Impact of Genetics on the Clinical Management of Channelopathies

Abstract

There are few areas in cardiology where the impact of genetics and of genetic testing on clinical management has been as great as in cardiac channelopathies, arrhythmic disorders of genetic origin related to the ionic control of the cardiac action potential. Among the growing number of diseases identified as channelopathies, three are sufficiently prevalent to represent significant clinical and societal problems and to warrant adequate understanding by practicing cardiologists: long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and Brugada syndrome.

This review will focus selectively on the impact of genetic discoveries on clinical management of these three diseases. For each disorder, we will discuss to what extent genetic knowledge and clinical genetic test results modify the way cardiologists should approach and manage affected patients. We will also address the optimal use of genetic testing including its potential limitations and the potential medico-legal implications when such testing is not performed. We will highlight how important can be to understand the ways by which genotype can impact clinical manifestations, risk stratification, and responses to the therapy. We will also illustrate the close bridge between molecular biology and clinical medicine, and will emphasize that consideration of the genetic basis for these hereditable arrhythmia syndromes, as well as the proper use and interpretation of clinical genetic testing, should remain the standard-of-care.

The discovery of the first three long QT syndrome (LQTS)-susceptibility genes in 1995-96 (1-3) had a transformative effect on the diagnosis and treatment of arrhythmias. It opened the way to the realization that molecular biology could no longer be regarded as “something weird, with an unfriendly jargon and of no interest for a clinician”; it allowed the understanding of how even simple aminoacid substitutions (missense mutations) due to a single nucleotide substitution could produce significant functional alterations in cellular electrophysiology. In addition, by showing that most of the disease genes for a number of arrhythmic disorders of genetic origin were involved in the ionic control of the cardiac action potential, it led to describe these disorders as “cardiac channelopathies”. Besides some very rare disorders, there are three truly important genetic heart rhythm diseases whose ignorance by practicing cardiologists could cost the life of the unfortunate patients seeking their medical advice. They are LQTS, catecholaminergic polymorphic ventricular tachycardia (CPVT), and Brugada syndrome (BrS).

Here, we will only touch briefly on the main features of these potentially life-threatening yet highly treatable diseases (at least LQTS and CPVT), as many thorough clinical reviews are available (4-8). Instead, our focus will be centered selectively on discussing the impact exerted on clinical management by the progressive unraveling of the genetic mechanisms underlying these diseases. Specifically, we will examine for each of them whether, how, and to what extent genetic test results should modify the way a cardiologist should approach and manage the mutation-positive (i.e. affected) patients.

The space given to the 3 diseases will be different. LQTS is undoubtedly the one that demonstrates best how close is the bridge between molecular biology and clinical medicine because it is the one in which the genotype-phenotype relationship has been best understood in terms of clinical manifestations, risk stratification, and response to the therapy. We trust that the readers will realize that, for patients with channelopathies, molecular genetics and clinical management should go hand in hand.

Long QT Syndrome

LQTS represents a leading cause of autopsy-negative sudden death in the young (9). It is characterized typically by a prolongation of the QT interval on the ECG and by the occurrence of syncope or cardiac arrest, mainly precipitated by emotional or physical stress; however, some deaths occur when patients are at rest or asleep. LQTS includes the relatively common Romano-Ward (RW) variant, which has a prevalence of 1:2000 live births (10), and the rare and extremely severe Jervell and Lange-Nielsen (JLN) syndrome accompanied by congenital deafness (11). The inheritance mode for RW is autosomal dominant or sporadic, whereas JLN shows autosomal recessive inheritance or sporadic cases of compound heterozygosity (11). LQTS contributes to Sudden Infant Death Syndrome (12) and even to stillbirths (13).

The ventricular tachyarrhythmia that underlies the cardiac events of LQTS is torsades-de-pointes (TdP). This highly specific type of ventricular tachycardia is often self-limiting, thus producing transient syncope, but TdP can also degenerate into ventricular fibrillation and cause cardiac arrest or sudden death. We still don't know why in certain patients TdP stops after few seconds whereas in others it continues with devastating consequences.

The morphology of the T wave is often useful for the diagnosis, and the lateral precordial leads are especially informative when they reveal biphasic or notched T waves (14). T-wave alternans in polarity or amplitude is a marker of major electrical instability and, when observed, is diagnostic (15). In the presence of syncopal episodes occurring under stressful conditions in an individual with marked prolongation of the QT interval, the diagnosis of LQTS is rather simple. For the more questionable situations, the so-called Schwartz score, originally proposed in 1993 (16) and updated more recently (6) (Table 1) can help. The standard and very effective therapy for LQTS is based on β-blockers [propranolol and nadolol, whereas metoprolol has been linked to frequent recurrences (17)] which should be administered also to still asymptomatic patients with QT prolongation, given the potential for sudden death as first disease manifestation. For patients on full-dose β-blocker therapy in the case of a first recurrence of syncope left cardiac sympathetic denervation (18,19) is the treatment of choice whereas in the case of cardiac arrest, recurrent syncope or of signs of very high risk an implantable cardioverter defibrillator (ICD) is appropriate (8,20).

Table 1. 1993-2012 LQTS Diagnostic Criteria.

			Points
ELECTROCARDIOGRAPHIC FINDINGS#
		≥480 ms	3
A	QTcˆ	460 – 479 ms	2
		450 – 459 (male) ms	1
B	QTcˆ 4th minute of recovery from exercise stress test ≥ 480 ms		1
C	TORSADE DE POINTES *		2
D	T WAVE ALTERNANS		1
E	NOTCHED T WAVE IN 3 LEADS		1
F	LOW HEART RATE FOR AGE @		0.5
CLINICAL HISTORY
A	SYNCOPE *	WITH STRESS	2
		WITHOUT STRESS	1
B	CONGENITAL DEAFNESS		0.5
FAMILY HISTORY
A	FAMILY MEMBERS WITH DEFINITE LQTS $		1
B	UNEXPLAINED SUDDEN CARDIAC DEATH BELOW		0.5
	AGE 30 AMONG IMMEDIATE FAMILY MEMBERS $

^#In the absence of medications or disorders known to affect these electrocardiographic features

^ˆQTc calculated by Bazett's formula where QTc = QT/√RR

^*Mutually exclusive

^@Resting heart rate below the 2^nd percentile for age

^$The same family member cannot be counted in A and B

Score: ≤ 1 point: low probability of LQTS

1.5 to 3 points: intermediate probability of LQTS

≥ 3.5 points high probability

(From ref. 6)

Updated Genetics

Sixteen genes have been identified so far as either responsible for or associated to LQTS (Table 2). The three main genes, KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3), account for approximately 75% clinically definite LQTS whereas the minor genes contribute an additional 5% collectively. An estimated 20% of LQTS remains genetically elusive.

Table 2. Molecular Basis of Cardiac Channelopathies.

Gene	Locus	Protein
LONG QT SYNDROME
Major LQTS Genes
KCNQ1 (LQT1)	11p15.5	IKs potassium channel alpha subunit (KVLQT1, KV7.1)
KCNH2 (LQT2)	7q35-36	IKr potassium channel alpha subunit (HERG, KV11.1)
SCN5A (LQT3)	3p21-p24	Cardiac sodium channel alpha subunit (NaV1.5)
Minor LQTS Genes (listed alphabetically)
AKAP9	7q21-q22	Yotiao
CACNA1C	12p13.3	Voltage gated L-type calcium channel (CaV1.2)
CALM1	14q32.11	Calmodulin 1
CALM2	2p21.3-p21.1	Calmodulin 2
CAV3	3p25	Caveolin-3
KCNE1	21q22.1	Potassium channel beta subunit (MinK)
KCNE2	21q22.1	Potassium channel beta subunit (MiRP1)
KCNJ5	11q24.3	Kir3.4 subunit of IKACH channel
SCN4B	11q23.3	Sodium channel beta 4 subunit
SNTA1	20q11.2	Syntrophin-alpha 1
ANDERSEN-TAWIL SYNDROME
KCNJ2 (ATS1)	17q23	IK1 potassium channel (Kir2.1)
ANKYRIN-B SYNDROME
ANKB	4q25-q27	Ankyrin B
TIMOTHY SYNDROME
CACNA1C (TS)	12p13.3	Voltage gated L-type calcium channel (CaV1.2)
CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA
RYR2 (CPVT1)	1q42.1-q43	Ryanodine Receptor 2
CASQ2 (CPVT2)	1p13.3	Calsequestrin 2
KCNJ2 (CPVT3)	17q23	IK1 potassium channel (Kir2.1)
CALM1	14q32.11	Calmodulin 1
TRDN	6q22.31	Triadin
BRUGADA SYNDROME
SCN5A (BrS1)	3p21-p24	Cardiac sodium channel alpha subunit (NaV1.5)
Minor BrS Genes (listed alphabetically)
CACNA1C	2p13.3	Voltage gated L-type calcium channel (CaV1.2)
CACNA2D1	7q21-q22	Voltage gated L-type calcium channel 2 delta 1 subunit
CACNB2	10p12	Voltage gated L-type calcium channel beta 2 subunit
DLG1	3q29	Synapse-associated protein 97
GPD1L	3p22.3	Glycerol-3-phosphate dehydrogenase 1-like
HCN4	15q24.1	Hyperpolarization-activated cyclic nucleotide-gated channel 4
KCND3	1p13.2	Voltage-gated potassium channel (Ito) subunit Kv4.3
KCNE3	11q13.4	Potassium channel beta subunit 3 (MiRP2)
KCNE5	Xq22.3	Potassium channel beta subunit 5
KCNJ8	12p12.1	Inward rectifier K(+) channel Kir6.1
MOG1	17p13.1	RAN guanine nucleotide release factor 1
SCN1B	19q13	Sodium channel beta 1
SCN3B	11q24.1	Sodium channel beta 3
S LMAP	3p14.3	Sarcolemma associated protein

KCNQ1 encodes the α-subunit of the K⁺ channel Kv7.1, generating I_Ks, which is physiologically increased by sympathetic activation and is essential for QT adaptation during heart rate increases. When I_Ks is diminished or dysfunctional, the QT interval fails to shorten appropriately during tachycardia, thus leading to a potentially arrhythmogenic condition. Heterozygous KCNQ1 mutations cause the dominant RW LQT1 syndrome and is the most common LQTS genotype accounting for 30-35% of LQTS. Homozygous mutations in KCNQ1, or compound heterozygous mutations, can cause the autosomal recessive JLN variant. Different effects may be produced by mutations in this multimeric K⁺ channel. If the mutation prevents the co-assembly such that only the wild-type subunit can tetramerize, then a mechanism of haploinsufficiency whereby I_Ks is reduced by 50% emerges. On the other hand, if the mutant-containing allele can tetramerize and “poison” the tetramer, then a dominant negative mechanism emerges resulting in a minimum residual of 6% current density. The dominant-negative effect of certain KCNQ1 mutations may manifest as a failure to modulate I_ks by (β-adrenergic signaling (21,22).

The second most common gene harboring LQTS mutations is KCNH2, encoding HERG, the α -subunit of the K⁺ channel conducting the I_kr current. I_kr and I_ks are two components of the delayed rectifier I_k current, the major determinant of phase 3 repolarization in ventricular cardiomyocytes. LQT2-causative mutations in KCNH2 provoke a reduction in I_kr current, most commonly by mechanisms involving impaired trafficking of the protein to the plasma membrane (23).

The third major LQTS gene is SCN5A, which encodes the α-subunit of the cardiac sodium channel (Na_V 1.5) that conducts the depolarizing inward sodium current. Few months after its identification as a LQTS gene in 1995 (1), it was shown that the SCN5A-ΔKPQ mutation produces the LQTS phenotype by increasing the persistent (or late) Na⁺ inward current and, therefore, prolonging action potential duration (24). This study provided the first evidence linking a mutation to a functional alteration in the ionic control of ventricular repolarization and paved the way to all subsequent functional studies that have become the gold standard to establish that a novel mutation in a LQTS patient is likely a disease-causing one.

After the identification of the first 3 major LQTS genes (1-3), several others were identified. KCNE1 and KCNE2 encode K⁺ channel auxiliary subunits that are associated with the α -subunits encoded by KCNQ1 and KCNH2. Mutations in KCNE1 may cause either the dominant RW (LQT5) or, if present in homozygosity or compound heterozygosity, the recessive JLN syndrome (11). There are few cases of KCNE2 mutations associated with LQTS and most of them represent acquired LQTS associated with specific drugs, almost all I_kr blockers.

Among the sodium channel interacting protein-coding genes, CAV3 (25), SCN4B (26), and SNTA1 (27) are regarded as additional LQTS genes (LQT9, LQT10, and LQT12) that essentially mimic LQT3. The AKAP9-encoded yotiao is involved in the phosphorylation of Kv7.1 and its mutation has been described in LQT11 which functionally mimics LQT1. Two missense mutations in CACNA1C, encoding a voltage-gated calcium channel, are linked to Timothy syndrome (TS; LQT8), a rare and extremely malignant LQTS variant. In a large Chinese family, a heterozygous mutation was identified in the inwardly rectifying K⁺ channel subunit Kir3.4, encoded by KCNJ5. The variant was present in all the 9 affected family members and was absent in >500 ethnically matched controls, suggesting a role in the pathogenesis of LQT13. On the other hand, the ANKB, KCNJ2, and CACNA1C genes, often referred to as LQT4, LQT7, and LQT8, are associated with complex clinical disorders: ankyrin B syndrome, Andersen-Tawil syndrome, and Timothy syndrome respectively. In the first two prolongation of the QT interval is modest. Until LQTS-causing mutations are found in these genes in patients with clinically definite LQTS, these 3 genes should not be strictly considered as part of LQTS.

Quite recently, a most malignant form of LQTS that causes recurrent cardiac arrest due to ventricular fibrillation manifesting in infancy has been found associated with mutations in CALM1 and CALM2, two of the three human genes encoding calmodulin (28). Calmodulin is a ubiquitous multifunctional Ca²⁺ binding protein and overexpression of calmodulin mutants with defective Ca²⁺ binding produces major prolongation of ventricular action potentials (29,30). In two unrelated infants with QT prolongation and very early occurrence of cardiac arrest due to ventricular fibrillation, whole exome sequencing revealed de novo mutations in either CALM1 or CALM2. A subsequent candidate gene screening in a cohort of 82 LQTS genotype-negative subjects identified two more CALM1 mutation carriers (28). All 4 patients share strikingly similar clinical manifestations: major QT prolongation (all > 600 ms), T wave alternans, cardiac arrest in infancy, multiple episodes of ICD-terminated ventricular fibrillation mostly triggered by sympathetic activation, poor response to pharmacological and non-pharmacological interventions.

Genetics and Arrhythmia Triggers

A study performed on almost 700 patients of known genotype and all with arrhythmic events demonstrated that the triggers for arrhythmias in LQTS are gene-specific (31). LQT1 patients are at risk especially during sympathetic activation, as with physical exercise or emotional stress (Fig. 1), and this stems from the fact that they have a lower-than-normal I_Ks and therefore their ability to shorten the QT interval when heart rate increases is impaired. LQT2 and LQT3 patients, who have a normal level of I_Ks, are at no special risk during physical exercise and sport activity. LQT2 patients are exquisitely sensitive to sudden noises, such as alarm clocks or telephone ringing whereas LQT3 patients tend to have their events while at rest or while asleep. The fact that in that large study 99% of the events occurring while swimming were in LQT1 patients and that 80% of the events triggered by sudden noises were in LQT2 patients allow the shrewd clinician to suspect the correct genotype based on simple clinical history and well before obtaining the genetic results.

Figure 1. Triggers for lethal cardiac events in LQT1, LQT2, and LQT3 patients.

The arrows point out the rare occurrence of these events during sympathetic activation in patients without mutations affecting the I_Ks current. (Modified from ref. 31)

Genetics and Risk Stratification

Since the early days of molecular genetics for LQTS attempts were made to correlate genotypes with outcomes. The first large study reported on 647 LQTS patients and suggested interactions between genotype, QTc and gender (32). The risk of cardiac events, higher for LQT2 females and LQT3 males, increased in the presence of marked QT prolongation (QTc >500 ms). LQT1 patients were less likely to experience events probably because of the very high percentage (36%) of patients with the disease-causing mutation but with a QTc<440 ms. The existence of these genotype-positive/phenotype-negative patients is related to the low penetrance existing in LQTS, which was postulated in 1980 (33) and demonstrated in 1999 (34).

A significant step forward came with the realization that besides genotype-based risk stratification, intragenic risk stratification was possible for LQT1 and LQT2 based on molecular/structural location and cellular function. In 2002 (35) and 2007 (36), Moss et al indicated first that LQT2 patients with pore-localizing mutations were at higher risk and then that in LQT1 patients both the transmembrane location of the mutations and their dominant-negative effect are independent risk factors for cardiac events. These studies indicated that not all mutations on the same gene produce a similar clinical phenotype and initiated a series of intriguing revelations on the complexity of the genotype-phenotype correlation. An evolution of these studies led to the realization that there are areas in the genes, such as the Kv7.1 cytoplasmic loops, not only associated to higher arrhythmic risk but also to a particularly good response to β-blocker therapy (21).

However, neither the localization of a mutation nor its cellular electrophysiological effect is sufficient to consistently predict the impact on clinical manifestations. The most striking example of mutation-specific behavior is probably that of KCNQ1-A341V, a relative hot-spot mutation characterized by unusual clinical severity demonstrated by 80% of the patients being symptomatic, with >30% experiencing cardiac arrest or sudden death (37,38). What is puzzling is that A341V is only a mildly dominant negative mutation producing a relatively modest I_Ks loss.

Genetics, Response to Therapy, and Clinical Management

Since the identification of the first LQTS genes high hopes were generated that understanding the molecular underpinnings of the disease would inspire novel therapeutic approaches. So far, this has been only partially true. Still, progress in the management of these patients based on genotype-phenotype studies is impressive and undeniable and so far, the diagnostic, prognostic, and therapeutic impact of genetic testing has been realized most fully for LQTS compared to all other genetically-mediated channelopathies and cardiomyopathies (7).

The response to β-blocker therapy is in part gene-specific, but not as much as previously thought. Clearly, β-blockers are extremely effective for LQT1 patients (31,39,40) and are also effective for LQT2 patients but LQT2 females are less fully protected. Contrary to some previous opinion, largely dependent on the inclusion in the analysis of a subgroup largely unresponsive to therapy and represented by patients with events in the first year of life (41), β-blockers are effective also for LQT3 patients as indicated by a study in > 400 patients (42).

Within months after the cellular demonstration, in August 1995, that the electrophysiological consequence of a SCN5A mutation is an increase in persistent Na⁺ current (24), clinical (43) and experimental (44) evidence was provided that a sodium channel blocker, mexiletine, could markedly shorten the QT interval in LQT3, but not in LQT1 and LQT2, patients and block the persistent Na⁺ current. It was immediately warned that mexiletine should have never been used instead of β-blockers but as potentially useful addition. Indeed, despite mexiletine-mediated clear evidence of benefit in certain patients there have been failures in others. The response to Na⁺ channel blockers is clearly mutation-specific and this dictates the correct clinical approach: to always test the QT shortening effect of mexiletine using the acute oral drug testing approach (45) with half of the daily dose while monitoring for two hours the patient's ECG: if the QTc shortens by more than 40 ms without undue PR lengthening then it is reasonable to add mexiletine to the β-blocker therapy. There is current interest as potential LQT3-specific therapy in ranolazine, a drug more selective for the persistent Na⁺ current, but the available clinical data are scanty and short term (46). It should not be forgotten that Na⁺ channel blockers have the potential to impair cardiac conduction, and vigilant ECG monitoring is necessary when LQT3 patients are treated with mexiletine to avoid serious consequences In LQT3 patients with specific mutations, mexiletine and propranolol may have beneficial synergistic effects in correcting major electrophysiological abnormalities (47). Flecainide should be, and is, seldom considered for LQTS patients because of its I_Kr blocking effect.

The major impact of genetics has been in the general management of the patients and of their families. As to the patients, the identification of specific triggers for the arrhythmic events (31) has led to rational attempts to avoid or counter the at-risk situations. LQT1 are advised to avoid excessive stress, be it physical or mental, and specific activities such as swimming unless under proper protection. For LQT2 patients it is important to minimize sudden noises, especially when resting; this means to avoid telephones and alarm clocks in the bedroom and to wake up the affected children without yelling. Also, as sleep deprivation and disruption is particularly bad for LQT2 women in the post-partum period it is advisable that fathers find the proper way to feed the infants nighttime without waking the mothers. LQT2 and LQT3 patients, because of their “normal” I_Ks, are not expected to be at special risk during physical activity.

As to the families, the issue is that of “cascade screening” (48), i.e. once the disease-causing mutation is identified in the proband the entire family should undergo testing for that specific mutation, which is rapid and inexpensive, in order to identify the mutation carriers with normal QT. This concept is the direct consequence of the fact that low penetrance is common in LQTS (34), which confirms the original hypothesis (33) that some patients may be affected by LQTS and nonetheless have a normal QT interval. This implies that normal findings on an electrocardiogram cannot be used to exclude LQTS and mandates the necessity to perform molecular screening in all family members once the disease-causing mutation had been identified in the proband.

Cascade screening allows the identification, as mutation positive, of individuals who would have been otherwise considered unaffected and therefore would have remained at risk for potentially life-threatening arrhythmias either as spontaneous event or more likely as provoked by a variety of drugs with an I_Kr blocking effect. This leads to prophylactic treatment in over 70% of mutation positive individuals (49). Also important is the fact that those family members who are found to be negative for the family's disease-causing mutation will be relieved to learn that they are not at risk and that they should not fear for their offspring.

The magnitude of the impact that genetic screening has on clinical cardiology is exemplified by the fact that not performing cascade screening could lead to a number of otherwise avoidable deaths among those genotype positive/phenotype negative family members of affected patients. These are deaths that could be prevented partly by therapy, when appropriate, and partly by providing on a regular basis an updated list of drugs to carefully avoid. Cascade screening forcefully demonstrates that molecular biology and genetics can no longer be regarded as tools for researchers, but nowadays represent an essential component of good medical care.

Catecholaminergic Polymorphic Ventricular Tachycardia

Akin to LQT1, catecholaminergic polymorphic ventricular tachycardia is characterized phenotypically by exercise-induced syncope, seizures, or sudden death in the setting of a structurally normal heart (50-52). However, in contrast to LQTS, the electrocardiogram at rest is typically normal with only subtle, non-diagnostic bradycardia and U waves occasionally present. Instead, provocative stress testing by treadmill, cycle, or isoproterenol is the key diagnostic test to elicit CPVT's trademark signature of exercise-induced bidirectional ventricular tachycardia. Importantly, although fairly specific for CPVT, exercise-induced bidirectional VT is insensitive as the majority of patients with mutation proven CPVT do not manifest this arrhythmia (52). Moreover, CPVT1 patients with a negative exercise stress test are not at zero risk (52). Rather, in the context of a positive personal or family history, CPVT should be suspected when a stress test exhibits the onset of premature ventricular contractions (PVCs) when the heart rate reaches around 110–130 beats per minute. At this work load, this exercise-induced ectopy will commence with single, intermittent PVCs and will progress typically to PVCs in bigeminy and couplets. Only occasionally will more complex ectopy ensue. Often the exercise-induced ectopy will burn out (return to normal sinus rhythm) at highest work load/peak heart rate and normal sinus rhythm almost always persists throughout the recovery phase.

Because CPVT is more arrhythmic than LQTS with higher estimated lethality and higher breakthrough rates during conventional β-blocker therapy, it is critical to distinguish CPVT from LQTS (53). Often, CPVT patients have been misdiagnosed as having “atypical” LQTS (54). As a diagnostic pearl, in the setting of an exercise-triggered cardiac event, a resting QTc < 460 ms, and a structurally normal heart, CPVT rather than “concealed” or “normal QT interval” LQTS, is far more likely to be the root cause.

Pathogenetically, approximately 50-60% of CPVT stems from heritable or sporadic mutations in the RYR2-encoded cardiac ryanodine receptor/calcium release channel, a critical regulator of intracellular calcium (55). RYR2 is one of the largest genes in the human genome with its 105 translated exons that encodes for a protein containing 4967 amino acids. However, there are 3 particular domains/clusters encoded by 16 exons where two-thirds of the current CPVT1-associated mutations localize and all published mutations currently reside within less than half of RYR2's exons (56,57).

Besides CPVT1, rare autosomal recessive subtypes of CPVT stem from mutations in CASQ2-encoded calsequestrin 2 (CPVT2) (58) or TRDN encoding the junctional protein triadin (59) (CPVT4). Recently, mutations in CALM1 encoding calmodulin were discovered in one family with autosomal dominant CPVT-like phenotype, and in a de novo single case, all genotype-negative for RYR2 or CASQ2 mutations (60).(CPVT5). Also, mutations in the KCNJ2-encoded Kir2.1 can express a clinical phenotype that mimics autosomal dominant CPVT (61) (CPVT3). Generally, loss-of-function KCNJ2 mutations cause type 1 Andersen-Tawil syndrome (ATS1), a heritable channelopathy readily distinguishable from CPVT with characteristic U waves, facial/skeletal stigmata, and with a much more benign prognosis. However, several KCNJ2 mutations have been identified in patients clinically diagnosed with CPVT because of complex ventricular ectopy including bi-directional VT with no clinical features to suggest ATS1.

CPVT genetic testing is recommended for any patient in whom a cardiologist has established a clinical index of suspicion for CPVT based on examination of the patient's clinical history, family history, and expressed electrocardiographic phenotype during provocative stress testing with cycle, treadmill, or catecholamine infusion and mutation-specific genetic testing is recommended for family members and appropriate relatives following the identification of the CPVT-causative mutation in an index case (62). Presently, the primary purpose of CPVT genetic testing is diagnostic, to genetically confirm a clinically suspected case of CPVT and establish the particular genotype and to identify the potentially at-risk relatives (63).

However, in stark contrast to the genotype-specific, region-specific, and even mutation-specific risk stratifying information that has emerged in LQTS, no definitive domain-specific or mutation-specific prognostication can be made for RYR2-mediated CPVT (i.e. CPVT1). Preliminary data suggest that relatives carrying a RYR2 mutation in the C-terminal channel-forming domain may be at greater arrhythmic risk than those with N-terminal domain (64).

Thus, all patients with clinically manifest CPVT1 are treated based upon their phenotype without regard to any details about the particular mutation. Here, the genotype does not guide therapy. Phenotype-guided therapy generally consists of β-blocker therapy and/or left cardiac sympathetic denervation therapy and if necessary, combination therapy with the addition of flecainide (53,65-68). Device therapy with an implantable defibrillator should be the last intervention (rather than the observed all too often first one) for only the highest risk CPVT subjects because of the uncommon but concerning issue of a CPVT-related ICD storm whereby the ICD ultimately fails to rescue the patient (69,70).

Although the chief purpose of CPVT genetic testing is diagnostic rather than either prognostic or therapeutic, the genotype influences management/treatment of a patient with genetically confirmed CPVT in two important ways. First, it is important to distinguish KCNJ2-mediated CPVT (CPVT3) from the more common CPVT1 as the treatment strategy is different for the two genotypes. In contrast to the phenotype-guided treatment strategy for either CPVT1 or genotype negative/phenotype positive CPVT, patients with KCNJ2-mediated CPVT may be more responsive to primary therapy with flecainide or mexiletine rather than β-blocker therapy (71). In addition, the protective, anti-fibrillatory effect of LCSD has been demonstrated much more clearly for patients with CPVT1 than CPVT3 patients (65).

Second, mutation-specific confirmatory testing in relatives enables prophylactic β-blocker therapy to be initiated at a young age if deemed necessary (64). Here, without the genotype, potentially at-risk family members would only be revealed after they are old enough to do a provocative stress test or if they manifest a concerning symptom. Considering that the first concerning symptom can be sudden death and that up to 15% of autopsy negative sudden unexplained death victims are CPVT1 positive (72), identifying a potentially vulnerable CPVT1-positive relative as early as possible is an indirect but potentially lifesaving therapeutic contribution of CPVT genetic testing.

Brugada Syndrome

The Brugada syndrome (BrS) is a hereditary disease characterized by its “signature sign”, a coved-type ST-segment elevation in the anterior precordial leads (V1 to V3), referred to as a type 1 Brugada ECG pattern, and by the presence of right ventricular conduction abnormalities and life threatening ventricular arrhythmias (73,74). The typical case is a 40-year-old resuscitated male without clear evidence for structural heart disease and with a family history for (nocturnal) sudden cardiac death (SCD). Indeed, up to 75% of those clinically affected are of male gender, and the mean age of onset of events is around 40 years but with a wide range. A family history of SCD is reported in 20-50% of cases. There is an autosomal dominant pattern of transmission with highly variable and often low penetrance. Several aspects are still unclear, especially the pathophysiology of the right precordial ST-segment elevation (75).

BrS is a genetically heterogeneous disease, with the involvement of at least 13 different genes (76,77). Most mutations occur in genes with impact on the function of cardiac Na⁺ channels. SCN5A, the gene encoding for the α-subunit of the cardiac sodium channel, is involved in 20% to 25% of patients. More than 200 SCN5A BrS-related mutations have been described to date (78). All mutations induce a reduction in the sodium current amplitude and do so through several mechanisms, including altered channel kinetics (e.g., faster inactivation or slower recovery from inactivation), trafficking defects and generation of truncated proteins. Recently, a pure, self-sufficient causative role of loss-of-function SCN5A mutations has been challenged as in several large SCN5A-related BrS families affected individuals did not carry the presumed familial disease causing mutation thus suggesting that SCN5A may actually represent a strong modifier (79).

Other genes with impact on sodium channel function are the sodium channel β-subunit genes (SCN1B, SCN3B) affecting channel kinetics, glycereol-3 phosphate dehydrogenase 1-like enzyme (GPD1L), MOG1 and SLMAP that affects trafficking of sodium channels. Potentially causative variants are also found in the calcium channel genes (CACNA1C, CACNB2B, CACNA2D1), in genes that affect the transient outward current (I_to) (KCNE3, KCND3, KCNE5) and in the gene that forms the pore forming unit of the ATP sensitive potassium current (I_KATP KCNJ8) (76). Involvement of most of these genes has been described in single patients and families with BrS, although mutations in CACNA1C and CACNB2B are reported to contribute to up to 11% of BrS. In basic electrophysiological studies, the calcium channel genes lead to loss of function of basal L-type calcium current (I_Ca,L), a mutation in KCNE3, KCND3, or KCNE5 lead to a gain of function of I_to and mutations in KCNJ8 increase I_KATP.

Genotype-phenotype correlation studies in Brugada syndrome are sparse. Initial studies indicate that SCN5A-associated BrS typically presents with longer conduction intervals in all cardiac compartments (80). Meta-analyses consistently show that the presence or absence of a SCN5A mutation does not impact clinical outcome (81). However, within the SCN5A cohort, the type of SCN5A mutation may be useful for risk stratification, with nonsense mutations giving rise to truncated protein leading to more severe conduction disorders and more symptoms (82). Calcium channel-related BrS seems to associate with shorter than normal QTc intervals, but it is not clear whether this impacts on prognosis (83).

As the diagnosis of BrS is made on clinical grounds, genetic testing is not required for this goal. Yet, the finding of a loss-of-function SCN5A mutation might help in a clinically uncertain diagnosis. As indicated above, knowledge of a mutation does not impact on prognosis, with the possible exception of specific findings in SCN5A. BrS genetic testing can be useful for any patient in whom there is reasonable suspicion for BrS based on examination of the patient's and of his/her family clinical history, and a clear electrocardiographic phenotype based on either resting 12-lead ECGs and/or provocative drug challenge testing. Because the presence of a disease-causing mutation does impact life style [e.g., fever and specific drugs should be avoided (84)], cascade genetic screening is recommended for family members following the identification of the BrS-causative mutation in an index case. In the setting of an isolated type 2 or type 3 Brugada ECG pattern genetic testing has no place (61).

Issues with Genetic Testing

Advances in deciphering the molecular basis for heritable cardiac arrhythmia susceptibility have re-shaped the diagnostic paradigm and clinical management of these three familial arrhythmia syndromes (cardiac channelopathies) to include genetic testing, gene specific considerations in therapy and increased awareness of the need to assess disease risk in family members. The accurate ascertainment of family history, the proper use and interpretation of genetic test results, and the identification and management of at-risk family members have become the standard-of-care in this field.

Optimal Use of Genetic Testing

Ascertaining a comprehensive family history of cardiac arrhythmias and unexpected death is of utmost importance when considering the diagnosis of a heritable arrhythmia syndrome. In performing a detailed family history, special attention should be paid to the occurrence of syncope or sudden unexpected death especially among young adults and children in the family, noting special circumstances surrounding unexpected death (e.g., drowning, seizures), and identifying any relatives with implanted cardiac devices along with the indication. A pedigree drawing is essential for the recognition of a mode of inheritance compatible with a monogenic disorder (e.g. autosomal dominant, autosomal recessive, X-linked). Information about the patient and close relatives is ultimately helpful in making final decisions about the use of genetic testing to confirm clinical suspicions. However, incomplete penetrance or subclinical disease expression may obscure the pattern of disease segregation in a family. This is unfortunately a common problem in families with BrS due to low penetrance (79) and may also obfuscate the recognition of inheritance patterns in LQTS (31). Especially severe and early-onset forms of LQTS and other syndromes may be caused by de novo mutations in which case no family history is expected (47,85,86).

Genetic testing is a specialized diagnostic procedure available for LQTS, BrS, and CPVT through commercial and research laboratories (87). In the United States, clinical genetic testing laboratories must meet stringent criteria for quality standards that conform to the federal Clinical Laboratory Improvement Amendments (CLIA) passed in 1988 (88). Further, unlike more commonly used laboratory tests, genetic testing should be performed after informing the patient regarding the potential risks, benefits and limitations. Involvement of a genetic counselor is ideal in circumstances where either physician time or knowledge is limited. Despite these requirements, genetic testing can have tremendous value in identifying mutations that help confirm clinical suspicions, select genotype-specific therapy and direct specific testing of at-risk relatives.

Pitfalls and Limitations of Genetic Testing

The current yield of genetic testing for each of these syndromes ranges from 25% (BrS) to 80% (LQTS). Further, the methods to identify mutations are not 100% sensitive and therefore a negative genetic test cannot exclude the disorder by itself. Also, certain detectable DNA sequence variants may not have a clear causal role in a patient's condition either because they are rare polymorphisms (89) or are located in regions of the channel protein that have unknown functional importance. Based upon several years of genetic testing experience in the academic and commercial sectors, we now know that most mutations are ‘private’ (i.e., occurring in a single family) missense mutations with uncertain functional or pathophysiological consequences (77,90). Therefore, interpreting genetic test results is often confounded by discovery of ‘variants of unknown significance’ for which there is insufficient data or predictive tools to assess accurately the likelihood that a particular variant predisposes to an arrhythmia or whether the change is merely a benign rare variant (91). Only a small fraction of all identified genetic variants in the myriad genes associated with LQTS, BrS, CPVT have been investigated functionally to elucidate a biologically plausible contribution to pathogenesis. Even fewer mutations have been studied in a genetically engineered animal model or native cardiac cell. Computational strategies have been developed to predict the functional consequences of mutations but none of these methods have been tested rigorously as valid clinical predictors. The lack of functional or biological validation of mutation effects remains the most severe limitation of genetic test interpretation in the cardiac channelopathies (88).

Interpretation of a negative genetic test in a symptomatic person is a challenge. Despite more than 18 years of genetic discovery in the cardiac channelopathies, there remain a substantial number of cases having classic symptoms and signs for one of the heritable arrhythmia syndromes who test negative for the many known genes. This may be explained by either a false negative or a true negative test result. One potential cause for a false negative genetic test is the location of a mutation outside the region of the gene normally interrogated by the test (92). Alternatively, certain types of mutation may be missed by standard testing strategies. For example, DNA sequencing can miss multi-exon deletion or duplication mutations (93-95). False negative results may sometimes be overcome by repeat testing in situations where the clinical diagnosis has a high level of certainty (96). A true negative test result may be a clue to the existence of an as yet undefined gene involved with arrhythmia susceptibility. These situations are excellent opportunities to pursue genetic discovery in a research setting as was recently done to identify novel calmodulin gene mutations in severe infantile presentations of LQTS (28). The potential negative impact of learning the results of a genetic test must also to be considered. Patients should be properly educated and carefully counseled about their long-term risks of cardiac arrhythmia without inciting excessive apprehension by implying that genotype is an absolute predictor of sudden death risk. Physicians should also be sensitive to the potential socioeconomic fallout (e.g., insurability) from a genetic diagnosis and vigorously guard confidentiality. Fortunately, in the United States, the recently enacted Genetic Information Non-discrimination Act (GINA) prohibits workplace and insurance discrimination based on genetic predisposition.

Genetic Modifiers

Another important conceptual barrier to extrapolating genetic test results to patient management is the variable disease expression and penetrance common among these disorders. For example, in congenital LQTS, not all individuals carrying disease associated mutations have equal risk for expressing the clinical manifestations of the disease (34,97). Clinical heterogeneity is a common feature in LQTS and BrS. Members of the same family that share the same mutation may have varying phenotypes, ranging from no symptoms to sudden death. In rare cases, multiple mutations or combinations of a mutation with a common variant have accounted for unusual severity of one member of a larger family. Compound mutations help explain exaggerated disease severity in 4-8% of LQTS probands (98,99). On the other hand, predicting the likelihood of life-threatening arrhythmias in an asymptomatic mutation carrier continues to be most challenging.

These and related observations have inspired the hypothesis that genetic factors other than the primary disease-associated mutation can modify the risk for disease-related morbidity and mortality. Conceptually, hypotheses proposed to explain variable penetrance in the genetic arrhythmias may be separated into two categories: 1) factors that modify the underlying arrhythmogenic myocardial substrate, and 2) factors that affect the probability and magnitude of arrhythmia-triggering events. Genetic factors that could impact on the myocardial substrate include genes that encode proteins that contribute to the balance of inward and outward currents operating during the cardiac action potential. Genetic factors responsible for inter-individual differences in sympathetic and parasympathetic tone may alter one's susceptibility to triggered arrhythmias. Similarly, the magnitude of catecholamine responses to stress and exercise vary among individuals and some of this variability may have a genetic basis (100). Therefore, genes that participate in autonomic responses are candidate genetic modifiers.

Sorting out the relative effects of genetic modifiers is very challenging. Demonstrating association of particular genetic variants with phenotype requires a large population, and there are sample size restrictions with any rare disease such as inherited arrhythmias. Exploiting unique cohorts such as founder populations may have particular value in identifying modifier genes (97,101). For example, common variants in NOS1AP originally tagged in association with variable QT interval duration in healthy adults have been demonstrated to be modifiers of QT interval and the probability of symptoms in LQTS (102,103). Also, common variants in the 3′ untranslated region of KCNQ1 modify disease severity in an allele specific manner (104).

Ideally, risk stratification schemes based on presence of a primary mutation and one or more modifier alleles will emerge to improve prediction of cardiac events. However, there are challenges to extrapolating from population-based results to predicting an individual's risk. Clearly, more work is needed before we can take full advantage of this information for the ultimate goal of assessing risk even in asymptomatic mutation carriers.

Genetics and Medico-Legal Implications

The effectiveness of cascade screening for the early identification of affected family members also carries medico-legal implications. Cascade screening requires positive genotyping of the proband because identification of the disease-causing mutation is the necessary first step. It follows that the physician who does not attempt to genotype the proband clinically affected by one channelopathy has willfully decided to ignore whether some of his or her family members are carriers of the disease and thereby exposed to the risk of life-threatening arrhythmias. Similarly, the physician who, after having obtained positive genotyping, does not propose to initiate cascade screening within the family of the proband has similarly willfully decided to leave the affected family members - approximately one-half of the first-degree relatives - uninformed about their status and unprotected.

Future Impact of Genetics

Advances in DNA sequencing technology have inspired a vision of widespread use of genome sequencing in clinical medicine. Exactly how this vision will be realized is uncertain at the present time, but there is enormous potential for impacting risk prediction for common and less common diseases and for predicting responses to drug therapy, including drug-induced TdP which can be favored by specific genetic variants (105,106). Whole genome sequencing will eventually achieve an accuracy level and price point that will supplant targeted genetic testing for rare diseases such as inherited arrhythmia syndromes. Although this may make diagnosing genetic disorders technically more feasible, the art of making a clinical diagnosis will remain important particularly when genetic information reveals unexpected findings. There is already information from large scale exome sequencing efforts to anticipate that many incidentally discovered genetic variants in disease-associated genes, including those associated with LQTS, will be discovered in many individuals (107). Many of these variants could be merely false positive results and strategies to properly interpret and handle these incidental findings will be critical to avoid evoking needless concern or implementing unnecessary therapies in an asymptomatic person.

Conclusion

The progress in understanding channelopathies, and the underlying molecular biology, proceeds at mind-boggling speed. It should be clear to everyone in cardiology and medicine that genetics and clinical management of these diseases are tied together and that nowadays it is seldom possible to efficiently treat the affected patients without keeping into account what has been learnt from genetic testing.

List of Abbreviations

BrS

Brugada Syndrome

CPVT

Catecholaminergic Polymorphic Ventricular Tachycardia

ICD

Implantable Cardioverter Defibrillator

JLN

Jervell and Lange-Nielsen syndrome

LQTS

Long QT Syndrome

PVCs

Premature Ventricular Contractions

RW

Romano Ward syndrome

SCD

Sudden Cardiac Death

TdP

Torsades-de-Pointes