RICERCA DI MUTAZIONI IN PROTEINE ASSOCIATE AI CANALI HCN IN PAZIENTI AFFETTI DA DISTURBI DEL RITMO CARDIACO ED EPILESSIA IDIOPATICA.

It is well known that HCN proteins, molecular correlates of the If current in the heart and Ih in neurons, interact with a system of proteins with regulatory activity and cytoskeleton anchoring. Among these proteins those that have been best studied to date are: caveolin 3, which influences the functionality of HCN4, filamin A which connects HCN1 to the cytoskeleton and MiRP1 that is involved in the modulation of current amplitudes and activation kinetics of HCN4 and 2. In vitro experiments have established that the non-functionality of these proteins can have profound effects on the activity and functional properties of HCN channels. On this basis we decided to see if mutations in the primary sequence of these proteins can be detected in patients with various heart rhythm disorders and epilepsy. Patients we considered: 38 tachycardia (IST), 107 effects by sinus bradycardia (BRA), 21 affected by sudden death syndrome (SUDS), 37 by sudden Antenatal Death Syndrome (SADS), 4 by sudden unespected infant death, 20 with paroxysmal atrial fibrillation (FAP), 110 by epilepsy (EPI) and 107 by sinus bradycardia. So far, we have highlighted some SNPs (Single Nucleotide Polymorphisms) in Caveolin 3: five of these do not lead to changes in amino acid sequence: L9L, N33N, F41F, V57V and S68S; instead another SNP leads to an amino acid change, T78M. All these SNPs have been already published on the NCBI data-Bese. The T78M mutation has already been linked to some cases of SIDS (4), we found it in four TSI, two FAP , oneBRA, and two SADS. The T78M mutation was not found in 418 alleles of control. Regarding Mirp1, Q9E mutations was found in cases of SUD and M54T in a case of seizure and none of them appeared in the control group. Furthermore, two polymorphisms were found:T8A and I57T that although in the literature, along with Q9E and M54T were related to drug induced atrial fibrillation , in our case there is no statistical significance in the association to diseases when compared with the results of the control group. The analysis of filanin A, in the same patients did not reveal significant genetic abnormalities. The mutations were found through the analysis techniques SSCP, DHPLC and sequencing of the gene, the statistical analysis is performed by exact Fisher Test. Later in the case of potentially interesting mutations we continued with genetic engineering techniques aimed at producing cells (HEK293) from which to draw electrophysiological data. The experiments performed to date have focused on functional characterization of the T78M mutation to determine its clinical relevance in the context of the conditions mentioned. Preliminary experiments of functional heterologous coexpression of HCN4 channel with caveolin-3 mutant and wild tipe, did not detect significant changes in current pacemakers. However, given the great variability of the data obtained so far, there is need for a further investigation of CAV3-T78M. Ultimately, the presence of CAV3-T78M is characterized by high statistical significance for groups TSI, FAP, and SADS, and this leads us to think that although the presence of CAV3-T78M is not correlated to a unique phenotype, is actually involved in some abnormalities even if not strictly dependent from HCN4. We therefore believe that CAV3-T78M can undertake activities dependent from the "genetic conditions" of the patient, such as the presence of mutations and polymorphisms, in other unknown interacting proteins and so, in many cases, to direct the expression to heterogeneous phenotypes.