Review. The Genetics of Cardiac Disease Associated with Sudden Cardiac Death

Transcription

1 ASIP 2012 JMD CME Program Review The Journal of Molecular Diagnostics, Vol. 14, No. 5, September 2012 Copyright 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved. The Genetics of Cardiac Disease Associated with Sudden Cardiac Death A Paper from the 2011 William Beaumont Hospital Symposium on Molecular Pathology Mark J. Perrin* and Michael H. Gollob* From the Division of Cardiology,* Department of Medicine, University of Ottawa Heart Institute, Ottawa, Ontario; and the Department of Cellular and Molecular Medicine, University of Ottawa, Ontario, Canada Sudden cardiac death due to ventricular arrhythmia most commonly occurs in the setting of coronary artery disease. However, a number of inherited syndromes have now been identified that carry a significant risk of sudden cardiac death and that are disproportionately represented in the young. Arrhythmia in such conditions may result from genetically mediated structural heart disease (eg, hypertrophic cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy) or from altered function of cardiac ion channels in the absence of overt structural disease (eg, Brugada syndrome and long QT syndrome). The past 15 years have seen considerable progress in our understanding of the genetic underpinnings of these disorders. With the advent of clinical genetic testing as a routine part of clinical care, a new knowledge base is required of practicing cardiologists and genetic testing facilities, particularly related to the rational ordering of genetic testing and the interpretation of results. This review addresses the latest findings in regard to the genetic causes of inherited syndromes associated with sudden cardiac death and summarizes recently published guidelines for the genetic testing of affected individuals and their families. ( J Mol Diagn 2012, 14: ; Each year in the United States approximately 350,000 people experience sudden cardiac death (SCD). Although coronary artery disease is the most common antecedent in those aged 40 years, thousands of younger people also die suddenly without warning. 1 Genetic 424 forms of structural heart disease and purely electrical disease of the heart are disproportionately represented among this group. Although structural heart diseases such as arrhythmogenic right ventricular cardiomyopathy (ARVC) have been clinically recognized for years, the genetic basis of these conditions has only come to light in recent years. Novel sudden death syndromes with a genetic basis continue to be described. Two purely electrical diseases of the heart known as catecholaminergic polymorphic ventricular tachycardia (CPVT) and short QT syndrome (SQTS) were only clinically recognized within the last 10 years. These conditions are commonly clinically silent and may manifest for the first time as a SCD in a previously well-functioning and healthy young individual. The emotional toll of such tragic deaths is felt not only by the surviving family members but also by the community at large. Genetics research has unraveled the genetic causes and enlightened our understanding of inherited cardiac syndromes associated with unexpected SCD. The routine use of genetic testing in clinical care is upon us and promises earlier and more certain diagnosis with the possibility to avert tragedy. However, this progress brings with it a considerable challenge. The cost of gene testing, Supported by a clinical fellowship from the University of Ottawa, Ontario, Canada (M.J.P.). Accepted for publication April 13, This article is partly based on material presented by the authors at the William Beaumont Hospital 20th Annual Symposium on Molecular Pathology: Clinical Applications of Genomic Medicine, which took place September 21 22, 2011, in Troy, Michigan. CME Disclosure: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interest to disclose. Address reprint requests to Michael H. Gollob, M.D., Inherited Arrhythmia Clinic and Arrhythmia Research Laboratory, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON, Canada K1Y 4W7. mgollob@ottawaheart.ca.

2 Genetics of Sudden Cardiac Death 425 though decreasing, remains high and is often not covered by medical insurance or publically funded health bodies. Therefore, there is a need for an understanding of a rational stepwise approach to testing (proceeding from common causes to uncommon) as advocated by recent genetic testing guidelines for SCD syndromes. 2 Although the yield of genetic testing is high in many conditions (eg, 80% in long QT syndrome [LQTS]), for other conditions the yield is considerably lower and judgment is required in considering the utility of genetic testing. The relative yield of testing for each condition is addressed in this review and must be known to provide adequate pretest counseling and to inform the interpretation of a negative result (ie, negative does not mean no disease). The benefits of a positive result, on the other hand, are significant and may confirm a diagnosis, guide therapy, and allow the efficient screening of family members for the disease. Of course, not all results are neatly negative or positive, and a clearly affected individual may be found to harbor a variant of unknown significance. In other cases, a disease-causing mutation may be identified in a family member without a phenotype of disease. A specific knowledge set is therefore required for the reasonable interpretation of these data in the context of a patient with a manifest clinical syndrome and a concerned family. We review each of the most common inherited cardiac syndromes associated with SCD and provide the relevant data to guide both the rational ordering of genetic testing and the interpretation of the findings. Familial cardiac syndromes associated with SCD may be divided into those associated with structural heart disease, including hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and ARVC, and the primary electrical disorders with structurally normal hearts, including LQTS, SQTS, Brugada syndrome (BrS), and CPVT. Each condition is discussed in turn in order of decreasing prevalence. Familial Cardiac Syndromes Associated with Structural Heart Disease Hypertrophic Cardiomyopathy HCM is an autosomal dominant disorder marked by unexplained and often asymmetric hypertrophy of the left ventricle (LV). Men are affected more commonly than women (3:2). It is the most common inherited cardiac disease, with a prevalence of approximately 1 in The symptoms of heart failure that may be associated with HCM are managed by the judicious use of -adrenergic blocking agents and diuretics. -Blockers may also be useful in the management of atrial arrhythmia. When outflow obstruction causes significant symptoms, either alcohol septal ablation or surgical myectomy is indicated. Patients at high risk for SCD are candidates for an implantable cardioverter defibrillator (ICD). 4 HCM is diagnosed by the presence of LV hypertrophy (maximal LV wall thickness 15 mm) without an alternative cause. Hypertensive heart disease, athlete s heart, and metabolic or infiltrative disorders can mimic the structural phenotype. 4 Symptoms of HCM may include shortness of breath on exertion, syncope, or SCD related to ventricular arrhythmia. When symptoms occur, several pathophysiologic mechanisms contribute to the clinical phenotype, including hypertrophy with or without obstruction of the left ventricular outflow tract (LVOT), diastolic dysfunction, myocardial ischemia, and mitral regurgitation. 5 LVOT obstruction occurs in one-third of patients at rest, occurs in another third with provocative maneuvers, and is absent in a third. 4 Similar to the presence or absence of LVOT obstruction, there is considerable variability between HCM patients in the degree, location, and morphologic features of hypertrophy, the amount of myocyte disarray and fibrosis, and the clinical progression of disease, ranging from asymptomatic to refractory cardiac failure or SCD at a young age. 6 HCM is a progressive disease, and death may occur from ventricular arrhythmia (51%), 7 progressive heart failure (36%), or stroke in the context of atrial arrhythmia (13%). 8 HCM is the most common cause of SCD in the young, including competitive athletes. Although initial reports quoted annual mortality of 4% to 6% in HCM, more recent studies examining unselected patients consistently report figures 1%. 8 In contrast to the marked phenotypic variability of the disease, most definite genetic causes of HCM occur due to disruption of the functional integrity of the cardiac sarcomere (Figure 1). Genetics of HCM Mutations in myofilament proteins. In 1989, linkage analyses on a large family affected by autosomal dominant HCM mapped the responsible gene to the long arm of chromosome Shortly after, the causative mutation, R403Q, was identified in the -myosin heavy chain gene (MYH7). 10 Since 1990, seven more myofilament encoding genes have been identified as definite (based on linkage analyses or strong cosegregation data) causes of HCM 4 (Table 1). Each gene encodes sarcomeric or sarcomereassociated proteins, in order of discovery: -tropomyosin (TPM1) and cardiac troponin T (TNNT2), 11 myosin-binding protein C (MYBPC3), 12 regulatory myosin light chain (MYL2) and essential myosin light chain (MYL3), cardiac troponin I (TNNI3), and actin (ACTC). 4 However, at most 65% of HCM may be explained by mutations in these genes, 13 with MYBPC3 and MYH7 accounting for 50% of HCM cases. 6 Nonsynonymous rare variants have been identified in MYH6 in isolated cases of HCM. 14 Similarly, mutations in troponin C (TNNC1) have been identified in approximately 0.4% of HCM with functional phenotypes of the mutant proteins similar to previously recognized HCM genes. 15 However, in neither case have the mutant genes been shown to definitively segregate with disease.

3 426 Perrin and Gollob Cardiac Action Potential Surface Electrocardiogram short QT long QT syndrome normal QT short QT syndrome long QT QT interval gain of function mutations in IK r (SQT1) shorten the cardiac action potential and the QT interval on the ECG NCX Z loss of function mutations in IK s (LQT1) and IK r (LQT2) prolong action potential duration thereby increasing the QT interval on the ECG sarcomere Na + Ca 2+ IK r IK s INa M sarcoplasmic reticulum DSP DSP Z loss of function of INa is associated with Brugada syndrome in ~ 20% of cases; an increase in late sodium current causes LQT3 DSC2 PKP2 PKP2 DSG2 truncation mutations in titin (TTN), the largest protein in the body, are the most common cause of DCM HCM is a disease of the sarcomere the contractile unit of the myocyte Figure 1. Mechanisms of familial cardiac disease associated with sudden cardiac death. Two myocytes are shown (bridged by a desmosome). A single sarcomere is represented with titin anchored to the Z-disk (squiggly lines) and spanning the full length of the contractile unit. The thin filaments (light gray) are attached at a single Z-disk, whereas the thick filaments (dark gray) span the M-line. Calsequestrin (CSQ) is shown anchored to the ryanodine receptor (RYR2) on the sarcoplasmic reticulum by junctin (jun) and triadin (tri). The three principal ionic currents responsible for SQTS and LQTS are pictured on the upper membrane (I Kr, rapid delayed rectifier potassium current; I Ks, slow delayed rectifier potassium current; I Na, voltage gated sodium current) along with representative cardiac action potentials (black action potential is control) and the phenotype of SQTS and LQTS on the surface ECG. DADs, delayed after depolarizations; DSC2, desmocollin 2; DSG2, desmoglein 2; DSP, desmoplakin; NCX, sodium-calcium exchanger channel; PKP2, plakophilin 2. RYR2 T-tubule abnormal calcium release from mutant RYR2 channels causes intracellular calcium overload and Na + influx into the cell (delayed after depolarizations) through NCX channels CSQ autosomal recessive CPVT (CPVT2) is caused by mutations in CSQ which modulates RYR2 ARVC is a disease of the desmosome which is crucial for cell-to-cell adhesion Mutations in Z-disk proteins. The exhaustion of sarcomeric gene candidates, combined with a significant burden of unexplained HCM, has encouraged a search for novel causes of HCM. Z-disks (Figure 1) mark the lateral border of the sarcomere and, although initially considered a mere structural component of the contractile apparatus, are now known to be involved in cellular signaling, mechanotransduction, and the modulation of cardiac hypertrophy. 16 Consistent with this understanding, nine genes encoding Z-disk proteins have been associated with HCM: myozenin-2 (MYOZ2), 17 -actinin 2 (ACTN2), 18 titin (TTN), muscle Lim protein (CSRP3), telethonin (TCAP), metavinculin (VCL), LIM domain binding 3 (LDB3), ankyrin repeat domain 1 (ANKRD1), and nexilin (NEXN). However, of the nine genes, only two (MYOZ2, 17 and ACTN2 18 ) have been shown to segregate with disease. Considering that each individual harbors approximately 5000 nonsynonymous variants, including radical mutations, the pathogenicity of the remaining seven genes cannot be established with certainty at this time. Interestingly, mutations in Z-disk proteins are more commonly identified in patients with DCM than HCM. 16 It is not immediately clear why certain mutations should favor one phenotype over the other or even whether Z-disk HCM is a phenotypically distinct disorder from sarcomeric HCM. 13 Mutations disrupting calcium handling proteins. Similar to Z-disk mutations, studies have identified rare nonsynonymous variants in genes involved in Ca 2 regulation, including JPH2-encoded junctophilin 2 19 and CALR3-encoded calreticulin and PLN-encoded phospholamban. 20 Recommendations for Genetic Testing in HCM Genetic testing in HCM is rarely used for the diagnosis of the condition which is usually achieved by diagnostic imaging and clinical history. The principal use of genetic testing is to aid in family screening in light of the variable clinical penetrance of this genetic disease. In the absence of an identified genotype, all family members of a proband must be followed lifelong for the developing

4 Genetics of Sudden Cardiac Death 427 Table 1. Genes Associated with HCM Listed According to Level of Evidence of Pathogenicity 4 Gene Protein Frequency, % Definite MYH7 -Myosin heavy chain MYBPC3 Myosin-binding protein C TPM1 Tropomyosin 2 5 TNNT2 Troponin T 2 5 TNNI3 Troponin I 2 5 MYL2 Ventricular regulatory myosin 1 light chain MYL3 Ventricular essential myosin 1 light chain ACTC -Cardiac actin 1 Probable MYOZ2 Myozenin-2 1 ACTN2 -Actinin 2 1 Possible MYH6 ( -myosin heavy chain), TNNC1 (troponin C), TTN (titin), CSRP3 (muscle Lim protein), TCAP (telethonin), VCL (metavinculin), LDB3 (LIM domain binding 3), ANKRD1 (ankyrin repeat domain 1), NEXN (nexilin) phenotype. A positive genotype, on the other hand, identifies those family members who truly require such medical surveillance and allows those without the genotype to be discharged from care. Because of the large number of genes implicated in HCM, genetic testing follows a tiered approach with initial screening for mutations in MYH7 and MYBPC3 (30% to 50% yield); this may be followed by testing for mutations in TNNT2 and TMP1 (5% to 10% yield). 2 Genetic testing of all other implicated genes is of such low yield and of uncertain clinical significance in most cases that the expense cannot be justified at this time. A question may arise as to how far genetic testing should be extended (ie, first-degree relatives alone). A recent study from Nannenberg and colleagues analyzed the mortality rates in untreated HCM families harboring a single MYBC3 truncation mutation: six large, 200-year, multigenerational pedigrees were constructed and compared with 140 small (first-degree relative) pedigrees. Overall mortality did not increase in the large kindreds, but an excess of mortality occurred in individuals aged 10 to 19 years. In the small kindreds, increased mortality was demonstrated from the age of 10 years, even up to the age of 59 years. 21 Thus, the results from this study support current testing recommendations for all first-degree relatives and extended testing in more distant relatives aged 10 to 19 years. Dilated Cardiomyopathy DCM is characterized by a dilated, poorly contracting left ventricle without evident cause after clinical testing. DCM is diagnosed in approximately 1 in 2500 of the population with equal male/female predilection. Typically, patients present with advanced disease manifested clinically by dyspnea, edema, and fatigue. Death may occur with 1 progressive heart failure or ventricular arrhythmia. Stroke is also common due to associated atrial fibrillation or thrombus in the left ventricle. Drug treatment of DCM includes angiotensin-converting enzyme inhibitors, -adrenergic blockade, and spironolactone with judicious use of diuretics and salt and water restriction to manage volume overload. Large trials now support the use of an ICD to prevent SCD when the LV ejection fraction is 35% 22 ; when a wide QRS of 150 milliseconds is present on the ECG, a cardiacresynchronization device combined with an ICD reduces mortality and improves functional status. 23 Genetics of DCM Familial DCM (FDCM) is diagnosed when DCM occurs in two or more first-degree relatives. The common mode of inheritance is autosomal dominant, but autosomal recessive and X-linked forms occur. DCM is often inherited with other phenotypes, both noncardiac (eg, sensorineural hearing loss and muscular dystrophy) and cardiac (eg, conduction disease). 24 Commonly, DCM is found in isolated cases without an apparent family history. In FDCM, genetic culprits may affect proteins in the nuclear envelope, sarcomere or desmosome, transcription factors, cytoskeletal complex, or even the mitochondria. 25 The more common genetic causes ( 1% to 2% of cases) identified to segregate with disease in affected families include TTN (encoding titin), 26 LMNA (encoding lamin A and lamin C), 27 MYH7 ( -myosin heavy chain), TNNT2 (troponin T), MYH6 ( -myosin heavy chain) and MYPBC3 (myosin binding protein C), 28 SCN5A (Nav1.5), 29 and RBM20 (ribonucleic acid binding motif protein 20). 30 Titin is the largest human protein (approximately 33,000 amino acids) and is an essential component in sarcomere assembly and force transmission. In 2012, using next-generation sequencing, Herman and colleagues identified radical mutations (nonsense/frameshift/splice site/copy number variant) in 54 of 203 patients with DCM (27%) with strong cosegregation data. 26 The frequency of these mutations was similar between FDCM (25%) and sporadic DCM (18%), whereas large control and HCM cohorts had a 1% frequency. Interestingly, another group of 149 DCM patients sequenced with traditional dideoxy-sequencing identified radical TTN mutations in a much lower proportion (9%). Penetrance for DCM was 95% in affected families and associated conduction or skeletal muscle disease was usually absent. Mutations in LMNA (encoding lamin A and lamin C, a nuclear envelope protein crucial for the structural integrity of the nucleus) are a common genetic cause of DCM, with a frequency of 4% in nonfamilial DCM and 8% with FDCM. 31 LMNA genetic defects often give rise to electrical conduction disease ( 90%), which may occur before the onset of heart failure 31 and sometimes with noncardiac phenotypes (Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy). 32 Patients with LMNA mutations have a high rate of heart failure (mostly appearing between 20 and 39 years of age) 25 and SCD: older than 40 years, approximately 50% have SCD or

5 428 Perrin and Gollob appropriate ICD therapy; 20 to 40 years old, approximately 20%; and younger than 20 years, 7%. 33 Sarcomeric mutations are identified in 10% or more of cases of FDCM 34 ; the more commonly identified genes include MYH7 (4.8% overall) ), TNNT2 (2.3% overall), 35,36 MYH6 (2.5%), and MYBPC3 (4.2%). 28 In families in whom the disease segregates clearly with the mutation, absence of a predilated hypertrophic phase has been established 34 ; however, it is possible that some identified DCM probands with sarcomeric mutations represent a final end-stage of HCM. The pleiotropism of sarcomeric mutations may be understood as mutationspecific interference with force generation (HCM) or force transmission (DCM). 13,37 Yet, it is not clear how a single mutation (eg, K247R in TNNT2) may alternatively cause HCM or DCM. Mutations in the SCN5A gene (encoding the voltagegated sodium channel, Nav1.5) may result in many phenotypes: BrS, LQT3, conduction disease, idiopathic ventricular fibrillation, and atrial fibrillation. Olson et al demonstrated by linkage analyses that a single mutation in SCN5A (D1275N) could cause DCM but also display marked phenotypic variability with isolated and combined phenotypes of DCM, atrial fibrillation, impaired automaticity, and conduction delay in mutation carriers in a single family. Interestingly, no mutation carrier in this study demonstrated prolonged QT intervals or ST elevation in the right precordial leads. 29 SCN5A mutations occur with a frequency approximately 2.5% in unrelated DCM probands. 29,36 Ribonucleic acid binding motif protein 20 (the RBM20 gene) is expressed strongly in the heart and hypothesized to regulate cardiac splicing. Brauch et al through linkage analysis identified a mutation in RBM20 segregating with the DCM phenotype and associated with a malignant clinical course. Subsequently, 278 unrelated probands were screened identifying mutations in RBM20 in six, all localizing to exon 9 (amino acids 634 to 638). 30 Li et al screened 312 DCM probands, identifying six novel mutations, with four localized to the exon 9 hotspot. 38 Again, a malignant clinical course demonstrated by heart failure, transplantation, and arrhythmia was noted. The overall frequency of mutations was 2%. This genotype-phenotype correlation was recently challenged by Refaat et al, who, while identifying mutations in 3% of probands, recorded a clinical course in affected individuals similar to other DCM probands. Unlike the previous two studies, only one mutation of eight identified localized to the hotspot. 39,40 Recommendations for Genetic Testing in DCM The yield of genetic testing is increased in FDCM and, excluding TTN, thought to be low in sporadic cases. Current guidelines recommend against testing in sporadic DCM (after establishment of no identified affected relatives). 2,41 Because of the marked genetic heterogeneity of the condition, targeted testing of the most commonly affected genes is advised, including TTN, LMNA, MYH7, SCN5A, MYBPC3, TNNT2, and MYH6. When genetic test results are negative, RBM20 and PLN (phospholamban), and possibly TPM1 and TNNC1, may be assessed. With the recent identification of TTN-associated DCM, a genetic cause of DCM may be identified in up to 40% of cases. Clinical features are of limited use in guiding genetic testing, but two clinical phenotypes may be helpful: DCM with prominent cardiac conduction disease is more common with LMNA or SCN5A mutations, whereas X-linked inheritance of DCM should prioritize testing of the dystrophin gene (DMD). 2 Arrhythmogenic Right Ventricular Cardiomyopathy ARVC is characterized pathologically by fibrofatty replacement of the RV myocardium and genetically as a disease of the desmosome. Males are more commonly diagnosed as having this condition (3:1). The estimated prevalence of ARVC is 1 in 5000, but some areas in Italy have a prevalence as high as 4.4 per Treatment of ARVC involves restriction of high-intensity exercise and the implantation of an ICD in the event of ventricular arrhythmia. 2 Radiofrequency ablation of putative electrical circuits in the RV may reduce ICD shocks in patients with incessant arrhythmia. Ventricular arrhythmia occurs early in the disease, preceding symptoms of RV failure by years or even decades. Of concern, the earliest concealed stage of disease is characterized by a normal ECG with minimal structural change and a risk of SCD during exercise. 42 ARVC is a significant cause of SCD in the young. Although traditionally considered a disease of the RV, careful investigation reveals involvement of the LV in many cases. 43 In some genetic forms, LV disease may be predominant To aid in the early diagnosis of ARVC, criteria have been devised (1994 task force) 47 that incorporate the structural, histologic, electrical, and familial features of the condition. These criteria, although highly specific, lack sensitivity. In 2010, the criteria were updated to include the identification of a pathogenic mutation as a major criterion for the diagnosis along with other changes that reflect a growing understanding of the disorder. 48 Genetics of ARVC Genetic causes of ARVC principally involve desmosomal proteins, which are essential for the structural integrity and normal function of cell-to-cell junctions. 48 Desmosomes are abundant in both the heart and skin. Consistent with this understanding, the first identified genetic cause of ARVC was a homozygous mutation in plakoglobin (JUP), causing Naxos syndrome (characterized by ARVC, palmoplantar keratoderma, and woolly hair). 49 By virtue of a nomenclature based on identified loci linked to ARVC, Naxos syndrome is sometimes referred to as ARVC12. Subsequent studies demonstrated further desmosomal protein genotypes: desmoplakin (DSP) inherited as an autosomal recessive disease, often associated with skin disease and woolly hair, 50 and an autosomal dominant form, 51

6 Genetics of Sudden Cardiac Death 429 plakophilin 2 (PKP2), 52 desmoglein 2 (DSG2), 53 and desmocollin 2 (DSC2). 54 In 2008, transmembrane protein 43 (TMEM43) was identified as the cause of ARVC5 (locus 3p23). 46 A single missense mutation (S358L) was shown to segregate completely with disease in 15 unrelated families from Newfoundland in Canada. ARVC5 displays 100% penetrance, a high risk of SCD, and a phenotype similar to desmosomal ARVC with the addition of prominent LV involvement. The mechanism of disease in ARVC5 is unknown. Nava et al described a family with juvenile SCD, effortinduced polymorphic ventricular tachycardia, and RV abnormalities, including fibrofatty replacement on autopsy. 55 On the basis of the RV disease, this condition has sometimes been called ARVC2. Linkage studies localized to the disease gene to 1q42, where a mutation in RyR2 was found to segregate with disease. 56 However, mutations in RyR2 are now understood as the common cause of catecholaminergic polymorphic ventricular tachycardia (CPVT). It is likely that the phenotype of ARVC2 resembled that of CPVT mimic those seen in ARVC. The causative genes at disease linked loci for ARVC3 (14q12-q22), ARVC4 (2q32.1-q32.3), ARVC 6 (10p12-14), and ARVC 7 (10q22) are yet to be identified. Recommendations for Genetic Testing in ARVC The yield for genetic testing of the five desmosomal genes in patients meeting task force criteria is 45% to 58%, with 67% to 90% of mutations identified in PKP2. 45,57 If a family history of ARVC is demonstrated, the yield of genetic testing may be as high as 90%. 57 Because ARVC commonly has incomplete penetrance or asymptomatic status, genetic testing of the proband is indicated primarily for the purpose of cascade family screening to ensure appropriate medical surveillance. Because of the predominance of PKP2 as a genetic cause of ARVC, current guidelines recommend a tiered approach to genetic testing. In those patients without a PKP2 mutation, additional testing in DSC2, DSG2, and DSP may identify a genetic cause in 5% to 10% of cases. 2 Select clinical data may guide genetic testing. When a family traces its ancestry to Newfoundland, TMEM43 testing is suggested. When LV involvement is predominant in a pedigree, genetic testing for DSP, DSG2, and TMEM43 mutations should be included in the first round of testing. Lastly, autosomal recessive inheritance of ARVC, especially when associated with a syndrome of skin disease and woolly hair, favors testing in JUP and DSP. The identification of a mutation requires interpretation and should not be considered as a positive or negative result. The strongest evidence of pathogenicity is the identification of a genetic mutation that segregates with disease in a large family either reported in the literature or observed through personal investigation. Kapplinger et al examined the background noise of genetic variability in desmosomal genes in a healthy control population. 58 Missense mutations in all desmosomal genes were common, especially in people of races other than white. Mutations in DSP, TMEM43, and DSC2 occurred with similar frequency between controls and ARVC patients. However, certain mutation characteristics were shown to strongly suggest pathogenicity: any radical mutation, any missense mutation in a white person, a missense mutation in PKP2 or DSG2 highly conserved across species, or a missense mutation in the N-terminal region of DSP or DSG2. 58 Familial Cardiac Syndromes without Structural Heart Disease Long QT Syndrome Congenital LQTS is marked by a prolonged QT interval, a peculiar form of polymorphic ventricular tachycardia torsades de pointes (twisting of the points) and a risk of SCD. LQTS is more commonly diagnosed in females (2:1). Population prevalence may reach 1:2000. LQTS is usually identified in the investigation of syncope in a young person and sometimes incidentally on an ECG performed for other reasons. Beta-adrenergic blockade is indicated for most patients with this condition, with an ICD reserved for those at very high risk or with breakthrough cardiac events while taking medication. Diagnosis is straightforward when typical cardiac symptoms (eg. syncope and SCD) accompany a very long corrected QT interval (QTc) (QTc 470 milliseconds in men and 480 milliseconds in women). However, up to 50% of LQTS gene carriers have a normal or borderline QTc. 59 T-wave morphologic features, which are specific for genotype, may suggest the diagnosis, 60 although provocation by epinephrine infusion or exercise treadmill testing 59 is often required to expose an abnormal QT adaptive response to increasing heart rate. Triggers to syncope or SCD in LQTS are genotype specific. In LQT1, cardiac events (eg, syncope and SCD) occur with exercise (55%) but also at sleep or rest (21%) and with arousal (loud noises or emotion) (14%). 61 In LQT2, the proportions reverse: arousal is the most common trigger (43%), followed by sleep or rest (29%) and exercise (13%). 62 Cardiac events in LQT3 occur predominantly during sleep or rest (64%). 62 Beta-adrenergic blockers are effective in reducing the risk of exercise-triggered cardiac events, including SCD, in LQT1 and LQT2. 61,63 They are, however, relatively ineffective in protecting against events triggered by sudden arousal or occurring at sleep or rest. 63 Because these triggers predominate in LQT2 and LQT3, the overall efficacy of -blockade in these subtypes is reduced. Therefore, in LQT2, a -blocker is administered along with advice to avoid possible arousal triggers (eg, loud telephones and door alarms); in LQT3, genotype-specific therapy with the sodium channel blocker mexiletine may reduce events. 64 All subtypes of LQTS are advised to avoid QT-prolonging drugs. Left cardiac sympathetic denervation reduces SCD in high-risk LQTS patients and may be considered where expertise for the procedure exists. 65 An ICD is reserved

7 430 Perrin and Gollob Table 2. Subtypes of LQTS Subtype Gene Protein/ionic current Pathophysiology Frequency, % LQT1 and JLN KCNQ1 Kv7.1, I Ks 2 I Ks LQT2 KCNH2 Kv11.1, I Kr 2 I Kr LQT3 SCN5A Nav1.5, I Na 1 Late I Na 5 10 LQT4 ANK2 Ankyrin B Loss of coordinated expression of NCX, 1 Na/K ATPase, and InsP 3 R LQT5 and JLN KCNE1 hmink, -subunit of I Ks 2 I Ks 1 LQT6 KCNE2 MiRP, -subunit of I Kr, possibly others 2 I Kr 1 LQT7 KCNJ2 Kir2.1, I K1 2 I K1 1 LQT8 CACNA1c Cav1.2, I CaL 1 I CaL 1 LQT9 CAV3 Caveolin-3 1 Late I Na 1 LQT10 SCN4B Na v 4, -subunit of I Na 1 Late I Na 1 LQT11 AKAP9 Yotiao 2 I Ks 1 LQT12 SNTA1 1 -Syntrophin 1 Late I Na 1 LQT13 KCNJ5 Kir3.4, I KACh 2 I KACh 1 1, increased; 2, decreased; I CaL, L-type calcium current; I Na, voltage-gated sodium current; I KACh, acetylcholine activated potassium current; I Kr, rapid delayed rectifier potassium current; I Ks, slow delayed rectifier potassium current; I K1, inward rectifier potassium current; InsP 3 R, inositol triphosphate receptor; JLN, Jervell and Lange-Nielsen; NCX, sodium/calcium exchanger. for patients with recurrent events despite treatment, where a contraindication to medical therapy exists, or in very high-risk individuals. Genetics of LQTS In 1991, linkage analyses of a large autosomal dominant LQTS kindred identified a disease locus at 11p15.15 (LQT1) 66 ; the discovery of other LQTS loci at 7q35-35 (LQT2) and 3p21-24 (LQT3) confirmed the genetic heterogeneity of the condition. 67 Shortly thereafter, mutations in the KCNH2 gene, encoding the pore-forming -subunit of the rapid delayed rectifier potassium channel (Kv11.1), were identified in 6 families with LQT2. 68 In the space of a year, KCNQ1 ( -subunit of the slow delayed rectifier potassium channel, Kv7.1) 69 and SCN5A ( -subunit of the voltage gated sodium channel, Nav1.5) 70 were revealed as the cause of LQT1 and LQT3, respectively. Mutations in these three genes are now known to account for 90% of genetically confirmed LQTS (Figure 1). 41 At present, mutations in 10 other genes have been implicated in autosomal dominant LQTS and are often referred to by their subtype number, reflecting the order of their discovery (Table 2). Each of LQT4 through LQT13 mutation accounts for 1% of genotype-positive LQTS. The autosomal recessive form of LQTS, Jervell-Lange Nielsen syndrome, characterized by congenital deafness and a very high risk of SCD, arises from mutations that affect both alleles of KCNQ1 71 or its -subunit, KCNE1. 72 Recommendations for Genetic Testing in LQTS When the diagnosis of LQTS is certain (eg, women with a QTc 480 milliseconds or men with a QTc 470 milliseconds), genetic testing is still useful to provide risk stratification, aid in family screening, and guide therapeutic decision making. Genetic testing may also diagnose LQTS in symptomatic patients with borderline QT intervals, particularly when T-wave morphologic features are abnormal 2 or when provocative epinephrine or exercise testing suggests the diagnosis. 59 LQT1 and LQT2 each account for 40% to 45% of genotype-positive LQTS, with LQT3 accounting for a further 5% to 10%. Together, LQT4 through LTQ13 represent 5% of positive genotypes. 41,73 Given this information, targeted genetic testing of KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 is currently recommended. 2 When this test result is negative and clinical suspicion remains high, testing for copy number variants (deletions and duplications) in KCNQ1 and KCNH2, representing 3% to 5% of genotypenegative LQTS, 73,74 and LQT4 and LQT7 through LQT13 may be performed. Even with comprehensive testing up to 20% of patients will remain genotype negative. Despite extensive research into LQTS, many identified mutations from such testing will be novel and return as a variant of unknown significance, depending on the identified gene and location. When published reports or family testing is not available to demonstrate segregation of the variant with disease, the type (missense or nonmissense) and location of the mutation may help determine pathogenicity. In general, mutations in LQTS are more common in LQTS patients than controls, 75 although the rarer a cause (eg, LQT4 through LQT13), the more likely that the variant of unknown significance represents a benign variant. The presence of a non-missense mutation, however, is 99% predictive of LQTS in LQT1 through LQT3. 75 Missense mutations, when pathogenic, are more likely to produce dominant-negative suppression of normal channel function and a more severe phenotype. However, in the absence of functional analysis, they may represent a benign variant, particularly when the phenotype is borderline or when segregation analysis is not possible. When deciding between these possibilities, location of the genetic variant may be very helpful. Kapa et al have reported, for instance, that mutations in the pore, transmembrane, and linker regions of KCNH2 are almost always pathogenic; similarly, mutations in the linker, pore, transmembrane, and C-terminus of KCNQ1 are usually pathogenic. In SCN5A, mutations appearing in the interdomain linker appear at similar frequencies in cases and controls; conversely, mutations in the transmembrane/linker of SCN5A are usually pathogenic. 75

8 Genetics of Sudden Cardiac Death 431 Brugada Syndrome BrS is characterized by ST elevation in the right precordial leads (V1 and V2) on a 12-lead ECG, a structurally normal heart, and a risk of SCD. 76 BrS usually manifests in adulthood and accounts for 4% of SCD overall and 20% of SCD where the heart is structurally normal. Events often occur at night or rest. 77 Population prevalence is estimated at 1 in 2000 but is likely much higher in southeast Asia. 77 Male incidence is 8 to 10 times that of women. Considerable controversy surrounds the treatment of asymptomatic patients with the observed ECG pattern of BrS. Notably, a family history of SCD does not seem to predict events. 78 Preliminary evidence indicates that quinidine may be an effective drug treatment, having shown efficacy in VF storms associated with BrS and in nonrandomized trials of symptomatic patients. 79 Genetics of BrS Loss-of-function mutations in SCN5A (voltage gated sodium channel, Nav1.5) are the most frequent genetic cause of BrS, identified in 11% to 28% of probands. 80,81 Typically, SCN5A mutation carriers compared with genenegative individuals have ECG evidence of impaired cardiac conduction and an exaggerated increase in QRS duration with sodium channel blocking drugs. 82 The cardiac L-type calcium channel in the heart is composed of at least three subunits, encoded by CACNA1C (pore-forming -subunit), CACNB2b ( -subunit), and CACNA2D1 ( 2 - subunit). Loss-of-function mutations in CACNA1C and CACNB2b in association with BrS and short QT intervals (three of seven patients with intervals 360 milliseconds) were first reported in A more comprehensive screening of 162 unrelated BrS probands revealed mutations in CACNA1C, CACNB2b, or CACNA2D1 in 12.3% of patients, although functional and/or familial data were not available for most mutations. 84 In all, 10 genetic types of BrS have been formally tabulated, although it may be better to consider this as a listing of BrS susceptibility genes. Many identified genetic causes are in isolated individuals, with no phenotypic data available to separate one type from the other (Table 3). Recommendations for Genetic Testing in BrS At present, genetic testing for SCN5A mutations is recommended with the highest yield in familial cases. 2 Consideration could also be given to testing the component proteins of the L-type calcium channel (CACNA1C, CACNB2b, and CACNA2D1) 84 ; however, because population frequency of rare variants in these genes is unknown, careful review of identified mutations is necessary (eg, assessment of level of conservation, segregation in the pedigree if available). The remaining susceptibility genes displayed in Table 3 have a yield 1%, and therefore testing is not indicated outside the research setting. Importantly, the rarer the association of the condition with a gene, the more challenging it is to interpret the genetic results. Negative genetic test results in BrS provide no reassurance to family members because of the low overall yield. However, the presence of a pathogenic mutation may aid in screening family members because the Brugada ECG pattern may be absent or intermittent in family members. Catecholaminergic Polymorphic Ventricular Tachycardia CPVT is characterized by sympathetically mediated ventricular arrhythmia in the absence of structural heart disease. CPVT is rare, with an estimated prevalence of 1:10,000. Symptoms typically begin in childhood, with a mean age of onset between 7 and 10 years and affecting men and women equally. 85,86 A family history of exerciseinduced syncope or SCD is present in 30%. 85 Exercise restriction and -blocker therapy are the mainstays of treatment. The resting ECG is usually normal in CPVT, but reproducible ventricular arrhythmia on exercise testing (premature ventricular beats and polymorphic ventricular tachycardia, often bidirectional) occurs in 75% to 100% of patients. 85,86 Bidirectional ventricular tachycardia, characterized by beat-to-beat alternation of the QRS morphologic features on the ECG, is specific for CPVT but may rarely occur in LQT4 (Ankyrin-B disease), in LQT7 (Andersen-Tawii syndrome), or with digoxin toxicity. A convergence of evidence supports adrenergicallytriggered abnormal calcium release from the sarcoplas- Table 3. BrS Susceptibility Genes Subtype Gene Protein/ionic current Pathophysiology Frequency, % BrS1 SCN5A Nav1.5, I Na 2 I Na BrS2 GPD1-L glycerol-3-phosphate dehydrogenase 2 I Na 1 BrS3 CACNA1C Cav1.2, I CaL 2 I CaL 5 BrS4 CACNB2B -Subunit of I CaL 2 I CaL 5 BrS5 SCN1B 1 -Subunit of I Na 2 I Na 1 BrS6 KCNE3 MiRP2 1 I to 1 BrS7 SCN3B 3 -Subunit of I Na 2 I Na 1 BrS8 KCNJ8 Kir6.1, I KATP 1 I KATP 1 BrS9 CACNA2D Subunit of I CaL 2 I CaL 2 BrS10 KCND3 Kv4.3, I to 1 I to 1 The strongest evidence for disease causation exists for BrS1 2 (linkage analyses), whereas causation for BrS3 6 is very likely (cosegregation of BrS with identified mutations in smaller kindreds) and not yet definite for the remaining subtypes. 1, increased; 2, decreased; I CaL, L-type calcium current; I Na, voltage-gated sodium current; I KATP, ATP sensitive potassium current; I Kr, rapid delayed rectifier potassium current; I Ks, slow delayed rectifier potassium current; I K1, inward rectifier potassium current; I to, transient outward potassium current.

9 432 Perrin and Gollob mic reticulum during cardiac diastole as the cause of arrhythmia in CPVT (Figure 1). Consequently, the foundation of treatment in CPVT is high-dose -adrenergic blockade and restriction of moderate- to high-intensity exercise. Large cohort studies report breakthrough arrhythmia in 23% to 46% of patients with -blockade. 85,87 Therefore, additional therapies have been advocated, of which flecainide is the most promising. 88 Left cardiac sympathetic denervation reduces the risk of SCD in patients with refractory arrhythmia despite medical therapy or where a contraindication to such therapy exists. 65 ICD implantation may be performed for refractory events. Genetics of CPVT In contrast to the other genetic cardiac electrical diseases, CPVT displays remarkable genetic homogeneity. RyR2 is currently the lone genetic cause of autosomal dominant CPVT. Priori et al first identified mutations in the RyR2 gene in 2001 in four of 12 probands with CPVT; each of the cases with CPVT had a de novo mutation in the single pedigree with a family history that identified mutation segregated with disease. 89 Since the initial description, 100 different mutations have been implicated in CPVT, 90 with most occurring in three discrete regions of RyR2: the N-terminus, central domain, and channel region. At present, 25% to 40% of patients diagnosed as having CPVT are genotype negative, 87,90 suggesting a degree of genetic heterogeneity. Interestingly, the clinical profile of genotype-negative patients is different than those with an identified RyR2 mutation, showing a later Table 4. SQTS Diagnostic Score Variable Points QTc and J point QTc 370 milliseconds 1 QTc 350 milliseconds 2 QTc 330 milliseconds 3 J-point and T-peak interval 120 milliseconds 1 Clinical history History of sudden cardiac death 2 Documented polymorphic ventricular 2 tachycardia or ventricular fibrillation Unexplained syncope 1 Atrial fibrillation 1 Family history First- or second-degree relative with high probability SQTS First- or second-degree relative with autopsynegative SCD 1 Sudden infant death syndrome 1 Genotype Genotype positive 2 Mutation of undetermined significance in a culprit gene A score of 4 points indicates high probability SQTS; 3 points, intermediate-probability SQTS; 2 points, low probability SQTS. The J-point and T-peak interval is measured in the precordial lead with the greatest amplitude of T wave. Points can only be received once for SCD, polymorphic ventricular tachycardia, or unexplained syncope. For family history, points may be received only once in this section. At least 1 point must be obtained from the QTc and J point section for the diagnosis of SQTS Table 5. Subtype SQTS Subtypes Gene median age of onset (20 versus 7 to 10 years) and female predominance but a similarly severe clinical phenotype and response to antiadrenergic therapy. 85 The possibility of undetectable structural disease mimicking a CPVT phenotype cannot be excluded in these cases. A smaller proportion of CPVT cases are inherited in an autosomal recessive manner. Initial linkage analyses mapped the culprit gene to chromosome 1, where a homozygous mutation in the calsequestrin-2 gene (CASQ2) was identified in affected individuals. 91 Recommendations for Genetic Testing in CPVT Targeted exon screening (ie, sequencing of exons where mutations are likely to be found) is currently advised because of the large size of the RyR2 gene (105 exons) and the clustering of pathogenic mutations in three distinct regions: N-terminus, central domain, and channel region. The estimated yield of RyR2 testing is approximately 50% to 60%. When the initial targeted exon screening result is negative and the clinical suspicion remains high, extended sequencing of the entire gene is advised. CASQ2 sequencing is indicated when an autosomal recessive mode of inheritance is observed. 2 When a causative gene is identified, genetic testing may be extended first to the parents (where possible) of the proband. If they do not carry the causative gene, genetic testing may be safely withheld from other family members. The possibility of germline mosaicism provides the only caution to this approach. 90 Exercise treadmill testing may be performed in the siblings of the proband with subsequent genetic testing if clinically indicated to identify this rare occurrence. Short QT Syndrome Protein/ionic current Pathophysiology Frequency, % SQTS1 KCNH2 Kv11.1, I Kr 1 I Kr SQTS2 KCNQ1 Kv7.1, I Ks 1 I Ks 5 SQTS3 KCNJ2 Kir2.1, I K1 1 I K1 5 1, increased; 2, decreased; I Kr, rapid delayed rectifier potassium current; I Ks, slow delayed rectifier potassium current; I K1, inward rectifier potassium current. SQTS is a very rare disease characterized by an abbreviated QT interval and a high risk of atrial and ventricular arrhythmia. Patients with SQTS tend to present early in life (median age of approximately 21 years), are predominantly male (75%), and have high rates of SCD. 92 The population prevalence is not known but is likely 1 in 100,000. Quinidine has shown some efficacy in management, but an ICD is often indicated. Similar to LQTS, the diagnosis of SQTS is confounded by marked overlap of QT intervals between affected and healthy individuals. The distribution of QT intervals in the population is bell-shaped; a short (or long) QT is defined arbitrarily as that falling outside two standard deviations

10 Genetics of Sudden Cardiac Death 433 from the mean: 350 milliseconds ( 450 milliseconds) for males and 360 milliseconds ( 460 milliseconds) for females. 93 However, because SQTS is rare, it is more likely that a short QT interval represents an outlier of the normal population rather than a person with this syndrome. The diagnosis of SQTS cannot, therefore, be made on the basis of QT interval alone except perhaps at the very extreme (QTc 320 milliseconds). Instead, diagnosis should be based on the identification of an arrhythmogenic substrate, which, although not directly measurable, may be inferred on the basis of an identified diseasecausing mutation or else by the self-declared history of the patient (eg, SCD) or their family. This general principle of diagnosis is now formalized in a SQTS diagnostic score incorporating the degree of QT shortening, clinical and family history, and identified genotype (Table 4). 92 Quinidine restores a short QT interval to near normal duration, particularly in individuals with KCNH2 mutations, and may reduce arrhythmic events. 94 When quinidine is ineffective or life-threatening events complicate SQTS, an ICD is recommended. Genetics of SQTS In 2004, Brugada et al identified mutations in KCNH2 (encoding the -subunit of the rapid delayed rectifier potassium channel, Kv11.1) in two of three families with SQTS, now called SQTS1 (Table 5). 95 Both mutations produced the same amino acid change, N588K, resulting in a marked increase (gain of function) in repolarizing current (a large repolarizing current serves to shorten the QT interval, whereas a diminished current prolongs the QT interval). Since the first report of Brugada et al, three other SQTS mutations in KCNH2 have been reported: R1135H, E50D, and T618I. 92 Gain-of-function mutations in KCNQ1 (encoding the -subunit of the slow delayed rectifier potassium channel, Kv7.1) 96 and KCNJ2 (encoding the -subunit of the inward rectifier potassium channel, Kir2.1) 97 have been likewise associated with SQTS. Loss-of-function mutations in components of the L-type calcium channel, CACNA1C (encoding the -subunit of Cav1.2), CACNB2b (encoding the -subunit of Cav1.2), 83 and CACNA2D1 (encoding the 2 -subunit of Cav1.2), 98 have been reported as causes of SQTS. Antzelevitch et al identified three probands with mutations in CACNA1C or CACNB2B who displayed mild shortening of the QT interval (mean QTc of 345 milliseconds) and right-sided precordial ST elevation typical of BrS. 83 Because the phenotype of BrS is predominant in these cases, we believe that such patients should be classified under that disorder. 92 In the report of CACNA2D1, a single proband with a QTc of 329 milliseconds had aborted cardiac arrest. The test results for BrS were negative; however, two family members with the genotype were clinically unaffected. 98 Recommendations for Genetic Testing in SQTS In a recent review of reported cases of SQTS, 17 of 42 genotyped individuals (40%) (excluding CACNA1C and CACNB2b) harbored a mutation in KCNH2 (n 14), KCNQ1 (n 1), or KCNJ2 (n 2). 92 These figures may overestimate the yield of genetic testing because of publication bias favoring a more severe phenotype. In the largest case series reported to date, which included asymptomatic individuals with short QT intervals, a causative gene was found in 23% of probands (4/22 in KCNH2 and 1/22 in CACNB2b). 94 Genetic testing cannot be recommended on the basis of a short QT interval alone. However, genetic testing for KCNH2/KCNQ1/KCNJ2 is reasonable when a diagnostic score for SQTS predicts a high probability of the condition. 92 In the instance where an ECG phenotype of BrS is associated with an abbreviated QT interval, genetic testing should follow the recommendations for BrS. Conclusions Through 20 years of genetics research and discovery, the advent of genetic diagnosis and management of familial cardiac syndromes has arrived. Although much has become known, the recent (2012) identification of titin (TTN) as the predominant genetic cause of DCM is a reminder that large impact discoveries remain to be made. Further research is required to determine the genetic underpinnings of the genotype-negative patients diagnosed, and to elucidate the role of gene-environment interactions and disease-modifying genes in predicting disease severity. Larger cohort studies are necessary to accurately ascertain genotype-phenotype correlation and the value of genetic information in directing treatment decisions. However, in the least, the available genetic knowledge has proven to be a powerful tool for the diagnostic confirmation of uncommon diseases and for cascade screening for the purpose of medical surveillance and prevention strategies in asymptomatic, at-risk family members. Perhaps most important, knowledge gained from understanding the genetic basis and molecular mechanisms of disease will ultimately lead to the development of targeted therapies through novel drug development and/or gene therapy. References 1. Liberthson RR: Sudden death from cardiac causes in children and young adults. N Engl J Med 1996, 334: Gollob MH, Blier L, Brugada R, Champagne J, Chauhan V, Connors S, Gardner M, Green MS, Gow R, Hamilton R, Harris L, Healey JS, Hodgkinson K, Honeywell C, Kantoch M, Kirsh J, Krahn A, Mullen M, Parkash R, Redfearn D, Rutberg J, Sanatani S, Woo A: Recommendations for the use of genetic testing in the clinical evaluation of inherited cardiac arrhythmias associated with sudden cardiac death: Canadian Cardiovascular Society/Canadian Heart Rhythm Society Joint Position Paper. Can J Cardiol 2011, 27: Maron BJ, Gardin JM, Flack JM, Gidding SS, Kurosaki TT, Bild DE: Prevalence of hypertrophic cardiomyopathy in a general population of young adults: echocardiographic analysis of 4111 subjects in the CARDIA Study. Circulation 1995, 92: Gersh BJ, Maron BJ, Bonow RO, Dearani JA, Fifer MA, Link MS, Naidu SS, Nishimura RA, Ommen SR, Rakowski H, Seidman CE, Towbin JA, Udelson JE, Yancy CW: 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary. J Am Coll Cardiol 2011, 58: