Munc13-4 Protein Research Articles

Atypical familial hemophagocytic lymphohistiocytosis type 3 in children: A report of cases and literature review.

Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is caused by UNC13D variants. The clinical manifestations of FHL3 are highly diverse and complex. Some patients exhibit atypical or incomplete phenotypes, making accurate diagnosis difficult. Our study aimed to broaden the understanding of the atypical FHL3 clinical spectrum. In our study, we analyzed in detail the clinical features of four Chinese patients with UNC13D variants. Additionally, we conducted a comprehensive review of the existing literature on previously reported atypical manifestations and summarized the findings. Two of our patients presented with muscle involvement, while the other two had hematological involvement; none of them met the diagnostic criteria for hemophagocytic lymphohistiocytosis (HLH). However, protein expression and functional analysis ultimately confirmed diagnostic criteria for FHL3 in all patients. From the literature we reviewed, many atypical FHL3 patients had neurological involvement, especially isolated neurological manifestations. At the same time, arthritis and hypogammaglobulinemia were also prone to occur. Our study highlights that the expression of the Munc13-4 protein may not fully indicate the pathogenicity of UNC13D variants, whereas CD107a analysis could be more sensitive for disease diagnosis. These findings contribute to a broader understanding of the FHL3 clinical spectrum and may offer new insights into the underlying pathogenesis of UNC13D variants. It is crucial to prioritize the timely and accurate diagnosis of atypical patients, as they may often be overlooked among individuals with rheumatic or hematological diseases.

Clinical, immunological and genetic findings in patients with UNC13D deficiency (FHL3): A systematic review.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive immune disorder that is caused by mutations in 6 different genes related to the formation and function of secretory lysosomes within cytotoxic T lymphocytes and natural killer (NK) cells. Thus, defect in these genes is associated with the accumulation of antigens due to defective cytotoxic function. FHL type 3 (FHL3) accounts for nearly 30-40% of FHL, and its underlying reason is mutation in UNC13D gene which encodes Munc13-4 protein. For the first time, we aimed to systematically review clinical features, immunologic data, and genetic findings of patients with FHL3. We conducted electronic searches for English-language articles in PubMed, Web of Science, EMBASE, and Scopus databases to collect comprehensive records related to patients with UNC13D mutations. A total of 279 abstracts were initially reviewed for inclusion. Among them, 57 articles corresponding to 322 individual FHL3 patients fulfilled our selection criteria. Finally, 73 and 249 patients were considered as severe and mild feature groups, respectively. Our results confirmed that fever, hepatosplenomegaly, and hemophagocytosis are common clinical features in the disease. Moreover, reduced fibrinogen and NK cell activity, as well as increased ferritin and triglycerides, are important markers for early diagnosis of the FHL3 disease. Investigation of genotype showed that the most prevalent type and zygosity of UNC13D are splice-site errors and compound heterozygous, respectively. FHL3 patients have a wide range of clinical manifestations, which makes it difficult to diagnose. Therefore, it seems that the sequencing of the entire UNC13D gene (coding and non-coding regions) is the most appropriate way to accurate diagnosis of FHL3 patients.

Lentiviral Gene Therapy for Familial Hemophagocytic Lymphohistiocytosis Type 3, Caused by UNC13D Genetic Defects.

Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a rare disease caused by mutations to the UNC13D gene and the subsequent absence or decreased activity of the Munc13-4 protein. Munc13-4 is essential for the exocytosis of perforin and granzyme containing granules from cytotoxic cells. Without it, these cells are able to recognize an immunological insult but are unable to execute their cytotoxic functions. The result is a hyperinflammatory state that, if left untreated, is fatal. At present, the only curative treatment is hematopoietic stem cell transplantation (HSCT), but eligibility and response to this treatment are largely dependent on the ability to control inflammation before HSCT. In this study, we describe an optimized lentiviral vector that can restore Munc13-4 expression and degranulation capacity in both transduced FHL3 patient T cells and transduced hematopoietic stem cells from the FHL3 (Jinx) disease model.

Retroviral UNC13D Gene Transfer Restores Cytotoxic Activity of T Cells Derived from Familial Hemophagocytic Lymphohistiocytosis Type 3 Patients In Vitro.

Familial hemophagocytic lymphohistiocytosis (FHL) is a group of life-threatening, autosomal recessive disorders of severe hyperinflammation. FHL type 3 (FHL-3) accounts for about 30% of FHL cases. It is characterized by mutations in the UNC13D gene that give rise to functionally impaired or absent Munc13-4 protein, resulting in impaired secretion of lytic granules by cytotoxic lymphocytes. Etoposide-based therapy is currently used as the standard of care that results in around 60% 5-year survival, illustrating the need for novel treatment approaches. Key problems include treatment toxicity and failure to induce or maintain remission of the hyperinflammation. Instead of immunosuppression, transplantation of autologous gene-corrected T cells can be envisaged as an approach to restore the impaired immune reaction. This study established a protocol that enabled hyperactivated, FHL-3 patient-derived T cells to be cultured and a codon-optimized UNC13D expression cassette to be delivered by either alpha- or gamma-retroviral gene transfer. The data demonstrate that the established protocol can be applied to FHL-3 patient cells with various genetic backgrounds and that gamma-retroviral UNC13D transfer restored expression of functional Munc13-4, as well as degranulation capacity and cell-mediated cytotoxicity of those patient-derived CD8+ T cells. Furthermore, the study shows that the co-introduction of a truncated low-affinity nerve growth factor receptor coding sequence enabled the therapeutic effect to be optimized by enriching transduced cells in a Good Manufacturing Practice-compliant manner. In conclusion, this study lays the foundation for an adaptive immune cell therapy approach aiming at immunological stabilization of FHL-3 patients with autologous, immune-competent T cells prior to hematopoietic stem-cell transplantation.

Human CTL-based functional analysis shows the reliability of a munc13-4 protein expression assay for FHL3 diagnosis

AbstractFamilial hemophagocytic lymphohistiocytosis (FHL) is the major form of hereditary hemophagocytic lymphohistiocytosis (HLH); as such, it requires prompt and accurate diagnosis. We previously reported that FHL type 3 (FHL3) can be rapidly screened by detecting munc13-4 expression in platelets using flow cytometry; however, the reliability of the munc13-4 expression assay for FHL3 diagnosis is unclear. Regardless of the type of UNC13D mutation, all reported FHL3 cases examined for the munc13-4 protein showed significantly reduced expression. However, the translated munc13-4 protein of some reportedly disease-causing UNC13D missense variants has not been assessed in terms of expression or function; therefore, their clinical significance remains unclear. The aim of this study was to determine the reliability of a munc13-4 expression assay for screening FHL3. Between 2011 and 2016, 108 HLH patients were screened by this method in our laboratory, and all 15 FHL3 patients were diagnosed accurately. To further elucidate whether munc13-4 expression analysis can reliably identify FHL3 patients harboring missense mutations in UNC13D, we developed an alloantigen-specific cytotoxic T lymphocyte (CTL) line and a CTL line immortalized by Herpesvirus saimiri derived from FHL3 patients. We then performed a comprehensive functional analysis of UNC13D variants. Transient expression of UNC13D complementary DNA constructs in these cell lines enabled us to determine the pathogenicity of the reported UNC13D missense variants according to expression levels of their translated munc13-4 proteins. Taken together with previous findings, the results presented herein show that the munc13-4 protein expression assay is a reliable tool for FHL3 screening.

682. Correction of CTLs Cytotoxic Function Defect by SIN-lentiviral Mediated Expression of Munc13-4 in Type 3 Familial Hemophagocytic Lymphohistiocytosis

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and it's defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. As Munc13-4's function is to allow proper cytotoxic activity in mature cytotoxic CD8+ T cells we proposed that these laters may constitute target for gene correction. We constructed a self-inactivating HIV-1 derived lentiviral vector encoding human Munc13-4 in two different pseudotypes, the high tropism VSV-G and the measles virus glycoproteins (H/F) envelope which target more efficiently lymphoid cells through the signaling lymphocyte activation molecule (SLAM) as described by Verhoeyen et al. 2011. We demonstrated that both vectors are able to stably transduce FHL3 CD8+ T cells resulting in correction of defective degranulation capacity of these cells. However comparative analysis showed that H/F pseudotyped vector was more efficient than VSV-G vector to transduce FHL3 T cells. Adoptive transfer of the gene-corrected FHL3 T cells in SCID mice bearing autologous B-LCL lymphoma led to significant tumor regression due to an efficient homing into the tumor mass and long persistence of corrected T cells in peripheral blood as compared to non-corrected T cells receiving mice. Our study shows for the first time that a lentiviral mediating gene transfer in T cells could be proposed to treat a hemophagocytic lymphohistiocytosis disorder.

759. Reduction of HLH-like Manifestations in Murine Model of Munc13-4 Deficiency Following Lentiviral Gene Transfer into Hematopoietic Stem and Progenitor Cells

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system which represents 25% of all FHLs. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and it's defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. In this study, we took advantages from a murine model of FHL3, the Jinx mice, to investigate the feasibility of HSPC gene therapy for this pathology. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus (LCMV). We generated and used a self-inactivated lentiviral vector to complement HSC from Unc13d -/- (Jinx) mice and transplanted them back into the irradiated Jinx recipients. This transplantation led to the complete reconstitution of the immune system at levels comparable to that of control mice. The recipients were then challenged with LCMV. While Jinx mice reconstituted with GFP expressing HSPC developed leukopenia, anemia and body weight loss, characteristic of HLH in this murine model, gene corrected Jinx recipients developed only mild or no HLH manifestations. This reduction in HLH manifestation correlated with a significant reduction of virus titer in the liver and serum level of IFN-g and inflammatory cytokines. All these ameliorations might be explained by the restoration of cytotoxic function of CTLs as demonstrated in an in-vitro degranulation assay. Overall, this study provides data supporting the potential of HSC gene therapy in a FHL immune dysregulation such as UNC13D deficiency.

Stem Cell and T-Cell Gene Therapy Using SIN-Lentiviral Vector In Type 3 Familial Hemophagocytic Lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is the genetic form of hemophagocytic lymphohistiocytosis with an autosomal recessive form of inheritance and early onset. FHL is characterized by prolonged fever, hepatosplenomegaly and pancytopenia. Type 3 of FHL (FHL3) which accounts for 30-35 % of all FHL patients, results from mutations in UNC13D gene encoding Munc13-4 protein. Munc13-4 controls fusion of lytic granules with the plasma membrane in cytotoxic lymphocytes and its defect leads to impaired cytotoxic activity in T and NK lymphocytes. The only curative method for FHL3 is allogenic hematopoietic stem cell (HSC) transplantation. For those patients without compatible bone marrow donor, gene therapy could represent a therapeutic option.Our study is based on a comparative analysis to investigate the efficacy and safety of stem cell and T-cell gene therapy for FHL3. To this end we have generated SIN-lentiviral constructs expressing Munc13-4 and used them to produce either VSVG or T-cell specific pseudotyped lentiviral vectors. We also could obtain functionally mature T-cells in vitro, derived from umbilical cord blood HSCs which respond to TCR stimulation and show cytotoxic effect. Using these approaches, first we demonstrated that our SIN-lentiviral constructs are able to transduce efficiently human T-cells and HSCs. We used then these vectors to complement the FHL3 patient's cells. Transduction of FHL3 CD8 effector cells restored their cytotoxic function that was comparable to that of control cells. In addition we noted that the overexpression of Munc 13-4 in normal human HSCs didn't alter in vitro differentiation of these cells towards T-cells. The effect of this overexpression on B-cells and myeloid differentiation is currently under investigation.These preliminary results will be followed by ex vivo gene transfer experiments in Munc13-4 deficient “Jinx” mice to further investigate the functional restoration as well as toxic effects. This strategy, if approved, could offer a safe therapeutic method not only for FHL3 patients but also for other genetic or acquired dysfunctions of T-lymphocytes. Disclosures:No relevant conflicts of interest to declare.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. MUNC13-4 is a RAB27A effector that coordinates exocytosis in hematopoietic cells, and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3. In this work, we have established an essential role for MUNC13-4 in selective vesicular trafficking, phagosomal maturation, and intracellular bacterial killing in neutrophils. Using neutrophils from munc13-4 knock-out (KO) mice, we show that MUNC13-4 is necessary for the regulation of p22(phox)-expressing granule trafficking to the plasma membrane and regulates extracellular ROS production. MUNC13-4 was also essential for the regulation of intracellular ROS production induced by Pseudomonas aeruginosa despite normal trafficking of p22(phox)-expressing vesicles toward the phagosome. Importantly, in the absence of MUNC13-4, phagosomal maturation was impaired as observed by the defective delivery of azurophilic granules and multivesicular bodies to the phagosome. Significantly, this mechanism was intact in RAB27A KO neutrophils. Intracellular bacterial killing was markedly impaired in MUNC13-4 KO neutrophils. MUNC13-4-deficient cells showed a significant increase in neutrophil extracellular trap formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of MUNC13-4 in the innate immune response of the neutrophil and have direct implications for the understanding of immunodeficiencies in patients with MUNC13-4 deficiency.

Abstract 228: Secretion of Bone Marrow--Derived Cells Contributes to Pressure-Overload--Induced Left Ventricular Hypertrophy

Bone marrow derived cells, especially inflammatory cells, may contribute to the pathological progression of pressure overload induced left ventricular (LV) hypertrophy and cardiac fibrosis. Previously, we reported that inflammatory cell accumulation and upregulation of cytokines in hearts of mice that had undergone transverse aortic constriction (TAC) surgery predicted later cardiac hypertrophic and fibrotic remodeling. However, the underlining mechanisms are still not clear. One potential mechanism for inflammatory cells to modulate their environment and affect surrounding cells is through release of cargo stored in granules. Jinx mice - which contain a single point mutation in Unc13d encoding the Munc13-4 protein, a limiting factor in vesicular priming and fusion - have defects in granular secretion in hematopoietic cells, such as platelets, NK cells, and neutrophils. In the current study, we investigated the role of bone marrow cell granule secretion in TAC-induced LV remodeling by creating with bone marrow transplantation chimeric mice specifically lacking Munc13-4 in marrow derived cells. Both wild-type mice (WT) that were transplanted with WT bone marrow and WT mice that received Jinx bone marrow developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at later times (5 weeks), mice lacking Munc13-4 in bone marrow-derived cells failed to sustain the cardiac hypertrophy observed in mice with WT bone marrow. No difference in cardiac fibrosis was observed at early or late times. These results suggest that sustained LVH in the setting of pressure overload depends on factor(s) secreted from bone marrow-derived cells, likely from either platelets, NK cells and/or neutrophils. Inhibiting granule cargo release may represent a novel therapeutic target to prevent the development of LVH.

Hematopoietic stem cell transplantation with reduced intensity conditioning from a family haploidentical donor in an infant with familial hemophagocytic lymphohistocytosis

Allogeneic hematopoietic stem cell transplantation is the only curative method for patients with familial hemophagocytic lymphohistiocytosis (FHL). We present a case of a 3-month-old girl with Munc13-4 mutation (FHL3), who underwent bone marrow transplantation (BMT) from her human leukocyte antigen-haploidentical mother following reduced intensity conditioning (RIC) with fludarabine, melphalan, and busulfan. Engraftment after BMT was generally uneventful, with only mild acute graft versus host disease. Munc13-4 protein was restored following BMT, and she is well and free of disease 14 months after BMT. These results suggest that BMT with RIC from a family haploidentical donor may sufficiently restore immune regulation in infants, while lessening treatment-related mortality and long-term sequelae.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein

AbstractFamilial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4–deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Multiple inherited sequence variations in two disease‐causing genes in familial haemophagocytic lymphohistiocytosis

Multiple inherited sequence variations in two disease‐causing genes in familial haemophagocytic lymphohistiocytosis

The non-neuronal syntaxin SYN-1 regulates defecation behavior and neural activity in C. elegans through interaction with the Munc13-like protein AEX-1

We have previously shown that the AEX-1 protein, which is expressed in postsynaptic muscles, retrogradely regulates presynaptic neural activity at the Caenorhabditis elegans neuromuscular junctions. AEX-1 is similar to vertebrate Munc13-4 protein, suggesting a function for vesicle exocytosis from a kind of cells. Compared to emerging evidences of the role of Munc13 proteins in synaptic vesicle release, however, the precise mechanism for vesicle exocytosis by AEX-1 and Munc13-4 is little understood. Here we have identified SYN-1 as a candidate molecule of AEX-1-dependent vesicle exocytosis from non-neuronal cells . The syn-1 gene encodes a C. elegans syntaxin, which is distantly related to the neuronal syntaxin UNC-64. The syn-1 gene is predominantly expressed in non-neuronal tissues and genetically interacts with aex-1 for presynaptic activity. However, the two proteins did not interact physically in our yeast two-hybrid system and mutational SYN-1 did not bypass the requirement of AEX-1 for the behavioral defects in aex-1 mutants, whereas mutant UNC-64 does in unc-13 mutants . These results suggest that a novel molecular interaction between the AEX-1 and syntaxin may regulate vesicle exocytosis for retrograde signal release.

Mutations affecting mRNA splicing are the most common molecular defect in patients with familial hemophagocytic lymphohistiocytosis type 3

Mutations of UNC13D have been described in patients affected by familial hemophagocytic lymphohistiocytosis (FHL3). The Munc13-4 protein contributes to the priming of the secretory granules. Mutation in this gene results in defective cellular cytotoxicity and the familial hemophagocytic lymphohistiocytosis clinical picture. Among reported mutations, few are predicted to impair splicing. Yet, functional impact of these mutations has not been addressed. We identified 18 out of 31 familial hemophagocytic lymphohistiocytosis families showing at least one mutation responsible for splicing error. We identified some known and three novel splicing mutations: one falls at the acceptor site of exon 11 and 2 are deep intronic mutations in IVS1 and in IVS30. We demonstrated that these deep intronic mutations affect regulatory sequences causing aberrant splicing. We report that UNC13D mutations leading to splicing errors represent the majority of mutations observed in familial hemophagocytic lymphohistiocytosis. This finding has implications for designing strategies for analysis of the families with suspected familial hemophagocytic lymphohistiocytosis.

Analysis of natural killer–cell function in familial hemophagocytic lymphohistiocytosis (FHL): defective CD107a surface expression heralds Munc13-4 defect and discriminates between genetic subtypes of the disease

AbstractNatural killer (NK) cells from patients with familial hemophagocytic lymphohistiocytosis because of PRF1 (FHL2, n = 5) or MUNC13-4 (FHL3, n = 8) mutations were cultured in IL-2 prior to their use in various functional assays. Here, we report on the surface CD107a expression as a novel rapid tool for identification of patients with Munc13-4 defect. On target interaction and degranulation, FHL3 NK cells displayed low levels of surface CD107a staining, in contrast to healthy control subjects or perforin-deficient NK cells. B-EBV cell lines and dendritic cell targets reveal the FHL3 NK-cell defect, whereas highly susceptible tumor targets were partially lysed by FHL3 NK cells expressing only trace amounts of Munc13-4 protein. Perforin-deficient NK cells were completely devoid of any ability to lyse target cells. Cytokine production induced by mAb-crosslinking of triggering receptors was comparable in patients and healthy control subjects. However, when cytokine production was induced by coculture with 721.221 B-EBV cells, FHL NK cells resulted in high producers, whereas control cells were almost ineffective. This could reflect survival versus elimination of B-EBV cells (ie, the source of NK-cell stimulation) in patients versus healthy control subjects, thus mimicking the pathophysiologic scenario of FHL.

Familial hemophagocytic lymphohistiocytosis with the MUNC13-4 mutation: a case report

A 44-day-old male infant with familial hemophagocytic lymphohistiocytosis (FHL) associated with the MUNC13-4 mutation is reported. He presented with fever and poor feeding, lymphocytosis with thrombocytopenia and CSF pleocytosis without virological explanation. On the basis of progressive hyperferritinemia (1323 ng/ml), anemia (hemoglobin: 5.2 g/dl), hypertriglyceridemia (547 mg/dl) and increased LDH (1063 IU/l) with hemophagocytosis in the bone marrow, hemophagocytic lymphohistiocytosis was diagnosed. He showed a good response to corticosteroid therapy and the disease was stable for more than 5 months. Thereafter, he suffered from central nervous system complications, and successfully underwent unrelated cord blood stem cell transplantation. A remission was observed for more than 2 years, with mild mental retardation. Genetic analysis revealed that he had a compound heterozygous mutation of MUNC13-4; namely a novel 2163G>A mutation resulting in W721X, and 754-1G>C resulting in a premature stop codon in this gene. Western blot analysis showed the complete loss of the MUNC13-4 protein, whereas other molecules associated with the SNARE systems were detected at normal levels. Conclusion. FHL may have a broad clinical spectrum, and further analysis on its phenotype-genotype association is required to establish an appropriate treatment strategy, including immunochemotherapy and stem cell transplantation in the future.