川崎氏症致病機轉之研究

Abstract

川崎氏病是一種急性全身性血管炎，主要發生於五歲以下幼童，尤其易侵犯心臟冠狀動脈，並引起血管瘤的形成。研究指出，未治療病患的冠狀動脈瘤發率約為所有病患的百分之二十五；因此，目前在文明國家中，川崎氏症已成為兒童後天性心臟病的主要原因。臨床與流行病學證據顯示，病原菌感染導致宿主發生不當免疫反應是誘發川崎氏症的決定性因子，宿主本身與免疫相關的遺傳標記亦參與疾病的發生。然而，與免疫相關的遺傳標記如何參與川崎氏症的發生，以及造成冠狀動脈病變的致病機轉，目前都尚未非常明瞭，因此本論文將針對此兩項課題做進一步的探討。 本論文第一部分將針對免疫相關遺傳標記如何參與川崎氏症的發生做深入的研究。BLK基因在過去台灣與日本的全基因組關聯分析(GWAS)中，已被證實其與川崎氏症的併發有高度關性。因此，本論文的第一部分研究將藉由整合不同族群的綜合性分析來更近一步證明BLK基因與川崎氏症病人之易感性有高度相關性。經由統計研究發現，位於BLK基因啟動子區域的單一核苷酸多型性位點rs2736340與台灣、日本、韓國、.歐洲的川崎氏症病人之易感性有明顯相關性；進一步利用聯鎖不平衡(linkage disequilibrium)分析顯示，與單一核苷酸多型性位點rs2736340相關連的區域︰包含啟動子與基因內區(intron)，皆與川崎氏症有顯著關聯性。此外，我們在此疾病相關的風險性基因位點rs2736340與急性期川崎氏症病人周邊血液B淋巴細胞的BLK基因表現量的相關聯性研究中發現，在川崎氏症病人的分離初級B淋巴球以及由人類皰疹病毒所轉染製造而成的B淋巴球細胞株中，帶有風險性基因位點rs2736340會明顯減少BLK基因以及蛋白質的表現量。由此基因關聯性研究顯示，B淋巴細胞在急性期川崎氏症的致病機轉中扮演重要角色，且其BLK基因的低表現將改變其正常性功能並造成個體對於川崎氏症的易感染性。因此，藉由瞭解基因多型性與B淋巴細胞功能性之間的連結將有助於瞭解川崎氏症的致病機轉，並發展新型川崎氏症診斷及治療方法。 本論文第二部分為找尋造成川崎氏症病患冠狀動脈病變的可能致病因子。我利用相對和絕對定量同位素標記(iTRAQ)蛋白質定量質譜分析技術，比較川崎氏症病人及非川崎氏症發燒病人血漿的蛋白質圖譜的差異性，藉此找尋導致冠狀動脈病變的致病因子。研究結果發現有505個表現差異性蛋白在川崎氏症病人血漿中表現，並且進一步利用系統生物學分析軟體暨生物資訊資料庫(Ingenuity Pathway Analysis)分析出141個蛋白與心血管疾病具有相關性。其中過去被認為與冠狀動脈疾病高度相關的類幾丁質酶蛋白CHI3L1亦被發現在川崎氏症病人血漿中高度表現。我們進一步分析此蛋白在33個川崎氏症病人及41個非川崎氏症發燒病人血漿中的含量，結果顯示CHI3L1蛋白在急性期及亞急性期川崎氏症病人血漿中有顯著性的增高。此外，分析曾經產生冠狀動脈瘤的川崎氏症病人在恢復期的CHI3L1蛋白表現量發現，其CHI3L1蛋白含量相對於同時期但未曾產生冠狀動脈瘤的川崎氏症病人之含量具有顯著性的增加。動物實驗中發現，在野生(wild-type)小鼠腹腔連續注射CHI3L1合成蛋白後28天，會造成小鼠的冠狀動脈有明顯增厚現象發生。經由定量即時聚合酶鏈鎖反應(Real-time PCR)實驗發現，CHI3L1合成蛋白腹腔注射後的小鼠，其病變的冠狀動脈有增強表現肌成纖維細胞蛋白及基質金屬蛋白酶現象。本論文的第二部分研究推論，CHI3L1蛋白在川崎氏症病人中高度表現將是造成冠狀動脈病變的重要因子，並可作為未來治療川崎氏症冠狀動脈病變的新藥摽的。

Kawasaki disease (KD) is an acute, self-limited vasculitis predominantly affecting children under the age of 5 years. Coronary artery aneurysms develop in 25% of untreated patients, making KD the leading cause of acquired heart disease in children in the developed countries. The clinical and epidemiological observations suggest that host immune dysregulation triggered by infectious agent(s) is the critical component for disease initiation, and genetic determinants in immune-related loci have also been identified to contribute to disease susceptibility. To date, the role of immune-associated genetic factors in disease progression and the pathological mechanism of coronary artery lesion in KD remain unclear; and my thesis focused on these two aspects. My first study focused on the further elucidation of the immune-associated genetic factors for KD. The BLK locus has been reported to be associated with Kawasaki disease in two genome-wide association studies (GWAS) conducted in a Taiwanese population of Han Chinese ancestry (Taiwanese) and in Japanese cohorts. In the first study, we build on these findings with replication studies of the BLK locus in populations of Korean and European descent. The BLK region was significantly associated with KD susceptibility in both populations. Within the BLK gene the rs2736340-located linkage disequilibrium (LD) comprising the promoter and first intron was strongly associated with KD, with the combined results of Asian studies including Taiwanese, Japanese, and Korean populations (2,539 KD patients and 7,021 controls) providing very compelling evidence of association (rs2736340, OR = 1.498, 1.354–1.657; P = 4.74×10-31). We determined the percentage of B cells present in the peripheral blood mononuclear cell (PBMC) population and the expression of BLK in the peripheral blood leukocytes (leukocytes) of KD patients during the acute and convalescent stages. The percentage of B cells in the PBMC population and the expression of BLK in leukocytes were induced in patients in the acute stage of KD. In B cell lines derived from KD patients, and in purified B cells from KD patients obtained during the acute stage, those with the risk allele of rs2736340 expressed significantly lower levels of BLK. These results suggest that peripheral B cells play a pathogenic role during the acute stage of KD. Decreased BLK expression caused by allele-restriction in peripheral blood B cells may alter B cell function and predispose individuals to KD. These associative data suggest that B cells homeostasis regulated by KD-associated SNPs may play a critical role in KD pathogenesis. Understanding the functional implications may facilitate the development of B cell-mediated therapy for KD. The second study of my thesis focused on the identification of potential biomarkers that might mediate the pathological change in the coronary arteries of KD. I globally analyzed the protein profiles of plasma samples obtained from KD patients in the acute phase and non-KD fever control subjects using isobaric tags for the relative and absolute quantitation (iTRAQ) labeling tandem mass spec¬trometry. Overall, 505 proteins were identified as differentially expressed proteins in KD, and 141 proteins were classified into the category of cardiovascular diseases by Ingenuity Pathway Analysis annotation. One of the notable upregulated proteins in the pathway was chitinase-3-like protein 1 (CHI3L1). Significant elevations of CHI3L1 were confirmed in a validation cohort of 33 KD patients during the acute and subacute phases as compared to non-KD fever controls (acute KD, 58.9 ± 22.4 ng/mL; subacute KD, 42.7 ± 23.1 ng/mL; control, 29.1 ± 15.5 ng/mL). In addition, convalescent KD subjects with coronary artery aneurysm had significantly higher CHI3L1 levels compared to KD subjects with normal coronary arteries. Notably, injection of recombinant CHI3L1 into wild-type mice caused intimal cell transition leading to coronary wall thickening. Coronary lesions in mice treated with CHI3L1 were enhanced by myofibroblast generation and matrix metalloproteinase activation. In the second study, these results suggest that CHI3L1 is crucial for the development of coronary artery abnormality in KD and potentially represents a novel pharmacological target for treating KD coronary lesions.