Hirokazu Kanegane

Clinical similarities and differences of patients with X-linked lymphoproliferative syndrome type 1 (XLP-1/SAP-deficiency) versus type 2 (XLP-2/XIAP-deficiency)

X-linked lymphoproliferative syndromes (XLP) are primary immunodeficiencies characterized by a particular vulnerability toward Epstein-Barr virus infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP type 1 (XLP-1) is caused by mutations in the gene SH2D1A (also named SAP), whereas mutations in the gene XIAP underlie XLP type 2 (XLP-2). Here, a comparison of the clinical phenotypes associated with XLP-1 and XLP-2 was performed in cohorts of 33 and 30 patients, respectively. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups. Epstein-Barr virus infection in XLP-1 and XLP-2 was the common trigger of HLH (XLP-1, 92%; XLP-2, 83%). Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in XLP-1 (XLP-1, 61%; XLP-2, 23%). Although only XLP-1 patients developed lymphomas (30%), XLP-2 patients (17%) had chronic hemorrhagic colitis as documented by histopathology. Recurrent splenomegaly often associated with cytopenia and fever was preferentially observed in XLP-2 (XLP-1, 7%; XLP-2, 87%) and probably represents minimal forms of HLH as documented by histopathology. This first phenotypic comparison of XLP subtypes should help to improve the diagnosis and the care of patients with XLP conditions.

X-linked lymphoproliferative disease due to SAP/SH2D1A deficiency: a multicenter study on the manifestations, management and outcome of the disease

X-linked lymphoproliferative disease (XLP1) is a rare immunodeficiency characterized by severe immune dysregulation and caused by mutations in the SH2D1A/SAP gene. Clinical manifestations are varied and include hemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinemia, often triggered by Epstein-Barr virus infection. Historical data published before improved treatment regimens shows very poor outcome. We describe a large cohort of 91 genetically defined XLP1 patients collected from centers worldwide and report characteristics and outcome data for 43 patients receiving hematopoietic stem cell transplant (HSCT) and 48 untransplanted patients. The advent of better treatment strategies for HLH and malignancy has greatly reduced mortality for these patients, but HLH still remains the most severe feature of XLP1. Survival after allogeneic HSCT is 81.4% with good immune reconstitution in the large majority of patients and little evidence of posttransplant lymphoproliferative disease. However, survival falls to 50% in patients with HLH as a feature of disease. Untransplanted patients have an overall survival of 62.5% with the majority on immunoglobulin replacement therapy, but the outcome for those untransplanted after HLH is extremely poor (18.8%). HSCT should be undertaken in all patients with HLH, because outcome without transplant is extremely poor. The outcome of HSCT for other manifestations of XLP1 is very good, and if HSCT is not undertaken immediately, patients must be monitored closely for evidence of disease progression.

Genetic analysis of patients with defects in early B‐cell development

Summary:  Approximately 85% of patients with defects in early B‐cell development have X‐linked agammaglobulinemia (XLA), a disorder caused by mutations in the cytoplasmic Brutons tyrosine kinase (Btk). Although Btk is activated by cross‐linking of a variety of cell‐surface receptors, the most critical signal transduction pathway is the one initiated by the pre‐B cell and B‐cell antigen receptor complex. Mutations in Btk are highly diverse, and no single mutation accounts for more than 3% of patients. Although there is no strong genotype/phenotype correlation in XLA, the specific mutation in Btk is one of the factors that influences the severity of disease. Mutations in the components of the pre‐B cell and B‐cell antigen receptor complex account for an additional 5–7% of patients with defects in early B‐cell development. Patients with defects in these proteins are clinically indistinguishable from those with XLA. However, they tend to be younger at the time of diagnosis, and whereas most patients with XLA have a small number of B cells in the peripheral circulation, these cells are not found in patients with defects in µ heavy chain or Igα. Polymorphic variants in the components of the pre‐B cell and B‐cell receptor complex, particularly µ heavy chain and λ5, may contribute to the severity of XLA.

The landscape of somatic mutations in Down syndrome–related myeloid disorders

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome–related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%).

Prognostic Factors for Chronic Active Epstein-Barr Virus Infection

Chronic active Epstein-Barr virus infection (CAEBV) is a high-mortality and high-morbidity disease. To clarify the prognostic factors, a national survey was performed in Japan, and data for 82 patients who met the criteria for CAEBV were analyzed. Of these 82 patients, 47 were alive and 35 had already died. Multivariate analysis revealed that thromobocytopenia and age at disease onset were correlated with mortality. The probability of 5-year survival was 0.45 for older patients (onset age, > or = 8 years), 0.94 for younger patients (P<.001), 0.38 for patients with thrombocytopenia (platelet count < 12 x 10(4) platelets/microL at diagnosis), and 0.76 for patients without thrombocytopenia (P=.01). Furthermore, patients with T cell infection by EBV had shorter survival times than patients with natural killer cell infection (probability of 5-year survival, 0.59 vs. 0.87; P<.009). Patients with CAEBV with late onset of disease, thrombocytopenia, and T cell infection had significantly poorer outcomes.

Functional Analysis of RUNX2 Mutations in Japanese Patients with Cleidocranial Dysplasia Demonstrates Novel Genotype-Phenotype Correlations

Cleidocranial dysplasia (CCD) is an autosomal dominant heritable skeletal disease caused by heterozygous mutations in the osteoblast-specific transcription factor RUNX2. We have performed mutational analysis of RUNX2 on 24 unrelated patients with CCD. In 17 patients, 16 distinct mutations were detected in the coding region of RUNX2: 4 frameshift, 3 nonsense, 6 missense, and 2 splicing mutations, in addition to 1 polymorphism. The missense mutations were all clustered within the Runt domain, and their protein products were severely impaired in DNA binding and transactivation. In contrast, two RUNX2 mutants had the Runt domain intact and remained partially competent for transactivation. One criterion of CCD, short stature, was much milder in the patients with the intact Runt domain than in those without. Furthermore, a significant correlation was found between short stature and the number of supernumerary teeth. On the one hand, these genotype-phenotype correlations highlight a general, quantitative dependency, by skeleto-dental developments, on the gene dosage of RUNX2, which has hitherto been obscured by extreme clinical diversities of CCD; this gene-dosage effect is presumed to manifest on small reductions in the total RUNX2 activity, by approximately one-fourth of the normal level at minimum. On the other hand, the classic CCD phenotype, hypoplastic clavicles or open fontanelles, was invariably observed in all patients, including those with normal height. Thus, the cleidocranial bone formation, as mediated by intramembranous ossification, may require a higher level of RUNX2 than does skeletogenesis (mediated by endochondral ossification), as well as odontogenesis (involving still different complex processes). Overall, these results suggest that CCD could result from much smaller losses in the RUNX2 function than has been envisioned on the basis of the conventional haploinsufficiency model.

Novel mutations in a Japanese patient with CD19 deficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by recurrent bacterial infections, hypogammaglobulinemia and low to normal numbers of circulating B cells. Mutations in the ICOS, TACI and CD19 genes have recently been identified in <10% of CVID patients. We, herein, describe two novel CD19 gene disruptions in an 8-year-old Japanese boy, who had been clinically diagnosed as having CVID at the age of 5 years. Flow-cytometric analysis demonstrated absence of CD19 and reduced CD21 expression on CD20-postive peripheral blood B cells. Mutation analysis of CD19 revealed a mutation in the splice acceptor site of intron 5 (IVS5-1G>T) of the maternal allele, resulting in skipping of exon 6, and a truncated protein product. The paternal allele was disrupted by a gross deletion encompassing at least the ATP2A1, CD19 and NFATC2IP genes. The patient had a small number of IgD− CD27+ memory B cells, in which somatic mutation were detected. His B cells showed substantial proliferation upon stimulation, but reduced IgG and IgA production in vitro. These findings extend the mutation spectrum of the CD19 deficiency to four, and confirm the homogeneity of the CD19 deficiency as a unique type of CVID.

XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles Quantification Using Neonatal Guthrie Cards

OBJECTIVE To assess the feasibility of T-cell receptor excision circles (TRECs) quantification for neonatal mass screening of severe combined immunodeficiency (SCID). STUDY DESIGN Real-time PCR based quantification of TRECs for 471 healthy control patients and 18 patients with SCID with various genetic abnormalities (IL2RG, JAK3, ADA, LIG4, RAG1) were performed, including patients with maternal T-cell engraftment (n = 4) and leaky T cells (n = 3). RESULTS TRECs were detectable in all normal neonatal Guthrie cards (n = 326) at the levels of 10(4) to 10(5) copies/microg DNA. In contrast, TRECs were extremely low in all neonatal Guthrie cards (n = 15) and peripheral blood (n = 14) from patients with SCID, including those with maternal T-cell engraftment or leaky T cells with hypomorphic RAG1 mutations or LIG4 deficiency. There were no false-positive or negative results in this study. CONCLUSION TRECs quantification can be used as a neonatal mass screening for patients with SCID.

Detection of Bruton's tyrosine kinase mutations in hypogammaglobulinaemic males registered as common variable immunodeficiency (CVID) in the Japanese Immunodeficiency Registry

CVID is frequently diagnosed in male and female individuals with hypogammaglobulinaemia of unknown aetiology. To examine the possibility that sporadic male cases with X‐linked agammaglobulinaemia (XLA), which is caused by mutations in the Brutons tyrosine kinase (Btk) gene, might be misregistered as having CVID, we employed a flow cytometric test to identify XLA in hypogammaglobulinaemic males registered as CVID in the Japanese Immunodeficiency Registry. From 30 male cases registered as having CVID between 1992 and 1998, we selected 21 males with low or unreported peripheral B cell counts. Blood samples could be obtained from 11 patients and their mothers. Using flow cytometric analysis, the Btk‐deficient status in monocytes was demonstrated in seven out of nine cases with decreased numbers of peripheral B cells. The diagnosis of XLA was confirmed in each of the seven patients by demonstration of Btk gene mutations in the patients or cellular mosaicism in the mother. This study demonstrates misregistration of XLA as CVID.