X-linked Lymphoproliferative Disease Type Research Articles

Highly sensitive detection of Epstein-Barr virus-infected cells by EBER flow FISH.

When Epstein-Barr virus (EBV) infection is suspected, identification of infected cells is important to understand the pathogenesis, determinine the treatment strategy, and predict the prognosis. We used the PrimeFlow™ RNA Assay Kit with a probe to detect EBV-encoded small RNAs (EBERs) and multiple surface markers, to identify EBV-infected cells by flow cytometry. We analyzed a total of 24 patients [11 with chronic active EBV disease (CAEBV), 3 with hydroa vacciniforme lymphoproliferative disorder, 2 with X-linked lymphoproliferative disease type 1 (XLP1), 2 with EBV-associated hemophagocytic lymphohistiocytosis, and 6 with posttransplant lymphoproliferative disorder (PTLD)]. We compared infected cells using conventional quantitative PCR methods and confirmed that infected cell types were identical in most patients. Patients with CAEBV had widespread infection in T and NK cells, but a small amount of B cells were also infected, and infection in patients with XLP1 and PTLD was not limited to B cells. EBV-associated diseases are believed to be complex pathologies caused by EBV infecting a variety of cells other than B cells. We also demonstrated that infected cells were positive for HLA-DR in patients with CAEBV. EBER flow FISH can identify EBV-infected cells with high sensitivity and is useful for elucidating the pathogenesis.

X-LINKED LYMPHOPROLIFERATIVE DISEASE: DIAGNOSTIC CHALLENGE IN A MIDDLE-AGED MALE

<h3>Introduction</h3> X-Linked Lymphoproliferative Disease Type 1 (XLP1) is a rare disease of males caused by mutation in the gene <i>SH2D1A</i> and is characterized by vulnerability to EBV infection, lymphoma and dysgammaglobulinemia. Fulminant infectious mononucleosis in early childhood is the most common clinical manifestation, but on rare occasions XLP1 does not present until adulthood. <h3>Case Description</h3> We present a 43-year-old male with a history of recurrent sinusitis and bronchitis and leukopenia since childhood. He had EBV infection when he was 18 years old complicated by jaundice and hepatitis that required treatment with corticosteroids. At the age of 42, he developed ITP and was found to have mild splenomegaly. Serum immunoglobulins were mildly abnormal with IgG of 575, IgM of 762 and IgA of 145 mg/dL. Lymphocyte immunophenotyping was within normal limits and serum protein electrophoresis and serum free light chains were normal. Bone marrow biopsy was unremarkable. Invitate PID panel was performed, which revealed a pathogenic variant in SH2D1A gene (c.2T>C). SAP expression by flow cytometry revealed absence in NK cells but present in 17% of CD8+ T cell population suggestive of a somatic reversion in this population. He had mildly decreased NK cell function and positive EBV serology. <h3>Discussion</h3> We present a case of XLP1 diagnosis in adulthood with a history of EBV infection of moderate severity and mild initial immunologic abnormalities. In patients with somatic reversion in CD8+ T cells, milder disease may be more common and genetic testing should be considered early. The management options including bone marrow transplant should be carefully considered.

Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1.

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

Successful Salvage Haploidentical Bone Marrow Transplantation in a Child With Hemophagocytic Lymphohistiocytosis, When the Previously Matched Unrelated Donor Tested Positive for SARS-CoV-2 on the Day of Stem Cells Collection

The coronavirus disease 2019 pandemic has made us adjust our standards and cope with unpredictable circumstances affecting the whole world, including the medical field.A 2-year-old boy diagnosed with X-linked lymphoproliferative disease type 2 with concomitant positive polymerase chain reaction test for Epstein-Barr virus–DNA was admitted to our transplant ward. His treatment scheme had to be modified at the last moment because of a donor disqualification due to a positive polymerase chain reaction result for severe acute respiratory syndrome coronavirus 2 just before the apheresis. We decided to perform salvage haploidentical bone marrow transplant from the patient's mother because it was the only possible option. Now, in a 5-month observation period after the hematopoietic stem cell transplantation, our patient is in good general condition. His case convinced us to redirect our approach to transplant procedure preparation. Following the European Group of Blood and Marrow Transplantation recommendations, we use cryopreserved apheresis materials to ensure the availability of stem cell products before the start of a conditioning regimen.

Hematopoietic stem cell transplantation with TCRαβ+/CD19+ - graft depletion for hemophagocytic lymphohistiocytosis

The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. We present the outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) with TCRab+/CD19+ graft depletion in patients with genetic hemophagocytic lymphohistiocytosis (HLH) at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology from 2012 to 2019. Thirty-six patients with various HLH: familial HLH (n = 20), X-linked lymphoproliferative disease (XLP) type 1 (n = 4), XLP type 2 (n = 9), Griscelli syndrome (n = 1), Chediak–Higashi syndrome (n = 2) received HSCT. Conditioning regimens were based on treosulfan in 9 patients, or on two alkylating agents: treosulfan with either melphalan, or thiotepa in 27 patients; all 36 patients received fludarabine and serotherapy. Thirty-two patients received rituximab 100 mg/m2 the day before stem cell infusion. Post- HSCT “graft versus host” disease (GvHD) prophylaxis was used in 29 patients. As a graft source peripheral blood stem cells from matched unrelated (n = 23), matched related (n = 3) and haploidentical family (n = 10) donors after TCRαβ+/CD19+ graft depletion were used. The cumulative incidence of primary and secondary graft failure in all patients was 0.11 (95% CI 0.04–0.29). The incidence of acute GvHD was limited to stage I-II. Overall survival was 0.91 (95% CI 0.82–1) without any significant differences in various donor groups (p = 0.33) as well as different conditioning regimens (p = 0.75). Allogeneic HSCT with TCRαβ+/CD19+ graft depletion after treosulfan-based conditioning is effective and safe technology for patients with genetic HLH.

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。

Detailed Phenotypic and Functional Characterization of a Rare, Antibody-Dependent SLAM-Associated Protein Expression Pattern.

SLAM-associated protein (SAP) is an adaptor molecule that facilitates critical effector functions in immune cells, and its deficiency causes X-linked lymphoproliferative disease type 1 in which effector responses directed against EBV are severely compromised. The primary objective of this study was to phenotypically and functionally characterize a rare, CD8 T cell-restricted bimodal SAP expression pattern observed in healthy, human donors with the widely used 1C9-SAP mAb clone. We initially observed this pattern during the clinical validation of our flow cytometry-based assay to diagnose X-linked lymphoproliferative disease type 1 in our laboratory. For this validation study, we used multiparameter flow cytometry to identify cytosolic SAP expression in lymphocyte subsets, and CD8 T cells from the donors displaying the rare SAP expression pattern mentioned above were separately further evaluated by intracellular cytokine and CD107a staining to examine polyfunctionality following PMA/ionomycin and HLA class I allele-restricted EBV peptide epitope-induced T cell activation. Our data revealed that SAP 1C9-hi CD8 T cells clearly displayed higher polyfunctional responses versus SAP 1C9-lo CD8 T cells following PMA/ionomycin stimulation. Furthermore, polyfunctional EBV-specific CD8 T cell responses segregated with the SAP 1C9-hi CD8 T cells and not the SAP 1C9-lo CD8 T cells. Additionally, and rather intriguingly, short- and long-term T cell stimulation selectively diminished the signal for the 1C9-hi subset. Overall, our data suggest that although rare, this unique SAP expression pattern merits further evaluation as it has the potential to provide some insight into fundamental processes as they might relate to host-pathogen dynamics.

Management of XLP-1 and ITK deficiency: The challenges posed by PID with an unpredictable spectrum of disease manifestations

The incorporation of next generation sequencing into routine immunological practice has enabled the identification of novel inborn errors of disease, helped define new categories of immune deficiency and extended the clinical spectrum associated with many long-recognised diseases. The family of EBV (Epstein Barr Virus)-sensitive primary immune deficiencies is one such group and in this paper we describe three families: two with X-linked lymphoproliferative disease type-1 (XLP-1) and one with deficiency of Interleukin-2 Inducible T-cell Kinase (ITK). Both diseases have a wide range of clinical manifestations and are united by an exquisite predisposition to EBV, dysgammaglobulinemia, hemophagocytic lymphohistiocytosis, and lymphoma. We detail our approach to diagnosis, treatment, and risk stratification in these diseases where both clinicians and patients must grapple with constant uncertainty.

Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes

Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn’s disease. Crohn’s disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn’s disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn’s Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn’s disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn’s Disease due to defects in autophagy.

X-Linked Lymphoproliferative Disease Type 1: A Clinical and Molecular Perspective

X-linked lymphoproliferative disease (XLP) was first described in the 1970s as a fatal lymphoproliferative syndrome associated with infection with Epstein–Barr virus (EBV). Features include hemophagocytic lymphohistiocytosis (HLH), lymphomas, and dysgammaglobulinemias. Molecular cloning of the causative gene, SH2D1A, has provided insight into the nature of disease, as well as helped characterize multiple features of normal immune cell function. Although XLP type 1 (XLP1) provides an example of a primary immunodeficiency in which patients have problems clearing primarily one infectious agent, it is clear that XLP1 is also a disease of severe immune dysregulation, even independent of EBV infection. Here, we describe clinical features of XLP1, how molecular and biological studies of the gene product, SAP, and the associated signaling lymphocyte activation molecule family receptors have provided insight into disease pathogenesis including specific immune cell defects, and current therapeutic approaches including the potential use of gene therapy. Together, these studies have helped change the outcome of this once almost uniformly fatal disease.

Analysis of Genes Associated With Monogenic Primary Immunodeficiency Identifies Rare Variants in XIAP in Patients With Crohn’s Disease

A few rare monogenic primary immunodeficiencies (PIDs) are characterized by chronic intestinal inflammation that resembles Crohn's disease (CD). We investigated whether 23 genes associated with 10 of these monogenic disorders contain common, low-frequency, or rarevariants that increase risk for CD. Common and low frequency variants in 1 Mb loci centered on the candidate genes were analyzed using meta-data corresponding to genotypes of approximately 17,000 patients with CD or without CD (controls) in Europe. The contribution of rare variants was assessed by high-throughput sequencing of 4750 individuals, including 660 early-onset and/or familial cases among the 2390 patients with CD. Variants were expressed from vectors in SW480 or HeLa cells and functions of their products were analyzed in immunofluorescence, luciferase, immunoprecipitation, and immunoblot assays. We reproduced the association of the interleukin 10 locus with CD (P= .007), although none of the significantly associated variants modified the coding sequence of interleukin 10. We found XIAP to be significantly enriched for rare coding mutations in patients with CD vs controls (P= .02). We identified 4 previously unreported missense variants associated with CD. Variants in XIAP cause the PID X-linked lymphoproliferative disease type 2, yet none of the carriers of these variants had all the clinical features of X-linked lymphoproliferative disease type 2. Identified XIAP variants S123N, R233Q, and P257A were associated with an impaired activation of NOD2 signaling after muramyl dipeptide stimulation. In a systematic analysis of variants in 23 PID-associated genes, we confirmed the association of variants in XIAP with CD. Further screenings forCD-associated variants and analyses of their functions couldincrease our understanding of the relationship between PID-associated genes and CD pathogenesis.

Two families of X-linked lymphoproliferative disease type 1 characterized by agammaglobulinemia

Objective: To investigate the clinical and immunological laboratory features, mutations in SH2D1A gene and SAP protein expression in four children of two families with X-linked lymphoproliferative disease type 1(XLP-1). Method: Four patients (Family A including Patient 1 and Patient 2, Family B including Patient 3 and Patient 4) and their maternal relatives were enrolled in this study. The clinical manifestation, EBV infection status and chest CT scan were analyzed. The absolute and relative numbers of lymphocyte subsets, T lymphocyte proliferative response, SAP protein expression were assessed by flow cytometry. Quantification of signal joint TCR rearrangementexcision circle (sjTRECs), CDR3 spectratyping of TCRvβ and gene mutation of SH2D1A were detected by PCR based on genomic DNA or cDNA. Result: Four male patients from two families were diagnosed with XLP-1. The ages of disease onset were more than 1 year, more than 1 year, more than 1 month and 6 months. The ages at diagnosis were nine years and ten months, sixteen years and eight months, fourteen years and ten months, four years and nine months. All patients had recurrent infections and EBV infection. Patients 1, 2, and 3 had agammaglobulinemia and Patient 4 had hypogammaglobulinemia. Chest CT scan showed all patients had atelectasis and pneumonia, and Patient 3 had bronchiectasis. Patient 3 was diagnosised as Burkitt lymphoma. For immunological function, all patients exhibited reduced CD4/CD8 ratios, increased numbers of exhausted T lymphocyte, decreased number of NK cell. The numbers of total B lymphocyte and naïve B lymphocyte were normal, but the number of memory B lymphocyte declined in all cases. Four patients' copy numbers of sjTRECs were low and CDR3 spectratypings of TCRvβ showed mildly skewed. But their T lymphocyte proliferative response was normal. SAP protein expression in four cases were measured by flow cytometry. Two patients from Family A were absent and two patients from Family B showed decreased values. SH2D1A gene sequence analysis showed that the patients of Family A harbored a nonsense mutation (c.163 C>T; p.R55X) in exon 2. Their mother and two sisters were carriers. A missense mutation of SH2D1A gene (c.278 G>A; p.G93D) in exon 3 was found in the patients of Family B. The mother was carrier. Four patients remain survived, Patient 3 gave up treatment, other three patients received IVIG therapy. Conclusion: Four patients with XLP-1 from two families characterized by agammaglobulinemia have an extreme vulnerability to Epstein-Barr virus (EBV) infection. The functions of T cell, B cell and NK cell are impaired at different stages. The detection of SAP protein and SH2D1A gene are the key methods for diagnosis of XLP-1.

How genetic testing can lead to targeted management of XIAP deficiency-related inflammatory bowel disease.

X-linked lymphoproliferative disease type 2 (XLP-2, OMIM 300635) is a primary immunodeficiency caused by the loss of X chromosome-linked inhibitor of apoptosis (XIAP), the X-linked inhibitor of apoptosis gene at Xq25. XLP-2 individuals are susceptible to several specific and potentially fatal infections, such as Epstein-Barr virus (EBV). Children with XIAP-related XLP-2 may present with either familial hemophagocytic lymphohistiocytosis, often triggered in response to EBV infection, or with a treatment-refractory severe pediatric form of inflammatory bowel disease (IBD) that might be diagnosed as Crohn disease. However, this monogenic cause of IBD is distinct from adult Crohn disease (a polygenic and multifactorial disease) in its etiology and responsiveness to therapy. XLP-2 and the associated IBD symptoms are managed by a reduced-intensity conditioning regimen with an allogeneic hematopoietic stem cell transplantation that causes resolution of gastrointestinal symptoms. Exome sequencing has enabled identification of XIAP-deficient diseased individuals and has altered their morbidity by providing potentially lifesaving strategies in a timely and effective manner. Here, we summarize XLP-2 IBD treatment history and patient morbidity/mortality since its original identification in 2006. Since XLP-2 is rare, cases are probably undergiagnosed or misdiagnosed. Consideration of XLP-2 in children with severe symptoms of IBD can prevent serious morbidities and mortality, avoid unnecessary procedures, and expedite specific targeted therapy.Genet Med 19 2, 133-143.

Reduced-Intensity Conditioning Hematopoietic Cell Transplantation Is an Effective Treatment for Patients with SLAM-Associated Protein Deficiency/X-linked Lymphoproliferative Disease Type 1

X-linked lymphoproliferative disease type 1 (XLP1) is a rare immune deficiency caused by mutations in SH2D1A. Allogeneic hematopoietic cell transplantation (HCT) is often performed because of the morbidity and mortality associated with XLP1. There is limited experience using reduced-intensity conditioning (RIC) regimens for these patients. Here we report our 8-year single-center experience. Sixteen consecutive patients diagnosed with XLP1 underwent allogeneic HCT between 2006 and 2013 after a RIC regimen consisting of alemtuzumab, fludarabine, and melphalan. Patient phenotypes included hemophagocytic lymphohistiocytosis (HLH) after Epstein-Barr virus (n = 5) or human herpesvirus 6 (n = 1), macrophage activation syndrome (n = 1), interstitial pneumonitis and encephalitis (n = 1), B cell lymphoma (n = 8), and hypogammaglobulinemia (n = 2). One patient was asymptomatic. Fourteen of 16 patients received 8/8 HLA-matched unrelated or related bone marrow grafts, whereas 2 patients received mismatched unrelated grafts. Acute graft-versus-host disease (GVHD) prophylaxis consisted of methylprednisolone and cyclosporine in all but 1 patient, who additionally received methotrexate. All patients had hematopoietic recovery. There were no cases of hepatic veno-occlusive disease or pulmonary hemorrhage. One patient (6%) developed acute GVHD and later also developed chronic GVHD (6%). Five patients (31%) developed mixed chimerism. Only 1 patient with mixed chimerism (6%) experienced a decline of donor chimerism to less than 50% but returned to full donor chimerism after infusion of donor lymphocytes and a CD34+ selected stem cell boost. Infectious complications were frequent, particularly viral reactivation. One-year survival estimated by Kaplan-Meier analysis was 80%, with long-term survival estimated at 71%. Survival was similar for patients with or without a history of HLH (86% versus 75%, respectively, P = .70). There were no occurrences of lymphoma or HLH after HCT. RIC HCT with alemtuzumab, fludarabine, and melphalan is an effective treatment for patients with XLP1, offering good survival rates regardless of prior disease manifestations, including HLH.

XIAP variants in male Crohn's disease

ObjectiveThe genetic basis of inflammatory bowel disease (IBD) is incompletely understood. The aim of this study was to identify rare genetic variants involved in the pathogenesis of IBD.DesignExome sequencing and...

Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients withSH2D1AandXIAP/BIRC4mutations

X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2.

Fatal EBV Infection and Variable Clinical Manifestations in an XLP-1 Pedigree – Rapid Diagnosis of Primary Immunodeficiencies may Save Lives

Two related boys who died from fulminant infectious mononucleosis were diagnosed with X-linked lymphoproliferative disease type 1 (XLP-1). Family screening (n=17) identified 6 female mutation carriers and 2 more XLP-1 patients in whom, despite recurrent infections, agammaglobulinemia, and Hodgkin's Disease, the genetic basis had been unknown; demonstrating that awareness and early genetic testing are crucial to reveal underlying primary immunodeficiencies and improve outcome. Furthermore, XLP should be included routinely in the differential diagnosis of severe hypogammaglobulinemia and/or lymphoma in males.