UNC13D Research Articles

The Spectrum of Clinical, Immunological, and Molecular Findings in Familial Hemophagocytic Lymphohistiocytosis: Experience From India.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of immune dysregulation characterized by hyperactivation of the immune system, excessive cytokine secretion and severe systemic inflammation. HLH is classified as familial (FHL) when associated with mutations in PRF1, UNC13D, STX11, and STXBP2 genes. There is limited information available about the clinical and mutational spectrum of FHL patients in Indian population. This study is a retrospective analysis of 101 molecularly characterized FHL patients over the last 10 years from 20 different referral centers in India. FHL2 and FHL3 together accounted for 84% of cases of FHL in our cohort. Patients belonging to different FHL subtypes were indistinguishable based on clinical and biochemical parameters. However, flow cytometry-based assays viz. perforin expression and degranulation assay were found to be specific and sensitive in diagnosis and classification of FHL patients. Molecular characterization of respective genes revealed 76 different disease-causing mutations including 39 (51%) novel mutations in PRF1, UNC13D, STX11, and STXBP2 genes. Overall, survival was poor (28%) irrespective of the age of onset or the type of mutation in our cohort. Altogether, this article sheds light on the current scenario of FHL in India. Our data reveal a wide genetic heterogeneity of FHL in the Indian population and confirms the poor prognosis of FHL. This study also emphasizes that though mutational analysis is important for diagnostic confirmation of FHL, flow cytometry based assays help significantly in rapid diagnosis and functional validation of novel variants identified.

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.

Whole Exome Sequencing for Mutation Screening in Hemophagocytic Lymphohistiocytosis

Background: Hemophagocytic lymphohistiocytosis (HLH) is an immune system disorder characterized by uncontrolled hyper-inflammation owing to hypercytokinemia from the activated but ineffective cytotoxic cells. Establishing a correct diagnosis for HLH patients due to the similarity of this disease with other conditions like malignant lymphoma and leukemia and similarity among its two forms is difficult and not always a successful procedure. Besides, the molecular characterization of HLH due to the locus and allelic heterogeneity is a challenging issue. Materials and Methods: In this experimental study, whole exome sequencing (WES) was used for mutation detection in a four-member Iranian family with children suffering from signs and symptoms of HLH disease. Data analysis was performed by using a multi-step in-house WES approach on Linux OS. Result: In this study, a homozygous nucleotide substitution mutation (c.551G>A:p.W184*) was detected in exon number six of the UNC13D gene. W184* drives to a premature stop codon, so produce a truncated protein. This mutation inherited from parents to a four-month female infant with an autosomal recessive pattern. Parents were carrying out the heterozygous form of W184* without any symptoms. The patient showed clinical signs such as fever, diarrhea, hepatosplenomegaly, high level of ferritin, and a positive family history of HLH disease. W184* has a damaging effect on cytotoxic T lymphocytes, and natural killer cells. These two types of immune system cells without a healthy product of the UNC13D gene will be unable to discharge toxic granules into the synaptic space, so the inflammation in the immune response does not disappear. Conclusion: According to this study, WES can be a reliable, fast, and cost-effective approach for the molecular characterization of HLH patients. Plus, WES specific data analysis platform introduced by this study potentially offers a high-speed analysis step. This cost-free platform doesn't require online data submission.

Analysis of clinical prognosis and mutant gene phenotype in children with hemophagocytic lymphohistiocytosis

Objective To investigate the clinical manifestations, prognosis and gene mutation phenotypes of hemophagocytic lymphohistiocytosis(HLH)in children of our hospital. Methods The clinical data of 42 patients with HLH from April 2013 to December 2018, and the genetic data of 8 patients with familial HLH(FHL)were collected retrospectively.The age, clinical manifestation, laboratory examination, prognosis and the characteristics of gene mutation phenotype of patients with HLH and FHL were analyzed emphatically.Furthermore, the clinical manifestations and prognosis of patients with HLH were analyzed according to whether EB virus was infected. Results Among these 42 patients with HLH, the onset age was ranged from 1 month to 13 years old and most of them were younger than 5 years old.The main clinical manifestations included cytopenia, prolonged fever, enlargement of liver and spleen and lymph nodes enlargement and serosal effusion.Laboratory examination showed that lactate dehydrogenas, ferritin, erythrocyte sedimentation rate and triglyceride increased significantly.The survival rate of the group in ferritin exceeding 4 500 μg/L and non-chemotherapy was lower than that of the group of ferritin less than 4 500 μg/L and chemotherapy in clinical prognosis(P<0.05). Ten patients of them survived after chemotherapy, and 2 patients survived for 5 to 6 months after hematopoietic stem cell transplantation in FHL.Patients with EB virus infection were older than those without EB virus infection.They had longer fever duration and higher proportion of lymph nodes enlargement and ferritin more than 4 500 μg/L(P values were 0.01, 0.04, 0.03, 0.03 respectively). However, there was no significant difference in survival time between the two groups.Eight patients had mutations in UNC13D(50.00%), PRF1(25.00%), PRKDC(12.50%)and IL2RG(12.50%)genes respectively, and most of the mutations were complex heterozygous mutations(62.50%). All the mutations were originated from their parents. Conclusion HLH is characterized by cytopenia, prolonged fever, enlargement of liver and spleen.HLH is more common in children under 5 years old.The clinical manifestations of HLH with EB virus infection are more severe while the prognosis is not statistically significant.The incidence of FHL is higher.There are more UNC13D gene mutations and complex heterozygous mutations.Children with HLH should be detected and treated with standardized therapy as soon as possible.Hematopoietic stem cell transplantation is a good treatment for HLH, especially for FHL patients. Key words: Hemophagocytic syndrome; Exon sequencing; Gene mutation; EB virus infection; Prognosis

Clinical features of the children with malignancy-associated hemophagocytic syndrome in Beijing

Objective To investigate the clinical features, treatment and prognosis of the children with malignancy-associated hemophagocytic syndrome (MAHS) in Beijing in recent decade. Methods The clinical data of the patients with MAHS under 18 years old from July 2007 to February 2018 collected by the Society of Beijing hemophagocytic syndrome were analyzed retrospectively. Results There were 46 patients under 18 years old with MAHS in all(male 27, female 19). The patients with MAHS took up 8.9% of the patients with hemophagocytic lymphohistiocytosis (HLH)(46/519 cases) from the area during that period.The median age of onset had 13.5 years (0.9-18.0 years). Thirty-five patients had lymphoma (76.0%), 9 cases had leukemia (19.6%), 1 case had myelodysplastic syndrome with refractory anemia with excess blast(RAEB-T), and 1 case had Epstein-Barr virus(EBV) associated lymphoproliferative disease (borderline tumor stage). All the patients had a fever.A half of them suffered from hepatosplenomegaly and 7 patients(15.2%) had neurological symptoms.The common laboratory abnormalities included cytopenias, hemophagocytosis in bone marrow (81.8%, 36/44 cases), elevated serum ferritin (87.8%, 36/41 cases), and elevated sCD25 (100.0%, 15/15 cases), decreased nature killer(NK) activity (61.1%, 11/18 cases), and plasma EBV-DNA positive (57.9%). Four patients did not receive treatment, the rest were treated by several chemotherapy protocols including the HLH-94/2004 protocol.Five patients (10.8%) received the allogeneic hematopoietic stem cell transplantation, and 1 case received the splenectomy therapy.The mortality was 58.7%.Four heterozygous mutations of SH2D1A, UNC13D and PRF1 genes were found in 2 patients. Conclusions MAHS in children progresses rapidly, with poor prognosis, and it is often complicated with EBV infection.The children with MAHS may develop genetic defects similar to primary hemophagocytic syndrome.Nowadays, there is no standard treatment for MAHS, so the individualized treatment is to be explored. Key words: Malignancy-associated hemophagocytic syndrome; Child; Clinical features; Treatment

2518 Transitory Hemophagocytic Lymphohistiocytosis (HLH)/Macrophage Activation Syndrome (MAS) in a Child With Very Early Onset (VEO) of Ulcerative Colitis (UC) Complicated With Primary Sclerosing Cholangitis (PSC)

INTRODUCTION: VEO - Inflammatory bowel disease (IBD), in children younger than 6 years of age, is frequently associated with a more severe course and distinct genetic profile compared with later onset IBD. We report the case of a child with VEO - UC complicated with PSC and HLH/MAS. CASE DESCRIPTION/METHODS: A 10-year-old male, presented with bloody diarrhea, hepatomegaly and elevated transaminases when he was 30 months old. He had moderate pancolitis associated with C difficile (Cdiff) infection. He improved after metronidazole and sulfasalazine (SFZ) treatment. Two months later, he had hematochezia and endoscopic worsening of his colitis, which improved after discontinuing SFZ and receiving steroids and 6-MP. MRCP was consistent with PSC (Figure 1) and he was started on ursodiol. At ∼ 7 years of age, while receiving treatment with 6MP and Infliximab, he was hospitalized due to fever, hepatomegaly, diarrhea and pancytopenia. Blood and stool cultures, and tests for Cdiff, EBV, CMV, rota and parvovirus were negative. Triglyceride and ferritin levels were normal. He improved clinically and his pancytopenia resolved after 5 days of empirical antibiotic treatment (AbTx). Bone marrow biopsy (BMBx) was deferred. Metabolites of 6MP were normal but 6MP was held temporarily. Six months later, he had fever and diarrhea associated with pancytopenia and high levels of ESR, CRP, ferritin, triglyceride and interleukin 2 receptor. Number and function of NK cells were decreased. Stools and blood cultures, and tests for EBV, HSV and CMV were negative. Cdiff stool antigen was positive but PCR was negative. BMBx showed histiocytosis and erythophagocytosis (Figure 2). Lymphocyte flow cytometry was negative for lymphoproliferative disorder. After AbTx he improved and pancytopenia and ferritin level corrected within one week. Routine immunodeficiency tests were normal. Gene mutations associated with familial HLH: PRF1, Rab27A, SH2D1A, STX11, STXBP2, UNC 13D and XIAP were negative. 6MP was held. Infliximab initially, and subsequently Adalimubab, were discontinued because of high antibodies and allergic reactions. Five months later 6MP was restarted. He has been in remission of UC for 16 months. DISCUSSION: HLH/MAS is a very rare, potentially fatal complication in IBD patients. Our patient has a history of VEO - UC, PSC and two episodes of pancytopenia not associated with EBV or CMV - one of which fulfilled criteria for HLH/MAS. Remarkably, he recovered within one week after empirical AbTx without steroids or other treatment for HLH/MAS.

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.

SAT-501 A Rare and Potentially Fatal Etiology of Hypercalcemia in an Infant

Background: Increased 1alpha-hydroxylase activity in granulomatous diseases is a well-defined cause of hypercalcemia. Disseminated fungal infections are a rare cause of granulomatous diseases in immunocompetent children. Clinical Case: A 7-month-old female presented with failure to thrive and vomiting for 2 months. Initial tests showed Hgb 10.9 g/dL, WBC 4.2k/uL (37% Neutrophil), total Ca 17.9 mg/dL, iCa 2.43 mmol/L, Mg 2.4 mg/dL, Phos 4.8 mg/dL, AST 102 IU/L, ALT 115 IU/L, albumin 4.4 g/dL, PTH 5.3 pg/mL (RR 10-65), 25-OHD 61.3 ng/mL, 1,25-(OH)2D 146 pg/mL (RR 19-79). Abdominal US showed an enlarged, heterogeneous liver parenchyma and splenomegaly. Calcium level did not respond to calcitonin. She received pamidronate IV 0.5 mg/kg total over 2 days. Initial workup for infectious, hematologic, oncologic and immunologic etiologies was negative. She had a G-tube and was discharged after achieving adequate weight gain and normal Ca (9.5 mg/dL). Three weeks later she returned with vomiting and hypercalcemia (13.5 mg/dL). She started prednisolone PO 1 mg/kg/day for hypercalcemia. Clinical and biochemical markers improved and she was discharged home on steroid. Six-weeks later, while on prednisolone, she presented with fever and hypotension. A stat CBC showed pancytopenia (Hgb 8 g/dL, WBC 2.4k/uL, Plt 23k/uL) and fungal elements in peripheral smear. Admitted to ICU for anti-fungal and supportive treatment. Serum Histoplasma antigen was mildly positive at 2.4 U/mL (negative <2). Bone marrow aspirate showed phagocytic histiocytes. Liver biopsy demonstrated lobular histiocytic infiltrate with rare granulomas and increased portal/periportal and pericellular fibrosis with bridging confirming the granulomatous disease. Extensive immunological tests did not identify any underlying immunodeficiency. She had a normal SNP microarray but a heterozygous mutation in the UNC13D gene, this was classified as a variant of unknown significance. No deletion or duplication was found in HLH gene panel. After one month of amphotericin B IV treatment, her clinical status improved and discharged home. She took itraconazole PO treatment for 9 months and made full recovery. Serum and urine Histoplasma antigens remained negative during and after completion of antifungal therapy. Steroid slowly tapered while Ca and vitamin D levels remained normal. Conclusion: Hypercalcemia can be the presenting sign of a potentially fatal disseminated fungal infection even in immunocompetent children. Although prednisolone has corrected hypercalcemia in this case, it has likely contributed to the dissemination of the unmasked fungemia. Extra caution should be used before considering steroid treatment for hypercalcemia and ideally, it should be delayed until after anti-fungal treatment begins once the correct diagnosis is reached.

Whole exome analysis of primary immunodeficiency

The human primary immunodeficiency diseases (PIDs) refer to a rare heterogeneous group of single-gene inherited disorders causing malfunctions in the immune system, and thus the affected patients have a predisposition to severe life-threatening infections. The heterogeneous nature of PIDs, which involves at list 300 different genes, makes diagnosis of the disease a complex issue. Although studies revealed that six million people have a kind of PID, but due to a complex diagnosis procedure many affected individuals have not gotten a correct diagnosis. However, thanks to advancing in the DNA sequencing method and availability of sophisticated sequencers molecular characterization of genetic disorders have been revolutionized. The whole exome sequencing (WES) method can help clinicians detect Mendelian disease and other complex genetic disorders. The presented study used WES to investigate two infants with symptoms of primary immunodeficiency including hemophagocytic lymphohistio­cytosis (HLH) and severe combined immunodeficiency (SCID). It has been shown that the HLH patient had a mutation in the UNC13D gene (NM_199242.2:c.627delT), and the SCID patient had a mutation in the RAG1 gene (NM_000448.2:c.322C&gt;G). It has been demonstrated that WES is a fast and cost-effective method facilitating genetic diagnosis in PID sufferers.

Synergistic defects of novo FAS and homozygous UNC13D leading to autoimmune lymphoproliferative syndrome-like disease: A 10-year-old Chinese boy case report

Autoimmune lymphoproliferative syndrome (ALPS) usually presents in childhood with fever, nonmalignant splenomegaly and lymphadenopathy along with hemocytopenia. This case report describes a 10-year-old boy presenting with signs of autoimmune disease, splenomegaly, hepatomegaly and resistant hemocytopenia. Sirolimus controlled the relapsed thrombocytopenia after splenectomy. Sequencing of the FAS gene identified two spontaneous heterozygous mutations (c.234 T > G, p.D78E) (c.236dupA, p.P80Tfs*26). The boy's homozygous missense variation (c.2588G > A, p.G863D) (rs140184929) in UNC13D gene had been identified as being related to familial hemophagocytic lymphohistiocytosis (FHL). TCRαβ + CD4/CD8 double-negative T cells (markers of ALPS) were not significantly increased from the outset. Elevated cytokines, such as interferon (IFN)-γ, interleukin (IL)-6 and tumor necrosis factor α decreased to normal levels after splenectomy whereas IL-10 remained high. Immunological analysis of the patient revealed a marked depletion of forkhead-box P3+ expressing regulatory T cells (Treg) and Th17 cells. The obtained data demonstrate that mutations to FAS and UNC13D which result in overwhelming T-cell and macrophage activation, one associated with inhibited Treg cell development and a severe ALPS-like symptom. Therefore, we propose that variations of UND13D may be a risk factor of ALPS development.

Identification of a novel nonsense mutation in the UNC13D gene from a patient with hemophagocytic lymphohistiocytosis: a case report

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a heterogeneous and potentially fatal disease that presents symptoms of persistent fever, splenomegaly and cytopenia. Primary HLH is identified as an autosomal recessive disorder with causative genes including HPLH1, PRF1, UNC13D, STX11 and STXBP2.Case presentationHere, we reported an 8-month-old female patient with compound heterozygosity in the UNC13D gene. The patient, who presented typical symptoms, was diagnosed with HLH based on HLH-2004 guidelines. High-throughput amplicon sequencing for the full-length exon, including a 5 bp padding region and 6 HLH-related genes, was performed to identify the pathogenic mutations in this patient. In all, 9 heterozygous variations were detected, namely, 7 nonpathogenic SNPs, one nonsense mutation (NM_199242.2:c.2206C > T, p.Gln736X), and one splicing mutation (NM_199242.2:c.2709 + 1G > A). These two mutations were considered pathogenic according to previous studies and functional prediction. A two-generation pedigree analysis based on Sanger sequencing was performed to confirm the result.ConclusionCompound heterozygosity in the UNC13D gene was identified in trans and considered a causative mutation in a female patient with HLH. The nonsense mutation (NM_199242.2:c.2206C > T, p.Gln736X) was novel in cases of HLH. Our data expand the spectrum of HLH-related mutations in China and demonstrate the potential of high-throughput amplicon sequencing in the diagnosis of HLH.

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory disease. This study aimed to investigate the frequencies and distributions of inherited variants in PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP genes in Chinese patients with HLH. A total of 265 patients diagnosed with HLH from January, 2010 to December, 2016 were recruited and analyzed for the 6 genes. Genetic variants were observed in 87 (32.83%) patients. 36 (13.58%) exhibited variants in UNC13D, 18 (6.79%) exhibited PRF1 variants, 10 (3.77%) had variants in XIAP, 9 (3.40%) exhibited variants in STXBP2, 6 (2.26%) carried variants in SH2D1A, 1 (0.38%) had STX11 variant, and 7 (2.64%) exhibited digenic variants. Monoallelic variants were the most common, which accounted for 49.43% of all cases with variants. All variants were confirmed to be germline-derived. The present study describes a distinct variant spectrum in Chinese patients with HLH, whereby UNC13D is the most frequently mutated gene with missense variants that are the most common molecular defects. The variant profile of Chinese HLH patients is quite different from that of Western cohorts but similar to that of Korean patients, yet showing its own uniqueness. This racial difference shows the role of genetic background in the occurrence of HLH.

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of PRF1 , UNC13D , STX11 , STXBP2 , SH2D1A , and XIAP

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: molecular analyses of <i>PRF1</i> , <i>UNC13D</i> , <i>STX11</i> , <i>STXBP2</i> , <i>SH2D1A</i> , and <i>XIAP</i>

Characterization of a large UNC13D gene duplication in a patient with familial hemophagocytic lymphohistiocytosis type 3

Familial hemophagocytic lymphohistiocytosis (FHL) type 3 is a life-threatening immune dysregulation syndrome caused by mutations in the UNC13D gene, encoding the munc13–4 protein, which is important for function of cytotoxic lymphocytes. FHL3 accounts for 30–40% of FHL cases, and more than 100 mutations in the UNC13D gene have been described to date. We describe the first case of FHL3 carrying an intragenic duplication of UNC13D, apparently mediated by recombination of Alu elements. NK cell degranulation and munc13–4 protein expression assays are useful for early identification of such mutations, which may be missed by analysis of genomic DNA alone.

Primary hemophagocytic lymphohistiocytosis in adults: the utility of family surveys in a single-center study from China

BackgroundThis study investigated the clinical characteristics of primary hemophagocytic lymphohistiocytosis (HLH) in adults, including immunological markers, pedigree findings, and conditions of allogeneic hematopoietic stem cell transplantation (Allo-HSCT).MethodsThe study included clinical data of 18 adult patients with primary HLH treated in our center from June 2010 to January 2017.ResultsOf these 18 cases, pathogenic variants were found in the following genes: PRF1 (n = 11), UNC13D (n = 5), SH2D1A (n = 2), RAB27a (n = 1), and LYST (n = 2). One patient had pathogenic variants in both PRF1 and UNC13D genes, one patient had pathogenic variants in both LYST and UNC13D genes and another patient had pathogenic variants in both PRF1 and SH2D1A genes. Additionally, 3 of the 18 cases involved homozygous pathogenic variants, while 2 cases involved hemizygous pathogenic variants. The remaining 13 cases involved compound heterozygous pathogenic variants. The natural killer (NK) cell activity test was conducted in all 18 cases where 14(77.8%)patients showed reduction in NK cell activity. Furthermore, this article presents 3 representative results of the pedigree findings from 12 patients who underwent family surveys. The 8 patients who underwent Allo-HSCT had a median survival of 27.2 months, as compared with the median survival of 7 months for the10 patients who did not undergo Allo-HSCT, a significant difference between the two groups of patients (p = 0.006).ConclusionPRF1 was one of the most commonly mutated gene in adult patients with primary HLH. Family surveys and immunological markers were important for the HLH diagnosis and the selection of an appropriate donor. Allo-HSCT was an effective therapy for adult primary HLH.

Characterization of a novel splicing mutation in UNC13D gene through amplicon sequencing: a case report on HLH

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a rare but fatal disease caused by uncontrolled proliferation of activated lymphocytes and macrophages. Six genes including SH2D1A, PRF1, UNC13D, STX11, STXBP2 and XIAP were reported as causative genes in most cases.Case presentationHere we report a novel splicing mutation in UNC13D gene, which was identified in an 18-year-old female. Patient was diagnosed as HLH base on HLH-2004 guidelines, no history of inherited diseases was revealed in this family, parents were healthy and non-consanguineous. Splenomegaly and hemophagocytosis in bone marrow were observed in clinical examination. Amplicon sequencing for the whole coding region of 6 HLH-related genes was performed on Ion S5XL genetic analyzer. In all, four heterozygous mutations were detected, including 2 nonpathogenic SNPs (PRF1:c.900C > T, STX11:c.*70G > A) and 2 splicing mutations in UNC13D gene (UNC13D:c.1299 + 1G > A and UNC13D:c.2709 + 1G > A), both of which were predicted to be potentially pathogenic by human splicing finder (HSF3) tool. The result was confirmed by two-generation pedigree analysis base on sanger sequencing.ConclusionsTwo compound heterozygous splicing mutations in UNC13D gene were identified and considered to be potential pathogenesis in a female patient of HLH. The mutation UNC13D:c.1299 + 1G > A was reported in HLH for the first time. The inheritance mode and source of the mutation in the proband was examined by family analysis. Our data suggest that further studies of the spectrum of HLH-related mutations in China are warranted.

Five cases of Epstein-Barr virus-associated extranodal natural killer/T cell lymphoma in children and lite-rature review

Objective To understand the clinical and pathological features as well as efficacy and prognosis of Epstein-Barr virus(EBV)-associated extranodal natural killer/T cell lymphoma in children. Methods Clinical, pathological and treatment data from 5 cases of sick children with natural killer/T cell lymphoma from June 2012 to August 2016, at Beijing Children′s Hospital, Capital Medical University, were clinically analyzed, and related literatures were reviewed. Results The 5 patients presented atypical initial clinical symptoms, such as fever, stuffy nose or runny nose, hearing loss, and so on.All of them had a long disease course (23 d-6 months) when seeking treatment, and eventually their diagnosis was confirmed based on pathological biopsy.One each patient was at the clinical stages Ⅰ, Ⅱ and Ⅲ, and 2 patients were at stage Ⅳ.One patient presented immune deficiency-associated gene mutation(UNC13D), and 1 patient was complicated with EBV-associated hemophagocytic syndrome on initial diagnosis.Che-motherapy with the SMILE(Methotrexate, Ifosfamide, Dexamethasone, Etoposide and L-Asparaginase) and VIDP (Ifosfamide, Dexamethasone, Etoposide and Cisplatin)regimens and local precision radiotherapy were given for treatment.The median follow-up time was 35 months (6-45 months). With the exception of 1 patient remaining on che-motherapy, all the rest 4 patients had discontinued chemo/radiotherapy and achieved persistent complete remission. Conclusions Pediatric EBV-associated extranodal natural killer/T cell lymphoma would present atypical clinical manifestations at the early stage.When they are suspected with this disease, pathological biopsy should be performed as early as possible to confirm the diagnosis, and chemo/radiotherapy should be introduced as early as possible after definite diagnosis.This therapy may have curative effect with SMILE regimen. Key words: Extranodal natural killer/T cell lymphoma; Epstein-Barr virus; Child

The significance of pedigree genetic screening and rapid immunological parameters in the diagnosis of primary hemophagocytic lymphohistiocytosis

目的探讨家系基因筛查及快速免疫学指标检测在原发性噬血细胞综合征（HLH）诊断中的意义。方法通过对伴有PRF1、UNC13D及SH2D1A基因突变的4例原发性HLH患者展开家系调查，分别完成基因筛查及各项免疫学指标检测（包括NK细胞活性、CD107a检测及HLH相关缺陷蛋白表达测定），评价各项检测指标在原发性HLH诊断中的意义并探讨各项指标间的相关性。结果4个家系基因突变分别为PRF1基因错义突变c.T172C（p.S58P）和非框架移码突变c.1083_1094del (p.361_365del）；PRF1基因错义突变c.C1349T（p.T450M）和框架移码突变c.1090_1091delCT (p.T364fsX93）；UNC13D基因错义突变c.G2588A（p.G863D）；SH2D1A基因半合子错义突变c.32T>G (p.I11S）。先证者及家系成员分别存在不同程度的NK细胞活性降低，其中PRF1基因及SH2D1A基因突变家系HLH相关基因编码穿孔素蛋白、信号淋巴细胞活化分子相关蛋白（SAP）表达水平下降，UNC13D基因突变先证者及与其存在完全相同突变位点的家系成员细胞毒脱颗粒功能（CD107a表达）显著减低。结论开展家系基因筛查及快速免疫学指标检测对诊断原发性HLH具有重要意义，两者具有较好的一致性，其中快速免疫学指标检测作为一种高效的检测手段，可为原发性HLH的早期诊断提供可靠依据。

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.