Europace (2010) 12, 424–429 doi:10.1093/europace/eup444

CLINICAL RESEARCH Clinical Trials and Registries

Polymorphisms associated with ventricular tachyarrhythmias: rationale, design, and endpoints of the ‘diagnostic data influence on disease management and relation of genomics to ventricular tachyarrhythmias in implantable cardioverter/defibrillator patients (DISCOVERY)’ study

1 Department of Cardiology, Elisabeth-Krankenhaus Essen, Klara-Kopp-Weg 1, 45138 Essen, Germany; 2Department of Cardiology and Pulmonology, Universita¨tsklinikum Charite´, Campus Benjamin Franklin, Berlin, Germany; 3Department of Cardiology, Rigshospitalet, Copenhagen, Denmark; 4Department of Cardiology, Hospital General Universitario de Alicante, Alicante, Spain; 5Department of Cardiology, Salzburger Landeskliniken, Paracelsus Private, Medical University, Salzburg, Austria; 6Department of Cardiovascular Research, Meilahden Sairaala, Helsinki, Finland; 7Department of Cardiology and Vascular Medicine, Hoˆpital Guillaume et Rene´ Lae¨nnec, Nantes, France; 8Department of Cardiology, Hospital General Yagu¨e, Burgos, Spain; 9Medtronic Inc., Meerbusch, Germany; 10Medtronic, Inc., Fridley, MN, USA; 11Department of Cardiac, Thoracic, and Vascular Sciences, University of Padua, Medical School, Padua, Italy; and 12Institute of Pharmacogenetics, University Clinics Essen, Germany

Received 3 September 2009; accepted after revision 23 December 2009; online publish-ahead-of-print 5 February 2010

Implantable cardioverter-defibrillator (ICD) therapy is effective in primary and secondary prevention for patients who are at high risk of sudden cardiac death. However, the current risk stratification of patients who may benefit from this therapy is unsatisfactory. Single nucleotide polymorphisms (SNPs) are DNA sequence variations occurring when a single nucleotide in the genome differs among members of a species. A novel concept has emerged being that these common genetic variations might modify the susceptibility of a certain population to specific diseases. Thus, genetic factors may also modulate the risk for arrhythmias and sudden cardiac death, and identification of common variants could help to better identify patients at risk. The DISCOVERY study is an interventional, longitudinal, prospective, multi-centre diagnostic study that will enrol 1287 patients in 80 European centres. In the genetic part of the DISCOVERY study, candidate gene polymorphisms involved in coding of the G-protein subunits will be correlated with the occurrence of ventricular arrhythmias in patients receiving an ICD for primary prevention. Furthermore, in order to search for additional sequence variants contributing to ventricular arrhythmias, a genome-wide association study will be conducted if sufficient a priori evidence can be gathered. In the second part of the study, associations of SNPs with ventricular arrhythmias will be sought and a search for potential new biological arrhythmic pathways will be investigated. As it is a diagnostic study, DISCOVERY will also investigate the impact of long-term device diagnostic data on the management of patients suffering from chronic cardiac disease as well as medical decisions made regarding their treatment.

----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords

Single nucleotide polymorphisms † Ventricular arrhythmia † Implantable cardioverter/defibrillator † G-protein † Genome-wide association study

* Corresponding author. Tel: þ492017234807; fax: þ492017235407, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].

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Heinrich Wieneke 1*, Sebastian Spencker2, Jesper Hastrup Svendsen 3, Juan Gabriel Martinez 4, Bernhard Strohmer 5, Lauri Toivonen 6, Herve´ Le Marec 7, Javier Garcia 8, Bernd Kaup 9, Orhan Soykan 10, Domenico Corrado 11, and Winfried Siffert 12

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Polymorphisms in arrhythmias: the DISCOVERY study

Introduction Implantable cardioverter-defibrillator (ICD) therapy is effective in primary and secondary prevention for patients at high risk of sudden cardiac arrest (SCA). However, the financial burden and potential risks associated with this therapy make better identification of patients with a propensity towards SCA mandatory. To date left ventricular function, clinical comorbidity, QRS duration, and various electrophysiological testing methods have been proposed as criteria for the screening of patients potentially at high risk for arrhythmic death.1 However, risk stratification as it exists today remains unsatisfactory as it is mainly performed using a single clinical marker, namely the left ventricular ejection fraction.2 Owing to the fact that diagnostic data of the device are used in clinical decision-making, the DISCOVERY study constitutes an ideal platform for the combination of various diagnostic markers, including information about tachyarrhythmia episodes, with genetic analyses to identify possible genetic markers for SCA.

Single nucleotide polymorphism

G-protein polymorphisms Common genetic variations may modify the susceptibility of individuals to certain diseases. Thus, genetic factors may also modulate the risk of arrhythmias and SCA, and identification of common variants could help to identify patients who are at risk. One potential genetic marker for SCA is located in a gene coding the G-protein. The G-protein is composed of three subunits (a, b, and g) and interacts with heptahelical transmembrane receptors, such as adrenoceptors, in intracellular signalling cascades relevant to various physiological functions, including those of the cardiovascular system.3 Thus, the G-protein is involved in signal transduction of receptors involved in the regulation of ventricular rhythm.4 Moreover, some evidence has indicated that the G-protein might be directly involved in the genesis of atrial and ventricular arrhythmias.5,6

Polymorphism in gene GNB3 The GNB3 gene consists of 12 exons localized on chromosomal position 12p13. It codes for the b3-subunit of the heterotrimeric

Polymorphisms in gene GNAQ The gene GNAQ codes for the Gaq-subunit of heterotrimeric Gproteins. The Gaq-protein transmits signals over a1-adrenoceptors (nor-adrenaline), endothelin receptors, as well as some other receptors. Gaq directly regulates many ion channels. Overexpression of Gaq in the heart leads to cardiac hypertrophy,13 whereas the knockout of Gaq (plus Ga11) counteracts the pressure-induced hypertrophy.14 Three new polymorphisms have recently been described in the promoter of gene GNAQ; these cause alterations in the expression of the Gaq protein— c.-909/-908GC . TT, c.-382G . A, and c.-387G . A.15

Polymorphisms in the GNAS gene The GNAS gene codes for the Gas-subunit of heterotrimeric Gproteins. Activation of Gas (formerly the stimulating G-protein) activates adenyl cyclase, leading to increases in cAMP levels. Gas is activated by many hormone receptors. The activation of ß1-adenoceptors is particularly important for the heart, as this leads to positive chronotropy and inotropy. Several somatic mutations in GNAS lead to rare endocrinological diseases.16 There is also a silent c.393C . T-polymorphism thought to influence the response to b-blockers.17 A series of polymorphisms in the promoter and intron-1 of gene GNAS has recently been described; these modify the transcription rate and protein expression (c.-1211G . A, c.2291T . C).18,19

Genome-wide association study The recent development of high-density genotyping arrays provides a unique opportunity for whole genome assessment of variants associated with common diseases. Using the gene-chip technique, up to 1 000 000 gene variants can be analysed simultaneously nowadays.20 These new methods allow not only the analysis of associations between SNPs and certain phenotypes, but also open the possibility for identification of new biological pathways.21 The chance to identify hitherto unsuspected loci that increase susceptibility to complex disease is one of the main strength of the genome-wide approach. After identifying associations between certain gene variants and the occurrence of ventricular arrhythmias, a careful analysis of potential causal mechanism will need to be done. Similar approaches have revealed aetiological pathways not previously implicated in other diseases, like the

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Human beings share 99.9% of their gene sequences. Given the approximate size of this genome being 3 billion base pairs, there can be as many as 3 million sequence differences between any two individuals. These base pair differences predominantly show up as polymorphisms, defined as variants that occur at a frequency .1% in the population. If these polymorphisms result from the substitution of one nucleotide for another in the DNA sequence, it is called a single nucleotide polymorphism (SNP). Polymorphisms affecting the coding region of a gene may influence the structure of the protein product, whereas others located within the regulatory sequences (also referred to as the promoter region) of a gene can influence the regulation of expression levels of the protein product. In some cases, these genetic variations may alter phenotypic expression following a change in physiological conditions, such as an ischaemic event or the administration of a medication.

G-proteins. The widely distributed c.825C . T polymorphism of this gene exhibits an exchange between Cytosine (C) and Thymine (T) in nucleotide position 825 of the cDNA.7 As Gproteins participate in signal transduction in almost all body cells, it was shown that this polymorphism is correlated with arterial hypertension,8 arteriosclerosis, and obesity9 along with changes in the response to hormones and drugs.10 Homozygotes of the 825T-allele exhibit changes in ion current in atrial cells11 and results in reduced risk of atrial fibrillation.6 Additionally, the results of a recent retrospective pilot study suggest that the c.825C . T polymorphism of the GNB3 gene may have a modifying effect on the propensity towards life-threatening arrhythmias in patients with ICD.12

426 autophagy pathway in inflammatory bowel disease22 and the complement pathway in macular degeneration.23 Furthermore, recent publications have shown that associations between genetic markers and SCA or surrogate markers for SCA, such as long-QT, can be discovered using whole genome association studies. However, these findings would need to be validated with independent studies such as DISCOVERY.24 – 26

Diagnostic data for disease management

Study objectives The DISCOVERY study is an interventional, longitudinal, prospective, multi-centre diagnostic study. It is composed of two parts: Part 1: This is the genetic part of the study. Blood samples are being collected and will be analysed in a double-blinded fashion. The genetic analysis will be performed using two distinct approaches: Part 1a: Candidate genes encoding for G-protein subunits will be analysed and correlated to ventricular and atrial tachyarrhythmias in patients with ICD for primary prevention of SCA. Analysis might also be carried out to validate the utility of markers that were previously identified by other studies. This type of analysis is also known as target gene approach. Part 1b: A genome-wide association study will be carried out to search for additional sequence variants contributing to ventricular arrhythmias in patients with indications for primary ICD therapy. Part 2: The second part of the study evaluates the influence of ICD-based diagnostic information on long-term patient management and treatment.

Study design The study protocol and informed consent forms have all been approved by the medical ethics committees at participating clinical centres. Patients are informed of the objectives of the study, the study organization, and the implications of their participation in

the study. Written informed consent is obtained from each patient prior to participation in the trial. The study will enrol 1287 patients in up to 80 sites in Europe. Enrolment began in April 2007 and was expected to last 36 months. Study follow-up for each patient will be at least 24 months, bringing the total study duration to 5 years. The primary endpoint of the study is defined as the occurrence of a sustained ventricular arrhythmia with a maximal cycle length of 400 ms. This cycle length was chosen to include the majority of true arrhythmias while rejecting most of the false detections. For example, in the Pain FREE RX II trial, the mean cycle length of ventricular tachycardia (VT) in primary prevention patients was 351 ms;28 hence, a cut-off value of 400 ms should safely capture the majority of ventricular arrhythmias. Conversely, in the EMPIRIC trial, a 400 ms cut-off resulted in the inappropriate treatment of supraventricular tachycardias at a rate of only 11.9%.29 Therefore, the maximum cycle length of 400 ms is expected to produce a high value for true positive detection while minimizing false-positive detections.

Study population The study inclusion and exclusion criteria are described in detail in Tables 1 and 2. Patients who already have an ICD implanted are excluded from the study. To date, 495 patients have been included in the study.

Device implantation and programming All enrolled patients receive a single- or dual-chamber ICD with Cardiac CompassTM for recording long-term clinical trends. ICD models include MarquisTM , MaximoTM , IntrinsicTM , Maximo II TM , EnTrustTM , VirtuosoTM , SecuraTM , and subsequent market-released single- and dual-chamber devices. The implantation of dual-chamber ICDs is recommended to the participating centres in order to assure reliable discrimination between ventricular and supraventricular tachyarrhythmias. Moreover, when a dual-chamber device is used, atrial sensing and collection of atrial rhythm data will enable additional analysis of the data to search for possible correlation

Table 1 Inclusion criteria First implantation of a market approved Medtronic single- or dual-chamber ICD with long-term clinical trends: Cardiac Compass (MarquisTM , MaximoTM , IntrinsicTM , Maximo IITM , EnTrustTM , VirtuosoTM , SecuraTM , and future release devices) Subjects requiring the implantation of an ICD for primary prevention according to the current AHA/ACC/ESC guidelines Subject is willing and able to comply with the Clinical Investigation Plan Subject is expected to remain available for follow-up visits Subject has signed the informed consent form within 10 days of implant The system implanted for this study is the first ICD implant for the patient

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With advancements in implantable device technology, new detection and therapy delivery algorithms that are present in modern ICDs are now enabling better patient-tailored therapies and also making increasingly differentiated diagnostic information available to physicians.27 The diagnostic memory of an implantable defibrillator in particular is a valuable source of information for making decisions regarding the long-term treatment and management of patients, most of whom are suffering from heart failure as an underlying disease. By storing the long-term trend data and electrogram recordings from arrhythmic events, the diagnostic memory of the ICD allows clinicians to better understand the cardiac rhythms of individual patients and better tailor drug and device therapies according to their individual needs. In addition, for research purposes, data that can be recovered from the diagnostic memory of the ICD allows an accurate classification of patients based on their well-documented arrhythmia history.

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Table 2 Exclusion criteria

Table 3 Required device programming

Women who are pregnant, or women of childbearing potential who are not on a reliable form of birth control

Parameter

Feature

Value

Subject is enrolled in a concurrent study that may confound the results of this study

VF

Detection Initial beats to detect (NID)

On 18/24

Subject is ,18 years of age, or the subject is under a minimum age that is required as defined by local law

VT

Detection Initial beats to detect (NID) V interval

On or monitor 16 400 ms

EGM 1 Source (AT and DR devices) EGM 1 Source (VR devices)

Atip—Aring

EGM 2 Source

Vtip—Vring

Subject has a life expectancy ,2 years Subject had post-heart transplant or awaiting heart transplantation Subject is anticipated to demonstrate poor compliance Subjects with syndromes known to be associated with ion channel pathologies such as: Long- or short-QT syndrome Brugada syndrome Catecholaminergic polymorphic ventricular tachycardia (CPTV)

Data collection and analysis Patients will be followed for at least 24 months, with required clinical visits at 6, 12, 18, and 24 months following the ICD implantation. During each follow-up, the device will be interrogated and all the data will be stored for later analysis. A blinded episode-reviewing committee will review and classify all reported spontaneous arrhythmias recorded by the devices during the study. The review of each episode will be performed independently by at least two experienced cardiologists.

Can—HVB (RVcoil)

EGM, intracardiac electrogram; VF, ventricular fibrillation; VT, ventricular tachycardia; AT, atrial tachycardia; DR, dual rate; VR, ventricular rate.

Blood sample collection and genetic analysis Blood samples will be collected from each patient during the initial implant procedure. A 20 mL sample of blood will be collected and shipped directly to the core laboratory (Eurofins Medigenomix GmbH, Ebersberg, Germany). Genotyping of the candidate gene polymorphisms will be performed at the end of the study according to modified polymerase chain reaction-based methods (Frey et al.31). Samples will be initially analysed for seven SNPs in the genes GNB3, GNAQ, and GNAS. For the analysis of the predictive power of the various SNPs sensitivity, specificity and positive and negative predictive values will be calculated as predictors of ventricular arrhythmia 400 ms. For the genome-wide association study, a commercially available high throughput system, such as the Illumina Human660W-Quad BeadChip array, will be used for obtaining the SNP data and copy number variation information. The resulting data will be analysed using established bioinformatics techniques, where one option might be the classification and regression tree analysis.32

Sample size calculation The sample size is determined based on the accuracy requirements for the positive predictive value (PPV) of the desired risk stratification test. A 95% confidence interval with a maximal width of +5% was deemed appropriate for the accuracy of the PPV. Assuming that the actual PPV is 40%, a sample size of 386 patients is needed to reach this level of accuracy. Here, the bisection method is used along with the proportion confidence interval formulas as described by Johnson and Kotz.33 DISCOVERY is sufficiently powered to evaluate markers expressed in more than one-third of all patients. Even though the reported incidence rates of appropriate therapy for VT and VF are between 20 and 25%,34,35 we anticipate that the incidence rates for ventricular arrhythmias in our study will be more than 30%, mostly because of the inclusion of all VT episodes in the analysis. Since 386 patients with arrhythmias are needed to reach the primary endpoint, at least 3  386 ¼ 1158 patients with a primary indication for ICD implantation should be enrolled. With an assumption that 10%

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between atrial arrhythmias and genetic markers. However, dualchamber ICDs are not obligatory and the decision is at the discretion of the local study investigator. Depending on the implanted device, bradycardia pacing parameters will be programmed to avoid ventricular stimulation. As far as dual-chamber ICDs are concerned, recently published data suggest that this device type is not associated with a negative outcome with respect to complications when programmed appropriately to avoid ventricular stimulation.30 Additionally, the availability of atrial electrograms from the dual-chamber ICDs will allow positive detection of atrial arrhythmias and confirmation of the source of ventricular disturbance. All settings regarding arrhythmia detection in the DISCOVERY study are similar to those applied in the EMPIRIC19 trial and it is recommended to programme the EMPIRIC settings for arrhythmia treatment. The required programming parameters that determine activation of the ICD detection for ventricular fibrillation (VF) and VT episodes are as follows. The detection interval for the VT-zone is set to be equal to 400 ms or more. In addition, programming allows the device to detect a VT episode when 16 consecutive intervals 400 ms are sensed and detect a VF episode when it senses 18 out of 24 of the most recent intervals that fall into the VF zone. The requirements regarding the intracardiac electrogram storage of arrhythmia episodes are also defined. The details of the required as well as the recommended programming parameters are listed in Tables 3 and 4.

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Table 4 Recommended device programming Parameter

Feature

Value

VF

V interval Redetect beats to detect (NID) Therapies

300 ms 9/12 6 times maximum energy (J)

FVT

Detection V interval Therapies

Via VF 240 ms Burst (1 sequence)a, 5 times maximum energy (J)

VT

Redetect beats to detect (NID) Therapies

8 Burst (2)a, Ramp (1)b, 20 J, 3 times maximum energy (J)

SVT criteria (where available)

PR Logic: AFib/AFlutter PR Logic: Sinus Tach 1:1 VT– ST boundary SVT limit Wavelet

On On 66% (except EnTrust, Virtuoso, Secura) 260 ms On

Pacing

MVP (where available) PAV ( where applicable) SAV ( where applicable)

On 230 ms 200 ms

...............................................................................................................................................................................

a

Burst ATP: 8 intervals, R-S1 ¼ 88%, 20 ms decrement. Ramp ATP: 8 intervals, R-S1 ¼ 81%, 10 ms decrement. FVT, fast ventricular tachycardia; SVT, supraventricular tachycardia; MVP, managed ventricular pacing; PAV, paced AV-delay; SAV, sensed AV-delay; ST, sinus tachycardia.

b

SNP

Frequency of minor allele

GNB3 c.825C . T

30% T

GNAQ c.-909/-908GC . TT

50% TT

GNAQ c.-382G . A GNAQ c.-387G . A

5% A 8% A

GNAS c.393C . T

50% T

GNAS c.2291C . T GNAS c.-1211G . A

30% T 25% T

................................................................................

of the patients may be lost during follow-up, a total of 100/90  1158 ¼ 1287 patients are required for the overall study. It is unlikely that the particular sample size chosen for the DISCOVERY study will be sufficient for the definitive identification of a correlation between SCA and a sequence variant other than the ones listed in Table 5. However, the DISCOVERY trial might identify novel associations in previously unrecognized loci, and that data itself might constitute a hypothesis that could be tested by future studies, as it was done recently to demonstrate the modulation of QT duration by genetic variants.

Conclusion The DISCOVERY study is the first prospective study to investigate the influence of SNPs in genes encoding for G-protein subunits on the occurrence of ventricular arrhythmias. In addition, a genomewide association study may be conducted in order to search for sequence variants contributing to the occurrence of ventricular

arrhythmias in patients receiving ICDs for primary prevention. The primary goal of the study is that known associations will be confirmed or disproved, and that new markers will be identified, paving the way for additional methods of risk stratification for SCA. Moreover, deeper insights into medical decision-making on the basis of diagnostic device data in patients with heart failure will be gained, which will eventually lead to better care and management of the growing number of patients with chronic heart disease. Conflict of interest: H.W. is a member of the speakers’ bureau at St. Jude. He is on the advisory board for Biotronic (Adverse event committee), and has received grants from Medtronic (Steering Committee compensation for the DISCOVERY study). B.K. is an employee of Medtronic Inc., Germany, and O.S. is an employee of Medtronic Inc., Fridley, MN, USA.

Funding This study is sponsored by a grant from Medtronic, Inc., Minneapolis, Minnesota, USA.

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Table 5 Prevalence of the SNPs analysed in the DISCOVERY study

Polymorphisms in arrhythmias: the DISCOVERY study

7.

8.

9. 10.

11.

12.

13.

14.

15.

17.

18.

19.

20.

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