| 摘要: | 中文摘要
CAPON是1998年首度在腦組織發現的蛋白，具有調節nNOS產生NO的重要功能。2006年4月，一項由美國約翰霍普金斯大學及德國慕尼黑大學主導的大規模人類基因體SNP association study，發表在Nature Genetics雜誌，此研究首度發現CAPON的基因變異會影響心臟再極化，反應在心電圖QT interval的變化。由於QT interval太長或太短，皆容易造成心因性猝死，因此，這項研究結果具有幾項重大意義，值得進一步探討：第一、CAPON可能是一項引起心因性猝死的嶄新危險因子；第二、CAPON本身並非離子通道蛋白，但卻能影響心臟離子通道的功能；第三、CAPON-nNOS-target protein-NO 之訊息作用途徑在心臟生理或病理狀況下，扮演重要的角色。但CAPON究竟在心臟扮演何種角色，它又如何影響心臟再極化，過去一無所知，我們從研究CAPON是否在心臟表達著手，再探討CAPON與NOS1的蛋白交互作用，並利用腺病毒作載體研究CAPON透過與NOS1的交互作用，當基因過度表達時，會抑制鈣離子通道電流，並進而縮短心肌細胞再極化的時間，經由此研究的結果可以印證人類族群CAPON基因變異和QT長短具有相關性的發現。然而，我們認為另有三大問題，急需解答：CAPON基因變異對國人心電圖QT interval的影響，當心臟處在病理狀態下CAPON扮演何種角色，及CAPON如何調控心臟電氣與收縮功能之交互作用。
針對第一個問題，我們假設CAPON基因變異確實會影響國人心電圖QT interval，這種影響可以反應在健康的個人，或當個人接受可引起QT延長的藥物治療時，帶有CAPON基因變異者可能進一步延長其QT interval，我們的策略是: 基因鑑定一群海洛因（heroin）成癮正在接受methadone治療的成年男女，初期測試兩個重要的CAPON SNP，rs10494366 與rs1415263，來看帶有基因變異者是否在服用methadone前、後影響其心電圖QT interval。關於這個假設，在精神分裂患者服用risperidone治療的族群（58人）上，我們初步發現帶有rs1415263 homozygous T alleles者，在接受risperidone治療後其心電圖QTc interval由396.7±26.7 ms延長為403.3±20.6 ms （∆6.7 ms），而帶有CC（407.7±29.8 ms vs. 397.7±17.4 ms）與CT alleles （401.0±17.1 ms vs. 394.5±18.2 ms）者在服用risperidone後QTc interval反而下降，而rs10494366這個在三大歐美族群研究皆會造成QTc延長的SNP，初步看來並不影響這個特定台灣精神分裂族群服用risperidone前、後的QTc interval。基於這個初步結果，促成我們想進一步探討藥癮患者在接受methadone治療的過程，帶有CAPON基因變異是否會造成嚴重QTc 延長，引發致命性心室不整脈而導致猝死。
另外，我們也計畫以大鼠急性或亞急性心肌梗塞的模式去探討CAPON在心肌梗塞部位與正常心肌表達是否不同，並且使用單一心肌細胞研究CAPON表達量不同時，對於心臟電氣與收縮功能之交互作用（excitation-contraction coupling）的影響。
在第一章，我們回顧心肌細胞膜上各種離子電流決定心電圖QT長短的機轉，說明研究QT的重要性，目前congenital long QT syndrome的分類，探討影響QT之大型genome-wide association study，藉由過去所知CAPON在神經系統的作用，並進而研究CAPON在心臟所扮演的角色。
在第二章中，在研究CAPON的分子機轉方面，我們利用腺病毒作載體以in vivo gene transfer的方式，將CAPON過度表達，探討CAPON的生理功能；在臨床研究CAPON變異與心電圖QT相關性方面，我們鑑定24項常見的CAPON基因變異，分析心電圖的QTc在單純methadone影響下與methadone加基因變異影響下的相關性。
在第三章中，在CAPON的分子機轉方面，我們呈現CAPON影響心臟再極化的分子機轉，另外我們也引用其他機構新近發表之心肌梗塞動物模式以探討CAPON之角色功能；在臨床證據方面，我們提出methadone或methadone加CAPON基因變異對心電圖QTc的影響的結果。
第四章，著重於討論現有CAPON分子機轉與臨床研究的結果，並和過去相關的研究作比較，以規劃進一步研究的方向，並舉出現有研究的限制。
第五章，則說明經由這些研究成果，可以讓我們從臨床證據與分子機轉全面釐清CAPON在心臟生理與病理狀況下所扮演的角色，確定CAPON基因變異對國人接受methadone治療之特定族群心臟再極化的影響，特別是，是否會造成嚴重QTc 延長，引發致命性心律不整，進而有助於防治心因性猝死。
Abstract
Congenital long or short QT syndrome caused by gene mutations is well recognized as capable of predisposing to ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in a neuronal nitric oxide synthase (NOS1) regulator, CAPON gene, have recently been associated with QT interval variations in a human whole-genome SNP association study in 2006; this association has been replicated in two different Caucasian populations in 2007. We have recently identified CAPON expression and interaction with NOS1-NO pathways to modulate cardiac repolarization in the heart, which provides the rationale for the association of CAPON gene variants and extremes of QT interval in Caucasian populations. However, there are at least three important issues remaining to be answered. Firstly, the association of CAPON gene variants and extremes of QT interval has not been documented in non-Caucasian populations; secondly, the role of CAPON in cardiac repolarization is not clear in pathological or diseased model, and lastly, CAPON-mediated modulation of excitation-contraction coupling has not been investigated.
To answer the first question, we hypothesize that CAPON gene variants affect healthy Taiwan Chinese population and the CAPON gene variants cause further QTc prolongation if the subjects are on QT-prolonging drugs. Our strategies are to genotype two important candidate SNPS, rs10494366 and rs1415263, in a heroin-addicted "healthy" Taiwan Chinese population who are undergoing methadone (a QT-prolonging drug via blocking IKr) treatment to assess the effects of gene variants on the pre- and the post-treatment QTc intervals. This approach is based on our preliminary data from a schizophrenic population (n=58). We found that when the schizophrenic patients carry the TT alleles at rs1415263, they tended to have QTc prolongation after receiving risperidone (another QT-prolonging drug via blocking IKr) treatment (396.7±26.7 ms vs. 403.3±20.6 ms), while those bearing the CC or CT alleles did not. Interestingly, rs1419263, a highly significant SNP associated with QTc prolongation in Caucasians did not affect the QT interval in this Taiwanese population both before and after risperidone treatment.
With the findings, we are motivated to investigate the influence of CAPON gene variants on the development of QTc prolongation and potential lethal ventricular tachyarrhythmias in a drug-addicted Taiwan Chinese population undergoing methadone treatment. This study might enable us to identify a novel genetic risk factor for life-threatening ventricular tachyarrhythmias occurring in this particular population while undergoing methadone treatment and lead to development of new therapeutic interventions to prevent sudden cardiac death.
In chapter 1, we stress the importance of studying QT interval and the underlying ionic mechanisms responsible for QTc prolongation. In addition to the well-known congenital long QT syndrome, Arking et al. has identified a new genetic marker, CAPON (NOS1AP), as a novel QT modifier in a recent genome-wide association study. This finding was soon replicated and confirmed by 3 other studies in different populations. As CAPON was previously unexpected to play a role in the heart, we designed an animal study to explore the molecular functions of CAPON in the heart. Additionally, we wanted to know whether the particular CAPON gene variants are also associated QTc prolongation in a Taiwanese population.
In chapter 2, we used in vivo gene transfer technique to overexpress CAPON in guinea pig heart then exploring the cellular electrophysiological remodeling after CAPON overexpression with and without pharmacological intervention. For the CAPON gene variants and QTc association study, we genotyped 24 CAPON single nucleotide polymorphisms (SNPs) using a high throughput SNP array technique in a heroin addiction population undergoing methadone treatment. We wanted to answer the effects of CAPON gene variants on the QTc with and without methadone.
In chapter 3, we found the expression of CAPON protein in the heart and when CAPON is overexpressed, it interacts with NOS1-NO pathway to affect the ICa,L and IKr currents causing acceleration of cardiac repolarization (shortening of action potential duration). The electrophysiological remodeling of CAPON could be reversed by L-NAME, attesting the mechanistic link of CAPON to NOS1-NO pathway. In clinical part, we found low maintenance dose of methadone is associated with QTc prolongation and methadone-associated QTc prolongation appears to be gender-dependent. In this study, men appear to be more susceptible than women to methadone associated QTc prolongation. For the CAPON SNP association study, we found rs10918594 C>G, CAPON variant but not rs10494366 T>G is associated QTc prolongation in this Taiwanese population and use of methadone in patients with rs10918594 homozygous variant might potentiate the QTc prolongation.
In chapter 4, we discussed the molecular mechanisms by which CAPON modulated cardiac repolarization and pointed out several potential limitations of the basic study. Indeed, future studies, particularly using a CAPON knock-out model are necessary to further elucidate CAPON function in the heart. For the clinical study, it seems that rs10918594 C>G is also associated QTc prolongation in this Taiwanese population, yet it needs to be confirmed in a larger population study and the underlying mechanisms of gender-dependent susceptibility of methadone associated QTc prolongation needs to be explored.
In chapter 5, we conclude that CAPON is a new QT modifier in the heart which exists not only in the western population but likely also in Aisan population. Therefore, people carrying the genetic variants might be at a greater risk of developing serious ventricular tachyarrhythmias and sudden death when superimposed with additional long QT conditions, such as drugs and/or electrolytes imbalance. |
