Espectro mutacional y caracterización funcional y clínica de nuevas mutaciones genéticas en canalopatías cardíacas

Resumo

Cardiac channelopathies are ion channels disorders in the absence of structural heart disease, and can lead to ventricular arrhytmias and sudden death, usually in young and previously healthy people. Among these disorders we find the Long QT Syndrome, Brugada Syndrome, Cathecolaminergic Polimorphic Ventricular Tachycardia and Idiopathic Ventricular Fibrillation. These diseases are caused by a disfunction of ion channels, mainly potassium, sodium and calcium, that are encoded by the following genes: KCNQ1, KCNH2 and SNC5A for Lond QT Syndrome, SCN5A for Brugada Syndrome and RyR2 for Cathecolaminergic Polimophic Ventricular Tachycardia. Mutations in these genes can produce an abnormal action potential and typical features in surface electrocardiogram, predisposing to polimorphic ventricular arrhytmias and sudden cardiac death. Numerous mutations in all these genes have been described, and the yield of genetic screening accounts for up to 70 % of long QT Syndrome patients, 30 % in Brugada Syndrome and 70 % in Cathecolaminergic Polimorphic V entricular Tachycardia. In Idiopathic Ventricular Fibrillation genetic testing has not been sufficiently studied yet. In some pathologies such as the Long QT Syndrome, previous studies suggests that depending on the genetic basis (basically the gen affected) response to treatment, arrhytmia triggers and outcome may be different. Most of the mutations found in these patients are ¿novel¿, so there are no data about their clinical impact and functional consequences. In our population there are no data about the genetic basis of cardiac channelopathies. Moreover, there are very few studies, and most of them are isolated cases, that describe natural history, clinical outcome and electrocardiographic features of patients with cardiac channelopathies. The aim of this Thesis is to analyze the mutation spectrum and clinical profile of series of patients with Long QT Syndrome, Brugada Syndrome and Idiopathic Ventricular Fibrillation. Besides, this Thesis provides the study of the functional effects of two novel mutactions related to Long QT Syndrome and Brugada Syndrome. In the Long QT Syndrome population the prevalence of genes mutations was 81 %, most of them in KCNH2, two of them in SCN5A and only one in KCNQ1. Three of these mutations had been previously associated to Long QT Syndrome in clinical or functional studies. In the Brugada Syndrome population the mutational prevalence was 30 %, and all the mutations had not been previously described. In the Idiopatic Ventricular Fibrillation population the yield of genetic screening for cardiac channelopathies was 22,2 %. The clinical study of these populations showed that less than a half of patientes fulfilled diagnostic criteria according to current guidelines for diagnosis of cardiac channelopathies. This was more relevant among first degree relatives, who only fulfilled diagnostic criteria in isolated cases. We describe clinical and functional effects of an heterozygous novel mutation in SCN5A affecting exon 28 and consisting of a nucleotidic insertion (D1816fs/g 98747-98748insT) in a Spanish family in which the index case presented as IVF. Previous studies have involved mutations in nearby locations on the protein both in LQTS and also in BS, but there was no evidence of this mutation causing cardiac ion-channel diseases or IVF. Our study demostrates that this mutation produces a Brugada phenotype in genetic carriers with a very low clinical penetrance, with only two out of eight gene carriers showing the typical electrocardiographic features. This finding was confirmed with a functional study where currents were recorded using the whole-cell patch-clamp in CHO cells transiently transfected with the gene encoding the native (Wild type) or the mutated Nav1.5 together with the ancillary Navß1 subunit. p.D1816fs significantly reduced the current density generated at -20 mV (-63.4±20 pA/pF) compared with Wild Type channels (-723±115 pA/pF). Coexpression of Wild type and mutated channels in a 0.5:0.5 ratio demonstrated that the mutation did not produce a dominant negative effect (-295±87 pA/pF). p.D1816fs shifted +/-15 mV towards more depolarized potentials the voltage- dependence of both the activation and the inactivation of the channel. Furthermore, recovery from fast inactivation was significantly faster for p.D1816fs than for Wild Type channels. In another paper we study the electrophysiological consequences of a novel missense mutations in KCNH2 found in a Spanish family with Long QT Syndrome. Wild-type (WT) and p.E637G Kv11.1 channels were transiently transfected in Chinese hamster ovary cells, and currents were recorded using the patch-clamp technique. The p.E637G channels lost inward rectification and K+ selectivity, generating small but measurable slowly activating, non inactivating currents. These important alterations were corrected neither by cotransfection with Wild Type channels nor by incubation at low temperatures or with pharmacological chaperones. As a consequence of its effects on channel gating, the mutation significantly reduced the outward repolarizing current during the action potential, resulting in a marked lengthening of the duration of a simulated human ventricular action potential. In summary, this Doctoral Thesis is the first description in our country of a serie of patients with cardiac channelopathies. Our data suggests that genetic screening had a high sensitivity fof the Long QT Syndrome with the most affected gene being different form previously reported. Clinical description sohwed a low penetrance in all of them with high prevalence of normal electrocardiograms in mutation carrriers and functional studies demonstrated a pathogenic effect of two novel mutations not previously reported and related to Long QT Syndrome and Brugada Syndrome.