Genomica funzionale, iPS cells, exome sequencing: strategia multidisciplinare per la gestione individualizzata delle malattie aritmogene ereditarie
Project Despite recent advancements of molecular genetic techniques, the basis of many types of arrhythmias, especially those resulting from anomalous activity of the leading pacemaker site or from ectopic pacemaker activity in working tissue (Sinus Bradycardia SB, Sick−Sinus Syndrome SSS, Inappropriate Sinus Tachycardia IST and Atrial Fibrillation AF) are still largely unresolved.
With this project we intend to investigate the genetic basis of inheritable sinus arrhythmias using a multi-task approach. The SAN is the primary site of cardiac pacemaking and the role of “funny” channels in the generation of spontaneous activity and heart rate control is well established. Thus, defective f-channels are obvious candidates in the search for the molecular basis of sinus node dysfunctions. In recent years the relevance of channellopathies arising from defective f-channels has emerged from some specific cases, suggesting the possible existence of a broader mechanim for sinus arrhythmias based on constitutive of f-channel modifications.
A first aim of our project will be therefore to investigate if familial forms of sinus node diseases are linked to genetic defects of the “funny” channels, the primary pacemaker impulse generators (whose molecular correlates are the HCN4 channels). This aspect will be addressed by a gene-candidate approach where sequencing of the Hcn4 gene and known modulatory proteins will be carried out. By use of this approach, our lab has identified for the first time, in a large Italian family with asymptomatic bradycardia, an HCN4 mutation whose functional modification could fully explain bradycardia (Milanesi et al., 2006).
A second aim will be to use the next generation exome sequencing technology to extend the range of investigation of the basic mechanisms responsible for the disease in those families presenting inherited forms of sinus arrhythmia but negative genotype for HCN4 channels and known interacting proteins. This approach aims to identify additional ion channels and structural/ancillary proteins whose function can affect the physiology/pathology of the SAN.
The functional evaluation of the mutations identified by both the candidate gene approach and by the exome capture and sequencing will clearly depend on the functional role of the mutated proteins. For HCN4 and other ion channels and associated modulatory proteins, our laboratory will carry out complete expression studies (patch-clamp electrophysiology and channel trafficking/ immunofluorescence detection) in HEK293 cells. These studies will provide basic functional information on the mutation-induced changes in the properties of channel/protein; our expectation is that these changes correlate with the arrhythmic phenotype.
A third aim will be to make cardiomyocytes from patient derived iPS cells. In all those cases where the function of the mutated protein cannot be easily interpreted as a simple action on the electrical properties of SAN cells, and therefore a more complex pathway of action must be assumed which is determined by other cellular mechanisms/factors (i.e. transcription factors, structural proteins, protein kinases), we will use an approach based on the production of pacemaker-like cells generated from patient-derived iPS cells, which will allow us to reproduce a cellular environment similar to that of the real SAN cells of the patient.
In conclusion the main objective of this study is to provide, by combining genetic investigation and functional characterization, a complete framework able to identify the genetic defects responsible for inheritable types of sinus node diseases, and propose a molecular/cellular interpretation of the disease.
With this project we intend to investigate the genetic basis of inheritable sinus arrhythmias using a multi-task approach. The SAN is the primary site of cardiac pacemaking and the role of “funny” channels in the generation of spontaneous activity and heart rate control is well established. Thus, defective f-channels are obvious candidates in the search for the molecular basis of sinus node dysfunctions. In recent years the relevance of channellopathies arising from defective f-channels has emerged from some specific cases, suggesting the possible existence of a broader mechanim for sinus arrhythmias based on constitutive of f-channel modifications.
A first aim of our project will be therefore to investigate if familial forms of sinus node diseases are linked to genetic defects of the “funny” channels, the primary pacemaker impulse generators (whose molecular correlates are the HCN4 channels). This aspect will be addressed by a gene-candidate approach where sequencing of the Hcn4 gene and known modulatory proteins will be carried out. By use of this approach, our lab has identified for the first time, in a large Italian family with asymptomatic bradycardia, an HCN4 mutation whose functional modification could fully explain bradycardia (Milanesi et al., 2006).
A second aim will be to use the next generation exome sequencing technology to extend the range of investigation of the basic mechanisms responsible for the disease in those families presenting inherited forms of sinus arrhythmia but negative genotype for HCN4 channels and known interacting proteins. This approach aims to identify additional ion channels and structural/ancillary proteins whose function can affect the physiology/pathology of the SAN.
The functional evaluation of the mutations identified by both the candidate gene approach and by the exome capture and sequencing will clearly depend on the functional role of the mutated proteins. For HCN4 and other ion channels and associated modulatory proteins, our laboratory will carry out complete expression studies (patch-clamp electrophysiology and channel trafficking/ immunofluorescence detection) in HEK293 cells. These studies will provide basic functional information on the mutation-induced changes in the properties of channel/protein; our expectation is that these changes correlate with the arrhythmic phenotype.
A third aim will be to make cardiomyocytes from patient derived iPS cells. In all those cases where the function of the mutated protein cannot be easily interpreted as a simple action on the electrical properties of SAN cells, and therefore a more complex pathway of action must be assumed which is determined by other cellular mechanisms/factors (i.e. transcription factors, structural proteins, protein kinases), we will use an approach based on the production of pacemaker-like cells generated from patient-derived iPS cells, which will allow us to reproduce a cellular environment similar to that of the real SAN cells of the patient.
In conclusion the main objective of this study is to provide, by combining genetic investigation and functional characterization, a complete framework able to identify the genetic defects responsible for inheritable types of sinus node diseases, and propose a molecular/cellular interpretation of the disease.