Masakatsu Yanagimachi

Influence of polymorphisms within the methotrexate pathway genes on the toxicity and efficacy of methotrexate in patients with juvenile idiopathic arthritis

AIMS We investigated whether several polymorphisms within the methotrexate (MTX) pathway genes were related to the toxicity and efficacy of MTX in 92 Japanese patients with articular-type juvenile idiopathic arthritis (JIA). METHODS Eight gene polymorphisms within the MTX pathway genes, namely, RFC, BCRP, MTHFR (two), FPGS, γ-glutamyl hydrolase (GGH; two) and ATIC, were genotyped using TaqMan assays. Liver dysfunction was defined as an increase in alanine transaminase to five times the normal upper limit. Non-responders to MTX were defined as patients refractory to MTX and were therefore treated with biologics. RESULTS The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction (P=0.028, odds ratio=6.90, 95% confidence interval 1.38-34.5), even after adjustment for the duration of MTX treatment. A longer interval from disease onset to treatment (8.5 and 21.3 months, P=0.029) and rheumatoid factor positivity (P=0.026, odds ratio=2.87, 95% confidence interval 1.11-7.39) were associated with lower efficacy of MTX. CONCLUSIONS The non-TT genotype at GGH T16C was associated with a high risk of liver dysfunction, presumably because the C allele of GGH C16T may reduce the activity of GGH. The time interval before MTX treatment and rheumatoid factor positivity were associated with the efficacy of MTX treatment. The pharmacogenetics of the MTX pathway genes affects the toxicity and efficacy of MTX in Japanese JIA patients.

Association of IRF5 Polymorphisms with Susceptibility to Macrophage Activation Syndrome in Patients with Juvenile Idiopathic Arthritis

Objective. Systemic-onset juvenile idiopathic arthritis (systemic JIA) and macrophage activation syndrome (MAS), the most devastating complication of systemic JIA, are characterized by abnormal levels of proinflammatory cytokines. Interferon regulatory factor 5 (IRF5) is a member of the IRF family of transcription factors, and acts as a master transcription factor in the activation of genes encoding proinflammatory cytokines. Polymorphisms in the IRF5 gene have been associated with susceptibility to autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. Our aim was to assess associations of IRF5 gene polymorphisms with susceptibility to systemic JIA and MAS. Methods. Three IRF5 single-nucleotide polymorphisms (rs729302, rs2004640, and rs2280714) were genotyped using TaqMan assays in 81 patients with systemic JIA (33 with MAS, 48 without) and 190 controls. Results. There were no associations of the IRF5 gene polymorphisms or haplotypes under study with susceptibility to systemic JIA. There was a significant association of the rs2004640 T allele with MAS susceptibility (OR 4.11; 95% CI 1.84, 9.16; p = 0.001). The IRF5 haplotype (rs729302 A, rs2004640 T, and rs2280714 T), which was reported as conferring an increased risk of SLE, was significantly associated with MAS susceptibility in patients with systemic JIA (OR 4.61; 95% CI 1.73, 12.3; p < 0.001). Conclusion. IRF5 gene polymorphism is a genetic factor influencing susceptibility to MAS in patients with systemic JIA, and IRF5 contributes to the pathogenesis of MAS in these patients.

Influence of CYP3A5 and ABCB1 gene polymorphisms on calcineurin inhibitor-related neurotoxicity after hematopoietic stem cell transplantation

Yanagimachi M, Naruto T, Tanoshima R, Kato H, Yokosuka T, Kajiwara R, Fujii H, Tanaka F, Goto H, Yagihashi T, Kosaki K, Yokota S. Influence of CYP3A5 and ABCB1 gene polymorphisms on calcineurin inhibitor‐related neurotoxicity after hematopoietic stem cell transplantation. 
Clin Transplant 2010: 24: 855–861.

A conditioning regimen of busulfan, fludarabine, and melphalan for allogeneic stem cell transplantation in children with juvenile myelomonocytic leukemia

Abstract:  A pilot study was undertaken using a myeloablative conditioning with fludarabine, busulfan, and melphalan to improve the outcome of HSCT in 10 children, aged six months to six yr, with JMML. All patients were conditioned with oral busulfan (560 mg/m2), fludarabine (120 mg/m2), and melphalan (180–210 mg/m2) prior to HSCT, and received stem cells from bone marrow in seven cases, and from cord blood in three cases. Engraftment was documented in eight patients, whereas graft failure occurred in two, one of whom had received HLA‐mismatched cord blood and other had received bone marrow from HLA‐mismatched mother. Three patients, including two in who graft failure had occurred, relapsed. Five patients developed acute GVHD and two developed chronic GVHD. Seven patients are alive and in remission 27–69 months after transplantation. Thus, our study showed that HSCT following conditioning with fludarabine, busulfan, and melphalan was well tolerated and appeared to be effective for JMML.

Association of IRF5 Polymorphisms with Susceptibility to Hemophagocytic Lymphohistiocytosis in Children

IntroductionHemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory syndrome and has a varied genetic background. The polymorphism of interferon regulatory factor 5 gene (IRF5) was reported to be associated with susceptibility to macrophage activation syndrome. IRF5 acts as a master transcription factor in the activation of pro-inflammatory cytokines. We assessed associations of IRF5 gene polymorphisms with susceptibility to secondary HLH.MethodsThree IRF5 single nucleotide polymorphisms (rs729302, rs2004640, and rs2280714) were genotyped using TaqMan assays in 82 secondary HLH patients and 188 control subjects.ResultsThere was a significant association of the GT/TT genotype at rs2004640 with secondary HLH susceptibility (p < 0.01). The IRF5 haplotype (rs729302 A, rs2004640 T, and rs2280714 T) was associated with secondary HLH susceptibility (p < 0.01).ConclusionsThese findings indicate that IRF5 is a genetic factor influencing the susceptibility to secondary HLH and that the IRF5-associated immune response contributes to the pathogenesis of HLH.

Graft versus leukemia effect against juvenile myelomonocytic leukemia after unrelated cord blood transplantation.

A 13‐month‐old female underwent unrelated cord blood transplantation (CBT) for juvenile myelomonocytic leukemia (JMML). In spite of progression of the disease after a conditioning regimen with high‐dose chemotherapy, a complete remission was induced in concordance with development of acute GVHD after reduction of the immunosupressant. She has been in complete remission for 1 year after transplantation. This case illustrates that CBT can provide a potent graft versus leukemia (GVL) effect against JMML. Pediatr Blood Cancer 2008;50:665–667.

Analysis of Gender Differences in Genetic Risk: Association of TNFAIP3 Polymorphism with Male Childhood-Onset Systemic Lupus Erythematosus in the Japanese Population

Background Systemic lupus erythematosus (SLE) is a systemic multisystem autoimmune disorder influenced by genetic background and environmental factors. Our aim here was to replicate findings of associations between 7 of the implicated single nucleotide polymorphisms (SNPs) in IRF5, BLK, STAT4, TNFAIP3, SPP1, TNIP1 and ETS1 genes with susceptibility to childhood-onset SLE in the Japanese population. In particular, we focused on gender differences in allelic frequencies. Methodology/Principal Findings The 7 SNPs were genotyped using TaqMan assays in 75 patients with childhood-onset SLE and in 190 healthy controls. The relationship between the cumulative number of risk alleles and SLE manifestations was explored in childhood-onset SLE. Logistic regression was used to test the effect of each polymorphism on susceptibility to SLE, and Wilcoxon rank sum testing was used for comparison of total risk alleles. Data on rs7574865 in the STAT4 gene and rs9138 in SPP1 were replicated for associations with SLE when comparing cases and controls (corrected P values ranging from 0.0043 to 0.027). The rs2230926 allele of TNFAIP3 was associated with susceptibility to SLE in males, but after Bonferroni correction there were no significant associations with any of the other four SNPs in IRF5, BLK, TNIP1 and ETS1 genes. The cumulative number of risk alleles was significantly increased in childhood-onset SLE relative to healthy controls (P = 0.0000041). Male SLE patients had a slightly but significantly higher frequency of the TNFAIP3 (rs2230926G) risk allele than female patients (odds ratio [OR] = 4.05, 95% confidence interval [95%CI] = 1.46–11.2 P<0.05). Conclusions Associations of polymorphisms in STAT4 and SPP1 with childhood-onset SLE were confirmed in a Japanese population. Although these are preliminary results for a limited number of cases, TNFAIP3 rs2230926G may be an important predictor of disease onset in males. We also replicated findings that the cumulative number of risk alleles was significantly increased in childhood-onset SLE.

Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children

Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

Suppressed neutrophil function in children with acute lymphoblastic leukemia

Infection is a major obstacle in cancer chemotherapy. Neutropenia has been considered to be the most important risk factor for severe infection; however, other factors, such as impaired neutrophil function, may be involved in susceptibility to infection in patients undergoing chemotherapy. In this study, we analyzed neutrophil function in children with acute lymphoblastic leukemia (ALL). Whole blood samples were obtained from 16 children with ALL at diagnosis, after induction chemotherapy, and after consolidation chemotherapy. Oxidative burst and phagocytic activity of neutrophils were analyzed by flow cytometry. Oxidative burst of neutrophils was impaired in ALL patients. The percentage of neutrophils with normal oxidative burst after PMA stimulation was 59.0 ± 13.2 or 70.0 ± 21.0% at diagnosis or after induction chemotherapy, respectively, which was significantly lower compared with 93.8 ± 6.1% in healthy control subjects (P = 0.00004, or 0.002, respectively); however, this value was normal after consolidation chemotherapy. No significant differences were noted in phagocytic activity in children with ALL compared with healthy control subjects. Impaired oxidative burst of neutrophils may be one risk factor for infections in children with ALL, especially in the initial periods of treatment.

Association of HLA-A * 02:06 and HLA-DRB1 * 04:05 with clinical subtypes of juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is one of the most common forms of pediatric chronic arthritis. JIA is a clinically heterogeneous disease. Therefore, the genetic background of JIA may also be heterogeneous. The aim of this study was to investigate associations between human leukocyte antigen (HLA) and susceptibility to JIA and/or uveitis, which is one of the most devastating complications of JIA. A total of 106 Japanese articular JIA patients (67 with polyarthritis and 39 with oligoarthritis) and 678 healthy controls were genotyped for HLA-A, -B and -DRB1 by PCR-sequence-specific oligonucleotide probe methodology. HLA-A*02:06 was the risk factor for JIA accompanied by uveitis after adjustment for clinical factors (corrected P-value <0.001, odds ratio (OR) 11.7, 95% confidence interval (CI) 3.2–43.0). On the other hand, HLA-DRB1*04:05 was associated with polyarticular JIA (corrected P-value <0.001, OR 2.9, 95% CI 1.7–4.8). We found an association of HLA-A*02:06 with susceptibility to JIA accompanied by uveitis, which might be considered a separate clinical JIA entity. We also found an association between HLA-DRB1*04:05 and polyarticular JIA. Thus, clinical subtypes of JIA can be classified by the presence of the specific HLA alleles, HLA-A*02:06 and DRB1*04:05.