Noriko Mitsuiki

Autoimmune lymphoproliferative syndrome-like disease with somatic KRAS mutation

Autoimmune lymphoproliferative syndrome (ALPS) is classically defined as a disease with defective FAS-mediated apoptosis (type I-III). Germline NRAS mutation was recently identified in type IV ALPS. We report 2 cases with ALPS-like disease with somatic KRAS mutation. Both cases were characterized by prominent autoimmune cytopenia and lymphoadenopathy/splenomegaly. These patients did not satisfy the diagnostic criteria for ALPS or juvenile myelomonocytic leukemia and are probably defined as a new disease entity of RAS-associated ALPS-like disease (RALD).

Quantification of κ-deleting recombination excision circles in Guthrie cards for the identification of early B-cell maturation defects

In conclusion, the associations among asthma, biofilmforming bacteria, and revision ESS are strong and robust after adjusting for other factors in patients with CRS from a tertiary medical center. Despite its limitations, this study may improve our understanding of refractory CRS pathogenesis, possibly leading to more effective treatment strategies, such as incorporating the treatments of asthma and biofilm infection into conventional CRS therapies. Prospective cohort studies in diverse populations are needed to assess the causality of these associations.

Common variable immunodeficiency classification by quantifying T-cell receptor and immunoglobulin κ-deleting recombination excision circles

In the United States the regulation of nonstandardized AEs presented some similaritieswith our approach.AEswere classified into 4 categories according to scientific data supporting their use in diagnosis and treatment, and the extracts were regularly evaluated by the regulatory agencies. The last updatewas conducted between 2003 and 2011, and the process was recently reviewed by Slater et al. It was shown that for nearly half of nonstandardized AEs there were, in fact, little or no data to support their effectiveness. We had similar results: 66 of 84 AEs were validated for diagnosis, but only for 29 of 66 was there at least 1 published piece of data to support their effectiveness for immunotherapy (Table I). Among those 66 authorized AEs, approximately one third are standardized. There is no consensus about the standardization methods, and the European approaches present some differences compared with the US approach (see Table E1 in this article’s Online Repository at www.jacionline.org). Briefly, in-house reference preparation (IHRP) AEs are standardized in vivo and in vitro. Each manufacturer has its own IHRP, and there is no national standard. Batch-to-batch standardization is performed in vitro through a comparison of the AEs with the IHRP. In the future, the NPP list will be updated every 5 years, and requests for MAwill be made and processed for standardized AEs produced industrially and frequently used for immunotherapy. In conclusion, for the first time in Europe, this work guarantees that available AEs are clinically relevant and safe. Moreover, it guarantees that all AEs comply with recent European guidelines on APs, including rare allergens for which it is not possible to obtain large clinical studies requested for MA. The process involved all the representatives of allergists and manufacturers and is still ongoing. Fr ed eric de Blay, MD Virginie Doyen, MD Evelyne Bloch-Morot, MD Daniel Caillot, MD Jacques Gayraud, MD Aymar de Laval, MD Alain Thillay, MD for the APSI group*

Phosphatase and tensin homolog (PTEN) mutation can cause activated phosphatidylinositol 3-kinase δ syndrome–like immunodeficiency

BACKGROUND Activated phosphatidylinositol 3-kinase δ syndrome (APDS) is a recently discovered primary immunodeficiency disease (PID). Excess phosphatidylinositol 3-kinase (PI3K) activity linked to mutations in 2 PI3K genes, PIK3CD and PIK3R1, causes APDS through hyperphosphorylation of AKT, mammalian target of rapamycin (mTOR), and S6. OBJECTIVE This study aimed to identify novel genes responsible for APDS. METHODS Whole-exome sequencing was performed in Japanese patients with PIDs. Immunophenotype was assessed through flow cytometry. Hyperphosphorylation of AKT, mTOR, and S6 in lymphocytes was examined through immunoblotting, flow cytometry, and multiplex assays. RESULTS We identified heterozygous mutations of phosphatase and tensin homolog (PTEN) in patients with PIDs. Immunoblotting and quantitative PCR analyses indicated that PTEN expression was decreased in these patients. Patients with PTEN mutations and those with PIK3CD mutations, including a novel E525A mutation, were further analyzed. The clinical symptoms and immunologic defects of patients with PTEN mutations, including lymphocytic AKT, mTOR, and S6 hyperphosphorylation, resemble those of patients with APDS. Because PTEN is known to suppress the PI3K pathway, it is likely that defective PTEN results in activation of the PI3K pathway. CONCLUSION PTEN loss-of-function mutations can cause APDS-like immunodeficiency because of aberrant PI3K pathway activation in lymphocytes.

A nationwide survey of Aicardi–Goutières syndrome patients identifies a strong association between dominant TREX1 mutations and chilblain lesions: Japanese cohort study

OBJECTIVES Aicardi-Goutières syndrome (AGS) is a rare, genetically determined, early onset progressive encephalopathy associated with autoimmune manifestations. AGS is usually inherited in an autosomal recessive manner. The disease is rare, therefore the clinical manifestations and genotype-phenotype correlations, particularly with regard to autoimmune diseases, are still unclear. Here we performed a nationwide survey of AGS patients in Japan and analysed the genetic and clinical data. METHODS Patients were recruited via questionnaires sent to paediatric or adult neurologists in Japanese hospitals and institutions. Genetic analysis was performed and clinical data were collected. RESULTS Fourteen AGS patients were identified from 13 families; 10 harboured genetic mutations. Three patients harboured dominant-type TREX1 mutations. These included two de novo cases: one caused by a novel heterozygous p.His195Tyr mutation and the other by a novel somatic mosaicism resulting in a p.Asp200Asn mutation. Chilblain lesions were observed in all patients harbouring dominant-type TREX1 mutations. All three patients harbouring SAMHD1 mutations were diagnosed with autoimmune diseases, two with SLE and one with SS. The latter is the first reported case. CONCLUSION This study is the first to report a nationwide AGS survey, which identified more patients with sporadic AGS carrying de novo dominant-type TREX1 mutations than expected. There was a strong association between the dominant-type TREX1 mutations and chilblain lesions, and between SAMHD1 mutations and autoimmunity. These findings suggest that rheumatologists should pay attention to possible sporadic AGS cases presenting with neurological disorders and autoimmune manifestations.

Pharmacokinetic monitoring is still required for intravenous busulfan in SCT for small children

Busulfan (BU) is an alkylating agent that is used in alter-native conditioning regimen for total body irradiation(TBI) before stem cell transplantation (SCT) especially toreduce its late toxicities such as growth impairment andsecond malignancy. Because its therapeutic window isnarrow and, inter- and intra-patient variability of plasmaexposure is wide in case of conventional oral formulation,therapeutic drug monitoring (TDM) is recommended toavoid severe toxicity or rejection, especially in youngerpatients in which its variability is much more [1, 2]. ByTDM, BU dose should be adjusted so that calculatedconcentration at steady state (Css) or area under the curve(AUC) is in the range of 600–900 ng/ml or 900–1500 mmol min/L. In this context, introduction of intra-venous formulation is expected to resolve these problems.For small children, recommended dose for intravenousformulation has been reported [3, 4]. However, there is arecent report that emphasizes the necessity of TDM even inintravenous formulation [5]. In this background, weinvestigated allogeneic SCT cases with BU regimen in ourinstitute retrospectively.Since 1995, 34 patients received allogeneic hematopoi-etic SCT by using BU regimen in our pediatric section.Former 29 patients received BU orally (four doses per dayfor four consecutive days; p.o.BU group), and latter fivepatients received BU intravenously (2-h infusion every 6 hfor four consecutive days; i.v.BU group).For pharmacokinetic (PK) analysis, blood samples wereobtained 1, 2, 3, 4, and 6 h after medication of BU. Inp.o.BU group, PK analysis were performed in the last 10cases, in which test dose (0.5–0.7 mg/kg) was performed2 weeks before conditioning chemotherapy, and dose wasadjusted according to the result of PK analysis up to themaximal dose of 2 mg/kg/dose (Table 1). PK analysis wasalso performed for the initial dose, and dose was adjustedin fourth day (13th dose) in case calculated Css of BUexceeded the range of 600–900 ng/ml [Only in case 3, dosewas reduced from 1.8 to 1.1 mg/kg/dose.]. In i.v.BU group,BU was administered according to the recommendation [3,4], and blood sample was taken and stored for later PKanalysis except for the last case (case 15). In case 15, testdose was performed, and dose was reduced from 1.0 to0.8 mg/kg/dose. Informed consent was obtained from all ofthe patients or patient’s guardians before conducting thetest dose. Also, we have approval for this procedure fromour IRB.Among 15 cases in which PK analysis was performed(Table 1), graft rejection was observed only in one case(case 5) of p.o.BU group, which was a patient withhemophagocytic lymphohistiocytosis. No hepatic veno-occlusive disease (HVOD) occurred in p.o.BU group, butwas observed in two out of five i.v. cases, in which onecase (case 13) had severe HVOD and died 60 days afterSCT because of hepatic failure. In case 13, the patientreceived first SCT from HLA identical sibling with TBI(12 Gy) and cyclophosphamide (60 mg/kg 9 2 days) reg-imen. After relapse in 3 months, he was treated withHperCVAD plus imatinib chemotherapy [6] and received

Multicolor Flow Cytometry for the Diagnosis of Primary Immunodeficiency Diseases

PurposePrimary immunodeficiency diseases (PIDDs) are rare inherited diseases that impair the human immune system. We established a multicolor flow cytometric assay to comprehensively evaluate the immune status and immunological characteristics of patients with PIDDs.MethodsFifty-nine normal controls and 75 patients with PIDDs, including X-linked severe combined immunodeficiency (X-SCID), X-linked agammaglobulinemia (XLA), X-linked hyper IgM syndrome (X-HIGM), ataxia telangiectasia (AT), Wiskott-Aldrich syndrome (WAS), hyper IgE syndrome (HIES), and chronic mucocutaneous candidiasis disease (CMCD), were enrolled in this study. Immunophenotyes were evaluated by multicolor flow cytometry using seven different panels that allowed the detection of major leukocyte populations in peripheral blood.ResultsMulticolor flow cytometry revealed distinct leukocyte populations and immunological features of patients with X-SCID, XLA, X-HIGM, AT, WAS, HIES, and CMCD.ConclusionsImmunophenotyping by multicolor flow cytometry is useful to evaluate immune status and contributes to the diagnosis and management of patients with PIDDs.

Mutations in Bruton’s tyrosine kinase impair IgA responses

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency caused by mutations in Bruton’s tyrosine kinase (BTK), and is characterized by markedly decreased numbers of blood B cells and an absence of all immunoglobulin isotypes. We performed whole exome sequencing in a male pediatric patient with dysgammaglobulinemia with IgA deficiency. Genetic analysis revealed a BTK missense mutation (Thr316Ala). To investigate whether a BTK mutation underlay this antibody deficiency with marked decrease of IgA in this patient, we performed functional analyses of B cells and phagocytes, and molecular analyses of somatic hypermutation and class switch recombination. The BTK missense mutation resulted in B cells with reduced BTK and high IgM expression. Equal proportions of CD19low and CD19normal fractions were observed, and both included naïve and memory B cells. Calcium influx and phospholipase Cγ2 phosphorylation upon IgM stimulation were marginally impaired in CD19low, but not in CD19+ B cells. Similar to XLA patients, IgA transcripts showed low SHM levels, whereas IgG transcripts were hardly affected. Our analyses suggest that the BTK mutation likely underlies the disease in this case, and that hypomorphic BTK mutations can result in normal circulating B cell numbers, but specifically impair IgA responses.

Irreversible leukoencephalopathy after reduced-intensity stem cell transplantation in a dyskeratosis congenita patient with TINF2 mutation.

Hematopoietic stem cell transplantation (HSCT) for dyskeratosis congenita (DC) is challenging due to severe treatment-related adverse effects. Development of pulmonary fibrosis or veno-occlusive disease is well described in DC. However, neurological complication after HSCT has not been reported. A 9-year-old Japanese male with DC harboring the TINF2 mutation received reduced-intensity HSCT. Unfortunately, patient developed posterior reversible encephalopathy syndrome–like symptoms plausibly result by combination of thrombotic microangiopathy, graft-versus-host disease, and persistent hypertension and has been persisted mental retardation. Therefore, to decrease risk in DC cases after HSCT, strict control of hypertension, graft-versus-host disease, and thrombotic microangiopathy is required.

A novel Wiskott-Aldrich syndrome protein mutation in an infant with thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) has not yet been reported to be associated with mutations in the Wiskott–Aldrich syndrome (WAS) gene. WAS is an X‐linked recessive disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. A broad spectrum of mutations in the WAS protein (WASP) gene have been identified as causing the disease. In this study, we report on a 2‐month‐old Japanese boy who presented with cytomegalovirus (CMV) infection and TTP. The activity of von Willebrand factor cleaving metalloproteinase, ADAMTS13 was low and the antibody against ADAMTS13 was positive (3.6 Bethesda U/mL). Although TTP was improved by plasma exchange and steroid pulse therapy, thrombocytopenia persisted and regular transfusions of irradiated platelets were needed. Tiny platelets were found on a peripheral blood smear. CMV genome was positive in peripheral blood by polymerase chain reaction and the CMV viremia continued to persist despite intravenous gancyclovir therapy. Through direct sequencing of genomic DNA of the WASP gene in the patient, we identified a novel mutation of WASP gene: the seventh nucleotide in exon 11 (G) had been deleted (1345delG). This mutation causes a frameshift and a stop codon at amino acid 470. Western blotting demonstrated a truncated WAS protein. To our knowledge, this is the first report describing TTP in WAS patients with novel mutation in the WASP gene.