Diagnostic Performance of Hemophagocytic Lymphohistiocytosis Criteria and HScore in Critically Ill Patients With Severe Hemophagocytic Syndrome.

OBJECTIVES: To assess whether critically ill hematologic patients without diagnosis of hemophagocytic lymphohistiocytosis may have features mimicking hemophagocytic lymphohistiocytosis according to both diagnostic scores. DESIGN: A retrospective case-control study. SETTING: Hemophagocytic syndrome diagnosis was standardized and based on a consensual diagnosis by at least two experts of a university hospital which is a reference center for hemophagocytic syndrome. PATIENTS: Cases (hemophagocytic syndrome+) consisted in a group of consecutive patients (n = 150) admitted in our ICU between 2007 and 2018. Control group (hemophagocytic syndrome-) consisted in patients included in a prospective multicenter cohort of hematologic patients in whom three independent experts ruled out the diagnosis of hemophagocytic syndrome (n = 1011). MEASUREMENTS AND MAIN RESULTS: Overall, 1,161 patients were included. Hospital mortality was 45.8% in hemophagocytic syndrome- patients (n = 66) and 38.8% in control patients (n = 392; p = 0.126). Median HScore was 235 (205-262) in hemophagocytic syndrome+ and 42 (18-62) in hemophagocytic syndrome- patients (p < 0.001); number of hemophagocytic lymphohistiocytosis criteria was 4 (4-5) vs 1 (0-1), respectively (p < 0.001). Diagnostic performances of both scores were excellent with area under receiver operating characteristic curve of 0.99 (95% CI, 0.99-0.99) and 0.99 (95% CI, 0.99-0.99) for hemophagocytic lymphohistiocytosis and HScore, respectively. After propensity score matching (n = 144 × 2), the median HScore was 234 (205-262) in hemophagocytic syndrome+ patients versus 49 (18-71) in hemophagocytic syndrome- patients (p < 0.001). Median number of hemophagocytic lymphohistiocytosis criteria was 4 (4-5) in hemophagocytic syndrome+ and 1 (0-1) in hemophagocytic syndrome- patients (p < 0.001). Area under receiver operating characteristic curve was then of 0.98 (95% CI, 0.96-0.99) for hemophagocytic lymphohistiocytosis criteria and 0.99 (95% CI, 0.99-1) for HScore. CONCLUSIONS: In ICU patients, several conditions share some similarities with hemophagocytic syndrome, explaining the poor predictive value of isolated biological markers such as ferritin level. Despite these potential confounding factors, our study suggests HScore and hemophagocytic lymphohistiocytosis criteria to be highly discriminant identifying hemophagocytic syndrome in critically ill patients.

Hemophagocytic lymphohistiocytosis in critically ill patients: diagnostic reliability of HLH-2004 criteria and HScore.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a rare though often fatal hyperinflammatory syndrome mimicking sepsis in the critically ill. Diagnosis relies on the HLH-2004 criteria and HScore, both of which have been developed in pediatric or adult non-critically ill patients, respectively. Therefore, we aimed to determine the sensitivity and specificity of HLH-2004 criteria and HScore in a cohort of adult critically ill patients. METHODS: In this further analysis of a retrospective observational study, patients ≥ 18 years admitted to at least one adult ICU at Charité - Universitätsmedizin Berlin between January 2006 and August 2018 with hyperferritinemia of ≥ 500 µg/L were included. Patients' charts were reviewed for clinically diagnosed or suspected HLH. Receiver operating characteristics (ROC) analysis was performed to determine prediction accuracy. RESULTS: In total, 2623 patients with hyperferritinemia were included, of whom 40 patients had HLH. We found the best prediction accuracy of HLH diagnosis for a cutoff of 4 fulfilled HLH-2004 criteria (95.0% sensitivity and 93.6% specificity) and HScore cutoff of 168 (100% sensitivity and 94.1% specificity). By adjusting HLH-2004 criteria cutoffs of both hyperferritinemia to 3000 µg/L and fever to 38.2 °C, sensitivity and specificity increased to 97.5% and 96.1%, respectively. Both a higher number of fulfilled HLH-2004 criteria [OR 1.513 (95% CI 1.372-1.667); p <  0.001] and a higher HScore [OR 1.011 (95% CI 1.009-1.013); p <  0.001] were significantly associated with in-hospital mortality. CONCLUSIONS: An HScore cutoff of 168 revealed a sensitivity of 100% and a specificity of 94.1%, thereby providing slightly superior diagnostic accuracy compared to HLH-2004 criteria. Both HLH-2004 criteria and HScore proved to be of good diagnostic accuracy and consequently might be used for HLH diagnosis in critically ill patients. CLINICAL TRIAL REGISTRATION: The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.

Outcomes in children with hemophagocytic lymphohistiocytosis treated using HLH-2004 protocol in Japan.

Recent advances in intensive chemo- and immunotherapy have contributed to the outcome of hemophagocytic lymphohistiocytosis (HLH); however, the prognosis of HLH in children differs by HLH subtype. In Japan, secondary HLH, particularly Epstein-Barr virus-associated HLH (EBV-HLH), is the most common HLH subtype. The prognosis of HLH has improved in recent years. We here conducted a prospective study of 73 patients who were treated with HLH-2004 protocol in Japan. EBV-HLH, familial HLH (FHL), and HLH of unknown etiology were seen in 41, 9, and 23 patients, respectively. Patients with resistant or relapsed disease after HLH-2004 treatment and those with FHL received hematopoietic stem cell transplantation (HSCT). The induction rate after initial therapy was 58.9%, and the 3-year overall survival (OS) rate of all patients was 73.9% and differed significantly among those with EBV-HLH, FHL, and HLH of unknown etiology. Of the 17 patients who received HSCT, the 3-year OS rates of those with and without complete resolution before HSCT were 83.3% and 54.5%, respectively. Outcomes in children with HLH who were treated with the same protocol differed among HLH subtypes. Appropriate strategy for each subtype should be established in future studies.

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

An effective diagnostic index for lymphoma-associated hemophagocytic syndrome.

BACKGROUND: Lymphoma-associated hemophagocytic syndrome (LAHS) is a highly fatal immune disorder. Poor prognosis is partly attributed to under diagnosis or delayed diagnosis. AIM: Early identification of LAHS patients based on the laboratory findings could improve the outcomes. DESIGN: Retrospective observational cross-sectional study. METHODS: From January 2011 to June 2016, 282 adult patients with hemophagocytosis in bone marrow were enrolled, and 114 hemophagocytic lymphohistiocytosis (HLH) patients with definite underlying cause were finally included for analysis. The HLH patients were further divided into LAHS (76 out of 114) and non-malignancy-associated HLH (38 out of 114) groups. RESULTS: Compared to non-malignancy-associated HLH, LAHS patients had significantly elevated lactate dehydrogenase (LDH) levels, increased thickness of spleen, higher proportion of patients with lymphadenopathy and significantly decreased peripheral blood cell count. In multivariate logistic regression model analysis, thickness of spleen ≥5 cm (OR = 17.9, 95%CI 1.35-236.6; P = 0.028), IL-6 level ≥55.1 pg/ml (OR = 12.01, 95%CI 1.03-138.9; P = 0.047) and IL-10 level ≥425.9 pg/ml (OR = 51.18, 95%CI 2.53-1035.1; P = 0.010) were independent predictors of LAHS diagnosis. Based on the regression parameters, we established a diagnostic index with weighted risk scores of 1 assigned to thickness of spleen and IL-6 level respectively, and a score of 3 assigned to IL-10 level. A diagnostic index ≥ 2 points had the best AUC value (0.889) with 84.2% of sensitivity and 93.7% of specificity for predicting LAHS. CONCLUSIONS: LAHS can be considered when HLH patients have a diagnostic index ≥2 points, so actively looking for evidence of lymphoma and effective chemotherapy may be necessary.

Understanding the spectrum of haemophagocytic lymphohistiocytosis: update on diagnostic challenges and therapeutic options.

The cytokine storm syndrome 'haemophagocytic lymphohistiocytosis' (HLH) is an under-recognized hyperinflammatory disorder, causing high morbidity and mortality risk in children and adults. It can be subdivided into a primary, genetic form and a secondary, acquired form that complicates diverse infections, malignancies and autoimmune or autoinflammatory disorders. Both subtypes present with the same spectrum of non-specific symptoms, making accurate diagnosis and rapid treatment initiation challenging. In the last decade, increased awareness and international collaborative efforts fuelled a marked progress in diagnostic protocols and novel treatment strategies for HLH and new diagnostic guidelines are being tailored to specific secondary HLH subtypes. Therapy is gradually shifting its focus from overall immunosuppression towards targeting specific cytokines, cell types or signalling pathways underlying pathophysiology. Nevertheless, continued research efforts remain indispensable to customize therapy to individual patient needs.

Current Updates on Classification, Diagnosis and Treatment of Hemophagocytic Lymphohistiocytosis (HLH).

Hemophagocytic lymphohistiocytosis (HLH) is a life threatening hyperinflammatory syndrome characterized by excessive activation of macrophages and T cells resulting from defective cytotoxicity. Severe hyperinflammation caused by uncontrolled proliferation of activated lymphocytes and histiocytes (macrophages) secreting high amounts of inflammatory cytokines threatens the life of the patient and may lead to death unless arrested by appropriate treatment. HLH can be caused either by certain underlying genetic diseases (familial HLH), or may also occur due to particular triggers in patients with no known inherited disorder (acquired HLH). Due to life threatening nature of the disease, early diagnosis and initiation of immunosuppressive therapy is extremely important. HLH diagnosis is based on constellation of clinical manifestations and laboratory parameters which often overlap with those of severe infection or sepsis. Identification of patients with familial HLH and their underlying genetic defects requires specialized laboratory tests and is important for predicting relapses and planning early therapeutic hematopoietic stem cell transplantation (HSCT). A high suspicion and thorough clinical, immunological and genetic work-up is required for diagnosis of HLH. Prompt initiation of adequate treatment is essential for the survival. Substantial progress has been made in exploring the complex cause and pathophysiology of HLH and also in management of HLH patients.

Hemophagocytic Syndrome and Critical Illness: New Insights into Diagnosis and Management.

Hemophagocytic lymphohistiocytosis (HLH) comprises a heterogeneous group of diseases that are characterized by a hyperinflammatory state due to uncontrolled T cell, macrophage, and histiocyte activation, accompanied by excessive cytokine production. This rare condition is almost uniformly fatal unless promptly recognized and treated. Much progress has been made in the last two decades in our understanding of the mechanisms underlying familial, and to a lesser extent, acquired cases of HLH. Recurrent mutations in more than 10 different genes have now been identified, involving biological pathways converging on intracellular vesicle trafficking and cytolytic granule exocytosis. Mechanisms underlying the majority of patients with acquired HLH, however, remain elusive, hampering both diagnostic evaluation and therapeutic management of these patients. Given that the majority of intensive care unit (ICU) patients with sepsis or multiorgan failure share many features of HLH, it is especially critical for pediatric and adult intensivists to be able to recognize patients with bona fide HLH and initiate treatment without delay. In this article, we review our current understanding of the pathophysiology, clinical testing, diagnosis, and treatment of patients with HLH, especially as it pertains to the care of critically ill patients in pediatric and medical ICUs.

Diagnosis and classification of adult Still's disease.

The cornerstone of adult onset Still's disease is the triad of daily fever, arthritis and rash. This syndrome remains enigmatic and most often a disease of exclusion. There are both musculoskeletal as well as systemic features. More importantly, reactive hemophagocytic syndrome may occur in patients. In this review we attempt to place this syndrome in perspective, including data on geoepidemiology, clinical and laboratory features.

[Hemophagocytic syndrome. Current concepts]. / Síndrome hemofagocítico. Conceptos actuales.

Hemophagocytic lymphohistiocytosis is a syndrome characterized by pathological immune activation that may occur as either a primary a familial disorder (associated with genetic mutations), or as a sporadic condition, associated to infections, malignancies or autoimmune diseases. The clinical picture is characterized by a disproportionate inflammation that causes fever, cytopenias, splenomegaly, bone marrow hemophagocytosis, hypertriglyceridemia and hypofibrinogenemia. Syndrome-related mortality is high, so it is important to maintain a high index of suspicion and start early treatment with immunochemotherapy and bone marrow transplantation in primary and refractory cases. In this article, we review the clinical manifestations, pathology, diagnosis and treatment of these patients.

[Type III familial hemophagocytic lymphohistiocytosis susceptibility gene UNC13D involves in homologous recombination repair].

This study was aimed to explore the pathogenesis of type III familial hemophagocytic lymphohistiocytosis (FHL3) via susceptibility gene UNC13D involving in homologous recombination repair (HRR) of DNA double-strand break (DSB). By means of DNA homologous recombination repair, the change of homologous recombination repair rate of normal control cells and DR-U2OS cells after down-regulation of UNC13D was detected; the UNC13D gene related function was explored. The results showed that DR-U2OS cells displayed a significant reduction in homologous recombination repair of DNA DSB after siRNA knockdown of UNC13D, compared to its normal control cell counterparts (P < 0.05), suggesting that UNC13D was involved in DNA double-stranded breakage repair. It is concluded that UNC13D gene mutation may be involved in the pathogenesis of FHL3 via its dual effects of both the cytotoxic granule exocytosis and decrease of homologous recombination repair rate after the DNA double-strand break, therefore, providing a new theoretical basis to reveal the pathogenesis of FHL3.

Gene-expression signatures differ between different clinical forms of familial hemophagocytic lymphohistiocytosis.

We performed gene-expression profiling of PBMCs obtained from patients with familial hemophagocytic lymphohistiocytosis (FHL) to screen for biologic correlates with the genetic and/or clinical forms of this disease. Unsupervised hierarchical clustering of 167 differentially expressed probe sets, representing 143 genes, identified 3 groups of patients corresponding to the genetic forms and clinical presentations of the disease. Two clusters of up- and down-regulated genes separated patients with perforin-deficient FHL from those with unidentified genetic cause(s) of the disease. The clusterscomprised genes involved in defense/immune responses, apoptosis, zinc homeostasis, and systemic inflammation. Unsupervised hierarchical clustering partitioned patients with unknown genetic cause(s) of FHL into 2 well-distinguished subgroups. Patterns of up- and down-regulated genes separated patients with "late-onset" and "relapsing" forms of FHL from patients with an "early onset and rapidly evolving" form of the disease. A cluster was identified in patients with "late onset and relapsing" form of FHL related to B- and T-cell differentiation/survival, T-cell activation, and vesicular transport. The resulting data suggest that unique gene-expression signatures can distinguish between genetic and clinical subtypes of FHL. These differentially expressed genes may represent biomarkers that can be used as predictors of disease progression.

Diagnostic evaluation of patients with suspected haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterized by severely disturbed immune homeostasis. It can affect all age groups. Diagnostic evaluation of the patient with suspected HLH has to address three main questions: (i) does the patient have HLH? There is no simple diagnostic test, but a number of clinical and laboratory criteria define this clinical syndrome. (ii) Can a trigger be identified? A variety of infections, malignant or autoimmune diseases can contribute to the disturbed immune homeostasis with important consequences for treatment. (iii) Does the patient suffer from a genetic disease predisposing to HLH? Recent advances in the understanding of the genetic and pathophysiological basis of HLH have enabled a better and more rapid answer to this question, which is relevant for prognosis and the decision to perform haematopoietic stem cell transplantation. This review summarizes the current diagnostic approach to the patient with HLH.

Distinct mutations in STXBP2 are associated with variable clinical presentations in patients with familial hemophagocytic lymphohistiocytosis type 5 (FHL5).

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.

[Reactive and neoplastic histiocytic diseases in the dog]. / Reaktive und neoplastische histiozytäre erkrankungen beim hund.

There are different histiocytic diseases in dogs that are characterized by the proliferation of histiocytic cells (macrophages and myeloid dendritic cells). Histiocytic diseases can be devided into neoplastic (cutaneous histiocytoma complex, histiocytic sarcoma, dendritic cell leukaemia) and reactive forms (reactive histiocytosis, haemophagocytic syndrome). All subtypes of the cutaneous histiocytoma complex (cutaneous histiocytoma, metastatic histiocytoma and Langerhans' cell histiocytosis) are of Langerhans' cell origin. Histiocytoma, which is a solitary tumour of the skin in young dogs, shows spontaneous regression in most cases. Occasionally, metastasis to lymph nodes can be seen (metastatic histiocytoma). Only one dog with Langerhans' cell histiocytosis has been described and was euthanized. Histiocytic sarcoma, which arises from myeloid dendritic cells, can be classified as localised histiocytic sarcoma or disseminated histiocytic sarcoma. Another form of histiocytic sarcoma - haemophagocytic histiocytic sarcoma - is derived from macrophages. Histiocytic sarcoma displays a very aggressive clinical course and has a poor prognosis. Breed predispositions have been reported for the disseminated and haemophagocytic form of histiocytic sarcoma in Bernese mountain dogs, Rottweilers and varoiusretrievers. In contrast, reactive histiocytosis (cutaneous and systemic forms) develops by reactive proliferation of interstitial dendritic cells. In systemic histiocytosis, breed predilections are similar to histiocytic sarcoma. Haemophagocytic syndrome develops as a consequence of proliferation of activated macrophages in different tissues. Prognosis in general is moderate to poor and depends on the origin of the underlying disease process.

Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive, often-fatal hyperinflammatory disorder. Mutations in PRF1, UNC13D, STX11, and STXBP2 are causative of FHL2, 3, 4, and 5, respectively. In a majority of suspected FHL patients from Northern Europe, sequencing of exons and splice sites of such genes required for lymphocyte cytotoxicity revealed no or only monoallelic UNC13D mutations. Here, in 21 patients, we describe 2 pathogenic, noncoding aberrations of UNC13D. The first is a point mutation localized in an evolutionarily conserved region of intron 1. This mutation selectively impairs UNC13D transcription in lymphocytes, abolishing Munc13-4 expression. The second is a 253-kb inversion straddling UNC13D, affecting the 3'-end of the transcript and likewise abolishing Munc13-4 expression. Carriership of the intron 1 mutation was found in patients across Europe, whereas carriership of the inversion was limited to Northern Europe. Notably, the latter aberration represents the first description of an autosomal recessive human disease caused by an inversion. These findings implicate an intronic sequence in cell-type specific expression of Munc13-4 and signify variations outside exons and splice sites as a common cause of FHL3. Based on these data, we propose a strategy for targeted sequencing of evolutionary conserved noncoding regions for the diagnosis of primary immunodeficiencies.

Review of haemophagocytic lymphohistiocytosis.

Haemophagocytic lymphohistiocytosis (HLH) describes a clinical syndrome of hyperinflammation resulting in an uncontrolled and ineffective immune response. It may develop subsequent to a number of recognised genetic mutations or in association with infection, malignancy, autoinflammatory or metabolic conditions. Even with the published diagnostic criteria it can be difficult to make the diagnosis of HLH. Patients presenting acutely to the general paediatrician or paediatric intensivist with a clinical picture of likely sepsis, ie fever, laboratory evidence of inflammatory response, coagulopathy and thrombocytopaenia should be appropriately investigated and managed for sepsis, but the possible diagnosis of HLH should be borne in mind, particularly in the child who deteriorates despite maximal therapy. This review discusses current knowledge on the classification, diagnosis and management of primary and secondary HLH, and suggests a pathway of investigation for the paediatrician faced with a potential case.

Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare disease of infancy or early childhood. To clarify the incidence and subtypes of FHL in Japan, we performed genetic and functional analyses of cytotoxic T lymphocytes (CTLs) in Japanese patients with FHL. DESIGN AND METHODS: Among the Japanese children with hemophagocytic lymphohistiocytosis (HLH) registered at our laboratory, those with more than one of the following findings were eligible for study entry under a diagnosis of FHL: positive for known genetic mutations, a family history of HLH, and impaired CTL-mediated cytotoxicity. Mutations of the newly identified causative gene for FHL5, STXBP2, and the cytotoxicity and degranulation activity of CTLs in FHL patients, were analyzed. RESULTS: Among 31 FHL patients who satisfied the above criteria, PRF1 mutation was detected in 17 (FHL2) and UNC13D mutation was in 10 (FHL3). In 2 other patients, 3 novel mutations of STXBP2 gene were confirmed (FHL5). Finally, the remaining 2 were classified as having FHL with unknown genetic mutations. In all FHL patients, CTL-mediated cytotoxicity was low or deficient, and degranulation activity was also low or absent except FHL2 patients. In 2 patients with unknown genetic mutations, the cytotoxicity and degranulation activity of CTLs appeared to be deficient in one patient and moderately impaired in the other. CONCLUSIONS: FHL can be diagnosed and classified on the basis of CTL-mediated cytotoxicity, degranulation activity, and genetic analysis. Based on the data obtained from functional analysis of CTLs, other unknown gene(s) responsible for FHL remain to be identified.

STXBP2 mutations in children with familial haemophagocytic lymphohistiocytosis type 5.

BACKGROUND: Familial haemophagocytic lymphohistiocytosis (FHL) is a rare immune deficiency with uncontrolled inflammation; the clinical course usually starts within the first years of life, and is usually fatal unless promptly treated and then cured with haematopoietic stem cell transplant. FHL is caused by genetic mutations resulting in defective cell cytotoxicity; three disease related genes have been identified to date: perforin, Munc13-4 and syntaxin-11. A fourth gene, STXBP2, has been identified very recently as responsible for a defect in Munc18-2 in FHL-5. AIMS: To describe the result of the screening of families with HLH and previously unassigned genetic defects. METHODS: Patients with HLH diagnosed according to current diagnostic criteria, and who lacked mutations in the PRF1, Munc13-4, and STX11 genes were sequenced for mutations in STXBP2. Functional study was performed when material was available. RESULTS: Among the 28 families investigated, 4 (14%) with biallelic STXBP2 mutations were identified. They originated from Italy, England, Kuwait and Pakistan. The p.Pro477Leu resulting from c.1430C>T, and p.Arg405Gln resulting from the single c.1214G>A nucleotide change are known, while we contribute two novel mutations: p.Glu132Ala resulting from c.395A>C, and p.Gly541Ser, resulting from c.1621G>A. The detrimental effect of the p.Gly541Ser mutation was documented biochemically and functionally in NK and CD8 cells. Additional polymorphisms are also described. CONCLUSION: These data expand current knowledge on the genetic heterogeneity of FHL and suggest that patients with FHL5 may have different results in degranulation assays under different conditions.