Long QT syndrome : the identification and verification of putative KCNE2-interacting proteins

Neethling, Annika (2013-12)

Thesis (MScMedSc)-- Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Long QT syndrome (LQTS) is a cardiac repolarization disorder affecting every 1:2000-1:3000 individuals. This disease is characterized by a prolonged QT interval on the surface electrocardiogram (ECG) of patients. Symptoms of LQTS range from dizziness and syncope to more severe symptoms such as seizures and sudden cardiac death (SCD). Clinical features of LQTS are a result of the precipitations of Torsades de Pointes, which is a polymorphic form of ventricular tachycardia. A number of genetic forms of LQTS have been identified with more than 700 mutations in 12 different genes leading to disease pathogenesis. However it has been estimated that approximately 25% of patients with compelling LQTS have no mutations within the known LQT genes. This proves to be problematic since treatment regimens depend on the genetic diagnosis of affected individuals. Of the known mutated genes, KCNE2 is associated with LQT6. KCNE2 encodes the beta-subunit of potassium ion channel proteins. These proteins contain cytoplasmic C-terminal domains in which many mutations have been identified. We hypothesize that genes encoding KCNE2-interacting proteins might be identified as disease-causing or modifying genes. The present study aimed to use yeast two-hybrid (Y2H) methodology to screen a pre-transformed cardiac cDNA library in order to identify putative interactors of the C-terminal of KCNE2. Through specific selection methods the number of KCNE2 ligands was reduced from 296 to 83. These interactors were sequenced and 14 were identified as putative interacting proteins. False positive ligands were excluded based on their function and subcellular location. Ultimately three strong candidate ligands were selected for further analysis: Alpha-B crystallin (CRYAB), Filamin C (FLNC) and voltage-dependent anion-selective channel protein 1 (VDAC1). Three-dimensional (3D) co-localization and co-immunoprecipitation were used to verify these proposed interactions and succeeded in doing so. The genes encoding verified interactors will be screened in our SA panel of LQT patients, to potentially identify novel LQT causative or modifying genes. Furthermore, the interactions verified in the present study may shed some light on the mechanism of pathogenesis of LQT causative mutations in KCNE2.

AFRIKAANSE OPSOMMING: Lang QT-sindroom (LQTS) is 'n hart her-polariserende siekte wat elke 1:2000-1:3000 individue affekteer. Hierdie siekte word gekenmerk deur 'n lang QT-interval op die oppervlak elektrokardiogram (EKG) van pasiënte. Simptome van LQTS wissel van duiseligheid en floutes tot meer ernstige simptome soos stuiptrekkings of aanvalle en skielike kardiale dood (SKD). Kliniese kenmerke van LQTS is 'n gevolg van die neerslag van Torsades de Pointes; 'n polimorfiese vorm van ventrikulêre tagikardie. Verskeie genetiese vorms van LQTS is geïdentifiseer met meer as 700 mutasies in 12 verskillende gene wat lei tot siekte patogenese. Dit is ergter beraam dat ongeveer 25% van pasiënte met dwingende LQTS geen mutasies in die bekend LQT gene besit nie. Dit is problematies aangesien siekte behandeling af hang van die genetiese diagnose van geaffekteerde individue. Een van die bekende gemuteerde gene is KCNE2 wat verband hou met LQT6. KCNE2 kodeer die beta-subeenheid van kalium ioonkanaal proteïene. Hierdie proteïene bevat sitoplasmiese C-terminale waarin baie mutasies alreeds geïdentifiseer is. Ons veronderstel dat gene wat proteïene kodeer wat met KCNE2 interaksie toon, geïdentifiseer kan word as siekte veroorsaakende of wysigings gene. Die huidige studie het die gis twee-hibried metode gebruik om 'n vooraf-getransformeerde hart cDNS biblioteek te sif om vermeende protein interaksies van die C-terminaal van KCNE2 te identifiseer. Deur middel van seleksie metodes is die aantal KCNE2 ligande verminder van 296 tot 83. Die identiteit van die proteïene is bekend gemaak deur volgorderbepaling waarna 14 geïdentifiseer is as proteïene wat moontlik interaksie kan toon met KCNE2. Vals positiewe ligande is uitgesluit op grond van hul funksie en subsellulêre lokasering. Drie kandidaat ligande is gekies vir verdere analise: Alfa-B crystallin (CRYAB), Filamin C (FLNC) en spanning-afhanklike anioon-selektiewe kanaal proteïen 1 (VDAC1). Drie-dimensionele (3D) mede-lokalisering en mede-immunopresipitasie tegnieke is gebruik om hierdie voorgestelde interaksies te verifieer en het geslaag om dit te doen. Die gene wat geverifieerde proteïene kodeer, sal gekeur word in ons Suid-Afrikaanse paneel van LQT pasiënte om sodoende potensieel nuwe LQT veroorsakende of wysigings gene te identifiseer. Verder kan die geverifieer interaksies in die huidige studie lig werp op die meganisme van die ontstaan van LQT veroorsakende mutasies in KCNE2.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/85718
This item appears in the following collections: