Familial Form Of Hemophagocytic Lymphohistiocytosis Research Articles

Whole exome sequencing identifies causative compound heterozygous variants in PRF1 in late-onset familial hemophagocytic lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare and life-threatening disease in which cells of the immune system are overactivated, leading to uncontrolled inflammation and tissue destruction. Inherited or familial forms of HLH (FHL) are further classified into FHL1 to 5, based on the underlying genetic etiology. The most common form, FHL2, is associated with mutations in the PRF1 gene encoding perforin, a pore-forming glycoprotein required for natural killer and cytotoxic T cell-mediated apoptosis. Importantly, diagnosis of FHL can be challenging, particularly in late-onset cases in which presentation is delayed beyond the first years of life. Aim: We report the essential role of whole exome sequencing in the diagnostic work-up of a patient with complex, late-onset FHL. Methods: A comprehensive retrospective chart review was performed. Results: Our patient presented at 11 years of age with recurrent fever, hepatosplenomegaly, and pancytopenia. In the following years, she was admitted to hospital on multiple occasions, including twice for febrile neutropenia, and once for febrile cytopenia. Serial immune evaluation revealed features of immune dysregulation. While HLH was suspected, she did not fulfil the diagnostic criteria. Initial genetic work-up involving a targeted primary immunodeficiency gene panel identified only a single novel variant of uncertain significance, c.T374C (p.I125T) in PRF1. Subsequently, research-based whole exome sequencing was performed which revealed a second variant, c.C272T (p.A91V), in the same gene. The expanded genetic findings, a set of compound heterozygous missense mutations in PRF1, strengthened the diagnosis of FHL. She later fulfilled the diagnostic criteria for HLH. Conclusion: Whole exome sequencing identified compound heterozygous mutations in the PRF1 gene in a patient with late-onset FHL. Statement of Novelty: We report the use of whole exome sequencing to identify compound heterozygous mutations in PRF1, including a novel p.I125T variant not previously identified in FHL.

Effective Control of Epstein-Barr Virus–Related Hemophagocytic Lymphohistiocytosis With Immunochemotherapy

The familial form of hemophagocytic lymphohistiocytosis (HLH) is a lethal disorder. Although the prognosis for Epstein-Barr virus-associated HLH (EBV-HLH) remains uncertain, numerous reports indicate that it can also be fatal in a substantial proportion of cases. We therefore assessed the potential of immunochemotherapy with a core combination of steroids and etoposide to control EBV-HLH in 17 infants and children who met stringent diagnostic criteria for this reactive disorder of the mononuclear phagocyte system. Treatment of life-threatening emergencies was left to the discretion of participating investigators and typically included either intravenous Ig or cyclosporin A (CSA). Five patients (29%) entered complete remission during the induction phase (1 to 2 months), whereas 10 others (57%) required additional treatment to achieve this status. In 2 cases, immunochemotherapy was ineffective, prompting allogeneic bone marrow transplantation. Severe but reversible myelosuppression was a common finding; adverse late sequelae were limited to epileptic activity in one child and chronic EBV infection in 2 others. Fourteen of the 17 patients treated with immunochemotherapy have maintained their complete responses for 4+ to 39+ months (median, 15+ months), suggesting a low probability of disease recurrence. These results provide a new perspective on EBV-HLH, showing effective control (and perhaps cure) of the majority of EBV-HLH cases without bone marrow transplantation, using steroids and etoposide, with or without immunomodulatory agents.

Hemophagocytic lymphohistiocytosis: An update on pathogenesis, diagnosis, and therapy.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening state of immune hyperactivation that arises in the setting of genetic mutations and infectious, inflammatory, or neoplastic triggers. Sustained, aberrant activation of cytotoxic CD8+ T cells and resultant inflammatory cytokine release are core pathogenic mechanisms. Key clinical features include high persistent fever, hepatosplenomegaly, blood cytopenia, elevated aminotransferase and ferritin levels, and coagulopathy. HLH is likely under-recognized, and mortality remains high, especially in adults; thus, prompt diagnosis and treatment are essential. Familial forms of HLH are currently treated with chemotherapy as a bridge to hematopoietic stem cell transplantation. HLH occurring in rheumatic disease (macrophage activation syndrome) is treated with glucocorticoids, IL-1 blockade, or cyclosporine A. In other forms of HLH, addressing the underlying trigger is essential. There remains a pressing need for more sensitive, context-specific diagnostic tools. Safer, more effective therapies will arise with improved understanding of the cellular and molecular mechanisms of HLH.

Hemophagocytic Lymphohistiocytosis in Children

Background: Hemophagocytic lymphohistiocytosis (HLH) is a disorder of the immune regulatory system with a high rate of mortality. The clinical presentation of HLH could be similar to those of many other conditions, making its diagnosis difficult. Methods: The current study was conducted on the children with HLH referred to the Pediatric Ward of Imam Reza Hospital, Mashhad, Iran during nine years from 2010 to 2018. The data collection tool included items on clinical presentations, physical examination, laboratory data, treatment strategy, and the disease outcome. The diagnosis of HLH was made based on the HLH-2004 Diagnostic Criteria. Data were analyzed using SPSS version 20. Results: Among the 17 cases with HLH, the mean age at the onset of HLH was 4.671 ± 4.60 years with a male to female ratio of 11:6. Fever and splenomegaly were the most common findings. The study of the etiology revealed that about 30 of the children had the familial form of HLH, 17.5 were previously diagnosed with rheumatologic disorders (i.e., systemic lupus erythematosus and systemic-onset juvenile idiopathic arthritis), and the same rate was affected by infectious diseases. Idiopathic factors, the Chediak-Higashi syndrome, and the Griscelli syndrome were responsible for the occurrence of HLH in 17.5, 12, and 6 of the cases, respectively. According to the obtained results of the study, 35.3 of the patients were cured and about 64.7 died. Conclusions: Due to the high risk of mortality from this disorder, the recognition of HLH symptoms, especially atypical presentations of the disorder, is critical both for pediatricians and subspecialists. © 2019, Author(s).

“Identification of a Deletion in Stxbp2 Causative of Familial Hemophagocytic Lymphohistiocytosis Type 5”

Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening immune deficiency, characterized by a hyper-inflammatory syndrome. The familial form of HLH (FHL) is caused by mutations in genes associated with lymphocyte granule-mediated cytotoxicity. Mutations in STXBP2 (Sintaxin binding protein 2) gene result in defect of Munc18-2 protein, the causative defect of the subtype defined as FHL5. Functional tests as intracytoplasmic expression of perforin and surface expression of CD107a, help to direct genetic analysis. Different mutations have been described in the FHL-related genes known so far (PRF1, UNC13-D, STX11, STXBP2): missense, nonsense, splicing, regulatory, small deletions/insertions. Recently a pathogenic inversion of 253 KB upstream of the 3’ UNC13D gene has been reported. Here we describe a new deletion causative of FHL5. We confirmed the deletion by Real-Time PCR and by CGHarray. We finally documented by western blot the absence of expression of Munc18-2 protein in our patient. These data shows the need to introduce new diagnostic strategies in order to screen mutations not detected by the methods classically used.

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.

Elucidating the molecular mechanism of Syntaxin11 function in cytotoxic T lymphocytes (P3032)

Abstract Cytotoxic T lymphocytes (CTL) play a crucial role in eliminating virus infected and cancer cells through exocytosis of perforin and granzymeB containing lytic granules (LGs). Soluble NSF Attachment Protein Receptors (SNAREs) function as executors of exocytosis. The importance of SNAREs and SNARE associated proteins in CTL function and LG exocytosis is evident in the form of Familial Hemophagocytic Lymphohistiocytosis (FHL). Mutations within Syntaxin11 (Stx11), a Qa-SNARE protein, result in FHL subtype-4, however the mechanism by which Stx11 malfunction leads to this severe phenotype remains unknown. Here, we investigate the molecular mechanism of Stx11 function in primary human CTLs. High resolution live cell imaging with CTLs before and after target cell contact showed sequential polarization of Stx11 followed by LGs to the immunological synapse. Using TIRF (total internal reflection fluorescence) microscopy we further showed an association of LGs with plasma membrane-bound Stx11 at the IS. Further, knockdown studies using Stx11 specific siRNA resulted in a strong reduction of both LG fusion and dwell time at the IS. We therefore propose that Stx11 is required for the final fusion of LG at the IS, explaining the severe FHL4 phenotype.

Familial and Acquired Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome in which an uncontrolled and ineffective immune response, triggered in most cases by infectious agents, leads to severe hyperinflammation. Familial forms of HLH (FHL), which are increasingly found also in adolescents and adults, are due to genetic defects leading to impaired function of natural killer cells and cytotoxic T cells. These mutations occur either in the perforin gene or in genes important for the exocytosis of cytotoxic granules. Cytotoxic granules contain perforin and granzymes, which induce apoptosis upon entering (infected) target cells. Additionally, perforin is important for the downregulation of the immune response. Acquired forms of HLH are encountered in association with (usually) viral infections, autoinflammatory/autoimmune diseases, malignant diseases, and acquired immune deficiency states (e.g., after organ transplantation). Treatment of HLH includes immune-suppressive and immune-modulatory agents, cytostatic drugs, and biological response modifiers. For patients with FHL, stem cell transplantation is indicated and can be curative.

Novel mutation in STXBP2 prevents IL-2–induced natural killer cell cytotoxicity

We have identified dizygotic twins with a novel syntaxin-binding protein 2 (STXBP2) mutation, where cytotoxicity cannot be restored with IL-2. This defines STXBP2 as an absolute requirement for NK cell cytotoxic function.

Molecular study of the perforin gene in familial hematological malignancies

Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein.

The role of BMT in childhood histiocytoses

Childhood histiocytoses comprise two main diseases, Langerhans cell histiocytosis (LCH) and hemophagocytic lymphohistiocytosis (HLH). LCH is a rare disorder with obscure pathogenesis. Data on clonality suggested neoplastic origin, yet were not convincing. Dysregulation of cytokines and of DC trafficking and cross-talk are documented. Clinical manifestations and course are highly variable, ranging from self-healing solitary bone lesion to disseminated, multi-organ involvement with 20% fatality rate despite standard chemotherapy. HSCT has been applied in less than 50 cases, outside any trial, with good disease control but elevated early toxicity. The familial form of HLH (FHL) has been recognized as congenital immune deficiency, with mutations of PRF1, Munc13-4, syntaxin11 genes resulting in defective cellular cytotoxicity machinery. Chemo-immunotherapy allows temporary disease control, but HSCT holds as the only procedure with potential for cure. Rapid identification of genetic defects allows differential diagnosis from transient, virus-associated HLH, thus indicating early HSCT. The role of HSCT in childhood histiocytoses is thus very important. Better understanding of the pathogenesis, in particular of genetic and immune function defects, will help to tailor indications and, possibly less toxic, conditioning regimens, reducing treatment-related mortality, and thus disclosing the way to final cure.

Mutations of the hemophagocytic lymphohistiocytosis–associated gene UNC13D in a patient with systemic juvenile idiopathic arthritis

The clinical syndromes of hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are both characterized by dysregulated inflammation with prolonged fever, hepatosplenomegaly, coagulopathy, hematologic cytopenias, and evidence of hemophagocytosis in the bone marrow or liver. While HLH is either inherited or acquired, children with severe rheumatic diseases, most notably systemic juvenile idiopathic arthritis, are at risk for MAS. The phenotypic similarity between HLH and MAS raises the possibility that they share common pathogenetic mechanisms. Familial forms of HLH have been attributed to mutations in the genes encoding perforin (PRF1) and Munc13-4 (UNC13D), among others, and are characterized by defective cytotoxic lymphocyte function. While some patients with systemic JIA have decreased levels of perforin protein expression and natural killer (NK) cell function, mutations of HLH-associated genes in patients with systemic JIA have not been reported. We report the case of an 8-year-old girl with systemic JIA without MAS who was found to have compound heterozygous mutations of UNC13D and reduced NK cell cytotoxic function. This case broadens the range of clinical phenotypes attributable to UNC13D mutations and offers new insights into the etiology and pathogenesis of systemic JIA.

Cutting Edge: Syntaxin 11 Regulates Lymphocyte-Mediated Secretion and Cytotoxicity

Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13-4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.

MUNC13–4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis Are Scattered over the Functional Domains of the Protein.

Mutations of PRF1 and MUNC13–4 genes, involved with cellular cytotoxicity, are associated with the familial form of Hemophagocytic Lymphohistiocytosis (HLH) type 2 (FHL2) and 3 (FHL3). In all patients with HLH, in which PRF1 mutations had been excluded, we screened MUNC13–4 mutations.The 32 exons and their proximal flanking regions of MUNC13–4 gene were sequenced by “cycle sequencing” approach (BigDye Terminator, Applied Biosystems).Of 60 patients, 21 had MUNC13–4 mutations. 5 were reported mutations: 753+1G>T, 817C>T (R273X); 1389+1G>A; 1822del 12 bp (del V608-A611); 2346–9delGGAG (R782FsX12). Of 16 novel mutations 2 (2212C>T and 2650C>T) introduce a stop codon (at Q738 and Q884); 4 cause frameshift: 441delA (P147fsX14), 532delC (Q178fsX70), 3082delC (L1028fsX), and 3226insG (H1076fsX51). Of 8 missense mutations [175G>A (A59T), 419T>C (I140T), 610A>G (M204B), 1241G>T (R414L), 1847A>G (E616G), 2039G>C (R680P), 2570T>G (F857C), 2782C>T (R928C)], 6 fall within the protein functional domains; each of the 2 falling outside was associated with 2 additional pathogenic mutations.The remaining novel 1992+5 G>A and 2448insC −12 fall outside the coding region but are close enough to induce alternative splicing. We identified polymorphisms 279C>T and 3198A>G. The 2599A>G (K867E) transition was found in several unrelated families, including one homozygous parent. To assess its frequency we studied 50 consecutive newborns, of which 64% were heterozygous.To understand the effects of the mutations we analysed Munc13–4 protein expression from CTL and/or NK cells by western blot using an antibody raised against amino acids 1–262. Trace amounts of protein could be detected only in 5 patients. Analysis of granule polarisation in 2 patients with trace amounts of Munc13–4 protein showed many more docked granules visible than in controls, consistent with a block in granule secretion in these patients.Median age at diagnosis of FHL3 was 6.5 months, but 8/21 (38%) patients were diagnosed when older than 5 years, with one young adult of 18 years. CNS disease was present in 10 patients; NK was markedly reduced or absent in all patients tested. MUNC13–4 mutations were found in 35% of our HLH patients non type-2. Mutations were almost entirely different from those reported so far and scattered over different exons, but in far most cases they fall within the protein functional domain. Since these patients may develop the disease during adolescence or even later on, not only pediatric but also as adults hematologists should include FHL-2 and 3 in the differential diagnosis of children and young adults with fever, cytopenia, splenomegaly and hypercytokinemia.

A proportion of patients with lymphoma may harbor mutations of the perforin gene

AbstractPerforin mutations have been demonstrated in a proportion of patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH). In the present study, we evaluated whether some patients with lymphoma sharing clinical characteristics with HLH might harbor mutations of the perforin gene. We analyzed 29 patients and found that 4 patients, who developed either Hodgkin or non-Hodgkin lymphoma, had biallelic mutations of the perforin gene. One of these 4 patients, a 19-year-old female with T-cell lymphoma, had a brother carrying the same mutations who developed HLH. In 2 of the 4 patients with biallelic mutations of the perforin gene, we evaluated perforin expression by flow cytometry and natural killer (NK) activity and both were found to be absent. Moreover, we documented the presence of monoallelic mutations of the perforin gene in 4 more patients. One of these 4 latter patients also carried a mutation of the Fas gene. These data indicate that perforin deficiency, either alone or in combination with other mutations of genes involved in lymphocyte survival or functional activity, may be present in patients with lymphoma. These findings suggest that perforin also plays a key role in the mechanisms of immune surveillance that prevent tumor growth and/or development.

Successful Treatment of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis with HLH-94 Protocol

Hemophagocytic lymphohistiocytosis (HLH) is a rare, fatal disorder of children, affecting predominantly the mononuclear phagocytic system. Previous reports indicate that Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (EBV-HLH) can also be fatal in many cases, although the prognosis for EBV-HLH is better than for the familial form of hemophagocytic lymphohistiocytosis. We treated four patients with EBV-HLH using immunochemotherapy including steroid, etoposide (VP-16), and cyclosporin, according to the HLH-94 protocol. All patients experienced persistent fever, cytopenia, and hypertriglyceridemia. Serological testing for EBV showed reactivated EBV infections in all patients. EBV DNA detected by PCR and EBV-encoded small RNA measured by in situ hybridization were confirmed in the patients' bone marrow specimens. Hemophagocytosis was shown in bone marrow aspirates and liver biopsy specimen. Complete remission was achieved in all patients after induction and continuation therapy for 4-10 months (median, 7 months) and was maintained for 15-27 months (median, 19 months) without the need for bone marrow transplantation. These results suggest that EBV-HLH can be effectively controlled by immunochemotherapy using the HLH-94 protocol.

Munc13-4 Is Essential for Cytolytic Granules Fusion and Is Mutated in a Form of Familial Hemophagocytic Lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.

Elevated circulating levels of interleukin-1 receptor antagonist but not IL-1 agonists in hemophagocytic lymphohistiocytosis.

The familial form of hemophagocytic lymphohistiocytosis (HLH) is an inherited disease with disturbed immunomodulation and characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, and coagulopathy, i.e., findings which are similar to many of the reported biological effects of the inflammatory cytokines. Due to the previously shown hypercytokinemia in active HLH with elevated levels of interleukin (IL)-6, tumor necrosis factor-alpha, and interferon-gamma, it has been suggested that cytokine dysregulation may be of pathophysiological importance. Here we have assayed the serum levels of the members of the IL-1 ligand family, the two agonists IL-1 alpha and IL-1 beta and the antagonist IL-1 receptor antagonist (IL-1ra), in nine children with HLH and cerebrospinal fluid (CSF) specimens from four children. Serum IL-1ra was elevated in all patients with active disease to a degree which correlated well with disease activity. Furthermore, the levels decreased day by day during treatment of a patient who suffered a relapse. Moreover, high levels of IL-1ra were also detected in CSF during active disease. However, IL-1 beta levels were all within normal limits and circulating IL-1 alpha levels were normal in all but two patients.