台灣地區遺傳性心因性猝死相關離子孔道基因突變的功能性研究

Abstract

遺傳性心因猝死症候群常造成非預期性的猝死，此疾病亦為年輕人死亡之重要原因之一。然而由於對其病理機轉的認知不足，目前可用的治療手段相當有限。在這一個世紀以來，多個基因已被發現與心律不整的致病機轉相關。本研究中，我們對台灣的QT過長症候群（Long QT syndrome, LQTS）以及Brugada syndrome患者進行基因分析後，在LQTS患者發現三個位於KCNH2的突變而在Brugada syndrome患者中找到三個位於SCN5A的基因突變，我們因而進一步研究其功能性變化以了解這些突變以及心律不整之間的關係。 KCNH2(或是hERG)的產物為一鉀離子通道（the rapid activating delayed rectifier potassium channel, IKr)。此鉀離子通道在心臟動作電位的後期扮演重要的角色，而我們所發現的三個KCNH2突變都會導致IKr減少，因而使動作電位延長以及心電圖上QT間距拉大。這三個KCNH2突變分別是p.N633D，p.R744fs， 以及 p.P923fs。 當將其表現在HEK293T細胞上時，p.N633D以及p.R744fs不會產生電流。p.P923fs所生成的電流密度則是比正常hERG離子通道有著顯著減少，而且p.P923fs的inactivation也較正常hERG離子通道來的快。在西方墨點法的分析中，我們發現p.R744fs無法被glycosylation，而這指出p.R744fs可能有著異常的細胞運送特性，而不會被細胞送上細胞膜。在共軛焦顯微鏡上我們也是看到同樣的現象。進一步地，我們發現當在p.R744fs的C端接上GFP時，glycosylation將可順利進行而如果GFP接在p.R744fs的N端時，glycosylation則不會進行。此外，藉由共同免疫沈澱法我們也發現p.R744fs無法有效地組合成四聚體。 SCN5A的產物是心臟的鈉離子孔道，它是目前已知造成Brugada syndrome的基因之一。至今已有超過百個SCN5A突變被報導 ，而一般認為Brugada syndrome的致病機轉便是來自於心臟鈉離子通道的功能異常。我們在台灣Brugada syndrome患者中找到三個SCN5A突變，分別是p.I848fs,p.R965C,以及 p.1876insM。其中p.I848fs產生的離子通道沒有功能，而p.R965C以及p.1876insM所產生的鈉離子通道具有與正常心臟鈉離子通道不同的電生理特性。p.R965C以及p.1876insM自inactivation回復的能力都比正常心臟鈉離子通道來的差，而且其steady state inactivation都比正常心臟鈉離子通道更偏向負電壓的方向（分別是9.4mV及 8.5mV）。此外p.1876insM的steady state activation也有改變。其比正常心臟鈉離子通道更偏正電壓的方向(7.69mV)。 隨著我們對於心律不整的治病基因的熟悉以及對致病機轉逐漸了解，將來我們將可以更容易也更加有效地診斷、治療甚至是預防心律不整。

Hereditary sudden cardiac death syndromes are major causes of unexpected death especially in young individuals. However, current treatment modalities for these syndromes are not satisfactory, presumably due to poor understanding of the underlying mechanisms that lead to the pathogenesis of these diseases. For the past decades, several responsible genes for these syndromes have been identified and studied. In our studies, we screened patients with LQTS or Brugada syndrome in Taiwan and identified three mutations in KCNH2 responsible for LQTS and three mutations in SCN5A responsible for Brugada syndrome. Functional studies were performed to elucidate the possible mechanisms of the disease-causing mutations. KCNH2, or hERG, encodes the pore forming subunit of the rapid activating delayed rectifier potassium channel (IKr), which plays important roles in the repolarization process during the late phase of cardiac action potential. The three LQTS-related mutations of the KCNH2 genes all lead to a reduced IKr and might be related to the prolongation of action potential duration and the prolongation of QT interval in ECG. These mutations are p.N633D, p.R744fs, and p.P923fs. When expressed in HEK293T cells, p.N633D and p.R744fs channels displayed no current while p.P923fs channel elicited current with significantly lower current density and faster inactivation kinetics. In western blotting analysis, pR744fs was the only one with glycosylation defect. In confocal microscopic studies, p.R744fs-GFP also revealed trafficking defect. However, p.R744fs-GFP differed from pR744fs in being fully glycosylated while p.R744fs fusion with GFP at the N-terminus revealed glycosylation defect. In co-immunoprecipitation studies, the assembling capacity of p.N633D, and p.P923fs were intact p.R744fs failed to interact with neither WT nor itself to form tetramers. SCN5A is the most well known responsible gene that causes Brugada syndrome. Until now, more than a hundred mutations in SCN5A responsible for Brugada syndrome have been described. Functional studies of some of the mutations have been performed and showed that a reduction of human cardiac sodium current accounts for the pathogenesis of Brugada syndrome. Here we reported three novel SCN5A mutations identified in patients with Brugada syndrome in Taiwan (p.I848fs, p.R965C, and p.1876insM). Their electrophysiological properties were altered in patch clamp analysis. The p.I848fs mutant generated no sodium current. The p.R965C and p.1876insM mutants produced channels with steady state inactivation shifted to a more negative potential (9.4mV and 8.5mV respectively), and slower recovery from inactivation. Besides, the steady state activation of p.1876insM was altered and was shifted to a more positive potential (7.69mV). With increasing understanding of the underlying mechanisms of hereditary sudden cardiac death, we expect advances in the diagnosis, treatment, and prevention of these syndromes.