Familial Hemophagocytic Lymphohistiocytosis Type Research Articles

Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Pituitary involvement in familial hemophagocytic lymphohistiocytosis type 5 associated with homozygous mutation in the syntaxin-binding protein-2 gene (STXBP2)

Pituitary involvement in familial hemophagocytic lymphohistiocytosis type 5 associated with homozygous mutation in the syntaxin-binding protein-2 gene (STXBP2)

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

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

Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality. We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV). We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection. Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH. Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth.

A Case of a Relapsing Remitting Macrophage Activating Syndrome After Covid-19 Vaccine In a Teenager with UNC13D Heterozygous Variant of Uncertain Significance

IntroductionMacrophage activation syndrome (MAS) is a severe hyper inflammatory condition caused by the over-activation and proliferation of T cells, NK cells and macrophages. It is often associated with complications of rheumatic/immune diseases. We present a case of a 15-year-old female who experiences recurrent episodes of MAS without any known definitive underlying etiology. Case PresentationA 15-year-old previously healthy female developed fatigue, fevers, myalgia, chest pain, splenomegaly and lymphadenopathy 10 days after receiving her first Pfizer COVID-19 vaccine. Her symptoms recurred 10 days after receiving the second dose. Her myocarditis, MIS-C, and infectious work up was negative except for positive EBV IgG. Laboratory studies revealed anemia, hypertriglyceridemia, hypofibrinogenemia, and hyperferritinemia. She initially responded to decadron; however, her symptoms recurred with steroid taper. Bone marrow biopsy revealed hemophagocytosis. Whole exome sequencing (WES) revealed a heterozygous variant of uncertain significance in UNC13D c.962C>A (p.Thr321Asn). She had multiple re-admissions with significantly elevated inflammatory markers, including extremely high IL2-R, IL-18 and CXCL9. Each episode was complicated by an acute viral infection. She responds to high dose steroids, anti-IL-1, and JAK inhibitors. Nonetheless, it has been difficult to wean decadron without triggering a flare. She continues to require increasing doses of baricitinib. DiscussionMAS may be seen as a complication of rheumatic diseases, as well as inborn errors of immunity. However, none of these conditions have been diagnosed in this patient despite extensive testing, including WES. The degree of her immune dysregulation has been very severe making her disease process unpredictable and extremely difficult to control. She has frequent flares precipitated by viral infections or attempts at adjusting her immunomodulators. Weaning her medications has been challenging as she continues to require increasing doses of baricitinib and corticosteroids. The UNC13D gene is associated with autosomal recessive familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Our patient is heterozygous for an UNC13D variant of uncertain significance. Additional genetic inquiries with whole genome sequencing to help elucidate the underlying etiology of her severe condition is being conducted. We hypothesize she developed MAS due to a combination of genetic predisposition, prior EBV infection, and immune stress associated with the COVID-19 vaccine. [Display omitted] [Display omitted] [Display omitted]

Granulomatous inflammation and hypogammaglobulinemia: Clinical conundrum of familial hemophagocytic lymphohistiocytosis type 5

Familial hemophagocytic lymphohistiocytosis (HLH) is an inherited disorder characterized by systemic hyperinflammation caused by an uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. Most children with familial HLH present within first 2 years of life and can have fatal disease unless hematopoietic stem cell transplant (HSCT) is performed (1). However, few patients may have late presentation and prolonged survival. With increasing awareness and facilities to identify HLH these disorders are being identified beyond infancy (2–4). Clinical and laboratory features are often similar to other primary immune deficiency diseases and pose diagnostic challenges (4–6). We report two patients who presented beyond the first decade of life with HLH, granulomatous inflammation, hypogammaglobulinemia, reduced B cells and were diagnosed to have familial HLH type 5 due to defect in STXBP2 gene.

Monogenic inflammatory bowel disease with STXBP2 mutations is not resolved by hematopoietic stem cell transplantation but can be alleviated via immunosuppressive drug therapy

STXBP2, encoding syntaxin-binding protein 2, is involved in intracellular organelle trafficking and is associated with familial hemophagocytic lymphohistiocytosis type 5. Although STXBP2 mutations reportedly cause monogenic inflammatory bowel disease, the clinical course and underlying pathogenic mechanisms remain unclear. We identified a novel mutation in STXBP2 [c.1197delC, p.Ala400fs] in a boy with congenital intractable diarrhea and hemophagocytic lymphohistiocytosis (HLH). HLH was treated with intravenous prednisolone, cyclosporine, and dexamethasone palmitate. Hematopoietic stem cell transplantation (HSCT) along with prophylaxis for graft-versus-host-disease was performed at 5 months of age. Additionally, colonoscopies done before and after HSCT showed mild colitis with cryptitis. The patient showed elevated fecal calprotectin levels and persistent diarrhea even after HSCT and required partial parenteral nutrition. While anti-inflammatory treatment reduced diarrhea, it was not completely normalized even after HSCT, suggesting that the pathogenesis of inflammatory bowel disease associated with STXBP2 mutations involves both hyperinflammation and functional epithelial barrier defects.

Familial Hemophagocytic Lymphohistiocytosis (Type 2) with solitary neurological presentation due to PRF1 gene mutation

Familial hemophagocytic lymphohistiocytosis (FHLH) clinically manifest with fever, hepatosplenomegaly, pancytopenia, hyperferritenemia, hypofibrinogenemia and/or neurological signs. We report a case of solitary neurological presentation and absence of systemic signs of inflammation which was initially thought to be genetic leukodystrophy but latter turn out to be FHLH type 2 due to mutation in PRF1 gene. Pediatricians should keep FHLH as a differential diagnosis in a patient with solitary neurological presentation due to neuroinflammation even if no signs of systemic inflammation or abnormal laboratory parameters.

Recurrent tandem duplication of UNC13D in familial hemophagocytic lymphohistiocytosis type 3

Familial hemophagocytic lymphohistiocytosis type 3 is a fatal inborn error of immunity due to abnormal cytotoxic activity of T and NK cells and is caused by variants in UNC13D, which encodes Munc13–4. One published case was reported to carry a tandem duplication of UNC13D exons 7–12, and we here present another case with the exact same duplication breakpoints. The patient carried the tandem duplication from maternal origin, and a c.2346_2349 variant on the paternal allele. Single nucleotide polymorphism analysis around UNC13D revealed that the allele with tandem duplication was most likely a founder allele. Transposable element analysis showed that the breakpoints occurred within Alu elements in introns 12 and 6. Multiple sequence alignment revealed that Alu elements containing the truncated points are highly homologous. Sequence homology was thought to be a factor predisposing to the tandem duplication variant.

Evaluation of CD107a and perforin expression detection in the diagnosis of primary hemophagocytic lymphohistiocytosis

Objective: To evaluate the efficacy of rapid immunological indicator-degranulation function (CD107a) and perforin expression in the diagnosis of primary hemophagocytic lymphohistiocytosis (pHLH). Methods: The clinical data of 295 HLH patients who underwent genetic screening from April 2015 to June 2020 in Beijing Friendship Hospital, Capital Medical University, Beijing Jingdu Children's Hospital and Beijing Children's Hospital, Capital Medical University was collected and analyzed. The fitness of CD107a and Perforin expression with genetic screening was compared to evaluate the sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of the two indicators, and the receiver operating characteristic (ROC) curves were generated and used to determine the optimal threshold (cut-off values) of CD107a and Perforin expression assays that would identify pHLH patients with a maximum sensitivity and specificity (Youden index). Results: In all 295 patients included, there were 156 males and 139 females, aged from 2 months to 70 years, with a median age of 18 years. In terms of distinguishing the type of pHLH associated with degranulation gene defect from all other genetic screening results, in the CD107a testing, the ROC curve was generated and showed an area under the curve (AUC) of 0.920 (P<0.001), and the optimal cut-off value was determined to be 7.15% with a sensitivity of 83.3% and specificity of 89.2% when the corresponding Youden index was maximized. The PPV and NPV were 33.3% and 98.8%, respectively. CD107a>10% had an accuracy of 81.6% in judging patients without degranulation-related gene defect and negative genetic screening results. In addition, in terms of distinguishing the type of familial hemophagocytic lymphohistiocytosis type 2 (FHL2) from all other genetic screening results, the sensitivity, specificity, PPV and NPV of the Perforin expression testing were 88.2%, 64.2%, 20.3% and 98.1%, respectively, based on the normal laboratory test value (≥ 81%). The ROC curve was established to further optimize the cut-off value. The AUC was 0.933 (P<0.001). The cut-off value corresponding to the maximum Youden index was 62.34%, and the sensitivity remained at 88.2%. While the specificity, PPV and NPV rose to 91.5%, 51.7% and 98.7%, respectively. Conclusions: CD107a and Perforin assays have good significance of early prediction for pHLH involved in impaired cytotoxic function. Selecting appropriate cut-off values can provide basis for accurate clinical diagnosis.

Exome sequencing detected an extremely rare case of foetal onset familial haemophagocytic lymphohistiocytosis type 5 presenting with hydrops foetalis

BackgroundFamilial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous autosomal recessive hyper-inflammatory syndrome which needs early accurate diagnosis and appropriate treatment to prevent complications and early mortality. Recently, it was reported that mutations in STXBP2 gene are linked to FHL type 5 (FHL-5).Case PresentationWe report a Sri Lankan neonate who presented with low Apgar scores at birth, abdominal distension, and hepatosplenomegaly, followed by lethargy, poor sucking and rapid decompensation with wide spread activation of inflammation within 48 h of birth. Her elder sibling also had a similar presentation during early neonatal period and deceased at two weeks of age with no diagnosis. Unfortunately, the index case deceased at 14 days of age following multi-organ dysfunction and severe metabolic acidosis. Targeted gene panel followed by reflex exome sequencing revealed a novel likely pathogenic homozygous variant in the STXBP2 gene (NM_001272034.1:c.1141-2A > G) which confirmed the diagnosis of autosomal recessive FHL-5.ConclusionEarly diagnosis of FHL type 5 using genetic analysis and timely treatment are difficult in the absence of family history due to a wide spectrum of clinical manifestations. However both early diagnosis and treatment doesn’t alter the long term prognosis. So genetic counselling would be the better option.

PRF1c.658G>Aとc.1090_1091delCTの複合ヘテロ接合体による新生児期発症の家族性血球貪食性リンパ組織球症の1乳児例

PRF1c.658G>Aとc.1090_1091delCTの複合ヘテロ接合体による新生児期発症の家族性血球貪食性リンパ組織球症の1乳児例

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.

Alternative UNC13D Promoter Encodes a Functional Munc13-4 Isoform Predominantly Expressed in Lymphocytes and Platelets.

Autosomal recessive mutations in genes required for cytotoxicity are causative of a life-threatening, early-onset hyperinflammatory syndrome termed familial hemophagocytic lymphohistiocytosis (FHL). Mutations in UNC13D cause FHL type 3. UNC13D encodes Munc13-4, a member of the Unc13 protein family which control SNARE complex formation and vesicle fusion. We have previously identified FHL3-associated mutations in the first intron of UNC13D which control transcription from an alternative transcriptional start site. Using isoform specific antibodies, we demonstrate that this alternative Munc13-4 isoform with a unique N-terminus is preferentially expressed in human lymphocytes and platelets, as compared to the conventional isoform that was mostly expressed in monocytes and neutrophils. The distinct N-terminal of the two isoforms did not impact on Munc13-4 localization or trafficking to the immunological synapse of cytotoxic T cells. Moreover, ectopic expression of both isoforms efficiently restored exocytosis by FHL3 patient-derived Munc13-4 deficient T cells. Thus, we demonstrate that the conventional and alternative Munc13-4 isoforms have different expression pattern in hematopoietic cell subsets, but display similar localization and contribution to T cell exocytosis. The use of an alternative transcriptional starting site (TSS) in lymphocytes and platelets could be selected for increasing the overall levels of Munc13-4 expression for efficient secretory granule release.

Novel mutation in perforin gene causing familial hemophagocytic lymphohistiocytosis type 2 in an Egyptian infant: case report

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a syndrome of pathological immune activation characterized by clinical signs and symptoms of extreme inflammation. It results from the uninhibited proliferation and activation of cells of the macrophage lineage and leads to the production of excess amounts of pro-inflammatory cytokines. The familial form of HLH disease is due to mutations in several genes necessary for natural killer (NK) cell and T cell granule-mediated cytotoxic function. These genes are involved in sorting, trafficking, docking, and fusion of cytotoxic granules containing granzymes A and B and perforin to the cell membrane of the target cell (using the proteins LYST, AP-3 complex, Rab27a, Munc 13–4, Munc 18–2, syntaxin 11). Defect in any of those proteins results in defective cytotoxicity. Consequently, genes included in these steps play valuable roles in the pathogenesis of familial HLH disease including perforin (PRF1) gene in which defect causes familial HLH type 2 (FHL2).Case presentationA 2-year-old boy suffered from hepatosplenomegaly and fever. He fulfilled the required criteria for the diagnosis of HLH according to HLH-2004 diagnostic criteria. We screened the patient for the presence of mutations in the coding exons and of PRF1 gene by PCR amplification of genomic DNA followed by direct sequencing of the PCR products. We report a novel homozygous deletion/insertion frameshift mutation in PRF1 gene (M28393: exon 2: c.536delAinsCG p.F178fs). We treated him with HLH 2004 protocol of treatment and showed a remarkable response with resolution of fever and decrement in the size of hepatosplenomegaly.ConclusionsOur study discovered a novel frameshift mutation in PRF1 gene in an infant with HLH disease, and it is the first report of this type of mutation in Egyptian patients with this disease.

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.

Clinical study of haploidentical hematopoietic stem cell transplantation on 15 cases of adult-onset primary hemophagocytic lymphohistiocytosis

目的探讨单倍型造血干细胞移植（haplo-HSCT）治疗成人原发性噬血细胞综合征（HLH）的疗效。方法对2013年1月至2019年10月期间于首都医科大学附属北京友谊医院接受haplo-HSCT的15例成人原发性HLH患者进行回顾性分析。结果全部15例患者中男10例、女5例，中位年龄21（18~52）岁。家族性噬血细胞综合征2型（FHL-2）8例，FHL-3 4例，Griscelli综合征2（GS-2）、X连锁淋巴组织增生综合征1型（XLP-1）、XLP-2各1例。确诊HLH至接受haplo-HSCT的中位时间为7（2~46）个月。所有患者移植前均接受HLH-94、HLH-2004或DEP方案诱导化疗，8例获得完全应答（CR），7例获得部分应答（PR）。移植预处理：Bu/Cy方案7例，TBI/Cy方案8例。输注单个核细胞12.6（9.2~20.3）×108/kg，CD34+细胞4.91（2.51~8.37）×106/kg。粒细胞植活中位时间为13（10~23）d，血小板植活中位时间为12（9~36）d。原发、继发性植入失败各1例。Ⅱ~Ⅳ度急性移植物抗宿主病（GVHD）发生率为71.4%（10/14），慢性GVHD发生率为30.8%（4/13）。移植后预期5年总生存（OS）率为65.5%（95%CI 34.9%~73.3%），移植相关死亡率为26.7%（4/15）。一线诱导治疗后立即接受移植（8例）、二线诱导治疗难治/复发患者（7例）的移植后预期5年OS率分别为87.5%（95%CI 38.7%~66.3%）、42.9%（95%CI 8.5%~65.2%）（χ2=2.387，P=0.122）。移植前CR（8例）、PR（7例）患者的移植后预期5年OS率分别为85.7%（95%CI 50.4%~89.8%）、42.9%（95%CI 6.4%~53.0%）（χ2=3.185，P=0.074）。结论haplo-HSCT是成人原发性HLH的有效治疗手段。

Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases: A Single Center Experience

PurposeWhile there is an urgent need for diagnosis and therapeutic intervention in patients with primary immunodeficiency diseases (PIDs), current genetic tests have drawbacks. We retrospectively reviewed the usefulness of flow cytometry (FCM) as a quick tool for immunophenotyping and functional assays in patients suspected to have PIDs at a single tertiary care institute.MethodsBetween January 2001 and June 2018, patients suspected of having PIDs were subjected to FCM tests, including lymphocyte subset analysis, detection of surface- or intracellular-target proteins, and functional analysis of immune cells, at Samsung Medical Center, Seoul, Korea. The genetic diagnosis was performed using Sanger or diagnostic exome sequencing.ResultsOf 60 patients diagnosed with definite or probable PID according to the European Society of Immune Deficiencies criteria, 24 patients were provided with useful information about immunological dysfunction after initial FCM testing. In 10 patients, the PID diagnosis was based on abnormal findings in FCM testing without genetic tests. The FCM findings provided strong evidence for the diagnosis of severe combined immunodeficiency (n = 6), X-linked chronic granulomatous diseases (CGD) (n = 6), leukocyte adhesion deficiency type 1 (n = 3), X-linked agammaglobulinemia (n = 11), autoimmune lymphoproliferative syndrome-FASLG (n = 1), and familial hemophagocytic lymphohistiocytosis type 2 (n = 1), and probable evidence for autosomal recessive-CGD (n = 2), autosomal dominant-hyper-immunoglobulin E (IgE)-syndrome (n = 1), and STAT1 gain-of-function mutation (n = 1). In PIDs derived from PIK3CD (n = 2), LRBA (n = 2), and CTLA4 mutations (n = 3), the FCM test provided useful evidence of immune abnormalities and a tool for treatment monitoring.ConclusionsThe initial application of FCM, particularly with known protein targets on immune cells, would facilitate the timely diagnosis of PIDs and thus would support clinical decisions and improve the clinical outcome.

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.

Deficiency of perforin and hCNT1, a novel inborn error of pyrimidine metabolism, associated with a rapidly developing lethal phenotype due to multi-organ failure

Pyrimidine nucleotides are essential for a vast number of cellular processes and dysregulation of pyrimidine metabolism has been associated with a variety of clinical abnormalities. Inborn errors of pyrimidine metabolism affecting enzymes in the pyrimidine de novo and degradation pathway have been identified but no patients have been described with a deficiency in proteins affecting the cellular import of ribonucleosides. In this manuscript, we report the elucidation of the genetic basis of the observed uridine-cytidineuria in a patient presenting with fever, hepatosplenomegaly, persistent lactate acidosis, severely disturbed liver enzymes and ultimately multi-organ failure. Sequence analysis of genes encoding proteins directly involved in the metabolism of uridine and cytidine showed two variants c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in SLC28A1, encoding concentrative nucleotide transporter 1 (hCNT1). Functional analysis showed that these variants affected the three-dimensional structure of hCNT1, altered glycosylation and decreased the half-life of the mutant proteins which resulted in impaired transport activity. Co-transfection of both variants, mimicking the trans disposition of c.1528C > T (p.R510C) and c.1682G > A (p.R561Q) in the patient, significantly impaired hCNT1 biological function. Whole genome sequencing identified two pathogenic variants c.50delT; p.(Leu17Argfs*34) and c.853_855del; p.(Lys285del) in the PRF1 gene, indicating that our patient was also suffering from Familial Hemophagocytic Lymphohistiocytosis type 2. The identification of two co-existing monogenic defects might have resulted in a blended phenotype. Thus, the clinical presentation of isolated hCNT1 deficiency remains to be established.