Detta, Nicola (2010) Molecular basis of cardiac arrhythmias: genetics of natural variants and electrophysiological investigation of mutant proteins. [Tesi di dottorato] (Unpublished)


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Item Type: Tesi di dottorato
Lingua: English
Title: Molecular basis of cardiac arrhythmias: genetics of natural variants and electrophysiological investigation of mutant proteins
Date: 30 November 2010
Number of Pages: 75
Institution: Università degli Studi di Napoli Federico II
Department: Biologia e patologia cellullare e molecolare "L. Califano"
Scuola di dottorato: Medicina molecolare
Dottorato: Genetica e medicina molecolare
Ciclo di dottorato: 23
Coordinatore del Corso di dottorato:
Date: 30 November 2010
Number of Pages: 75
Uncontrolled Keywords: Cardiomyopathy; Long QT syndrome; Brugada syndrome; arrhythmias; genetics; electrophysiology; ion channels; molecular biology; mutant proteins
Settori scientifico-disciplinari del MIUR: Area 06 - Scienze mediche > MED/03 - Genetica medica
Area 05 - Scienze biologiche > BIO/18 - Genetica
Additional Information: Tesi svolta presso il Dipaertimento di Biochimica e Biotecnologie Mediche, Napoli e presso il CEINGE-Biotecnologie avanzate, Napoli.
Date Deposited: 13 Dec 2010 22:33
Last Modified: 30 Apr 2014 19:44
DOI: 10.6092/UNINA/FEDOA/7981


Channelopathies are diseases caused by deranged functioning of ion channel subunits or the proteins that regulate them. Long QT and Brugada syndrome are included in this group. In particular, long QT syndrome (LQTS) is a familial autosomal dominant disease characterized by prolongation of the QT interval on the surface ECG, syncope, torsade de pointes and sudden cardiac death in young patients. Each type of heritable LQTS (LQTS 1-12) is linked to mutations in a specific gene. Mutations occur more frequently in the cardiac ion channel coding genes (SCN5A, KCNH2,KCNQ1) and ancillary β-subunits (KCNE1 and KCNE2). Differently, BrS is an inherited cardiac disease characterized by ST segment elevation in the right precordial leads (V1 to V3), susceptibility to ventricular tachyarrhythmia and sudden cardiac death, typically during rest or sleep. BrS is inherited as an autosomal dominant trait and its prevalence in Caucasians is 5/1000. The disorder is linked to mutations in the SCN5A gene. Our project was designed to functionally characterize the novel mutations found in genes related to LQTS and BrS to better understand the pathogenesis of pathological phenotypes . To this aim, we firs amplified by PCR all coding exons, 5’ and 3’ UTR of the SCN5A, KCNQ1, KCNH2, KCNE1 and KCNE2 genes and analyzed them by dHPLC and automatic sequencing. The mutants were generated by QuickChange site-directed mutagenesis. Mutants were transiently transfected in mammalian cells for in vitro analysis. We characterized the LQT3 associated p.ΔN1472 mutation that we found in SCN5A gene. The electrophysiological studies demonstrated that the hH1 mutation had a shift in the voltage-dependence of inactivation, a positive shift in the voltage dependence of activation and a slower recovery from inactivation compared to WT channel. Moreover, the persistent current levels were much higher in SCN5A-p.ΔN1472 than in the WT channel. We also studied mutations KCNH2-p.C108Y and KCNQ1-p.R583H. Interestingly, only subjects carrying both mutations manifested severe LQTS. The biophysical studies showed that in the homozygous condition, KCNH2-p.C108Y, led to a non-functional KCNH2 channel, whereas, in the heterozygous condition, mutant KCNH2 had a significantly reduced current density and a negative shift in the voltage dependence of activation compared to the WT. Furthermore, mutant KCNQ1-p.R583H had a significantly reduced tail current density compared to the WT channel, but no significant changes in activating current density and in voltage-dependence of activation. In conclusion, we demonstrate that the SCN5A-p.ΔN1472 and KCNH2-p.C108Y mutants exhibit characteristic biophysical properties causing LQTS; whereas KCNQ1-p.R583H, in combination with KCNE1-WT, does not exhibit striking biophysical defects, but in combination with mutant KCNH2 it results in a more severe phenotype. Our results allow to better understand the pathogenesis of LQTS phenotype and to increase the knowledge of ion channel behavior in the pathological conditions.


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