Yoichi Mashimo

A genome-wide association study identifies RNF213 as the first Moyamoya disease gene.

Moyamoya disease (MMD) shows progressive cerebral angiopathy characterized by bilateral internal carotid artery stenosis and abnormal collateral vessels. Although ∼15% of MMD cases are familial, the MMD gene(s) remain unknown. A genome-wide association study of 785 720 single-nucleotide polymorphisms (SNPs) was performed, comparing 72 Japanese MMD patients with 45 Japanese controls and resulting in a strong association of chromosome 17q25-ter with MMD risk. This result was further confirmed by a locus-specific association study using 335 SNPs in the 17q25-ter region. A single haplotype consisting of seven SNPs at the RNF213 locus was tightly associated with MMD (P=5.3 × 10−10). RNF213 encodes a really interesting new gene finger protein with an AAA ATPase domain and is abundantly expressed in spleen and leukocytes. An RNA in situ hybridization analysis of mouse tissues indicated that mature lymphocytes express higher levels of Rnf213 mRNA than their immature counterparts. Mutational analysis of RNF213 revealed a founder mutation, p.R4859K, in 95% of MMD families, 73% of non-familial MMD cases and 1.4% of controls; this mutation greatly increases the risk of MMD (P=1.2 × 10−43, odds ratio=190.8, 95% confidence interval=71.7–507.9). Three additional missense mutations were identified in the p.R4859K-negative patients. These results indicate that RNF213 is the first identified susceptibility gene for MMD.

The GSTP1 Gene Is a Susceptibility Gene for Childhood Asthma and the GSTM1 Gene Is a Modifier of the GSTP1 Gene

Background: Bronchial asthma is a chronic airway disorder characterized by bronchial inflammation. Oxidative stress is a key component of inflammation. Glutathione S-transferase P1 (GSTP1), the abundant isoform of glutathione S-transferases (GSTs) in lung epithelium, plays a key role in cellular protection against oxidative stress. Several studies have shown that the GSTP1 geneis involved in the pathogenesis of asthma and a gene-gene interaction may occur within the GST gene superfamily. Methods: We screened single-nucleotide polymorphisms (SNPs) at the GSTP1 locus and performed an association study in the Japanese population using two independent case-control groups (group 1: 391 pediatric patients with asthma, 462 adult patients with asthma, and 639 controls, and group 2: 115 pediatric patients with asthma and 184 controls). The effect of GSTM1 null/present genotype on the association between GSTP1 Ile105Val and asthma was also investigated. Results: We identified 20 SNPs at this locus and found this region consisted of one linkage disequilibrium block represented by four SNPs (tag SNPs). The association between the Ile105Val polymorphism in the GSTP1 gene and childhood asthma was significant in both groups (p = 0.047 in group 1, and p = 0.021 in group 2). This association was only significant in patients with GSTM1-positive genotype in both groups (group 1: GSTM1 present p = 0.013 and GSTM1 null p = 0.925, and group 2: GSTM1 present p = 0.015 and GSTM1 null p = 0.362). Conclusions: These findings suggest that the GSTP1 gene is a childhood asthma susceptible gene, and the GSTM1 gene is a modifier gene of GSTP1 for the risk of childhood asthma in the Japanese population.

IL‐10 gene polymorphism, but not TGF‐β1 gene polymorphisms, is associated with food allergy in a Japanese population

The regulatory IL‐10 and TGF‐β1 cytokine gene polymorphisms have been associated with allergic diseases in different populations, like Caucasian, Chinese and Indians. We investigated the association between the polymorphisms IL‐10 A−1082G, C−819T, C−627A and TGF‐β1 T+869C, G+915C, C−509T and food allergy in Japanese children. One hundred and eleven children with food allergy and 115 atopic control children without food allergy were recruited. DNA samples from these subjects were genotyped by using PCR. The odds ratio of IL‐10 −1082 AA genotype was 2.5 (95% CI, 1.0–6.4) for food allergy risk when compared with atopic control subjects (p = 0.03). There were no significative differences in the frequency of TGF‐β1 gene polymorphisms between both groups. Our results indicate that IL‐10 A−1082G gene polymorphism is associated with food allergy susceptibility in atopic Japanese children.

Replication of genetic association studies in asthma and related phenotypes

In asthma genetics, the association of highly replicated susceptibility genes lacks consistency across populations. To identify genuine associations, we investigated the reproducibility of the 23 most promising asthma and asthma-related candidate genes in a moderately sized sample from the Japanese population. We compared the frequency of 33 polymorphisms in unrelated cases and controls and tested for their association with asthma, atopy and serum total IgE levels using allele frequency, codominant, dominant and recessive genotype models. On the basis of the consistency of our findings with previous meta-analyses and large replication studies, IL13, TNF, ADAM33, IL4RA and TBXA2R might represent common major asthma and asthma-related trait genes. Individual gene assessment was extended to the interactions between two polymorphisms using our original method. Interactions between TBXA2R and ADAM33, and IL4RA and C3 were suggested to increase the risk for childhood and all asthma (adult and childhood asthma combined). The confirmation of previously reported associations between gene polymorphisms and phenotypes was problematic when as few as several hundred samples per group were used. Stratification of the subjects by environmental factors or other confounding factors may be necessary to improve the sensitivity and reliability of association results.

Methyl-donor deficiency in adolescence affects memory and epigenetic status in the mouse hippocampus.

DNA methylation is one of the essential factors in the control of gene expression. Alteration of the DNA methylation pattern has been linked to various neurological, behavioral and neurocognitive dysfunctions. Recent studies have pointed out the importance of epigenetics in brain development and functions including learning and memory. Nutrients related to one‐carbon metabolism are known to play important roles in the maintenance of genomic DNA methylation. Previous studies have shown that the long‐term administration of a diet lacking essential one‐carbon nutrients such as methionine, choline and folic acid (methyl donors) caused global DNA hypermethylation in the brain. Therefore, the long‐term feeding of a methyl‐donor‐deficient diet may cause abnormal brain development including learning and memory. To confirm this hypothesis, 3‐week‐old mice were maintained on a folate‐, methionine‐ and choline‐deficient (FMCD) or control (CON) diet for 3 weeks. We found that the methyl‐donor deficiency impaired both novel object recognition and fear extinction after 3 weeks of treatment. The FMCD group showed spontaneous recovery of fear that differed from that in CON. In addition, we found decreased Gria1 gene expression and specific CpG hypermethylation of the Gria1 promoter region in the FMCD hippocampus. Our data suggest that a chronic dietary lack of methyl donors in the developmental period affects learning, memory and gene expressions in the hippocampus.

Association study of the C3 gene with adult and childhood asthma

AbstractBronchial asthma (BA) is a multifactorial disorder, the development of which is affected by both environmental and genetic factors. The complement system plays an important role in immunological response against invading microorganisms. It has been shown that complement-C3-deficient mice have reduced inflammation of asthmatic airways. Previously, we reported the association of four single nuclear proteins (SNPs) in the exons of the C3 gene with childhood and adult BA. The C3 gene, however, is a large gene, and functional SNPs associated with susceptibility to BA have not yet been identified. We analyzed 26 SNPs in the C3 gene and its promoter region to narrow down the regions showing association with childhood and adult BA. Childhood and adult atopic BA patients and healthy child and adult controls were recruited from urban cities in Japan and genotyped. In SNP analysis, an SNP (SNP24, rs11569562) located in intron 31 of the C3 gene was associated with adult BA [corrected P (Pcor) = 0.030]. In linkage disequilibrium (LD) block 4 spanning exons 24-41, the frequency of the CCC haplotype in adult BA was significantly higher than that in adult controls (Pcor = 0.038). Neither the SNP nor the haplotype showing association with adult BA demonstrated a significant association with serum total immunoglobulin E (IgE) level in BA patients and controls. Our results suggest that LD block 4 confers susceptibility to adult BA with mechanisms relevant to the effector phase of allergic inflammation.

Functional variants in the thromboxane A2 receptor gene are associated with lung function in childhood‐onset asthma

The thromboxane A2 receptor (TBXA2R) gene is associated with asthma, but no functional genetic variations are known to associate with the disease or its related phenotypes.

Association of the MMP9 gene with childhood cedar pollen sensitization and pollinosis

Matrix metalloproteinase 9 (MMP9) gene has been shown to be involved in the pathogenesis of allergic rhinitis (AR) and asthma. Previous studies suggested that single-nucleotide polymorphisms (SNPs) of the MMP9 gene conferred a risk for childhood asthma. However, whether the SNPs confer a risk for AR has not been previously investigated. The objective of this study was to investigate whether SNPs of the MMP9 gene are associated with risk of seasonal AR (pollinosis), perennial AR and allergen sensitization. A total of 670 school children were recruited in Japan and genotyped for functional polymorphism in the promoter (–1590C/T: rs3918242) and three amino-acid substitutions (R297Q: rs17576; P574R: rs2250889; R668Q: rs17577). Serum levels of total and specific IgE were determined. Disease status and other clinical characteristics of the subjects were investigated using a questionnaire. Associations between the MMP9 SNPs and both AR and serum IgE levels were evaluated. –1590C/T showed significant association with cedar pollinosis (corrected P (Pcor)=0.039). R668Q was in strong linkage disequilibrium (LD) with –1590C/T and showed significant association with cedar pollinosis (Pcor=0.023) and serum cedar pollen-specific IgE level (Pcor=0.022). A haplotype associated with –1590T and 668Q showed a significant association with cedar pollinosis, orchard grass pollinosis and cedar pollen-specific IgE (Pcor=0.0012, Pcor=0.0059 and Pcor=0.0041, respectively). R297Q and P574R were in weak LD with the rest of the SNPs and did not show significant association with disease. Compared with wild-type MMP9 protein (279R–574P–668R), a variant enzyme (279R–574P–668Q) that showed association with pollinosis had lower activity. However, lower enzyme activity was not associated with disease risk because another variant (279Q–574R–668R) showed lower enzyme activity but was not associated with pollinosis. The –1590T allele and its corresponding haplotype was associated with higher promoter activity and with pollen-specific IgE levels and pollinosis, suggesting that –1590C/T may have more impact on sensitization and disease development than R668Q. Our results suggest that the MMP9 gene confers susceptibility to cedar pollinosis in Japanese children. The MMP9 gene may be associated with pollinosis through sensitization processes.

CD14 and IL4R gene polymorphisms modify the effect of day care attendance on serum IgE levels

normal eosinophil levels, the patient’s eosinophilia worsened during late 2008 (Fig 1). A single osseous plasmacytoma was found and irradiated but progressed to multiple lytic lesions and multiple myelomas confirmed by means of bone marrow biopsy. The patient died in December 2008 as a result of this disease. HES can occur as a myeloproliferative, lymphoproliferative, or, most frequently, idiopathic variant. Some myeloproliferative patients respond to imatinib mesylate and possess a mutant gene on chromosome 4. Deletion of approximately 800 kB on chromosome 4 results in a FIP1L1/PDGFRA fusion gene and formation of a kinase potently inhibited by imatinib mesylate. In our patient the FIP1L1/PDGFRA fusion gene was not detected, and imatinib mesylate failed to control eosinophilia. In the lymphoproliferative HES variant, T-lymphocyte clones produce cytokines, especially IL-5, that stimulate eosinophil production in the bone marrow. Mepolizumab, an anti–IL-5 drug, is used to inhibit eosinophil proliferation stimulated by IL-5; however, for unknown reasons, 16% of patients do not respond to mepolizumab. In this case mepolizumab had no effect, suggesting that eosinophilia was not IL-5 dependent or that other cytokines, such as IL-3 or GM-CSF, were supporting eosinophil growth. Alternatively, the patient might have been producing so much IL-5 that the levels might have outpaced mepolizumab injections. IFN-a treatment is often effective because of a shift in the cytokine milieu from TH2, which is supportive of eosinophil growth, to a TH1type response. In this case IFN-a caused a decrease in eosinophil counts, although not to normal levels, and the patient experienced the side effects of IFN-a. Alemtuzumab is an anti-CD52 antibody that can bind to both eosinophils and T cells, potentially inhibiting either the myeloproliferative, lymphoproliferative, or idiopathic variant. CD52 is a glycosylphosphatidylinositol-anchored molecule expressed on human eosinophils, lymphocytes, macrophages, and monocytes but not on neutrophils. Alemtuzumab is approved for the treatment of B-cell chronic lymphocytic leukemia and is also used to treat small lymphocytic lymphoma and mantle cell lymphoma in conjunction with other treatments. Side effects include infusion reactions (often severe), lymphopenia, anemia, thrombocytopenia, and infections. Alemtuzumab was used successfully as a treatment for HES in 2 prior cases, 1 lymphoproliferative and 1 myeloproliferative, both of which did not respond to imatinib mesylate or IFN-a and that were not tested for the FIP1L1/ PDGFRA fusion. Alemtuzumab controlled our patient’s eosinophilia for 11⁄2 years, and the patient’s quality of life appeared improved. Our patient most likely had the idiopathic HES variant. However, the occurrence of thromboembolism and an increased B12 level point to a possible myeloproliferative HES variant. The patient had a plasmacytoma and then multiple lytic lesions and multiple myelomas, suggesting involvement of 2 cell lineages by a single mutation or possibly independent mutations. Overall, the results in our patient and the previously reported cases suggest that alemtuzumab might be an effective treatment for the myeloproliferative, idiopathic, and lymphoproliferative HES variants. Lori A. Wagner, PhD Stephen Speckart, MD Bruce Cutter, MD Gerald J. Gleich, MD From the Departments of Dermatology and Medicine, the University of Utah School of Medicine, Salt Lake City, Utah, and Montana Oncology, Missoula, Mont. E-mail: lori.wagner@hsc.utah.edu. Disclosure of potential conflict of interest: L. A. Wagner has received research support from the National Institutes of Health. G. J. Gleich has received research support from the National Institutes of Health and Dyax. The rest of the authors have declared that they have no conflict of interest.

Association of TNFRSF4 gene polymorphisms with essential hypertension

Background Essential hypertension is a complex disorder that results from the interaction of a number of susceptibility genes and environmental factors. The TNFRSF4 (tumor necrosis factor receptor superfamily, member 4) gene was one of the genes that showed altered renal expression in long-term salt loading in mice. Moreover, association of the TNFRSF4 and TNFSF4 (tumor necrosis factor (ligand) superfamily, member 4) genes with myocardial infarction was recently reported. Since essential hypertension is a well-known risk factor for myocardial infarction, we hypothesized that TNFRSF4 could be a susceptibility gene for essential hypertension. Methods We performed a case–control study of TNFRSF4 in two independent population. Results Extensive investigation of single nucleotide polymorphisms of the entire gene suggested that it resided in one linkage disequilibrium block, and four single nucleotide polymorphisms in the 5′ flanking region sufficiently represented major haplotypes. In the combined population, the frequency of the most frequent haplotype, C-C-A-A, was significantly lower (P = 8.07 × 10−5) and that of the second most frequent haplotype, C-T-G-A, was significantly higher (P = 6.07 × 10−4) in hypertensive subjects than in control subjects. This difference was observed only in female patients. The C-T-G-A haplotype showed a lower promoter activity than other haplotypes, suggesting a relationship with disease susceptibility. Conclusion Our results suggest that TNFRSF4 is a female-specific susceptible gene for essential hypertension.