Familial Hemophagocytic Lymphohistiocytosis Research Articles

An A91V SNP in the Perforin Gene Is Frequently Found in NK/T-Cell Lymphomas

NK/T-cell lymphoma (NKTCL) is the most frequent EBV-related NK/T-cell disease. Its clinical manifestations overlap with those of familial haemophagocytic lymphohistiocytosis (FHLH). Since PERFORIN (PRF1) mutations are present in FHLH, we analysed its role in a series of 12 nasal and 12 extranasal-NKTCLs. 12.5% of the tumours and 25% of the nasal-origin cases had the well-known g.272C>T(p.Ala91Val) pathogenic SNP, which confers a poor prognosis. Two of these cases had a double-CD4/CD8-positive immunophenotype, although no correlation was found with perforin protein expression. p53 was overexpressed in 20% of the tumoral samples, 80% of which were of extranasal origin, while none showed PRF1 SNVs. These results suggest that nasal and extranasal NKTCLs have different biological backgrounds, although this requires validation.

Familial Hemophagocytic Lymphohistiocytosis Type 3 Diagnosed at School Age

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening condition characterized by immune hyperactivation and clinical signs of extreme inflammation. We describe a 7-year-old male who presented with fever resistant to antibiotic therapy, pancytopenia, splenomegaly, hypertriglyceridemia, and hyperferritinemia. Bone marrow aspirate showed hemophagocytosis. Epstein-Barr virus genome was positive in blood. Functional screening showed reduced capacity of cytotoxic degranulation. Mutation analysis of the FHL-related genes revealed compound heterozygous for UNC13D mutations: c. 753+1G>T, and the novel c.544C>T (p.P182S). Patients with a clinical presentation of HLH, even if older than typically seen, should be screened for familial HLH by mutation analysis.

The 253‐kb inversion and deep intronic mutations in UNC13D are present in North American patients with familial hemophagocytic lymphohistiocytosis 3

The mutations in UNC13D are responsible for familial hemophagocytic lymphohistiocytosis (FHL) type 3. A 253-kb inversion and two deep intronic mutations, c.118-308C > T and c.118-307G > A, in UNC13D were recently reported in European and Asian FHL3 patients. We sought to determine the prevalence of these three non-coding mutations in North American FHL patients and evaluate the significance of examining these new mutations in genetic testing. We performed DNA sequencing of UNC13D and targeted analysis of these three mutations in 1,709 North American patients with a suspected clinical diagnosis of hemophagocytic lymphohistiocytosis (HLH). The 253-kb inversion, intronic mutations c.118-308C > T and c.118-307G > A were found in 11, 15, and 4 patients, respectively, in which the genetic basis (bi-allelic mutations) explained 25 additional patients. Taken together with previously diagnosed FHL3 patients in our HLH patient registry, these three non-coding mutations were found in 31.6% (25/79) of the FHL3 patients. The 253-kb inversion, c.118-308C > T and c.118-307G > A accounted for 7.0%, 8.9%, and 1.3% of mutant alleles, respectively. Significantly, eight novel mutations in UNC13D are being reported in this study. To further evaluate the expression level of the newly reported intronic mutation c.118-307G > A, reverse transcription PCR and Western blot analysis revealed a significant reduction of both RNA and protein levels suggesting that the c.118-307G > A mutation affects transcription. These specified non-coding mutations were found in a significant number of North American patients and inclusion of them in mutation analysis will improve the molecular diagnosis of FHL3.

An N-Terminal Missense Mutation in STX11 Causative of FHL4 Abrogates Syntaxin-11 Binding to Munc18-2

Familial hemophagocytic lymphohistiocytosis (FHL) is an often-fatal hyperinflammatory disorder caused by autosomal recessive mutations in PRF1, UNC13D, STX11, and STXBP2. We identified a homozygous STX11 mutation, c.173T > C (p.L58P), in three patients presenting clinically with hemophagocytic lymphohistiocytosis from unrelated Pakistani families. The mutation yields an amino acid substitution in the N-terminal Habc domain of syntaxin-11 and resulted in defective natural killer cell degranulation. Notably, syntaxin-11 expression was decreased in patient cells. However, in an ectopic expression system, syntaxin-11 L58P was expressed at levels comparable to wild-type syntaxin-11, but did not bind Munc18-2. Moreover, another N-terminal syntaxin-11 mutant, R4A, also did not bind Munc18-2. Thus, we have identified a novel missense STX11 mutation causative of FHL type 4. The syntaxin-11 R4A and L58P mutations reveal that both the N-terminus and Habc domain of syntaxin-11 are required for binding to Munc18-2, implying similarity to the dynamic binary binding of neuronal syntaxin-1 to Munc18-1.

Perforin and human diseases.

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use a highly toxic pore-forming protein perforin (PFN) to destroy cells infected with intracellular pathogens and cells with pre-cancerous transformations. However, mutations of PFN and defects in its expression can cause an abnormal function of the immune system and difficulties in elimination of altered cells. As discussed in this chapter, deficiency of PFN due to the mutations of its gene, PFN1, can be associated with malignancies and severe immune disorders such as familial hemophagocytic lymphohistiocytosis (FHL) and macrophage activation syndrome. On the other hand, overactivity of PFN can turn the immune system against autologous cells resulting in other diseases such as systemic lupus erythematosus, polymyositis, rheumatoid arthritis and cutaneous inflammation. PFN also has a crucial role in the cellular rejection of solid organ allografts and destruction of pancreatic β-cells resulting in type 1 diabetes. These facts highlight the importance of understanding the biochemical characteristics of PFN.

A novelPRF1gene mutation in a fatal neonate case with type 2 familial hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) occurs in the primary form (genetic or familial) or secondary form (acquired). The familial form of HLH (FHL) is a potentially fatal autosomal recessive disorder that occurs because of constitutional defects in cell-mediated cytotoxicity. Here, we report a fatal neonatal case of type 2 FHL (FHL2) that involved a novel frameshift mutation. Clinically, the newborn presented with severe sepsis-like features and required mechanical ventilation and continuous venovenous hemodiafiltration. Flow cytometry analysis showed marked HLH and complete absence of intracytoplasmic perforin expression in cytotoxic cells; therefore, we performed molecular genetic analyses for PRF1 mutations, which showed that the patient had a compound heterozygous mutation in PRF1, that is, c.65delC (p.Pro22Argfs*2) and c.1090_1091delCT (p.Leu364Glufs*93). Clinical and genetic assessments for FHL are required for neonates with refractory fever and progressive multiple organ failure, particularly when there is no evidence of microbiological or metabolic cause.

Remission and Relapse of Hemophagocytic Lymphohistiocytosis in a Patient Harboring a PRF1 Homozygous Mutation

The aim of this paper was to describe a case of familial hemophagocytic lymphohistiocytosis (HLH) in a pediatric patient with a PRF1 homozygous mutation. An 8-year-old boy diagnosed with HLH was in remission after undergoing nonspecific treatment; however, merely 2 months later, he was presented at our hospital with a relapse of HLH. His genetic analysis showed that he had a homozygous mutation c.1066C>T in the PRF1 gene. Timely distinction of primary HLH from secondary HLH is critical.

Defective UNC13D Gene-associated Familial Hemophagocytic Lymphohistiocytosis Triggered by Visceral Leishmaniasis

Visceral leishmaniasis (VL) triggered genetic hemophagocytic lymphohistiocytosis (HLH) is clinically challenging. One-year-old VL-HLH patient improved after liposomal-amphotericin-B therapy, but subsequently deteriorated, although bone marrow amastigotes disappeared. Symptoms resolved after 8 weeks of HLH-2004 therapy but recurred upon cessation. Homozygous UNC13D gene 627delT mutation was identified however stem cell donor was unavailable. The patient died at age 4 years after central nervous system attacks and HLH recurrences. VL in HLH patients does not exclude a genetic etiology and requires structured clinical management. VL should be excluded in all HLH patients in endemic regions before immunochemotherapy, which is recommended for VL-HLH patients unresponsive to VL treatment and/or reactivated.

肝不全を合併し急激な経過を辿った家族性血球貪食症候群の1乳児例

肝不全を合併し急激な経過を辿った家族性血球貪食症候群の1乳児例

Syntaxin11 serves as a t‐SNARE for the fusion of lytic granules in human cytotoxic T lymphocytes

CTLs kill target cells via fusion of lytic granules (LGs) at the immunological synapse (IS). Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs in CTL function is evident in the form of familial hemophagocytic lymphohistiocytosis type 4 that is caused by mutations in Syntaxin11 (Stx11), a Qa-SNARE protein. Here, we investigate the molecular mechanism of Stx11 function in primary human effector CTLs with high temporal and spatial resolution. Downregulation of endogenous Stx11 resulted in a complete inhibition of LG fusion that was paralleled by a reduction in LG dwell time at the IS. Dual color evanescent wave imaging suggested a sequential process, in which first Stx11 is transported to the IS through a subpopulation of recycling endosomes. The resulting Stx11 clusters at the IS then serve as a platform to mediate fusion of arriving LGs. We conclude that Stx11 functions as a t-SNARE for the final fusion of LG at the IS, explaining the severe phenotype of familial hemophagocytic lymphohistiocytosis type 4 on a molecular level.

Hemophagocytic lymphohistiocytosis: pathogenesis and treatment

Hemophagocytic lymphohistiocytosis (HLH) is not an independent disease but rather a life-threatening clinical syndrome that occurs in many underlying conditions and in all age groups. HLH is the consequence of a severe, uncontrolled hyperinflammatory reaction that in most cases is triggered by an infectious agent. Persistent stimulation of lymphocytes and histiocytes results in hypercytokinemia, leading to the characteristic symptoms of HLH. Genetic defects in familial HLH and in immunodeficiency syndromes associated with albinism affect the transport, processing, and function of cytotoxic granules in natural killer cells and cytotoxic T lymphocytes. This leads to defective killing of target cells and a failure to contract the immune response. The defects are increasingly found also in adolescents and adults. Acquired HLH occurs in autoinflammatory and autoimmune diseases (macrophage activation syndrome) and in patients with iatrogenic immunosuppression or with malignancies, but also in otherwise healthy persons with infections. Treatment of HLH aims at suppressing hypercytokinemia and eliminating the activated and infected cells. In genetic HLH, hematopoietic stem cell transplantation (HSCT) is needed for the correction of the immune defect. Treatment modalities include immunosuppressive, immunomodulatory, and cytostatic drugs; T-cell antibodies; and anticytokine agents. Using immunochemotherapy, familial HLH, which had been invariably fatal, has become a curable disease with more than 50% survivors. Reduced intensity conditioning for HSCT, which is associated with less transplantation-related mortality, will further improve cure rates.

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.

Syntaxin binding mechanism and disease-causing mutations in Munc18-2.

Mutations in either syntaxin 11 (Stx11) or Munc18-2 abolish cytotoxic T lymphocytes (CTL) and natural killer cell (NK) cytotoxicity, and give rise to familial hemophagocytic lymphohistiocytosis (FHL4 or FHL5, respectively). Although Munc18-2 is known to interact with Stx11, little is known about the molecular mechanisms governing the specificity of this interaction or how in vitro IL-2 activation leads to compensation of CTL and NK cytotoxicity. To understand how mutations in Munc18-2 give rise to disease, we have solved the structure of human Munc18-2 at 2.6 Å resolution and mapped 18 point mutations. The four surface mutations identified (R39P, L130S, E132A, P334L) map exclusively to the predicted syntaxin and soluble N-ethylmaleimide-sensitive factor accessory protein receptor binding sites of Munc18-2. We find that Munc18-2 binds the N-terminal peptide of Stx11 with a ~20-fold higher affinity than Stx3, suggesting a potential role in selective binding. Upon IL-2 activation, levels of Stx3 are increased, favoring Munc18-2 binding when Stx11 is absent. Similarly, Munc18-1, expressed in IL-2-activated CTL, is capable of binding Stx11. These findings provide potential explanations for restoration of Munc18-Stx function and cytotoxicity in IL-2-activated cells.

Association of Nonimmune Hydrops Fetalis With Familial Hemophagocytic Lymphohistiocytosis in Identical Twin Neonates With Perforin His222Arg (c665A>G) Mutation

This is the first study demonstrating that nonimmune hydrops fetalis (NIHF) in identical twin neonates is associated with biallelic gene defect causing familial hemophagocytic lymphohistiocytosis. Preterm male twins (31(4/7) wk) with NIHF and hepatosplenomegaly gradually developed pancytopenia, hyperferritinemia, hyponatremia, hypoalbuminemia, and elevated alanine aminotransferase, aspartate aminotransferase, bilirubin, and lactate dehydrogenase levels. Suspected sepsis led to antibiotic therapy. Upon detection of hemophagocytosis in bone marrow, multiorgan failure and pulmonary bleeding led to death. Homozygous His222Arg (c665A>G) mutation was identified in Perforin. Familial hemophagocytic lymphohistiocytosis should be considered in first days of NIHF cases to have chance for HLH-2004 therapy. Missense mutations of Perforin codon His222 may lead to intrauterine presentation.

Stem cell transplantation for primary immune deficiency

In this article, we summarize the recent advances in treating primary immune deficiency (PID) disorders by stem cell transplantation (SCT); we have focused on articles published in the past 2 years since the last major review of SCT for PID. Analyses of the outcomes of SCT for PID by specific molecular defect have clarified which conditions are receptive to unconditioned transplants and which require more myeloablative conditioning. Improved outcomes for 'difficult' conditions [adenosine deaminase-severe combined immunodeficiency (ADA-SCID), major histocompatibility complex class II deficiency] and potential advantages of using cord blood as a stem cell source have also been described. Newborn screening for SCID identifies well babies with SCID: the optimal SCT protocol for such young infants remains to be determined. Reduced toxicity conditioning has been successfully used to treat conditions such as Wiskott-Aldrich syndrome and chronic granulomatous disease, offering curative engraftment with reduced transplant-related mortality. Similarly, treating children with familial hemophagocytic lymphohistiocytosis using reduced intensity conditioning SCT results in much improved outcomes. Advances in next generation sequencing have identified new diseases amenable to SCT, such as DOCK8 deficiency, resulting in improved quality of life and protection from malignancy. Recent studies suggest that further improvements in treating PID with SCT are possible with a greater understanding of the genetics and immunobiology of these diseases, facilitating the matching of donor type and conditioning regimens, or indeed alternative therapies (such as gene therapy) to specific PID disorders.

Mutation of FAS, XIAP, and UNC13D Genes in a Patient With a Complex Lymphoproliferative Phenotype

This article presents a case report for a child presenting with mixed clinical features of autoimmune lymphoproliferative syndrome (ALPS), familial hemophagocytic lymphohistiocytosis (FHL), and X-linked lymphoproliferative (XLP) disease. From 6 months, he exhibited splenomegaly and lymphoadenopathy and from 4 years, he showed recurrent severe autoimmune hemocytopenia and sepsislike bouts of fever, from which he eventually died at the age of 12. Intriguingly, the patient carried mutations in FAS, XIAP, and UNC13D genes, which are involved in ALPS, XLP disease, and FHL, respectively. These mutations were inherited from the mother, who had rheumatoid arthritis but no signs of ALPS. A role for other modifying genes was suggested by the finding that the healthy father exhibited defective Fas function, without mutation of the FAS gene, and had transmitted to the patient an osteopontin (OPN) gene variant previously associated with ALPS. Therefore, several genes might influence the disease outcome in this family. In vitro analyses revealed that the FAS and the XIAP mutations decreased expression of the corresponding proteins, and the UNC13D mutation decreased granule secretion and Munc interaction with Rab-27a. These findings suggest that overlap may exist between ALPS, FHL, and XLP disease, in accordance with the notion that FHL and XLP disease are due to defective natural killer (NK)/NK T-cell function, which involves Fas. Therefore, we propose that NK cell defects should be evaluated in patients with ALPS-like characteristics, and hematopoietic stem cell transplantation should be considered in individuals with severe refractory cytopenia and FHL-like manifestations.

Down-regulation of CD5 expression on activated CD8+ T cells in familial hemophagocytic lymphohistiocytosis with perforin gene mutations

Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of T cells and macrophages with overproduction of cytokines. Familial HLH type 2 (FHL2) is the most common form of primary HLH and is caused by mutations in PRF1. We have recently described a significant increase in the subpopulation of CD8+ T cells with clonal expansion and CD5 down-regulation in Epstein-Barr virus associated-HLH, which represented a valuable tool for its diagnosis. However, this unusual phenotype of CD8+ T cells has not been investigated fully in patients with FHL2. We performed immunophenotypic analysis of peripheral blood and measured serum pro-inflammatory cytokines in five patients with FHL2. All patients showed significantly increased subpopulations of activated CD8+ T cells with down-regulation of CD5, which were negligible among normal controls. Analysis of T-cell receptor Vβ repertoire suggested the reactive and oligoclonal expansion of these cells. The proportion of the subset declined after successful treatment concomitant with reduction in the serum levels of cytokines in all patients except one who continued to have a high proportion of the subset and died. These findings suggest that down-regulation of CD5 on activated CD8+ T cells may serve as a useful marker of dysregulated T cell activation and proliferation in FHL2.

Hemophagocytic Lymphohistiocytosis: Advances in Pathophysiology, Diagnosis, and Treatment

emophagocytic lymphohistiocytosis (HLH) is a potentially life-threatening disorder characterized by immune dysregulation, overwhelming immune activation, and inflammation. HLH has been categorized as primary or familial HLH (FHLH), when there is a family history of HLH or known underlying genetic defects. Reactive or secondary HLH occurs in the setting of infection or underlying rhematologic disorders or malignancy. HLH occurring in a setting of rheumatologic illness is commonly referred to as macrophage activation syndrome (MAS). Most patients with FHLH have defects in lymphocyte cytotoxicity, leading to ineffective infection control and immune dysregulation resulting in massive activation and expansion of cytotoxic T cells and macrophages. This immune activation results in marked elevation of inflammatory cytokines, including interferon (IFN)-g, interleukin (IL)-1, IL-6, and IL-10, and an ensuing extreme hyperinflammatory state. Clinically, patients with FHLH or secondary HLH typically present with high-grade fever, progressive cytopenias, liver dysfunction, coagulopathy, and variable degrees of neurologic symptoms. Depending on the predominant organ system involved in HLH, patients may be seen by different clinical subspecialists. Initial manifestations of HLH may include fulminant hepatic failure or isolated neurologic involvement presenting with seizures and altered sensorium. Over the last decade, research on the genetics and pathophysiology of HLH has greatly improved our understanding of this condition. Importantly, increasing awareness and availability of better treatment options have improved the prognosis of HLH from a fatal disorder to a treatable condition with good long-term survival. In this review, we present recent advances in genetic and pathophysiological research, rapid diagnostic modalities, and clinical management of HLH.

Feasibility of reduced-intensity conditioning followed by unrelated cord blood transplantation for primary hemophagocytic lymphohistiocytosis: a nationwide retrospective analysis in Japan

A nationwide retrospective analysis was performed on patients who received allogeneic hematopoietic stem cell transplantation for primary or familial hemophagocytic lymphohistiocytosis (HLH) in Japan. The present analysis investigated whether reduced-intensity conditioning (RIC) followed by cord blood transplantation (CBT) (RIC-CBT) is feasible, compared to the outcomes of myeloablative conditioning and bone marrow transplantation. Based on the JSHCT data, 53 patients were analyzed. The overall survival rate (OS) was 65.4±6.6%. RIC-CBT (n=13) was not inferior to other methods. Patients with a performance status of PS 4 (ECOG scale) with HLH-associated severe organ dysfunction during the initiation of conditioning had extremely poor outcomes. The OS rate in the RIC-CBT patients, excluding those with a performance status 4, was 80.0±12.6%. RIC may reduce treatment-related mortality; in addition, patients with engraftment failure, which is the main adverse event following RIC-CBT, were successfully rescued with secondary CBT. Unrelated cord blood may represent an alternative source if a patient has no related donor. As a RIC regimen for CBT, 140mg/m(2) melphalan with fludarabine and anti-lymphocyte globulin or anti-thymocyte globulin may be feasible, but further dosage optimization should be performed in controlled clinical trials.

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations

AbstractFamilial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.