杉野英彦(大阪大学) 2010年 日本リウマチ学会総会(神戸)

W-3-F-1-5 4月24日(土) 8:30〜9:30 F会場(布引+北野)

GWASから得られた関節リウマチ感受性遺伝子のDNAマイクロアレイによる発現検討

杉野英彦1,李慧敏1,安達康雄2,青木千惣子2,
西本憲弘1,2
1大阪大学大学院生命機能研究科, 2和歌山県立医科大学免疫制御学講座

【目的】Genome-Wide Assocjat・Study(GWAS)により現在,リスクの高い6個の関節リウマチ(RA)感受性遺伝子(CD244,PADI4,SLC22A2,PTPN22,CTLA4,STAT4)が報告されている。一方我々はDNAマイクロアレイを用いて、RAにおける遺伝子発現異常の網羅的解析を行った。そこでRA感受性遺伝子と,DNAマイクロアレイによる遺伝子発現異をDNAの関遅を検討する。【方法】RA患者112人の末梢血のmRNAの発現量をDNAマイクロアレイで測定した。対象群として健常成人45人の末梢血を用いた。【結果】感受性遺伝子6個中4個,CD244、PADI4、SLC22A2、CTLA4、PTPN22は有意に発現が上昇していた。CTL4は発現変動がなく,STAT4は有意に発現が低下していた。発現が上昇していた4個は,変異遺伝子を導入した細胞培養実験の結果と一致していた。

参考文献
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2888179/

Abstract
Arthritis Res Ther. 2010; 12(2): 401. Published online 2010 Mar 12
DNA microarray analysis of rheumatoid arthritis susceptibility genes identified by genome-wide association studies
Hidehiko Sugino(杉野英彦),1 Hooi-Ming Lee,1 and Norihiro Nishimoto1,2
Author information
1Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamada-Oka, Suita-City, Osaka 565-0871, Japan
2Laboratory of Immune Regulation, Wakayama Medical University, 105 Saito Bio Innovation Center, 7-7-20 Saito-Asagi, Ibaraki-city Osaka, 567-0085, Japan

In a recent interesting review, Alex Clarke and Timothy Vyse described the genetics of rheumatic disease [1]. In the past several years, genome-wide association studies (GWAS) have led to the identification of six high-risk rheumatoid arthritis (RA) susceptibility genes – namely,CD244, PADI4, SLC22A2, PTPN22, CTLA4, and STAT4 (summarized in [2]). In vitro studies using mutant alleles and cultured cells have revealed the individual upregulation of CD244,PADI4, SLC22A2, and PTPN22 [2-6]; however, studies on the expression of RA susceptibility genes in RA patients are rare. We therefore investigated the expression of the above-mentioned six RA susceptibility genes in 112 RA patients using DNA microarray analysis. This study aims to clarify whether DNA microarray analysis and GWAS produce comparable results with respect to RA susceptibility genes.

Total RNA extracted from total peripheral blood cells obtained from 112 RA patients and 45 healthy individuals was used to prepare aminoallyl RNA. As a reference, mixed RNA from 45 healthy individuals was used. The aminoallyl RNA of each individual and the reference was subjected to Cy3 and Cy5 labeling, respectively, and was hybridized with an oligonucleotide-based DNA microarray. The data obtained were analyzed by nonparametric statistical group comparison. The intensities of the noprobe spots were used as the background. The median and standard deviation of the background intensity were calculated. The genes with an intensity value that was less than the median plus 2 standard deviation of the background intensity were identified as null. The Cy3/Cy5 ratios of all spots on the DNA microarray were normalized using the global ratio median. Only gene expression data that were collected from at least 80% of samples from each group were selected for further analysis. The unpaired Mann-Whitney test was used to determine statistically significant differences in the mRNA expression levels between the RA and healthy groups. Statistical significance was set at P < 0.05. The results of our DNA microarray analysis showed that the expressions of four out of the six RA susceptibility genes were significantly higher in RA patients than in healthy individuals (1.0 × 10-16 to 2.32 × 10-5) (Table ?(Table1).1). As described above, the upregulation of these four genes (CD244, PADI4, SLC22A2, and PTPN22) has been previously confirmed in in vitro studies. We found, however, that CTLA4 expression levels were similar between the RA and control groups, whereas STAT4 expression was significantly downregulated in the RA group (1.38 × 10-8). We investigated the expression of other RA susceptibility genes – namely, TRF1/C5 [7], CD40 [8], and CCL21 [8] – and found that their expressions were similar in both groups. The genetic risk factors for RA were recently reported to differ between Caucasian and Asian (Korean) populations [9]. The samples used in our microarray analysis were derived from the same Asian (Japanese) cohort. The expression profiles for these three genes may therefore not be consistent with the profiles determined by GWAS. Candidate genes identified from rheumatoid arthritis genome-wide association studies
In this study, we revealed the correlation between five out of the six high-risk RA susceptibility genes using DNA microarray analysis. Prostate cancer susceptibility genes identified by GWAS were recently reported to be consistent with those identified by microarray analysis [10]. We therefore concluded that the combination of microarray analysis and GWAS would be a more effective approach for gene identification than the analysis of individual datasets. Moreover, the simultaneous use of both methods would allow for more accurate identification of RA candidate genes.

W-3-F-1-5 4月22日(土) 8:30〜9:20 C会場(偕楽2)

全身型JIA患者におけるDNAチップを用いた
トシリズマブの治療効果予測の可能性

西本憲弘1, 河田祐−2,李慧敏3,育木千憲子2,安連康雄1,
杉野英彦3,今川智之4,森雅亮4,寓板美奈子4,岩田疸美4,村田卓士4,三好麻里4,相原雄幸4,武井修治4,
横田俊平4

1和歌山県立医科大学医学部免疫制御学講座,2中外製薬株式会社,
3大阪大学大学院生命機能研究科,4 Japanese MRA study group for JIA

【目的】DNAチップを用いて、治療前の患者末梢血中の遺伝子発現を網羅的に解析し、
トシリズマブ(TCZ)による48週後の臨床効果ならびに血中IL6正常化を予測する。
【方法】143例のsJIA患者末梢血の治療前のDNAチップデータ(3万分子)を用い、TCZ治療によるJIA70、ステロイド減量(5mg以下)、IL-6正常化(35pg/ml以下)の達成の有無を識別する分子を決定した。【結果】JIA70、ステロイド減量、IL・6正常化の予測に有用な143、40、86分子をそれぞれ決定した。これらの分子による各項目の識別の正確度は、各々84%、84%、72%であった。【結論】治療前の遺伝子発現プロフィールから48週後の効果予測が可能である。IL-6正常化は急激な再燃なしにTCZを中止できる可能性を示唆する。

リンク先
http://rnavi.ndl.go.jp/books/2010/08/000010817107.php

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