川崎病病童的冠狀動脈病變 -由高危險基因到動脈病變之重塑與逆轉

Abstract

川崎病(Kawasaki disease, KD)是一種原因還不太清楚的血管炎，好發於年紀較小的兒童，它有以下五個特徵：發燒持續五天以上、兩側非化膿性結膜炎、口腔黏膜發紅、四肢肢端皮膚脫皮、紅疹及頸部淋巴結。川崎病之所以愈來愈受到重視，有下列幾個原因：(1)隨著公共衛生的進步，川崎病已取代以往的風溼熱及風溼性心臟病成為兒童後天性心臟病的頭號要犯;(2)川崎病的血管炎，特別喜歡攻擊中型血管，尤其是冠狀動脈，由於冠狀動脈是心臟收縮時能量和氧氣供應的重要通道，若未得到適時而正確的治療，會有像老人家的心肌梗塞甚至猝死的風險;(3)此病在台灣的發生率高居世界第三，僅次於日本與韓國，根據健保局的資料，每一年在台灣新增的病例約有800人左右，雖然沒有感冒那麼常見，但也不算太少。 在過去這三十幾年來，人們雖然很努力的在找尋川崎病的致病機轉，但很遺憾的，截至目前為止仍然沒有突破性的進展；一般相信川崎病是擁有某些特定基因型體質的病童受到特定或一般病原體感染之後，進而造成宿主的免疫系統過度反應及失調，導致血管炎性反應，進而引起此病。 從過去的文獻我們擷取出1996到2002年之間，國民健康保險的資料庫中川崎病的流行病學資料，將其中未滿18 歲住院且符合川崎病診斷標準 (ICD: 446.1) 的病人列入統計，同時將這些資料轉型成累積分佈曲線(cumulative distribution curve)；我們應用Kuiper’s test 來分析這條曲線並研究其是否有明顯的季節性分布。結果發現在研究期間，每一年的季節集中性均大於百分之九十九，意即有明顯的季節趨勢；這也支持了過去的理論，川崎病可能是由某些病原體所誘發的；有趣的現象是在台灣川崎病的高峰似乎在4-6月間較溫暖的春夏交界之際，然而在日本卻總是在最寒冷的冬天，同在東亞的兩個地域上的孩子，為何有著截然不同的發病型態，是不同的遺傳背景抑或是與環境中的微生物不同的互動模式所導致，目前仍然不明白。接下來我們就要跨進分子生物學的領域，從這個角度切入川崎病的致病機轉。 從流行病學調查結果知道川崎病在日本有全世界最高的發生率，這種情形於日本裔的移民身上也可以發現；更有其他的family based的證據發現一個家庭裏若有一個孩子得了川崎病，他(她)的兄弟姐妹未來發作川崎病的概率是同年齡孩子風險的10-20倍，而且小時候得過川崎病的病患其兒女再得川崎病的機率也較一般民眾高出兩倍之多；這些都暗示某些基因在川崎病的致病機轉上扮演著重要的角色。所以許多的基因相關性研究(genetic association study)已被廣泛的運用在搜尋像川崎病這樣的複雜性多基因疾病(complex trait disease)的致病基因(susceptible genes)。我們經過了文獻的搜尋和考慮，選擇了Dr. Onouchi在2008年所發表的川崎病風險基因ITPKC作為我們這個研究切入的開始。 我們一方面選定了這個ITPKC基因上的rs28493229作為研究的目標，另一方面也思考這個SNP如果能影響T淋巴球的活化，造成免疫系統的過度反應，那它是否與川崎病的某種臨床表現有關，這個部分的相關性是沒有文獻提過的，特別是冠狀動脈的影響或是卡介苗接種部位的紅腫與這個SNP的相關性如何，我們將一併來探討。這個部分的研究總共收錄了280位典型川崎病的病患，其中男性佔了61.8％，年齡的中位數是21.5個月大；其中有162位(佔57.9％)曾患有冠狀動脈病變 (coronary lesions)；這162位曾有冠狀動脈影響的病人包含了51位中型或巨大冠狀動脈瘤(coronary aneurysm)的病人，以及111位曾有冠狀動脈輕度擴張或是小型動脈瘤者；在rs28493229這個SNP上共有三種基因型分別是GG、GC和CC，這三種基因型在我們研究的280位川崎病人的分布是GG:GC:CC是236:43:1，對照組的分布則是454:37:1，若以對偶基因頻率(Allele frequency)的角度來看，C allele的出現頻率在病人組及對照組分別為8.04％及3.96％，兩組間的差異，不論是基因型頻率或是對偶基因頻率，在統計上都非常顯著(勝算比分別為2.23及2.12，p值均小於0.001)。在這些病人的臨床表現，我們依照rs28493229上基因型的不同分成兩組(GG組及GC＋CC組)，觀察他們在臨床表現(六大診斷基準，diagnostic criteria)及卡介苗接種部位是否紅腫及膿尿(pyuria)的比例是否在兩組間有所不同，結果發現GC＋CC這一組(也就是carrier of C allele)的病患相較於GG組有較高比例有卡介苗接種部位紅腫的現象(19/44 vs. 66/236; OR: 1.96，p＝0.044)；我們也進一步的分析了這個研究中病患年齡小於20個月的150位病人，結果發現帶有C allele的幼童(小於20個月)發病時卡介苗紅腫的比例高達71.4％(15/21)，遠高於相同年齡群未帶有C allele(即GG基因型)的病童(56/129, 43.4％)，兩者之間的差異更大，更顯著(勝算比3.26，95％，信賴區間：1.19∼8.94，p＝0.017)。 我們同時也將病人依照冠狀動脈是否受到影響分成兩組，各有162及118人，利用χ2 test檢驗C allele的有無是否與冠狀動脈病變的產生有關，結果發現沒有顯著相關(勝算比：0.85，95％信賴區間：0.45∼1.63)，接著再依據冠狀動脈病變的嚴重度，區分病人為無影響、輕度影響及重度影響三類，利用3×2 χ2 test(卡方)分析，結果依然發現這個ITPKC基因的rs28493229多型性變化與冠狀動脈病變的嚴重度無關。 在這個部份的研究中，我們首先發現基因ITPKC的多型性(SNP)與川崎病急性期的表現之一：卡介苗接種處紅腫有關，這是從來沒有人注意到或報告過的，同時我們也証實了，即使基因頻率遠較日本為低(8.04％相對於日本22.6％)，這個遺傳變異(genetic variant)在台灣地區確實與川崎病的發生有關，也因為這個C allele與卡介苗紅腫相關，似乎也暗示了這個ITPKC的多型性，是經由過度激發的免疫功能，進而促進川崎病的發生。 最後一個部份則是關於川崎病冠狀動脈病變重塑的探討；已經有報告顯示川崎病病人即使在病程晚期，血管周圍及心肌的纖維化及細胞間質的重組仍持續活躍的進行，所以我們猜測纖維化的相關指標可能可以作為偵測相關心臟事件的指標。 在這部份的研究中，我們收錄了35位青少年及年輕人[25位為川崎病-無冠狀動脈病變(KD-N(on)CAL)組，10位為川崎病-冠狀動脈持續病變(KD-P(ersistent)CAL)組] 和25位類似年齡(age-matched)的控制組參與這個研究。冠狀動脈持續病變的病人均接受過電腦斷層檢查(佔8/10或80％個案數)或血管攝影(佔7/10或70％個案數)來確認冠狀動脈損傷狀態，並藉由影像學來定義和定量冠狀動脈損傷情形，包括冠狀動脈瘤的數目、冠狀動脈的鈣化負擔(calcium burden)、在冠狀動脈瘤中是否有血栓形成以及冠狀動脈的狹窄(stenosis)程度。 基本的實驗室測量包括血清中的膽固醇、高密度脂蛋白、低密度脂蛋白、三酸甘油脂、肝臟酵素[(包括天門冬胺酸轉胺酶(AST)及丙胺酸轉胺酶(ALT) ]以及高感度的C反應蛋白等，至於纖維化的指標則以血漿中的amino-terminal propeptide of type III procollagen (PIIINP)、matrix metalloproteinase-9 (MMP-9)及tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)來代表。我們分析了這些標記在三組病人間的分佈有無差異，也評估所選定的纖維化指標是否與影像學上定量的指標呈現某種有意義的相關。結果發現KD-PCAL組的PIIINP的濃度明顯地比從來沒有冠狀動脈問題的病患(KD-NCAL)組來得高；更有趣的是這兩群病人PIIINP值均比同年齡的對照組要來的高；MMP-9、TIMP-1的濃度以及MMP-9/TIMP-1的比值則低於對照組；與電腦斷層上各個冠狀動脈異常分數相關的生物指標中以PIIINP的相關性最好，如果進一步分析可以發現PIIINP與冠狀動脈狹窄分數(stenosis score)和血栓形成分數(thrombus score)的相關性最好(r值分別是0.72與0.64，p＝0.01)；運用趨勢分析(trend test)調整(adjust)了年紀、性別、身體質量指數(BMI)、血壓及HDL-cholesterol的濃度等可能的干擾因子(confounding factor)之後，P IIINP的濃度仍然與電腦斷層上冠狀動脈病變嚴重度分數呈現有意義的相關，特別是狹窄分數(coronary stenosis score，p＝0.03)與血栓分數(coronary thrombus score，p＝0.05)。 根據文獻，這篇研究應該是第一篇研究川崎病在發病十餘年後細胞間介質(extracellular matrix)相關的生物指標(biomarker)變化的文章，我們有以下叁個重要的發現：(1) 膠原蛋白(collagen)合成的指標之一，PIIINP，在有川崎病病史的青少年或年輕人是高於一般的健康人，特別是在有持續性冠狀動脈病變的病人，(2)更重要的是這個PIIINP的濃度高低是與病人定量化的冠狀動脈病變指標(包括狹窄分數與血栓分數)呈現正相關；而且(3) 看似沒有冠狀動脈病變的病人，這個膠原蛋白合成的指標PIIINP還是處於不正常的狀態。整體而言，我們可以推論在川崎病發病的晚期(十數年之後) 膠原蛋白的合成(turnover)仍處於不正常的狀態，而且可能與冠狀動脈病變的嚴重度、細微血管病變或是心肌的纖維化都有關係。 總結來說，由這三個部份的研究，我們證實了川崎病的確有明顯的季節性分布；再由遺傳的角度切入，肯定了與ITPKC 基因相關的T淋巴球活化是川崎病發生的重要步驟；最後應用間質重塑的指標監測，我們發現在川崎病的晚期，纖維化的相關指標仍然不正常，這個新發現不但呼應了過去的病理報告，也為日後川崎病的追蹤與治療開啟了一個新的研究方向。

Kawasaki disease (KD) is an acute systemic vasculitis that occurs predominantly in infants and young children. KD is characterized by fever, bilateral nonexudative conjunctivitis, erythema of the lips and oral mucosa, changes in the extremities, rash and cervical lymphadenopathy. Coronary artery aneurysms or ectasia develop in 5% to 10% of affected children even after the recommended intravenous immunoglobulin therapy. With an ever increasing incidence almost worldwide, KD is now the leading cause of acquired heart disease in children. The annual incidence of KD in Taiwan has increased steadily since 1976 and is now 66-69 patients per 100,000 children <5 years old, the third highest rate in the world. The etiology and pathogenesis of KD are still uncertain. It is generally agreed that KD is related to ubiquitous infectious agents that cause the disease in genetically predisposed individuals. In the present doctoral thesis, we first sought to examine the seasonality of KD in Taiwan by using the epidemiologic data of KD in Taiwan between 1996 and 2002. Basically, the database came from the National Health Insurance data and hospitalized patients who were under 18 years old and met criteria listed for KD (ICD-9-CM code: 446.1) were selected. The data were transformed and analyzed in the form of cumulative distribution curve. Kuiper’s test was applied to test the seasonality, and we found the results of Kuiper’s test for seasonality in each year between 1996 and 2002 showed statistical significance for all years examined, (p <0.01) and ascertained the presence of seasonality for KD. This will support the theory that certain infectious agents may cause KD. The most interesting is the peak rmonth of patient numbers is April to June in Taiwan, rather than January (in Japan). The reasons for such differences between Japan and Taiwan remain unclear. But, the possibility that different interaction between infectious agents and genetic predisposing factors may be elucidated in the future studies. The highest incidences of KD are found in three countries in Japan. The risk of KD in siblings of affected children is ten times higher as compared to the general population, while it is twice as high in children born to parents with a history of KD compared to the general population. Echoing these epidemiological data, previous studies have demonstrated the associations of genetic markers with a susceptibility to KD and/or increased risk of developing CAL in KD patients. Of the studied genetic markers, we picked up the functional single nucleotide polymorphism (SNP) rs28493229 of ITPKC (inositol 1,4,5-trisphosphate 3-kinase C) gene as the candidate and studied its association with susceptability to KD. Furthermore, if the C allele of ITPKC may lead to immune hyper-reactivity and susceptibility to KD, its presence may be also associated with different clinical manifestations (ex: erythema at BCG scar) during the acute phase of KD. Based on a Taiwanese KD cohort with complete medical data, we sought to define the role of the C allele of ITPKC rs28493229 polymorphism played in the disease susceptibility, clinical manifestation and the development and severity of CAL in KD patients. We enrolled 280 unrelated Taiwanese children with KD and 492 healthy ethnically and gender-matched controls. Genotyping for the ITPKC polymorphism was conducted and the clinical manifestations and laboratory data were systemically collected. We found that the GC and CC genotypes of ITPKC gene SNP rs28493229 were over-represented in KD patients (GG:GC:CC was 236:43:1, C allele frequency: 8.04%) than in the controls (GG:GC:CC was 454:37:1, C allele frequency: 3.96%; OR:2.23, p=0.001). In KD patients, patients with GC or CC genotypes of SNP rs28493229 (19/44) were more likely to have reactivation at the Bacille Calmette-Gu&eacute;rin (BCG) inoculation site than those with GG genotypes (66/236; OR= 1.96, p=0.044). Such association was particularly strong in patients aged < 20 months (OR= 3.26, p=0.017). The other clinical manifestations were not related to this SNP. In this cohort, there were 160 (57.1%) patients with coronary arterial lesions (CAL). The development and the severity of CAL was not associated with this SNP either. Comparison between patients with and without BCG reactivation revealed only one difference: patients with reactivation were younger. Therefore, we concluded, in a cohort from a population with the world’s third highest incidence of KD, the C-allele of ITPKC SNP rs28493229 is associated with KD susceptibility and BCG scar reactivation during the acute phase, though its frequency is lower than that in Japanese cohort (22.6%), suggesting this SNP contributes to KD susceptibility via induced hyperimmune function reflected in the BCG reactivation. The final part of the doctoral thesis is regarding to the research on matrix remodeling of coronary lesions in KD patients. Pathological data from the hearts of KD patients who suffered from sudden death showed thickening of coronary arteries and arterioles as well as perivascular and myocardial fibrosis. Alterations in the build-up and breakdown of arterial extracellular matrix are key features in vascular remodeling. Therefore, serum biomarkers of fibrosis may be sensitive indices for late cardiac complications of KD. We studied a cohort of 60 adolescents and young adults, which comprised 10 KD patients with persistent coronary artery lesions (CAL) 14.5 ± 4.4 years since onset, 25 KD patients with no CAL since onset and 25 healthy age-matched volunteers. The lipid profile, liver function, amino-terminal propeptide of type III procollagen (PIIINP), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1) and the MMP-9/TIMP-1 ratios were compared. Severity of CAL was based on computer tomography determinations of the frequency of aneurysms, the extent of coronary stenosis/occlusion, thrombosis and calcification. We found elevated PIIINP, and decreased MMP-9, TIMP-1 and MMP-9/TIMP-1 ratios not only in KD patients with persistent CAL but also in those without, although to a lesser extent in the latter group. In KD patients, the concentrations of PIIINP were positively associated with the severity of coronary stenosis/occlusion (r=0.72, p= 0.011) and with the extent of coronary thrombus (r=0.64, p=0.014). The concentrations of high sensitivity C reactive protein, however, were not found different across groups. This is the first study of profiling for biomarkers of the extracellular matrix in KD adult patients more than ten years after disease onset. We report the following novel findings: (1) the index of collagen synthesis, PIIINP, was elevated and the concentrations of MMP-9, TIMP and the MMP-9/TIMP ratio were diminished in KD adolescents and young adults, especially in patients with persistent CAL. (2) In patients with KD, the concentrations of PIIINP were closely associated with quantifiable characteristics of severity of CAL (coronary stenosis/occlusion and coronary thrombosis). (3) In patients without gross coronary complications, the collagen turnover was still abnormal. Together these findings suggest that late after the onset of KD, the collagen turnover is still far from normal and may be related to the severity of CAL, microangiopathy or myocardial fibrosis. In conclusion, the present doctoral thesis first confirmed the seasonality of KD in Taiwan. With the genetic analysis, we then demonstrated the ITPKC-realted T cell activation were associated with the development of KD and BCG scar reactivation. Finally, in adolescents and young adults late after the onset of KD, we novelly found alterations in biomarkers of extracellular matrix and turnover of collagen, not only echoing to the previous pathology reports, but also opening a new direction for the future monitoring and treatment of Kawasaki disease.