Genetic predisposition to sudden cardiac death Lia Crotti University of Pavia and IRCCS Fondazione Policlinico San Matteo, Pavia, Italy Correspondence to Lia Crotti, MD, PhD, Department of Cardiology, IRCCS Fondazione Policlinico San Matteo. Piazzale Golgi 19, 27100 Pavia, Italy Tel: +39 0382 501323; fax: +39 0382 501322; e-mail: [email protected], [email protected] Current Opinion in Cardiology 2011, 26:46–50

Purpose of review Sudden cardiac death (SCD) is a major public health burden, and evidence from family history and from molecular studies on inherited arrhythmogenic syndromes indicates that genetic factors are important contributors to the risk of SCD. This review discusses recent advances on the genetic predisposition to SCD, with a specific focus on primary ventricular fibrillation and channelopathies. Recent findings Coronary artery disease is the major determinant of SCD, and its predisposing genetic background is complex. Very recently, a first genome-wide association study on primary ventricular fibrillation was published but the results are not conclusive and further studies with greater numbers are needed. Among channelopathies, long QT syndrome and Brugada syndrome are those in which more significant advances have been reported in the last year. Of note is the recently described early repolarization syndrome and the proposed classification of J wave syndromes. Revision of current guidelines for autopsy investigation has introduced molecular autopsy as a standard requirement for adequate assessment of SCD. Summary Interesting data on the genetic basis of sudden cardiac death have been published in the past year, and, whereas in the field of channelopathies research findings have been partially recognized by current guidelines and translated into clinical practice, in the field of coronary artery disease further advances are still needed. Keywords channelopathies, genetic, modifier genes, primary ventricular fibrillation, sudden cardiac death Curr Opin Cardiol 26:46–50 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins 0268-4705

Introduction Sudden cardiac death (SCD) is the single leading cause of mortality in the Western world and represents a major health challenge. In 80% of cases SCD occurs in the context and often as the first manifestation of coronary artery disease. Structural heart diseases (e.g. hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right-ventricular dysplasia) and genetically mediated primary arrhythmia syndromes account for most of the remaining 20%. Although epidemiological risk factors such as age, prior myocardial infarction (MI) and low ejection fraction are well established, the contribution of genetic factors has been inferred by the importance of a positive family history for SCD and by advances in delineating the genetic basis for arrhythmic risk in channelopathies and familial cardiomyopathies. The review will highlight recent advances in the understanding of the genetic basis of SCD in different predis0268-4705 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

posing conditions. The main focus will be on primary ventricular fibrillation and channelopathies.

Primary ventricular fibrillation Sudden cardiac death from ventricular fibrillation during acute MI, ‘primary ventricular fibrillation’, is a leading cause of total and cardiovascular mortality. The evidence for a major genetic contribution to SCD, especially in association with acute MI, is strong and supported by multiple studies. In the Paris Prospective Study parental SCD was found to be an independent risk factor for sudden death [1]. Whereas the SCD of one parent increased the relative risk of SCD by 2.6 times after correction for conventional cardiovascular risk factors, the SCD of both parents increased the risk by 9.4 times [1]. A Dutch case–control study showed that familial SCD occurred significantly more frequently among patients with a first MI complicated by ventricular fibrillation than in patients with a first MI without ventricular fibrillation [odds ratio (OR) 2.72] [2]. A Finnish DOI:10.1097/HCO.0b013e32834138dd

Genetic predisposition to sudden cardiac death Crotti 47

study with an almost identical design provided the same results [3]. Different strategies can be followed to identify the genetic basis of SCD during MI and two main strategies have been used so far. The first strategy aims at the identification of an association between genetic variants and markers of SCD risk, such as the length of the QT interval. Indeed, as a prolonged QTc is associated with an increased risk of SCD among patients with coronary artery diseases [4,5], genetic variants associated with a prolonged QT could be genetic markers of SCD. Outstanding genome-wide association studies (GWAS) that identified the association between different single-nucleotide polymorphisms (SNPs) and the length of the QT have been published in the past 4 years [6–8,9,10]. Interestingly, all these studies demonstrated a robust association between NOS1AP, which encodes a nitric oxide synthase adaptor protein, and QT-interval duration in the general population [6–8,9,10]. Among these studies, only three evaluated the association of NOS1AP SNPs and SCD [11–13]. In the study by Aarnoudse et al. [11] the Rotterdam study population was evaluated, and, whereas an association between NOS1AP and QTc was confirmed, no association with SCD was observed. By contrast, in the study by Kao et al. [12] a significant association was observed between NOS1AP rs16847548 and rs12567209 and SCD in white but not in black patients [12]. A subsequent meta-analysis, including the Rotterdam study population, showed an association between genetic variations within NOS1AP and SCD [13]. However, as all these studies were not designed to specifically test the genetic basis of SCD, in order to better understand the role of NOS1AP variants in primary ventricular fibrillation we tested rs16847548 and rs4657139 in 123 patients with a first MI complicated by ventricular fibrillation and in 216 controls (patients with a first MI not complicated by ventricular fibrillation) [14]. NOS1AP rs4657139 was observed with a minor allele frequency (MAF) of 0.386 in both cases and controls. NOS1AP rs16847548 was observed with a MAF of 0.248 in cases and of 0.243 in controls. Therefore, no statistically significant associations were observed between NOS1AP variants and the risk of ventricular fibrillation during a first myocardial infarction [14]. A more direct but also more difficult strategy to follow is aimed at testing directly an association between genetic variants and SCD risk. The most difficult part of this approach is the collection of an adequate number of patients with primary ventricular fibrillation. Very recently, Bezzina et al. [15] published the first GWAS study for ventricular fibrillation during acute MI and they compared 515 patients with ventricular fibrillation during

a first MI and 457 with uncomplicated MI. Similarly to our data [14], they also found no association between NOS1AP SNPs and SCD risk. The most significant association with ventricular fibrillation was found at 21q21 [rs2824292, OR ¼ 1.78, 95% confidence interval (CI) 1.47–2.13, P ¼ 3.3  1010]. The association of this SNP with ventricular fibrillation was replicated in an independent case–control study, consisting of 146 outof-hospital cardiac arrest individuals with MI and 391 individuals who survived a MI (OR ¼ 1.49, 95% CI 1.14– 1.95, P ¼ 0.004). The closest gene to this SNP is CXADR, which encodes a viral receptor previously implicated in myocarditis, dilated cardiomyopathy and cardiac conduction disorders, but never implicated in arrhythmia susceptibility [15]. This study represents a very significant starting point, but further studies with more cases and controls are needed to better elucidate the genetic basis of primary ventricular fibrillation. To add further insights in the area of SCD, we have started a multicentre Italian study, ‘PREDESTINATION’ (PRimary vEntricular fibrillation and suDden dEath during a firST myocardIal iNfArcTION), aimed at enrolling 2000 patients with a first MI complicated by ventricular fibrillation and 2000 controls, that is, patients with a first MI and no lifethreatening arrhythmias.

Channelopathies Channelopathies, being pure electrical disorders, have always been considered as good models for understanding the genetic basis of SCD, due to the absence of macroscopic modifications of the cardiac function that could interfere with arrhythmic risk. Dealing with channelopathies, two main genetic components predisposing to sudden cardiac death should be taken into account: disease-causing genes, that are prerequisite and sufficient to cause the disease; modifier genes, that are neither necessary nor sufficient to cause the disease, but that can significantly modify the risk of sudden cardiac death in affected patients. Only the identification and the better comprehension of these two genetic components will allow a thorough understanding of the genetic basis of sudden cardiac death. The long QT syndrome (LQTS) is the oldest and best studied model. Many genes have been identified at the basis of the disease, a few of them quite recently; however, these new genes contribute only a handful of patients, and the vast majority of the genotyped-positive LQTS patients are carrying a disease-causing mutation in the three originally described genes: KCNQ1, KCNH2 and SCN5A [16]. Therefore, despite many efforts, in the past 15 years no other important disease-causing genes have

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been identified and the percentage of genotyped-positive patients did not significantly increase. Possible explanations differ, going from technical limitations to novel genes/pathways not yet explored; however, two hypotheses are worthy of discussion: the first hypothesis is that intronic variants or exonic variants not changing the amino acid sequence, in already known LQTS genes, could cause the disease by interfering with the expression of the encoded gene or with all the steps leading from gene to protein. This hypothesis is supported by the recent evidence that an intronic mutation of the KCNH2 gene (IVS9-28A/G) is able to cause the LQTS in a large family, disrupting the acceptor splice site definition and causing an intron retention able to disrupt the normal structure of the protein [17]. Another hypothesis could be that a combination of ‘minor genetic variants’ prolonging the QT interval could be the cause of LQTS in some sporadic cases. The identification of the disease-causing mutation, however, is not the whole story. Incomplete penetrance and variable expressivity, typical of LQTS, suggest the existence of factors other than the primary mutation that can modify the probability of symptoms. Identification of genetic modifiers of LQTS would lead to improved risk stratification among mutation carriers and could also provide information about the risk of life-threatening arrhythmias in more common conditions, such as acute myocardial infarction and congestive heart failure. In the past 5 years an increasing number of publications have focused on the contribution of common genetic variants in the LQTS. ‘The first study to convincingly establish a causal role for a common SNP in a long QT gene in determining the severity of a LQTS phenotype’ [18] was the one published in 2005, in which we provided evidence that the KCNH2-K897T polymorphism was able to modify the clinical expression of a latent LQT2 mutation [19]. Very recently, this observation was confirmed by Nof et al. [20] in another LQT2 family in which the KCNH2-K897T markedly accentuated the loss of function of mildly defective human ether-a-go-go-related gene (HERG) channels, leading to LQTS-mediated arrhythmias and sudden infant death. Whether the effect of the K897T is limited to some KCNH2 mutation or to all LQT2/LQTS patients still needs to be defined in large populations of LQTS patients. One of the problems when dealing with modifier genes is the identification of a well suited population. We chose to test candidate LQTS modifier genes in a large group of patients carrying the same disease-causing mutation. This unique study design eliminates the confounding effect of genetic heterogeneity that is present when a study involves multiple different disease-causing mutations known to carry widely different arrhythmic risk. We specifically studied a South African LQT1 founder popu-

lation harbouring a mutation in KCNQ1 (A341V) that exhibits a wide range of QTc values and clinical manifestations [21,22]. In this population we tested the hypothesis that NOS1AP could act as a genetic modifier influencing not only the length of the QTc, but also the risk for SCD [23]. Interestingly, we observed that rs4657139 and rs16847548 were significantly associated not only with the occurrence of symptoms but also with a greater probability of life-threatening arrhythmias, defined as cardiac arrest and SCD (rs4657139, P ¼ 0.028; rs16847548, P ¼ 0.014) [23]. Very recently, a study on a more heterogeneous LQTS cohort was published [24], supporting the role of NOS1AP as a genetic modifier of LQTS. Among channelopathies, while no significant advances have been reported in the past year on the genetic basis of short QT syndrome (SQTS) and catecholaminergic polymorphic ventricular tachycardia (CPVT), worthy of note is the recently described early repolarization syndrome [25,26]. Early repolarization, present in 3–5% of controls, was long considered an unremarkable and essential benign pattern. In 2008 Haı¨ssaguerre et al. [25] observed that an early repolarization pattern was significantly more frequent in case participants with idiopathic ventricular fibrillation (IVF) than in controls (31 vs. 5%; P < 0.001). Interestingly, in the same issue of the same journal, Nam et al. [26] in a ‘letter to the editor’ provided data in a different population supporting the same observation, suggesting that an early repolarization pattern is not always benign, as previously thought. Patients with early repolarization and IVF had dramatic but transient accentuation of J waves before the development of electrical storms, and quinidine, isoproterenol and pacing at rapid rate suppressed accentuated J waves and prevented ventricular fibrillation [26]. These observations led the authors to propose that this unique phenotype may represent a variant of a much broader syndrome that should include Brugada syndrome, in which the appearance of prominent J waves underlies the development of arrhythmogenicity. In a subsequent publication this broader syndrome was defined as J-wave syndrome [27]. In 2009 Haı¨ssaguerre et al. [28] identified the first gene implicated in IVF and early repolarization, the KCNJ8 gene, coding for the subunit Kir6.1 of KATP channel. Related to this, and in support of the ‘J-wave syndrome view’, the same mutation was very recently identified also in one of our genotype-negative Brugada syndrome patients and in another patient with ventricular fibrillation and early repolarization [29]. The mutation expressed in COS-1 cells induced, as expected, a significant increase of the KATP current [29]. These observations are adding new pieces to the Brugada puzzle, which, among the different channelopathies, is probably the most heterogeneous and difficult to

Genetic predisposition to sudden cardiac death Crotti 49

understand. So far, including KCNJ8, there are eight genes observed in association with this syndrome, but among them only SCN5A has been identified in a significant but still small number of patients, being responsible for 13–28% of cases [30]. Mutations in SCN5A can also be responsible for overlapping phenotypes, and a good example is the SCN5A-E1784K mutation, the most frequently described Brugada syndrome mutation [30], which is also a highly prevalent mutation among LQT3 patients, able to cause Brugada pattern, long QT, sinus node dysfunction and life-threatening ventricular arrhythmias [31]. The second most frequently implicated channel in Brugada syndrome is the voltage-dependent L-type cardiac calcium channel, initially described in patients with Brugada pattern, short QT and risk of SCD [32]. To add further complexity, in a study by Probst et al. [33] 13 large Brugada syndrome families with 115 carriers of a mutation in SCN5A were studied, and in five of these families eight individuals mutation-negative, but affected with Brugada syndrome, were identified. The authors concluded that SCN5A mutations may not be the whole of the story in Brugada syndrome, and they suggested that genetic background may play a powerful role in the pathophysiology of this disease. Despite some limitations [33], the findings of this study, the observation that Brugada syndrome families with SCN5A mutations usually have low penetrance and heterogeneous clinical manifestations, and the great difficulties in finding Brugada syndrome families large enough to perform linkage analysis are all data strongly suggesting that Brugada syndrome phenotype is probably the consequence of more complex genetic interactions and environmental factors, which need to be identified.

Molecular autopsy in sudden cardiac death cases Sudden cardiac death can be the first clinical manifestation of an inherited arrhythmogenic cardiac disease and autopsy may represent the first and certainly the last opportunity to make the proper diagnosis, extremely important for those left behind, as a correct diagnosis in the proband would allow the identification of affected family members, potentially at risk of SCD if not correctly recognized and treated [34,35]. A standard autopsy is sufficient to identify monogenic structural heart diseases, but in the case of channelopathies a ‘negative autopsy’ would be the only result. This is no longer acceptable, given that one-third of unexplained SCD cases are of genetic origin [36,37]. Furthermore, the role of channelophaties has been recently enlarged, being able to cause a significant percentage of sudden infant death syndrome (SIDS) cases [38–40] and stillbirth cases [41], other examples of ‘mors sine materia’.

Recently, Basso et al. [42], on behalf of the Association for European Cardiovascular Pathology, strongly addressed this point, revising guidelines for autopsy investigation of sudden cardiac death. The most important novelty introduced was the molecular screening in both structural and nonstructural genetically determined heart diseases, as part of the requirements for the adequate assessment of SCD [42,43]. The introduction of molecular autopsy in SCD victims with normal heart [43] represents a critical step forward for the understanding of SCD and for the establishment of early preventive measures in affected family members. Hopefully, these concepts will be extended by the indication to perform a molecular autopsy in all cases of SIDS and of unexplained stillbirths, given the significant role that channelopathies play in these conditions [38–41].

Conclusion Sudden cardiac death has a strong familial component; however, our understanding of its genetic basis varies significantly according to the underlying causes. When coronary artery disease is involved, and this happens in the majority of the cases, the predisposing genetic background is complex, and despite some progress it remains largely unknown. Quite different is the case of monogenic structural and nonstructural heart diseases, which are responsible for 30–40% of SCD cases in the young. As SCD can be the first clinical manifestation of inherited syndromes, it is extremely important to make the correct diagnosis with autopsy, in order to identify affected family members in whom preventive measures should be established. Accordingly, current guidelines for autopsy investigation of SCD have been revised to include molecular autopsy, the only way to make a post-mortem diagnosis in channelopathies. Overall, these data, together with the inclusion in current guidelines of molecular screening for diagnosis and/or risk stratification of specific inherited cardiac diseases [44], exemplify how research on the genetic basis of SCD is deeply translational and is already progressively improving everyday practice.

Acknowledgement I express my gratitude to Peter J. Schwartz for constructive criticism.

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