Ignite Creation Date:
2024-05-06 @ 4:00 PM
Last Modification Date:
2024-10-26 @ 2:01 PM
Study NCT ID:
NCT04832126
Status:
UNKNOWN
Last Update Posted:
2021-04-06
First Post:
2021-03-30
Brief Title:
Genetic Analysis of Heart Channelopathies in Brazilian Patients and Their Relatives
Sponsor:
DOr Institute for Research and Education
Organization:
DOr Institute for Research and Education
Study Overview
Official Title:
Genetic Analysis of Heart Channelopathies in Brazilian Patients and Their Relatives
Status:
UNKNOWN
Status Verified Date:
2021-04
Last Known Status:
RECRUITING
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
several genes have been associated with ion channel diseases but a large number of families do not yet have an identified genetic cause There is a lack of information on the genetic characteristics of channelopathies in Brazilians affected by these diseases This study aims to carry out a comprehensive genetic analysis of cardiac channelopathies in Brazilian patients and their families The study will involve 20 patients and 80 family members a total of 100 individuals accompanied by the Rede DOr arrhythmia group in Rio de Janeiro Individuals will be recruited and subjected to DNA sequencing and phenotypic evaluation including clinical evaluation echocardiography 24-hour Holter or longer electrocardiographic monitoring An integrated analysis of phenotype-genotype will be made in all individuals included in the study Patients and their families will be followed up annually for 2 to 5 years through clinical evaluations and the same complementary methods described The DNA sequencing of patients and their families may contribute to improve the diagnosis of channelopathies and allow the determination of the pattern of occurrence of the disease in the cases involved Besides this study may lead to the discovery of new genetic variants associated with channelopathies that will serve as a basis for designing and carrying out broader molecular epidemiological studies The study of the molecular genetics of channelopathies is important mainly so that patients can avoid sudden death but also for the medical community researchers laboratories companies involved in the production of medical devices and public health authorities
Detailed Description:
Inherited arrhythmias are characterized by variable expressiveness and incomplete penetrance Extensive genotype-phenotype studies are needed to elucidate the genetic basis of these diseases There are several reasons for studying the molecular genetics of channelopathies such as determining the molecular epidemiology of these diseases offering evidence for a better understanding of the molecular basis of diseases determining the genetic patterns of occurrence inherited or again and improving the diagnosis and genetic counseling Besides the genetic study of these diseases can lead to the discovery of new genetic variants associated with these diseases and to the creation of experimental models
Genetic testing is recommended Class I for any patient with a strong clinical suspicion of genetically inherited channelopathies including long QT syndrome catecholaminergic polymorphic ventricular tachycardia Brugada syndrome and short QT syndrome Specific genetic testing for the mutation identified in the index case is also recommended Class I for family members
The molecular genetics of cardiac channelopathies has been extensively studied in populations in developed countries In the past the most frequently used methods were the sequencing of a single or a few suspect genes More recently wider panels or even the sequencing of the complete exome have been increasingly used This procedure has allowed the discovery of an increasing number of genetic variants
Although the genetic basis for many cases of these diseases is known many families do not yet have a definite genetic cause Besides it is difficult to predict the pathogenicity and clinical evolution of individual mutations Recent advances in the generation of induced pluripotent stem cells iPS may allow considerable progress in the translational research of arrhythmias
Since the discovery of the first channelopathy in 1995 the use of genetic tests for diagnostic and prognostic purposes has evolved considerably and is now being used in clinical practice and not just for research purposes Despite all these advances the use of DNA sequencing to determine the presence or absence of some diseases is a challenge since all these clinical syndromes can be associated with different mutations many of which have not yet been described The genetic sequencing of patients with genetically inherited arrhythmias is essential not only to provide clinical information related to well-characterized mutations but also to contribute to the discovery of new genetic alterations In Brugada syndrome for example although 12 genotypes have been described so far genetic changes have been identified in only 30 of cases The reason for these discrepancies is related to still incomplete knowledge of all the pathways involved in the function and regulation of cardiac ion channels
Brief descriptions of the most studied channelopathies are presented below Long QT syndrome LQTS This disease has been identified in different areas of the world in different ethnic groups Although at least 13 forms of the disease have been described so far the cause in approximately 20 of cases is not yet defined
The disease is characterized by a prolongation of the QT interval and arrhythmic events The arrhythmias usually found in these patients are episodes of polymorphic ventricular tachycardia torsades de pointes that causes dizziness and syncope and may progress to ventricular fibrillation and sudden death Individuals affected by LQTS are more susceptible to atrial fibrillation than the general population This being the most studied channelopathy the correlation between genetic variants and clinical evolution is very high Currently it is known that exercise and emotional stress trigger arrhythmias in patients with LQT1 and that patients with LQT3 usually have episodes of arrhythmia during sleep It is also known that LQT8 is extremely aggressive with symptoms appearing early and a poor response to treatment At least 20 of genetically proven LQTS cases have normal ECG For all these reasons LQTS is an example of how genetic characterization can help in understanding the pathophysiology of a clinical condition and improve treatment
Brugada Syndrome BRS This clinical syndrome is characterized by spontaneous episodes of polymorphic ventricular tachycardia that can degenerate into ventricular fibrillation and a typical electrocardiographic pattern of elevation of the ST segment from V1 to V3
The prevalence of this disease is higher in Asia where in some locations it is considered the most common cause of natural death among men under 50 years old
The syndrome usually begins in adulthood being rare in children Several genotypes have already been described typically involving a decrease in the currents of sodium Na or an increase in the currents of potassium K The pattern of inheritance is autosomal dominant so half of the family members are expected to be affected As mentioned earlier for LQTS the molecular genetics of several cases with clinical criteria for Brugada syndrome has not yet been elucidated
The treatment of choice is the implantation of a defibrillator No pharmacological treatment is safe although isoproterenol or quinidine can be used in cases of electrical storms Ablation is being investigated to control some of these patients
The diagnosis is made from the electrocardiogram ECG with a typical pattern associated with the clinical evidence of the disease Occasionally a provocative drug test may be necessary as the typical pattern may be transient Blockers of Na channels such as ajmaline can help to identify these patients
Catecholaminergic Polymorphic Ventricular Tachycardia CPVT This disease is characterized by arrhythmic events of bidirectional or polymorphic ventricular tachycardia triggered by an adrenergic stimulus Baseline ECG and imaging tests are nonspecific Ventricular arrhythmias are observed exclusively during efforts so that the stress test and the Holter are important diagnostic methods Two types of mutations have been described so far One at the ryanodine receptor and the other at the gene encoding calsequestrin Only 60 of diagnosed individuals carry one of these mutations and therefore other genes must be involved Other conditions Several other arrhythmic genetic syndromes have been described such as short QT syndrome sudden unexplained death syndrome idiopathic ventricular fibrillation early repolarization and some genetic forms of atrial fibrillation Many patients are diagnosed after an episode of sudden aborted death Better genetic categorization of these cases could help to better understand the mechanisms involved in these diseases and improve treatment Family screening and adequate genetic counseling are essential
OBJECTIVES PRIMARY OBJECTIVE This study aims to carry out a comprehensive genetic analysis of cardiac channelopathies in Brazilian patients and their families
SECONDARY OBJECTIVES Discover new genetic variants that are causal or associated with arrhythmias Determine the molecular epidemiology of these diseases Find evidence for a better understanding of the molecular basis of these diseases
Allow the creation of experimental models Determine the genetic patterns of occurrence inherited or new among others Allow an improvement in the diagnosis Enable an improvement in genetic counseling
HYPOTHESES Genetic analysis will improve the diagnosis and genetic counseling of patients and their families Early diagnosis can prevent sudden death in some cases There is a possibility to detect new genetic variants associated with channelopathies
METHODS STUDY DESIGN AND POPULATION 20 patients and 80 family members a total of 100 individuals accompanied by the arrhythmia group of Rede DOr will be recruited The total number of individuals to include in the study refers to a convenience sample based on patients currently followed by the group of specialists from Rede DOr who are part of the study and their families The choice of up to 4 family members per proband is justified by the fact that this number of related individuals is in most cases sufficient to determine the pattern of occurrence and segregation of phenotypes
The specification of the family members of each proband that will be included will be made on a case-by-case basis depending on the pattern of genetic inheritance the family composition and the availability of samples from other family members The inheritance pattern of causal genetic variants or those associated with phenotypes jointly defined as cardiac channelopathies is more commonly autosomal dominant In this context the genetic testing of both parents and whenever possible of first-degree relatives affected or not by the phenotype is sufficient for the establishment or exclusion of causality Although less common sporadic cases can also be seen In such cases the pathogenic genetic variant should not be detected in the parents but testing them is necessary to confirm the sporadic pattern In these cases siblings andor first-degree relatives should only be tested in the absence of the parents
This is a pilot study that will provide information on the population affected by these diseases in our country All eligible patients affected by channelopathies and followed up in hospitals and clinics belonging to Rede DOr São Luiz in Rio de Janeiro and selected family members will be invited to participate in the study DNA sequencing will be performed 3 2 months after inclusion The phenotypic analysis will be performed 2 1 months after inclusion and will consist of a clinical evaluation echocardiography 24-hour Holter or prolonged electrocardiographic monitoring data collection form - Appendix B A complete and integrated genotype-phenotype analysis of the cases included will be performed Patients and their families will be followed for a minimum of 2 years and a maximum of 5 years and they will be evaluated annually with clinical consultations and with the same complementary methods described above
PROCEDURES Collection and purification of DNA samples Genomic DNA will be purified from peripheral blood or mouth swabs Whole blood will be collected by peripheral phlebotomy in tubes containing K2EDTA as an anticoagulant Blood samples will be stored at 4-8 oC for up to a week before DNA purification Mouth swabs will be collected using the ORAcollect DNA kit OCR-100 DNA Genotek Inc Canada Genomic DNA from whole blood or mouth swab samples will be purified using DNeasy Blood Tissue Kit QIAGEN
The analysis of germline genetic variants inherited or de novo through DNA sequencing either by the Sanger method or by new generation sequencing is not influenced by the tissue or body fluid from which the DNA is purified Therefore peripheral blood or oral smearssaliva can be used as a source of DNA without any technical compromise or introduction of bias Thus the choice of blood or oral swab will be determined essentially by the comfort and convenience for the research participant and aiming at the rational use of the available resources
DNA quantification and quality control The purified genomic DNA will be quantified by ultraviolet spectrophotometry using the NanoDrop microvolume spectrophotometer ThermoFisher Scientific DNA integrity will be measured by agarose gel electrophoresis and using the Agilent 4200 TapeStation system
Genetic analysis strategy In the initial discovery phase 20 probands will be selected for analysis of sequence and copy number variants within a panel of selected candidate genes The suspicious causal variables identified in this initial phase will later be analyzed in up to 4 family members or first-degree relatives for each proband
Genetic panel and gene selection criteria Candidate genes included in the panel were selected from searches in the Online Mendelian Inheritance in Man OMIM database by reviewing the literature and consensus recommendations and guidelines issued by panels and specialist societies The final list of candidate genes was established to include clinically informative genes ie which assist in diagnostic or therapeutic decisions and experimental evidence indicative of genetic involvement in the pathophysiology of arrhythmias
New generation sequencing Candidate genes will be analyzed for the detection of sequence and copy number variants using new generation sequencing The entire coding region and intron-exon boundaries will be sequenced using Ampliseq technology The oligonucleotides for the amplification of the regions of interest will be designed using the online tool Ion AmpliSeq Designer The sequencing libraries will be generated using the Ion AmpliSeq Library Kit on the Ion OneTouch 2 System ThermoFisher Scientific The sequencing will be performed on an Ion Personal Genome Machine PGM sequencer using the Ion 318 Chip v2 BC and the Ion PGM Hi-Q View Sequencing reagents ThermoFisher Scientific Sequencing analysis will be performed using the Ion Reporter software ThermoFisher Scientific
Interpretation and reporting of sequence variants The interpretation and registration of sequence variants will be performed according to the consensual recommendation of the American College of Medical Genetics and Genomics the Association for Molecular Pathology and the College of American Pathologists Briefly to describe variants identified in genes that cause Mendelian disorders this consensus recommends the use of standard terms pathogenic probably pathogenic uncertain meaning likely benign and benign According to this recommendation the process of classifying variants in these five categories is based on a series of different types of evidence such as population data computer simulations functional data and genetic segregation data
The evaluation of the pathogenicity of the genetic variants that are detected will be done independently ie blind by the specialists The clinical evaluation of the patients and determination of the phenotype will be carried out by the arrhythmia specialists in the group under the supervision of Dr Nilson Araújo The genetic evaluation will be done in a blind way by Dr Marcelo Reis In case of divergence between the variant found and the phenotype the findings will be reviewed by Dr Luciana Sacilotto specialist in arrhythmia with experience in clinical genetics and by Dr Carolina Bustamante specialist in molecular genetics
Computational prediction of the functional effect of genetic variants To estimate the functional effects of the sequence variants a series of publicly available tools will be used These algorithms consider the evolutionary conservation of amino acid residues and conserved protein domains protein structure and function Hiden Markov models and position-dependent logic Whenever available functional experimental data in vitro on the effect of specific sequence variants will also be considered for interpretation
Genetic testing is recommended Class I for any patient with a strong clinical suspicion of genetically inherited channelopathies including long QT syndrome catecholaminergic polymorphic ventricular tachycardia Brugada syndrome and short QT syndrome Specific genetic testing for the mutation identified in the index case is also recommended Class I for family members
The molecular genetics of cardiac channelopathies has been extensively studied in populations in developed countries In the past the most frequently used methods were the sequencing of a single or a few suspect genes More recently wider panels or even the sequencing of the complete exome have been increasingly used This procedure has allowed the discovery of an increasing number of genetic variants
Although the genetic basis for many cases of these diseases is known many families do not yet have a definite genetic cause Besides it is difficult to predict the pathogenicity and clinical evolution of individual mutations Recent advances in the generation of induced pluripotent stem cells iPS may allow considerable progress in the translational research of arrhythmias
Since the discovery of the first channelopathy in 1995 the use of genetic tests for diagnostic and prognostic purposes has evolved considerably and is now being used in clinical practice and not just for research purposes Despite all these advances the use of DNA sequencing to determine the presence or absence of some diseases is a challenge since all these clinical syndromes can be associated with different mutations many of which have not yet been described The genetic sequencing of patients with genetically inherited arrhythmias is essential not only to provide clinical information related to well-characterized mutations but also to contribute to the discovery of new genetic alterations In Brugada syndrome for example although 12 genotypes have been described so far genetic changes have been identified in only 30 of cases The reason for these discrepancies is related to still incomplete knowledge of all the pathways involved in the function and regulation of cardiac ion channels
Brief descriptions of the most studied channelopathies are presented below Long QT syndrome LQTS This disease has been identified in different areas of the world in different ethnic groups Although at least 13 forms of the disease have been described so far the cause in approximately 20 of cases is not yet defined
The disease is characterized by a prolongation of the QT interval and arrhythmic events The arrhythmias usually found in these patients are episodes of polymorphic ventricular tachycardia torsades de pointes that causes dizziness and syncope and may progress to ventricular fibrillation and sudden death Individuals affected by LQTS are more susceptible to atrial fibrillation than the general population This being the most studied channelopathy the correlation between genetic variants and clinical evolution is very high Currently it is known that exercise and emotional stress trigger arrhythmias in patients with LQT1 and that patients with LQT3 usually have episodes of arrhythmia during sleep It is also known that LQT8 is extremely aggressive with symptoms appearing early and a poor response to treatment At least 20 of genetically proven LQTS cases have normal ECG For all these reasons LQTS is an example of how genetic characterization can help in understanding the pathophysiology of a clinical condition and improve treatment
Brugada Syndrome BRS This clinical syndrome is characterized by spontaneous episodes of polymorphic ventricular tachycardia that can degenerate into ventricular fibrillation and a typical electrocardiographic pattern of elevation of the ST segment from V1 to V3
The prevalence of this disease is higher in Asia where in some locations it is considered the most common cause of natural death among men under 50 years old
The syndrome usually begins in adulthood being rare in children Several genotypes have already been described typically involving a decrease in the currents of sodium Na or an increase in the currents of potassium K The pattern of inheritance is autosomal dominant so half of the family members are expected to be affected As mentioned earlier for LQTS the molecular genetics of several cases with clinical criteria for Brugada syndrome has not yet been elucidated
The treatment of choice is the implantation of a defibrillator No pharmacological treatment is safe although isoproterenol or quinidine can be used in cases of electrical storms Ablation is being investigated to control some of these patients
The diagnosis is made from the electrocardiogram ECG with a typical pattern associated with the clinical evidence of the disease Occasionally a provocative drug test may be necessary as the typical pattern may be transient Blockers of Na channels such as ajmaline can help to identify these patients
Catecholaminergic Polymorphic Ventricular Tachycardia CPVT This disease is characterized by arrhythmic events of bidirectional or polymorphic ventricular tachycardia triggered by an adrenergic stimulus Baseline ECG and imaging tests are nonspecific Ventricular arrhythmias are observed exclusively during efforts so that the stress test and the Holter are important diagnostic methods Two types of mutations have been described so far One at the ryanodine receptor and the other at the gene encoding calsequestrin Only 60 of diagnosed individuals carry one of these mutations and therefore other genes must be involved Other conditions Several other arrhythmic genetic syndromes have been described such as short QT syndrome sudden unexplained death syndrome idiopathic ventricular fibrillation early repolarization and some genetic forms of atrial fibrillation Many patients are diagnosed after an episode of sudden aborted death Better genetic categorization of these cases could help to better understand the mechanisms involved in these diseases and improve treatment Family screening and adequate genetic counseling are essential
OBJECTIVES PRIMARY OBJECTIVE This study aims to carry out a comprehensive genetic analysis of cardiac channelopathies in Brazilian patients and their families
SECONDARY OBJECTIVES Discover new genetic variants that are causal or associated with arrhythmias Determine the molecular epidemiology of these diseases Find evidence for a better understanding of the molecular basis of these diseases
Allow the creation of experimental models Determine the genetic patterns of occurrence inherited or new among others Allow an improvement in the diagnosis Enable an improvement in genetic counseling
HYPOTHESES Genetic analysis will improve the diagnosis and genetic counseling of patients and their families Early diagnosis can prevent sudden death in some cases There is a possibility to detect new genetic variants associated with channelopathies
METHODS STUDY DESIGN AND POPULATION 20 patients and 80 family members a total of 100 individuals accompanied by the arrhythmia group of Rede DOr will be recruited The total number of individuals to include in the study refers to a convenience sample based on patients currently followed by the group of specialists from Rede DOr who are part of the study and their families The choice of up to 4 family members per proband is justified by the fact that this number of related individuals is in most cases sufficient to determine the pattern of occurrence and segregation of phenotypes
The specification of the family members of each proband that will be included will be made on a case-by-case basis depending on the pattern of genetic inheritance the family composition and the availability of samples from other family members The inheritance pattern of causal genetic variants or those associated with phenotypes jointly defined as cardiac channelopathies is more commonly autosomal dominant In this context the genetic testing of both parents and whenever possible of first-degree relatives affected or not by the phenotype is sufficient for the establishment or exclusion of causality Although less common sporadic cases can also be seen In such cases the pathogenic genetic variant should not be detected in the parents but testing them is necessary to confirm the sporadic pattern In these cases siblings andor first-degree relatives should only be tested in the absence of the parents
This is a pilot study that will provide information on the population affected by these diseases in our country All eligible patients affected by channelopathies and followed up in hospitals and clinics belonging to Rede DOr São Luiz in Rio de Janeiro and selected family members will be invited to participate in the study DNA sequencing will be performed 3 2 months after inclusion The phenotypic analysis will be performed 2 1 months after inclusion and will consist of a clinical evaluation echocardiography 24-hour Holter or prolonged electrocardiographic monitoring data collection form - Appendix B A complete and integrated genotype-phenotype analysis of the cases included will be performed Patients and their families will be followed for a minimum of 2 years and a maximum of 5 years and they will be evaluated annually with clinical consultations and with the same complementary methods described above
PROCEDURES Collection and purification of DNA samples Genomic DNA will be purified from peripheral blood or mouth swabs Whole blood will be collected by peripheral phlebotomy in tubes containing K2EDTA as an anticoagulant Blood samples will be stored at 4-8 oC for up to a week before DNA purification Mouth swabs will be collected using the ORAcollect DNA kit OCR-100 DNA Genotek Inc Canada Genomic DNA from whole blood or mouth swab samples will be purified using DNeasy Blood Tissue Kit QIAGEN
The analysis of germline genetic variants inherited or de novo through DNA sequencing either by the Sanger method or by new generation sequencing is not influenced by the tissue or body fluid from which the DNA is purified Therefore peripheral blood or oral smearssaliva can be used as a source of DNA without any technical compromise or introduction of bias Thus the choice of blood or oral swab will be determined essentially by the comfort and convenience for the research participant and aiming at the rational use of the available resources
DNA quantification and quality control The purified genomic DNA will be quantified by ultraviolet spectrophotometry using the NanoDrop microvolume spectrophotometer ThermoFisher Scientific DNA integrity will be measured by agarose gel electrophoresis and using the Agilent 4200 TapeStation system
Genetic analysis strategy In the initial discovery phase 20 probands will be selected for analysis of sequence and copy number variants within a panel of selected candidate genes The suspicious causal variables identified in this initial phase will later be analyzed in up to 4 family members or first-degree relatives for each proband
Genetic panel and gene selection criteria Candidate genes included in the panel were selected from searches in the Online Mendelian Inheritance in Man OMIM database by reviewing the literature and consensus recommendations and guidelines issued by panels and specialist societies The final list of candidate genes was established to include clinically informative genes ie which assist in diagnostic or therapeutic decisions and experimental evidence indicative of genetic involvement in the pathophysiology of arrhythmias
New generation sequencing Candidate genes will be analyzed for the detection of sequence and copy number variants using new generation sequencing The entire coding region and intron-exon boundaries will be sequenced using Ampliseq technology The oligonucleotides for the amplification of the regions of interest will be designed using the online tool Ion AmpliSeq Designer The sequencing libraries will be generated using the Ion AmpliSeq Library Kit on the Ion OneTouch 2 System ThermoFisher Scientific The sequencing will be performed on an Ion Personal Genome Machine PGM sequencer using the Ion 318 Chip v2 BC and the Ion PGM Hi-Q View Sequencing reagents ThermoFisher Scientific Sequencing analysis will be performed using the Ion Reporter software ThermoFisher Scientific
Interpretation and reporting of sequence variants The interpretation and registration of sequence variants will be performed according to the consensual recommendation of the American College of Medical Genetics and Genomics the Association for Molecular Pathology and the College of American Pathologists Briefly to describe variants identified in genes that cause Mendelian disorders this consensus recommends the use of standard terms pathogenic probably pathogenic uncertain meaning likely benign and benign According to this recommendation the process of classifying variants in these five categories is based on a series of different types of evidence such as population data computer simulations functional data and genetic segregation data
The evaluation of the pathogenicity of the genetic variants that are detected will be done independently ie blind by the specialists The clinical evaluation of the patients and determination of the phenotype will be carried out by the arrhythmia specialists in the group under the supervision of Dr Nilson Araújo The genetic evaluation will be done in a blind way by Dr Marcelo Reis In case of divergence between the variant found and the phenotype the findings will be reviewed by Dr Luciana Sacilotto specialist in arrhythmia with experience in clinical genetics and by Dr Carolina Bustamante specialist in molecular genetics
Computational prediction of the functional effect of genetic variants To estimate the functional effects of the sequence variants a series of publicly available tools will be used These algorithms consider the evolutionary conservation of amino acid residues and conserved protein domains protein structure and function Hiden Markov models and position-dependent logic Whenever available functional experimental data in vitro on the effect of specific sequence variants will also be considered for interpretation
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None