed to reveal thiamine status in obese subjects and examine the expression of SLC19A2/3 genes encoding thiamine transporters and Sp1 transcription factor. METHODS AND RESULTS: Thirty-five adult obese subjects and 11 healthy controls were included in this cross-sectional study. Small intestine epithelial cells were used for quantitative RT-PCR analysis of the gene expression. The daily thiamine and energy intake were assessed with a food frequency questionnaire. Thiamine phosphate esters were hydrolyzed to free thiamine, and liquid chromatography with a tandem mass spectrometry-based method was used to measure total thiamine in whole blood. Daily energy intake according to body weight and daily carbohydrate intake were not significantly different between groups after adjustment for sex. Although daily thiamine intake was significantly lower in the obesity group (p = 0.015), obese subjects had significantly higher whole blood thiamine levels than controls (44.96 ± 14.6 ng/mL and 33.05 ± 8.6 ng/mL, p = 0.002). There was a significant positive correlation between whole blood thiamine and BMI (r = 0.342, p = 0.020). SLC19A2 gene expression was lower in those with BMI ≥35 kg/m2 (p = 0.036). A significant positive correlation was found between SLC19A2 expression and whole blood thiamine level (r = 0.310, p = 0.038). CONCLUSION: A possible association between intestinal thiamine intake and total thiamine in whole blood was determined. The transcriptional changes of genes encoding the high-affinity membrane thiamine transporters, especially SLC19A2, probably play a role in this relationship.

Immunodeficiency associated with a novel functionally defective variant of SLC19A1 benefits from folinic acid treatment.

Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.

Defective Treg generation and increased type 3 immune response in leukocyte adhesion deficiency 1.

In 15 Turkish LAD-1 patients and controls, we assessed the impact of pathogenic ITGB2 mutations on Th17/Treg differentiation and functions, and innate lymphoid cell (ILC) subsets. The percentage of peripheral blood Treg cells, in vitro-generated induced Tregs differentiated from naive CD4+ T cells were decreased despite the elevated absolute counts of CD4+ cells in LAD-1 patients. Serum IL-23 levels were elevated in LAD-1 patients. Post-curdlan stimulation, LAD-1 patient-derived PBMCs produced more IL-17A. Additionally, the percentages of CD18-deficient Th17 cells expanded from total or naïve CD4+ T cells were higher. The blood ILC3 subset was significantly elevated in LAD-1. Finally, LAD-1 PBMCs showed defects in trans-well migration and proliferation and were more resistant to apoptosis. Defects in de novo generation of Tregs from CD18-deficient naïve T cells and elevated Th17s, and ILC3s in LAD-1 patients' peripheral blood suggest a type 3-skewed immunity and may contribute to LAD-1-associated autoimmune symptoms.

A Novel Biallelic LCK Variant Resulting in Profound T-Cell Immune Deficiency and Review of the Literature.

Lymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4+ and CD8+ αßT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency.

Characterization of peripheral blood T follicular helper (TFH) cells in patients with type 1 Gaucher disease and carriers.

BACKGROUND: Gaucher disease (GD) is the most common autosomal recessive lipid storage disease. In this study, the changes in TFH cells and IL-4 and IL-21 cytokines in blood samples of GD patients, carriers and healthy volunteers were investigated. METHODS: Two pretreatment type 1 GD patients, 20 currently treated type 1 GD patients, 6 carriers, and 27 healthy volunteers were enrolled in the study. TFH cell (CD45RA-CD4+CXCR5+) number, phenotype (PD1, ICOS expression), and cytokine production (IL-21, IL-4) were assessed via flow cytometric assays. RESULTS: No significant differences were found between the groups with respect to the number, frequency and PD1 or ICOS expression of TFH cells between healthy controls, patients and carriers. However, IL-4+ TFH cells were significantly reduced both in percent and number in the treated GD patients compared with healthy controls (p < 0.05). Interestingly, the IL-21+ TFH cell number was increased in treated GD patients. When TFH cells were examined based on CXCR3 expression, the frequency of the PD1+Th17-Th2-like fraction (CXCR3-) was found to be significantly increased in treated GD patients. CONCLUSION: To our knowledge, this is the first study to assess TFH cells in GD patients, and to show that the production of IL-4 and IL-21 by TFH cells and their subsets may be altered in type 1 GD patients.

The Beneficial Clinical Effects of Teriflunomide in Experimental Autoimmune Myasthenia Gravis and the Investigation of the Possible Immunological Mechanisms.

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disease characterized by skeletal muscle weakness exacerbated with exercise. There is a need for novel drugs effective in refractory MG. We aimed to test the potential of teriflunomide, an immunomodulatory drug currently used in rheumatoid arthritis and multiple sclerosis treatment, in a murine experimental autoimmune myasthenia gravis (EAMG) model. EAMG was induced by immunizations with recombinant acetylcholine receptor (AChR). Teriflunomide treatment (10 mg/kg/day, intraperitoneal) was initiated to one group of mice (n = 21) following the third immunization and continued for 5 weeks. The disease control group (n = 19) did not receive medication. Naïve mice (n = 10) received only mock immunization. In addition to the clinical scorings, the numbers of B cells and T cells, and cytokine profiles of T cells were examined by flow cytometry. Anti-AChR-specific antibodies in the peripheral blood serum were quantified by ELISA. Teriflunomide significantly reduced clinical disease scores and the absolute numbers of CD4+ T cells and some of their cytokine-producing subgroups (IFN-γ, IL 2, IL22, IL-17A, GM-CSF) in the spleen and the lymph nodes. The thymic CD4+ T cells were also significantly reduced. Teriflunomide mostly spared CD8+ T cells' numbers and cytokine production, while reducing CD138+CD19+lambda+ plasma B cells' absolute numbers and CD138 mean fluorescent intensities, probably decreasing the number of IgG secreting more mature plasma cells. It also led to some selective changes in the measurements of anti-AChR-specific antibodies in the serum. Our results showed that teriflunomide may be beneficial in the treatment of MG in humans.

Synthesis and Comprehensive in Vivo Activity Profiling of Olean-12-en-28-ol, 3ß-Pentacosanoate in Experimental Autoimmune Encephalomyelitis: A Natural Remyelinating and Anti-Inflammatory Agent.

Multiple sclerosis (MS) treatment has received much attention, yet there is still no certain cure. We herein investigate the therapeutic effect of olean-12-en-28-ol, 3ß-pentacosanoate (OPCA) on a preclinical model of MS. First, OPCA was synthesized semisynthetically and characterized. Then, the mice with MOG35-55-induced experimental autoimmune/allergic encephalomyelitis (EAE) were given OPCA along with a reference drug (FTY720). Biochemical, cellular, and molecular analyses were performed in serum and brain tissues to measure anti-inflammatory and neuroprotective responses. OPCA treatment protected EAE-induced changes in mouse brains maintaining blood-brain barrier integrity and preventing inflammation. Moreover, the protein and mRNA levels of MS-related genes such as HLD-DR1, CCL5, TNF-α, IL6, and TGFB1 were significantly reduced in OPCA-treated mouse brains. Notably, the expression of genes, including PLP, MBP, and MAG, involved in the development and structure of myelin was significantly elevated in OPCA-treated EAE. Furthermore, therapeutic OPCA effects included a substantial reduction in pro-inflammatory cytokines in the serum of treated EAE animals. Lastly, following OPCA treatment, the promoter regions for most inflammatory regulators were hypermethylated. These data support that OPCA is a valuable and appealing candidate for human MS treatment since OPCA not only normalizes the pro- and anti-inflammatory immunological bias but also stimulates remyelination in EAE.

Evolution and long-term outcomes of combined immunodeficiency due to CARMIL2 deficiency.

BACKGROUND: Biallelic loss-of-function mutations in CARMIL2 cause combined immunodeficiency associated with dermatitis, inflammatory bowel disease (IBD), and EBV-related smooth muscle tumors. Clinical and immunological characterizations of the disease with long-term follow-up and treatment options have not been previously reported in large cohorts. We sought to determine the clinical and immunological features of CARMIL2 deficiency and long-term efficacy of treatment in controlling different disease manifestations. METHODS: The presenting phenotypes, long-term outcomes, and treatment responses were evaluated prospectively in 15 CARMIL2-deficient patients, including 13 novel cases. Lymphocyte subpopulations, protein expression, regulatory T (Treg), and circulating T follicular helper (cTFH ) cells were analyzed. Three-dimensional (3D) migration assay was performed to determine T-cell shape. RESULTS: Mean age at disease onset was 38 ± 23 months. Main clinical features were skin manifestations (n = 14, 93%), failure to thrive (n = 10, 67%), recurrent infections (n = 10, 67%), allergic symptoms (n = 8, 53%), chronic diarrhea (n = 4, 27%), and EBV-related leiomyoma (n = 2, 13%). Skin manifestations ranged from atopic and seborrheic dermatitis to psoriasiform rash. Patients had reduced proportions of memory CD4+ T cells, Treg, and cTFH cells. Memory B and NK cells were also decreased. CARMIL2-deficient T cells exhibited reduced T-cell proliferation and cytokine production following CD28 co-stimulation and normal morphology when migrating in a high-density 3D collagen gel matrix. IBD was the most severe clinical manifestation, leading to growth retardation, requiring multiple interventional treatments. All patients were alive with a median follow-up of 10.8 years (range: 3-17 years). CONCLUSION: This cohort provides clinical and immunological features and long-term follow-up of different manifestations of CARMIL2 deficiency.

A Novel Intronic Mutation Reduces HAX1 Level and is Associated With Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a rare disease. Autosomal recessive forms of SCN are more frequent in countries where consanguineous marriages are common. In this report, we describe a 54-day-old female with neutropenia who presented with ecthyma gangrenosum. Clinical exome sequencing was used to identify the mutation. HAX1 messenger RNA and isoforms were examined by real-time quantitative and conventional polymerase chain reaction. Bone marrow aspiration was stained by hematoxylin and eosin. Granulocytes were tested for apoptosis upon H2O2 exposure. T-cell proliferation was tested by flow cytometry. Clinical exome sequencing revealed a novel homozygous acceptor splice site mutation in intron 3 of HAX1 (c.505-1G>C), which reduced both isoforms A and B of HAX1 messenger RNA. The Western blot studies showed a complete absence of HAX1 protein. The purified neutrophils from the patient showed increased apoptosis upon H2O2 exposure, whereas T-cell proliferative responses to various stimuli were intact. The patient was treated with combined antibiotics, filgrastim, and placed on antibiotics prophylaxis. To the best of our knowledge, our data provide the first experimental evidence for HAX1 deficiency because of a splice site mutation. Although 3 other splice site variants have been deposited in databases, functional studies were missing. This novel variant of HAX1 may explain the SCN and secondary infections in our patients.

Circulating fibrocyte level in children with cystic fibrosis.

BACKGROUND: This study aimed to evaluate circulating fibrocyte levels in cystic fibrosis (CF) patients during stable and exacerbation periods of the condition. METHODS: The study group consisted of 39 patients diagnosed with CF and 20 healthy controls. Individuals included in the study were divided into three groups: CF, CF exacerbated, and a healthy control group. Their circulating fibrocyte levels were compared. Findings from a pulmonary function test and high-resolution computed tomography of the lung were evaluated and compared. RESULTS: The circulating fibrocyte count was found to be significantly higher in patients with CF compared with the exacerbated and control groups. No correlation was found between the forced expiratory volume in 1 s and forced vital capacity values in the pulmonary function test and the circulating fibrocyte count. The circulating fibrocyte count in patients (in the CF group) with positive findings in the high-resolution computed tomography was statistically significantly lower. CONCLUSIONS: The circulating fibrocyte level in the peripheral blood of the patients with CF was increased.

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis.

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

Common Variable Immunodeficiency, Autoimmune Hemolytic Anemia, and Pancytopenia Associated With a Defect in IKAROS.

OBJECTIVE: Mutations in IKZF1, which encodes Ikaros family zinc finger 1 (IKAROS) transcription factor, are associated with recurrent infections, cytopenia, autoimmune diseases, and hematologic malignancies. Diverse clinical phenotypes resulting from IKZF1 mutations include pulmonary fungal infections, cytopenia, autoimmune hemolytic anemia (AIHA), and malignancies. In this study, we aimed to assess the DNA-binding ability and pericentromeric (PC) localization of a variant of IKZF discovered in a patient. MATERIALS AND METHODS: DNA-binding ability of a pathogenic IKZF variant was tested using electrophoretic mobility shift assay and PC localization of the variant was assessed by immunofluorescent microscopy in NIH3T3 cells. RESULTS: Clinical features of a 3-month-old male infant who underwent hematopoietic stem cell transplantation because of an IKZF1 mutation-associated common variable immunodeficiency, AIHA, and pancytopenia are described. DNA studies revealed a heterozygous missense variant (IKZF1 NM_006060 c.427C>T; p.R143W). Cotransfection studies revealed that mutant R143W has a partial dominant-negative effect over PC targeting and DNA binding. CONCLUSIONS: IKZF1 mutation must be kept in mind if neonatal AIHA, common variable immunodeficiency, and pancytopenia are observed.

ILC3 deficiency and generalized ILC abnormalities in DOCK8-deficient patients.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency is the main cause of the autosomal recessive hyper-IgE syndrome (HIES). We previously reported the selective loss of group 3 innate lymphoid cell (ILC) number and function in a Dock8-deficient mouse model. In this study, we sought to test whether DOCK8 is required for the function and maintenance of ILC subsets in humans. METHODS: Peripheral blood ILC1-3 subsets of 16 DOCK8-deficient patients recruited at the pretransplant stage, and seven patients with autosomal dominant (AD) HIES due to STAT3 mutations, were compared with those of healthy controls or post-transplant DOCK8-deficient patients (n = 12) by flow cytometry and real-time qPCR. Sorted total ILCs from DOCK8- or STAT3-mutant patients and healthy controls were assayed for survival, apoptosis, proliferation, and activation by IL-7, IL-23, and IL-12 by cell culture, flow cytometry, and phospho-flow assays. RESULTS: DOCK8-deficient but not STAT3-mutant patients exhibited a profound depletion of ILC3s, and to a lesser extent ILC2s, in their peripheral blood. DOCK8-deficient ILC1-3 subsets had defective proliferation, expressed lower levels of IL-7R, responded less to IL-7, IL-12, or IL-23 cytokines, and were more prone to apoptosis compared with those of healthy controls. CONCLUSION: DOCK8 regulates human ILC3 expansion and survival, and more globally ILC cytokine signaling and proliferation. DOCK8 deficiency leads to loss of ILC3 from peripheral blood. ILC3 deficiency may contribute to the susceptibility of DOCK8-deficient patients to infections.

Genetic Deficiency and Biochemical Inhibition of ITK Affect Human Th17, Treg, and Innate Lymphoid Cells.

PURPOSE: Interleukin-2-inducible T cell kinase (ITK) is an important mediator of T cell receptor signaling. Loss of function mutations in ITK results in hypogammaglobulinemia and CD4+ T cell loss in humans, and the patients often present with EBV-associated B cell lymphoproliferative syndrome. Itk-deficient mice show loss of T cell naivety, impaired cytolytic activity of CD8+ T cells, and defects in CD4+ T cell lineage choice decisions. In mice, Itk mutations were shown to affect Th17-Treg lineage choice in favor of the latter. In this study, we explored whether human ITK reciprocally regulates Th17-Treg balance as its murine ortholog. METHODS: Whole Exome Sequencing was used to identify the mutation. ITK-deficient peripheral blood lymphocytes were characterized by FACSAria III-based flow cytometric assays with respect to proliferation, apoptosis, cytokine production, and innate lymphoid cell (ILC) frequency. Sorted T cells from healthy donors were exposed to ibrutinib, an irreversible ITK inhibitor, to assess ITK's contribution to Th17 and Treg cell generation and functions. RESULTS: In this study, we report a child with a novel ITK mutation who showed impaired CD3/CD28 induced proliferation in T cells. ITK-mutant cells were more apoptotic irrespective of TCR activation. More importantly, T cells produced less Th17-associated cytokines IL-17A, IL-22, and GM-CSF. Conversely, Th1-associated IFN-γ production was increased. An irreversible inhibitor of ITK, ibrutinib, blocked ex vivo Th17 generation and IL-17A production, conversely augmented FOXP3 expression only at low doses in Treg cultures. Finally, we analyzed peripheral ILC populations and observed a relative decrease in ILC2 and ILC3 frequency in our ITK-deficient patient. CONCLUSIONS: To our knowledge, this is the first report showing that both genetic and chemical inhibition of ITK result in reduced Th17 generation and function in humans. We also report, for the first time, a reduction in ILC2 and ILC3 populations in an ITK-deficient human patient.