Prognostic impact of misdiagnosis of cardiac channelopathies as epilepsy.

INTRODUCTION: Cardiac channelopathies are a frequent cause of sudden cardiac death (SCD) and often manifest with convulsive syncope, leading to a misdiagnosis of epilepsy. We aim to evaluate the clinical impact of epilepsy misdiagnosis in a cohort of patients with cardiac channelopathies.
METHODS: Fifty probands/families with a cardiac channelopathy were included. We retrospectively collected information from medical records to identify all patients who presented with convulsive syncope and were diagnosed with epilepsy after neurological evaluation. Clinical data and outcome were compared with those of patients without a previous epilepsy diagnosis.
RESULTS: Eight patients had a previous diagnosis of epilepsy. At first episode, 3 of them presented a positive family history of SCD and 5 showed a pathological electrocardiogram; half presented with sudden cardiac arrest (SCA) and the rest with recurrent syncope despite treatment with 1 or more anti-epileptic drugs. Five patients had long QT syndrome, 2 had catecholaminergic polymorphic ventricular tachycardia, and 1 had Brugada syndrome. Epilepsy misdiagnosis was associated with an increased risk of SCA/SCD (OR 6.92, P = .04), a delay of 12 years (P = .047) in correct diagnosis, and a delay from first symptom to channelopathy diagnosis of 18.45 years (P < .0001).
CONCLUSION: Cardiac channelopathy patients can be misdiagnosed with epilepsy. This involves a delayed diagnosis, a delay from the first symptom to a correct diagnosis, and an increased risk of SCA/SCD.

Introduction

The causes of sudden cardiac death (SCD) among pediatric and young individuals (under 35 years of age) are predominantly primary arrhythmia syndromes (channelopathies) and cardiomyopathies.[1] Sudden cardiac death from channelopathies is estimated to account for 10–15% of SCD in individuals without structural heart disease at autopsy, often called sudden arrhythmic death syndrome (SADS).[2] Channelopathies are caused by mutations in genes encoding cardiac ion channel subunits or proteins that interact with, or regulate, ion channels. These genetic variants result in gain or loss of channel function, modifying ventricular action potential generation and leading to life-threatening arrhythmias. Long QT syndrome (LQTS), Brugada syndrome (BS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and short QT syndrome (SQTS) are the most common channelopathies. These genetic conditions are often identified by specific ECG abnormalities either at baseline or in particular circumstances, such as during exercise (e.g., CPVT and LQTS), fever (e.g., BS), or pharmacological challenge.[3,4]

Warning symptoms may precede the SCD episode, one of the most common events being syncope.[5] Cardiogenic syncope is secondary to a rapid self-terminating polymorphic ventricular tachycardia, frequently accompanied by epileptiform activity (myoclonic seizures). These episodes are recurrent and are sometimes misdiagnosed as seizure disorders.[6] Up to 20% of patients with convulsive syncope could be misdiagnosed with an epileptic disorder.[7] As a result, patients at risk of SCD suffer a delay in diagnosis and are commonly exposed to anti-epileptic drugs (AED) with potential pro-arrhythmogenic effects.

We aim to evaluate the prevalence and clinical impact of epilepsy misdiagnosis in a well-characterized, single-center cohort of channelopathy patients.

Methods

Population and study groups

All subjects signed the written informed consent form. The local Ethics Committee approved the study (Virgen de las Nieves Hospital Ethical Committee. All index cases from 50 families screened for suspicion of channelopathy or during SCD evaluation at our Inherited Cardiac Disease Unit between 2012 and 2018 were included. Probands were diagnosed according to current international criteria[8] and classified in two groups according to the presence or absence of a previous diagnosis of epilepsy. A positive epilepsy diagnosis was defined as the presence of either a personal history of seizures, epilepsy or a history of AED therapy after a neurologist assessment. We assessed the presence of a diagnosis of epilepsy or seizure-related disorder in medical records from all included patients. We collected clinical information, neuroimaging and electroencephalogram (EEG) findings, and details of treatment for those individuals. Exclusion criteria included all acquired causes of seizures comprising traumatic/vascular injury, fever or metabolism disbalance.

Clinical, genetic, and ECG variables

Individual clinical information was collected retrospectively, including personal history (especially regarding prior neurological evaluations), positive family history of SCD, symptomatology and triggers at first manifestation, 12-lead electrocardiogram (ECG), bidimensional echocardiography, exercise test and genetic evaluation according to phenotype, clinical presentation, and physician criteria. Selected patients underwent pharmacological challenge. Some patients presenting with idiopathic ventricular fibrillation (VF) were diagnosed based on a previous published protocol.[9] Clinical information on arrhythmic events was collected for all the participants. We analyzed time and age from first event to SCA/SCD episode, as well as age at diagnosis.

First available ECGs were retrospectively collected. Baseline ECGs from probands were performed at 25 mm/s and blind-reviewed by two clinical cardiac electrophysiologists. A QT interval was considered abnormally prolonged when QTc was greater than 460 ms in females and 440 in males, paying special attention to T-wave amplitude and shape; BS was diagnosed when type 1 Brugada pattern appeared spontaneously or after ajmaline or flecainide challenge. A diagnosis of CPVT required at least three premature ventricular complexes of different morphologies during exercise test or epinephrine challenge. Short QT syndrome was diagnosed according to current criteria.[8,10]

Sudden cardiac arrest was defined as unexpected circulatory arrest reversed by successful resuscitation maneuvers. The definition of SCD applied when no obvious extra-cardiac causes had been identified by post-mortem examination and therefore an arrhythmic event was a likely cause of death.[11] Sudden unexpected death in epilepsy (SUDEP) was defined as a “sudden, unexpected, witnessed or unwitnessed, non-traumatic, and non-drowning death in patients with epilepsy with or without evidence for a seizure, and excluding documented status epilepticus, in which postmortem examination does not reveal a structural or toxicological cause of death.[11]

Genetic evaluation was performed by Sanger sequencing according to physician criteria, phenotype suspicious and, in more recent cases, by a next-generation-sequencing (NGS) panel including 80 genes related to cardiac arrhythmia and SCD (S1 Table). Pathogenicity of identified genetic variants was established according to current recommendations[12], clinical evaluation and family pedigree. A positive NGS analysis was considered when a potential pathogenic (likely pathogenic or pathogenic) variant was present in the proband. On the other hand, a negative result from NGS analysis was considered when no potential pathogenic variant was detected. Finally, patients presenting a complex genotype (co-existence of two mutations in different genes) or a potential pathogenic variant in a rare gene (such as CALM2, CASQ2 or KCNJ2 genes) after NGS analysis, were considered in the “other group”. Detailed information on genetic evaluation is included in S1 Methods.

Statistical analysis

The data were analyzed with SPSS software version 21.0 (Chicago, IL, USA). Results are expressed as mean plus or minus standard deviation (SD) or frequencies and percentages. Clinical characteristics were compared using χ2 or Fisher’s exact test for categorical variables and unpaired Student-t test or Mann-Whitney U test for continuous variables. A P value of less than 0.05 was considered to be statistically significant. Variables related to SCA or SCD at first presentation were selected by univariable analysis (p <0.1) and contrasted by multivariable logistic regression with selection of variables through exclusion by steps.

Results

Of the 50 index cases, 29 were male, with an age of 34.5±17.2 years. Median age at diagnosis was 32.5 years (12.7–43.2). Family history of SCD was present in 15 cases. Overall, 9 individuals experienced SCA/SCD and 18 cardiac syncope at the time of diagnosis. We identified a typical trigger in 31 probands, but the most prevalent circumstances of arrhythmic events were during rest and during exercise. The primary arrhythmia syndrome spectrum included LQTS (n = 19), BS (n = 15), CPVT (n = 15) and SQTS (n = 1). For 40 of 50 cases, genetic analyses were performed. For 10 BrS patients genetic evaluation was not performed based on absent of a positive SCD family history and the low yield of genetic testing in this syndrome. Overall, genetic testing led by phenotype and physician criteria identified a disease-causing mutation in 31 of 40 patients. Distribution of genes was 8 of 50 for RYR2 6 of 50 for KCNH2, 5 of 50 for KCNQ1, 4 of 50 for SCN5A and 8 of 50 for other uncommon genotypes. Fig 1 represents phenotype-genotype distribution in all probands. Genotype details are added in the S2 Table.

Fig 1

Primary inherited arrhythmic syndromes and genotype distribution among all probands (panel A), probands without previous epileptiform diagnosis (panel B) and probands previously misdiagnosed (panel C).

Clinical features of the 50 cases, according to the previous diagnosis of epilepsy, are summarized in Table 1. Eight patients were evaluated for convulsive syncope and misdiagnosed with epilepsy before a channelopathy diagnosis was reached. All these patients with epilepsy misdiagnosis presented as generalized tonic-clonic seizures described in the in the emergency admission report as shock-like and irregular movements of both arms and legs. In a more detailed examination in the Inherited Cardiovascular Disease Clinic, it could be concluded that they had actually presented a cardiogenic syncope prior to seizures.

Table 1

Clinical features of probands.

BS: Brugada syndrome; CPVT: catecholaminergic polymorphic ventricular tachycardia; ECG: electrocardiogram; FHx: family history; LQTS: long QT syndrome; SCA: sudden cardiac arrest; SCD: sudden cardiac death; SQTS: short QT syndrome.

	Patients without previous epilepsy diagnosis (n = 42)	Patients with previous epilepsy diagnosis (n = 8)	P value
Male sex, n	24	5	.54
Age at diagnosis (y), median (interquartile range)	29.5 (12–41.5)	41.5 (30.2–53)	.02
Delay from first symptom (cardiac syncope or SCD) to (y) diagnosis, median (interquartile range)	0.1 (1–1.15)	18.5 (10.6–36.6)	< .0001
SCD FHx, n	12	3	.61
Channelopathy diagnosis, n
BS	14	1	.23
LQTS	14	5	.23
SQTS	1	0	.84
CPVT	13	2	.70
Genetic background, n
SCN5A	3	1	.51
KCNQ1	4	1	.60
KCHN2	3	3	.07
RYR2	7	1	.62
Others	8	0	.31
Negative	7	2	.31
Not performed	10	0
Triggers, n
Exercise	9	3	.37
Emotion	4	1	.60
Rest	28	2	.23
Auditory stimuli	1	2	.29
Symptoms at diagnosis, n
Palpitations	6	0	.57
Vasovagal syncope	7	0	.58
Cardiogenic syncope	14	4	.43
Other	10	0	.18
SCA / SCD	5	4	.02
ECG, n
First ECG Diagnostic	20	5	.70

Regarding the EEG findings, there were no abnormalities in seven out of the eight patients; just in one patient (patient VI in Table 2) the EEG analysis showed “unspecific findings that could be related to Temporal Lobe Epilepsy”, but a definitive diagnosis was not achieved. Further evaluations with neuroimaging techniques, as brain magnetic resonance, showed no abnormalities in the misdiagnosed patients. Clinical characteristics of these 8 patients are summarized in Table 2. All patients underwent neuroimaging and EEG, Median age of misdiagnosed patients with a seizure-related disorder was 41.5 years (30.2–53) and 5 were male. A final diagnosis of a channelopathy was made after recurrent cardiac syncope in 4 probands after SCA with electrocardiographically proven VF in 3, and post-mortem in 1. Three patients had a positive family history of SCD and as many as 6 presented a characteristic trigger of the sentinel events such as exercise, emotional stress, and auditory stimuli. There was a similar gender distribution between groups, but probands with epilepsy were significantly older. Presence of a positive family history of SCD was similar (Table 1 and Fig 2A).

Table 2

Clinical features of patients with cardiac channelopathy and prior neurological diagnosis.

AED: anti-epileptic drug; BS: Brugada syndrome; CPVT: catecholaminergic polymorphic ventricular tachycardia; ECG: electrocardiogram; LQTS: long QT syndrome; SADS: sudden arrhythmic death syndrome; SCA: sudden cardiac arrest; SCD: sudden cardiac death; SQTS: short QT syndrome; F: female; M: male; y: year.

Patient	Sex	Age at diagnosis	Previous neurological diagnosis	Number of AEDs	Cardiac channelopathy	Gene test	Family history of SCD	Retrospective diagnostic first ECG	Trigger	Diagnostic delay (y)	Event at diagnosis
I	F	22	Epilepsy	1	CPVT	Negative	No	No	Exercise	9.5	Recurrent syncopes
II	M	30	Cryptogenic generalized epilepsy	3	LQTS2	KCNH2 +	No	Yes	Auditory stimuli	16.1	SCA
III	M	50	Generalized epilepsy	1	BS	SCN5A +	No	No	Rest	21	Recurrent syncopes
IV	M	39	Epilepsy	1	LQTS	Negative	Yes	Yes	Emotional stress	34.3	Recurrent syncopes
V	M	57	Cryptogenic generalized epilepsy	2	LQTS1	KCNQ1 +	Yes	Yes	Rest	39	Recurrent syncopes
VI	F	44	Temporal lobe epilepsy	2	LQTS2	KCNH2 +	No	Yes	Auditory stimuli	48.4	SCA
VII	F	31	Generalized epilepsy	3	CPVT	RYR2 +	Yes	No	Exercise	6.2	SCA
VIII	M	54	Cryptogenic generalized epilepsy	2	LQTS2	KCNH2 +	No	Yes	Exercise	11.8	SADS

Fig 2

Age at diagnosis in years in the two different groups showing a significantly delayed diagnosis in the previously misdiagnosed patients (Panel A). Compared distribution of primary inherited arrhythmic syndromes among probands (Panel B). Distribution of typical triggers within probands (Panel C). Percentage of patients presented with sudden cardiac arrest at time of diagnosis (Panel D). *number above bars represent the percentage among the group. †* means significance p value <0.05.

Patients with a prior epilepsy diagnosis constituted 5 of 19 for LQTS probands (3 KCNH2 and 1 KCNQ1), 2 of 15 for CPVT probands (1 RYR2), and 1 of 15 for BS probands (1 SCN5A) (Fig 2B). Yield of genetic testing was high for both groups: 25 of 31 in probands without prior epilepsy diagnosis and 6 of 8 in patients with epilepsy diagnosis. Similarly, we did not find differences in distribution of disease-causing genes (Table 1 and Fig 1B), but there was a tendency towards a higher incidence of KCHN2 variants in patients with a prior epilepsy diagnosis (OR 4.7, P = .06). At time of first ECG, the two groups presented similar diagnostic findings of particular channelopathies in each case (P = .44). The groups showed a comparable prevalence of cardiac channelopathy spectrum, but out of 8 misdiagnosed patients, 5 had LQTS. These patients all showed a QTc interval greater than 470 ms in the first ECG with a characteristic notched T wave (Fig 3); 4 of them were KCNH2 mutation carriers.

Fig 3

Representative ECG traces of misdiagnosed probands, showing typical LQTS features.

Panels A to C show basal ECG from three KCNH2 mutation carriers with extremely prolonged QT intervals and abnormal T-wave shape (A: notched T wave, B: inverted and wide T wave, and C: widened T wave). Panel D shows an ECG recording during one of the syncopal attacks with convulsive status suffered by the patient in panel A, who corresponds to patient II in Table 2, demonstrating a torsade de pointes episode that required external defibrillation.

Overall, the two groups presented a comparable distribution of identifiable event triggers (Fig 2C), but patients with a previous diagnosis of epilepsy were more likely to experience their sentinel arrhythmic event as an SCA/SCD episode (50% vs 10%; P = .02) (Fig 2D). For those misdiagnosed with epilepsy, the median age of channelopathy diagnosis was 41.5 years, 12 years later than those with a correct diagnosis from the beginning, and in those patients who presented cardiac syncope or SCD as first symptom, the median time from first symptom to diagnosis was extremely delayed, much more than in those without a epilepsy diagnosis (Table 1).

Influence of misdiagnosis on clinical outcome

In patients with misdiagnosed epilepsy, being treated with two or more drugs (OR 8, P = .029) was significantly associated with SCA or SCD at presentation. Comparing both groups, univariable analysis revealed that a previous diagnosis of epilepsy (OR 6.6, P = .02), QTc of more than 460 ms at first event (OR 6, P = .02), and presence of KCNH2 mutation (OR 4.7, P = .06) were significantly associated with (or showed a tendency towards) SCA or SCD at first presentation. After multiple logistic regression analysis we identified epilepsy misdiagnosis as an independent predictor of an SCA or SCD event at time of channelopathy diagnosis (OR 7.46, P = .03 (Table 3).

Table 3

Univariable and multivariable predictors.

QTc: corrected QT interval.

Predictors	Univariate analysis OR	P value	Multivariate analysis OR	P value
Prior epilepsy diagnosis	6.6	.02	7.46	.03
QTc > 460 ms	6	.02		NS
KCNH2 mutation	4.7	.06		NS

Discussion

Previously reported series demonstrated that individuals with epilepsy have an increased risk of sudden unexpected death, especially in the young.[13] This tragic event is often referred to as sudden unexpected death in epilepsy, or SUDEP.[11,14] Cardiac arrhythmias due to unrecognized cardiac channelopathies have been described as one of the possible underlying causes that might provoke SUDEP.[15] The diagnostic error usually occurs in patients presenting with arrhythmic syncope and a secondary convulsive episode caused by brain hypoxia, but the central issue is low cardiac output due to a polymorphic ventricular arrhythmia.[16] Whether clinical misdiagnosis of epilepsy has significant implications for patient outcomes has not been defined before. In this single-center cohort of 50 patients with a clinical diagnosis of probable or definite cardiac ion channelopathy, we show that this epilepsy misdiagnosis has a negative impact on clinical outcomes of patients with underlying channelopathies, emphasizing the need for accurate cardiological evaluation in cases with syncope or seizures of unclear origin.

Real etiology for this sudden death cases still remains a matter of debate. Risk factors for this tragic event among patients with a clear diagnosis of epilepsy include young age and frequent generalized tonic-clonic seizures[17]. However, up to 20% epilepsy diagnosis, according to previous data, are wrong due to an underlying cardiovascular condition[18]. Whether all SUDEP cases are caused by this misdiagnosis of an underlying cardiac channelopathy, as a result of malignant ventricular arrhythmias, cannot be concluded from our work. Recent studies have deeply investigated the pathophysiology of SUDEP, and there is agreement that cardiac dysfunction plays a major role.[19] These studies have demonstrated that a severe depression of the cardiac and respiratory centers may arise after a generalized tonic-clonic seizure, particularly among young patients, and this fact can provoke severe hypoxemia and/or hypotension, leading to an instant death[20]. On the other hand, among cardiac dysfunction causes there is lack of evidence for the existence of malignant ventricular arrhythmias in the context of epilepsy.[21-22] Similarly to our findings, McCormick et al6 reported that some patients were classified as epileptic for many years until a final LQTS diagnosis was reached. Some of that patients suffered serious arrhythmias during follow up, highlighting the needing of a high degree of suspicious when facing patients with a not completely typical epilepsy.

Another possible link between these two apparently unrelated entities is the presence of common ion channels that are present either in central nervous system or in cardiac conduction system. In fact, some mutations associated with epilepsy have been identified in cardiac arrhythmia genes, like SCN5A, KCNH2 and KCNQ1[23-25]. These data have not been validated in larger cohorts, and the hypothesis remains controversial. There is evidence of patients with coexistence of epilepsy and cardiac arrhythmias with a single mutation, but definitive clinical or functional data demonstrating a unique cardio-cerebral channelopathy are absent.[26] In our study, KCNH2 mutations showed no statistically significant relationship with the previous diagnosis of epilepsy. KCNH2 mutation carriers are at increased arrhythmic risk compared to other gene loci, particularly for mutations at the pore[27]; in our families genotype-phenotype segregation was observed, with no evidence for neurological impairment in any of the carriers. In fact, in our cohort, after thorough review, none of the misdiagnosed cases showed typical traces of epilepsy, as neurological imaging exams and EEGs were normal. Furthermore, a clear diagnosis of channelopathy was patent in ECG traces so our data do not support the shared cardio-cerebral channels hypothesis.

Sudden unexpected death in epilepsy, which is likely underestimated, usually occurs during the night.[28] These data are different from our series, where episode triggers were more similar to those observed for arrhythmia in cardiac channelopathies, such as exercise, auditory stimuli, and adrenergic situations. The presence of this clinical context should be a warning sign for the presence of a cardiac channelopathy such as LQTS or CPVT. Moreover, a family history of sudden death, which was present in more than a third of the misdiagnosed patients in our cohort, should raise suspicions of the underlying genetic cardiac condition. Finally, correct interpretation of ECG traces in doubtful epilepsy cases is critical, especially in patients with normal neurological tests and poor response to anticonvulsant treatment; 6 out of 8 patients showed clear evidence of LQTS or BS in the ECG. Besides, comparison of ECG features between cases with correct and incorrect diagnosis was similar, suggesting an interpretation mistake. This confirms the need for close collaboration between neurologists and cardiologists to avoid diagnostic delay when facing with atypical epileptic cases, syncope, or SUDEP.[29]

Clinical outcome was poor in misdiagnosed cases. McCormick et al study reported that LQTS patients labeled as epileptic experienced a particularly long diagnostic delay and that ECGs were frequently requested but interpretation errors were common.6 Our data develop and extend this demonstration of clinical impact: there was not only a long diagnostic delay in misdiagnosed cases, but also a higher incidence of VF/SCD. Syncope and absence of medical therapy (particularly betablockers in LQTS and CPVT cases) are strong predictors of impaired survival in cardiac channelopathies.10 These two features were present in all the misdiagnosed cases. Of particular interest is the finding that the use of more than 1 AED is an independent predictor of adverse outcome, given that some of these drugs have a potential arrhythmogenic role in LQTS due to their ability to prolong the action potential and the QT interval. These results have a strong clinical impact in daily practice.

As a retrospective study, it is susceptible to many limitations. First, although neurological medical records were rigorously evaluated, the analyzed data may be incomplete in some patients. Second, genetic evaluation was not performed in all probands and this could underestimate genetic testing yield. Third, patients are a highly selected population from a single referral center limitating the representation of these patients. Finally, the sample size is modest, albeit for an uncommon clinical entity such as cardiac channelopathies results can be clinically interpreted with enough accuracy.

Conclusion

Cardiac channelopathy patients can be misdiagnosed with epilepsy. This involves diagnostic delay, prolonged absence of correct antiarrhythmic therapy, and the use of AEDs with a potential proarrhythmic effect, resulting in increased risk of malignant ventricular arrhythmias and SCD.

Detailed genetic evaluation.

(DOCX)

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Genes included in the NGS panel.

(DOCX)

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Mutations identified in patients diagnosed with a cardiac channelopathy.

(DOCX)

Click here for additional data file.

Database with anonymized patients data, for independent replication of the results.

(SAV)

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31 Dec 2019

PONE-D-19-31902

Prognostic impact of misdiagnosis of cardiac channelopathies as epilepsy

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Reviewer #1: The authors report their experience about fifty families with channellopathy and they collected information about their epilepsy. This study focuses attention on a very interesting topic. The manuscript has some inaccuracies that I have detailed below:

- Population and study Group: the weak point of this part is the selection of the patients: I suggest it could be more helpful if the authors mentione more about the inclusion and exclusion criteria for the enrollment of the patients.

- Genetic evaluation: this section must be expanded because more details about the methods used must be reported.

- Results: it is essential to describe the clinical symptoms and EEG abnormalities found in the patients in order to support the main message of the study. Please add these details.

- Discussion: The discussion of the manuscript is inconclusive; the authors must discuss better the genetic of sudden unexpected death (see the paper by Manolis TA, et al. Seizure. 2019;64:65-73). Moreover, the authors must dedicate more room to the possible link between cardiac channellopathies and epilepsy (see the papers by Li MCH, et al. Epilepsia. 2019;60:1753-1767 and by Iannetti P et al. Eur Rev Med Pharmacol Sci. 2017;21:5523-55269). All these references should be quoted and briefly but incisively discussed.

In summary, the paper would acquire greater interest and attraction by giving emphasis to the possible genetic links between cardiac and neurological channellopathies.

Reviewer #2: This is a well written and clear manuscript with a clinically important finding about the significance of misdiagnosis in Long QT syndrome. Although the study group is not large the central finding has not been examined in other larger published Long QT cohorts, and there is only one previous comparable paper in the area from 2009.

The paper would benefit from some revisions, Major:

Discussion - although the paper is concerned principally with the the misdiagnosis of inherited cardiac channelopathies as epilepsy a substantial early part of the discussion (para 2) focuses on SUDEP - the occurrence of sudden death in individuals with a correct diagnosis of epilepsy. This includes a consideration of whether cardiac channels are also expressed in the CNS and contribute to epilepsy. This seems to be perpetuating the confusion that the paper is aiming to clarify - namely that these patients didn't have epilepsy and that this misdiagnosis was evident in the majority of cases from their first ECG.

I recommend that the discussion of SUDEP should be given less emphasis and the more relevant discussion relating to the clinical distinction between cardiac syncope and true seizures be expanded.

Logistic regression - the information on the regression analysis is not well presented and more detail is needed. What was the full list of variables considered? Age at diagnosis appears to be an important difference between the two groups, were the influence age and gender examined? Treatment with >1 AED was the most significant variable in the univariate analysis but it is not included in table 3 and no mention is made of what happened to this variable in the multiple logistic regression.

Minor:

Intro para 2: Therefore, patients at risk of SCD... suggest: As a result patients at risk of SCD...

Results para 2: "All patients underwent neuroimaging and EEG, showing no definite evidence of neurological disease" - this statement is vague. Were all the investigations actually normal? If not please describe any abnormalities.

Table 1 "First diagnostic ECG" - better labelled, 'First ECG diagnostic'

Discussion - last para - discussion of AE Drugs. Do you have any information about the specific drugs that individuals were on at the time of the SCA/SCD events? Were they on known QT prolonging meds?

Supplementary table S1 - please provide transript details (i.e. NM_ and NP_ numbers) that you have used to annotate these variants and ensure that you are using the standard (cardiac) transcripts. For instance Patient 2 is annotated form NM_000238.3, which is standard for KCNH2 but patient 1 is annotated from a different transcript.

It isn't clear to me how some of these variants reach a classification of likely pathogenic/pathogenic on standard ACMG criteria. For instance, based on current information our centre would only classify KCNH2 I30F as a VUS. Can you provide more detail about your classification pathway?

Reviewer #3: The topic of the paper (possible misdiagnosis of epilepsy in the context of cardiac channelopathy) is timely and of interest. The problem with the manuscript is the very modest number of subjects studied and some inaccuracies in the presentation of the numbers.

General comments:

The material consists of 50 patients (as such, quite limited number), and genetic analyses were performed only in 40 cases. Moreover, DNA diagnosis could be established in only 31 of these 40 cases, leaving 10 + 9 cases somewhat unkown in nature. The authors should seriously take these figures into account when they sum their conclusions e.g. on the increased risk of SCA/SD. One may even ask whether they should collect more cases before publication. The least they need to do is to provide a critically written paragraph "Study limitations" at the end of Discussion.

Specific comments:

1. It is irritating to give percentage values with decimals when analysing numbers of <40, in particular those of <8 (epilepsy cases). Please correct.

2. Fig. 1: Does panel A, combined with the text, indeed show that 10 patients with Brugada syndrome could not be verified with DNA? This should be explained if this is the case.

3. There appears to be no legend for Fig. 2D.

4. Results, 1st para: "Distribution of genes was..." suggests 8 + 6 + 5 + 4 = 23 but the previous sentence tells that there 31 disease-causing mutations. Reason for this discrepancy?

5. Table 1: Again, there is some problem with the n values. At "Genetic background" the numbers and %values are OK for the epilepsy cases (n = 8) but remain obscure for the other group (n = 31). It is impossible to get the sum 31 by any inspection of the data. And what is the difference between "Negative" and "Others" in this portion of the Table? My concern is that if there is some confusion with the figures, should there perhaps also be concern on the conclusions drawn?

**********

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17 Feb 2020

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Response: Dear Editor. It was not our aim to literally replicate any text from another publication. Certainly, we have read with interest the paper you mention by Coll et al, and some of our data are concordant. Perhaps we have involuntarily reproduced some similar information from this paper., We have carefully reviewed the discussion and reformulated some very similar phrases. We are very thankful for your thorough review, as this is something we had completely missed.

Reviewer #1: The authors report their experience about fifty families with channellopathy and they collected information about their epilepsy. This study focuses attention on a very interesting topic. The manuscript has some inaccuracies that I have detailed below:

- Population and study Group: the weak point of this part is the selection of the patients: I suggest it could be more helpful if the authors mentione more about the inclusion and exclusion criteria for the enrollment of the patients.

Response: Dear reviewer, thank you for your comment. Clearly it helps to improve the manuscript. Information regarding criteria for considering the presence or absence of epilepsy have been added in the method section (population and study groups). For considering an epilepsy diagnosis we contemplated the presence of either a personal history of seizures, epilepsy or a history of AED therapy after a neurologist assessment, including EEG and neuroimaging. Furthermore, we provide exclusion criteria among probands. We excluded all acquired causes of seizures comprising traumatic/vascular injury, fever or metabolism disbalance.

- Genetic evaluation: this section must be expanded because more details about the methods used must be reported.

Response: Thanks for your comment. We are aware that genetic evaluation methodology was succinctly described, mainly due to words account restriction. Methods on genetic evaluation have been briefly expanded in the main manuscript and further description is available in the supplementary data. We also attach a table including all genes analyzed in the NGS panel (Table 1 in supplementary material). The vast majority of genetic tests were performed by a multidisciplinary and experienced team heading by Dr. Lorenzo Monserrat, and finally interpreted in the patient/family context by our group.

- Results: it is essential to describe the clinical symptoms and EEG abnormalities found in the patients in order to support the main message of the study. Please add these details.

Response: According to the emergency admission reports, all the patients misdiagnosed with epilepsy presented generalized tonic-clonic seizures, described as shock-like and irregular movements of both arms and legs.

After a thorough evaluation in the Inherited Cardiovascular Disease Clinic and a primary arrhythmia syndrome was diagnosed, we can suspect that patients presented a cardiogenic syncope prior to seizures, and that these could be a consequence of the cerebral hypoxia that occurs in syncopes.

- Discussion: The discussion of the manuscript is inconclusive; the authors must discuss better the genetic of sudden unexpected death (see the paper by Manolis TA, et al. Seizure. 2019;64:65-73). Moreover, the authors must dedicate more room to the possible link between cardiac channellopathies and epilepsy (see the papers by Li MCH, et al. Epilepsia. 2019;60:1753-1767 and by Iannetti P et al. Eur Rev Med Pharmacol Sci. 2017;21:5523-55269). All these references should be quoted and briefly but incisively discussed.

In summary, the paper would acquire greater interest and attraction by giving emphasis to the possible genetic links between cardiac and neurological channellopathies.

Response: Thanks for this useful comment. As the reviewer states, etiology of SUDEP remains a matter of debate. It´s likely that there is not a single explanation for these unexplained deaths. We agree genetics play a role in SUDEP cases, but there is some heterogeneity and many doubts about the real cause in some of SUDEP cases. Our work focuses in those cases with a clear underlying cardiac genetic condition. Whether always there is a pitfall in diagnosis or there is a true cardio-cerebral channelopathy cannot be concluded from our data. In the revised version of the manuscript, we have gone deeper inside this issue, reviewing and quoting the interesting references the reviewer suggested. The article by Manolis TA et al is a thorough review about this possible cardio-neural connection, suggesting but not confirming the co-existence of both disorders. They suggest that many of SUDEP cases are really caused by this neuro-cardio-respiratory disfunction that leads to severe cardiorespiratory depression after a tonic-clonic generalized seizure. Our work focuses in a different clinical scenario, with a not definitive epilepsy diagnosis in all cases, so we cannot fully support this cardio-cerebral syndrome with our data. This is in agreement with the interesting editorial you have suggested, by Ianneti. There is not definitive evidence for the real co-existence of brain and Heart channelopathies, in the same patients, and for the same mutations. We have re-formulated this issues and cited these suggested references.

Reviewer #2: This is a well written and clear manuscript with a clinically important finding about the significance of misdiagnosis in Long QT syndrome. Although the study group is not large the central finding has not been examined in other larger published Long QT cohorts, and there is only one previous comparable paper in the area from 2009.

The paper would benefit from some revisions, Major:

Discussion - although the paper is concerned principally with the the misdiagnosis of inherited cardiac channelopathies as epilepsy a substantial early part of the discussion (para 2) focuses on SUDEP - the occurrence of sudden death in individuals with a correct diagnosis of epilepsy. This includes a consideration of whether cardiac channels are also expressed in the CNS and contribute to epilepsy. This seems to be perpetuating the confusion that the paper is aiming to clarify - namely that these patients didn't have epilepsy and that this misdiagnosis was evident in the majority of cases from their first ECG.

I recommend that the discussion of SUDEP should be given less emphasis and the more relevant discussion relating to the clinical distinction between cardiac syncope and true seizures be expanded.

Response: we are thankful for this useful comment, and we certainly agree in your statement. We are aiming to describe the poor clinical outcome in cases of underlying real channelopathy with no evidence of definitive epilepsy. We have reformulated the discussion giving more emphasis to cardiac conditions. We have not completely deleted other possible explanations for SUDEP as the cardiac autonomic system depression or the cardio-cerebral syndrome as we feel that they might explain some SUDEP cases, but we made a statement that our work do not support this hypothesis, albeit we cannot fully exclude it.

Logistic regression - the information on the regression analysis is not well presented and more detail is needed. What was the full list of variables considered? Age at diagnosis appears to be an important difference between the two groups, were the influence age and gender examined? Treatment with >1 AED was the most significant variable in the univariate analysis but it is not included in table 3 and no mention is made of what happened to this variable in the multiple logistic regression.

Response: Dear reviewer, the variables considered for the multivariate analysis were those which showed a tendency towards (p<0.1) or were significantly associated (p<0.005) with SCA or SCD at first presentation. Age at diagnosis was different in both groups because an epilepsy misdiagnosis led to a delay of the channelopathy diagnosis in this group. However, it was not associated with an increased risk of SCA or SCD event at time of channelopathy diagnosis (p=0.57) just as it wasn't the gender (0.78).

So both were considered for statistical analysis and none of them was were associated with an increased risk of SCA or SCD event at time of channelopathy diagnosis.

Treatment with >1 AED is a variable just present in those patients with epilepsy misdiagnosis. Therefore, we performed an univariate analysis in the patients (n=8) who were treated with AED, that is a completely different analysis that the univariate analysis that we perform with the variables present in all patients (n=50) so it could not be included in the multivariate analysis performed with those variables which showed a tendency towards (p<0.1) or were significantly associated (p<0.005) with SCA or SCD at first presentation in the group of 50 patients.

Minor

Intro para 2: Therefore, patients at risk of SCD... suggest: As a result patients at risk of SCD...

Response: Thank you for your suggestion, we changed the sentence.

Results para 2: "All patients underwent neuroimaging and EEG, showing no definite evidence of neurological disease" - this statement is vague. Were all the investigations actually normal? If not please describe any abnormalities.

Response: Dear reviewer, as it is described in result section, there were no relevant abnormalities at EEG evaluation. However, in one patient (patient VI in table 2) the EEG analysis showed “unspecific findings that could be related to Temporal Lobe Epilepsy”, bur not a definite diagnosis was performed . This information is reflected in table 2. Further evaluations with neuroimaging techniques, as brain magnetic resonance, showed no abnormalities in none of the misdiagnosed patients

Table 1 "First diagnostic ECG" - better labelled, 'First ECG diagnostic'

Response: The sentence has been changed, as suggested by the reviewer.

Discussion - last para - discussion of AE Drugs. Do you have any information about the specific drugs that individuals were on at the time of the SCA/SCD events? Were they on known QT prolonging meds?

Response: Dear reviewer, six of the eight patients were on valproic acid, two patients were on carbamazepine and lamotrigine, respectively. Until now, there is no evidence for an increased risk of acquired QT prolongation (https://crediblemeds.org).

This is an important issue of our paper. Here we point that not only misdiagnosed patients are at risk of taking potentially hazardous drugs, but not responding to one AEDs or the need for several AEDs may arise the suspicion of an underlying cardiac channelopathy. This fact was independently associated with an adverse outcome.

Supplementary table S1 - please provide transript details (i.e. NM_ and NP_ numbers) that you have used to annotate these variants and ensure that you are using the standard (cardiac) transcripts. For instance Patient 2 is annotated form NM_000238.3, which is standard for KCNH2 but patient 1 is annotated from a different transcript. It isn't clear to me how some of these variants reach a classification of likely pathogenic/pathogenic on standard ACMG criteria. For instance, based on current information our centre would only classify KCNH2 I30F as a VUS. Can you provide more detail about your classification pathway?

Response: Dear reviewer, genetic analysis were performed in a worldwide recognized center in cardiovascular genetics (Health in Code, Dr. Lorenzo Monserrat).

These analyisis are performed by a multidisciplary team, according to the guidelines of EuroGentest and the American College of Medical Genetics (ACMG) appropriately with the requirements of the UNE-EN ISO 15189 and CLIA-88 standards as quality standards for clinical laboratories.

The potential pathogenicity of the identified variants in probands is initially evaluated based on previous description in clinical literature, as well as published in-vitro or in-vivo studies and bioinformatics. This information is analyzed and interpreted by experienced cardiologists and geneticists. Further information regarding genetic evaluation is available in the supplemental material.

Finally the information from genetic analysis is evaluated in the context of the patient’s clinical scenario and family screening. For example the I30F KCNH2 you mentioned, was interpreted as likely pathogenic based on previously described pathogenic variants affecting the same aminoacidic residue and gene region (PAS domain), as well as, bioinformatics predictors. Proven cosegregation in this extensive family supported its pathogenicity. We provide the family pedigree and ECG of three cases from two different generations.

Reviewer #3: The topic of the paper (possible misdiagnosis of epilepsy in the context of cardiac channelopathy) is timely and of interest. The problem with the manuscript is the very modest number of subjects studied and some inaccuracies in the presentation of the numbers.

Response: we are thankful for your positive comments. We agree the numbers are not high. We are dealing with uncommon clinical entities such as cardiac channelopathies and 50 seems to be an acceptable number to get some conclusions. However, we have added a limitation statement to accept this limitation, and all your considerations about the number presentations and statistics have been taken into account.

General comments:

The material consists of 50 patients (as such, quite limited number), and genetic analyses were performed only in 40 cases. Moreover, DNA diagnosis could be established in only 31 of these 40 cases, leaving 10 + 9 cases somewhat unkown in nature. The authors should seriously take these figures into account when they sum their conclusions e.g. on the increased risk of SCA/SD. One may even ask whether they should collect more cases before publication. The least they need to do is to provide a critically written paragraph "Study limitations" at the end of Discussion.

Response: we agree with this limitation. In this unicentric cohort of patients it´s unlikely to be able to add more patients. We feel statistics can help us understand and conclude some interesting points in studies with such limitation. In our opinion, and adding your suggested limitation paragraph, we feel these data are of clinical value and help the reader understand the poor clinical outcome in cases of clear cardiac channelopathy, but wrongly labelled as epileptic.

Specific comments:

1. It is irritating to give percentage values with decimals when analysing numbers of <40, in particular those of <8 (epilepsy cases). Please correct.

Response: Thank you for your suggestion, we changed the numbers presentation in the manuscript and tables.

2. Fig. 1: Does panel A, combined with the text, indeed show that 10 patients with Brugada syndrome could not be verified with DNA? This should be explained if this is the case.

Response: Dear reviewer, as you correctly remark, genotype analysis of 10 Brugada Syndrome (BrS) patients were not performed.

As previously reported in the HRS/EHRA Expert Consensus Statement on the State of Genetic Testing for the Channelopathies and Cardiomyopathies (Ackerman MJ, 2011), the diagnosis of BrS is clinical: based on ECG and clinical presentation, however genetic evaluation is not involved.

Since genetic evaluation is not implicated for neither diagnosis or risk stratification, in the context of the absence of a positive family history of SCD, we do not routinely perform genetic evaluation. It could lead to misinterpretations (Risgaard B; Clin Genet 2013) and unjustificable alarm and morbidity associated with unnecessary medical interventions among patients and relatives.

Moreover, although genetic testing costs are decreasing and is widely available, genetic testing yield in BrS is very low, about 30% (Priori SG. Circulation, 2002), limiting its benefice. Based on this reasons, we feel that genetic testing in BrS is recommended in selected patients. We have included and explained this point in results section, and added the information in Figure 1.

3. There appears to be no legend for Fig. 2D.

Response: Thank you for your comment, it was not properly indicated. It has been corrected.

4. Results, 1st para: "Distribution of genes was..." suggests 8 + 6 + 5 + 4 = 23 but the previous sentence tells that there 31 disease-causing mutations. Reason for this discrepancy?

Response: Thank you for your observation. In order to expose a clearer presentation of the data, we described the most common genes in this peculiar population. Certainly, probands with a positive genotype are 31:

8 RYR2 + 6 KCNH2 + 4 SCN5A + 5 KCNQ1 + 8 OTHERS = 31. “Others” group includes probands with mutation in CALM2 (protein calmodulin 2) (2 probands) (Jiménez-Jáimez J. PLoS One. 2016), CASQ2 (protein calsequestrin 2) (3 probands) and KCNJ2 (protein Kir2.1) (2 probands), responsible for the development of CPVT and Andersen-Tawil Syndrome, respectively. Furthermore one patient carrying pathogenic variants in KCH2 and KCNQ1 genes was considered in the group “others”.

This “others” group has been added to the revised main manuscript (results, 1st para). Furthermore, genotype details from the 31 probands are available in the supplementary material.

5. Table 1: Again, there is some problem with the n values. At "Genetic background" the numbers and %values are OK for the epilepsy cases (n = 8) but remain obscure for the other group (n = 31). It is impossible to get the sum 31 by any inspection of the data. And what is the difference between "Negative" and "Others" in this portion of the Table? My concern is that if there is some confusion with the figures, should there perhaps also be concern on the conclusions drawn?

Response: Dear reviewer, since “others” group was not properly described in the manuscript, information in text and tables was confusing. Thank you for your comment, we have improved the presentation of our data.

In table 1, there were various errors in the numbers, I would like to deeply apologize for that. These mistakes have been resolved and we have also added “not performed” row for a clearer presentation of data.

For clarification, a negative result from NGS analysis was considered when no potential pathogenic variant was detected. On the other hand, patients who presented a complex genotype (multiple mutations in different genes) or a potential pathogenic variant in a rare gen (in our case, CASQ2, CALM2 and KCNJ2) after NGS analysis, were considered in the “other group”.

Furthermore, in the “method/5 para” and “results/1st para” sections, information regarding genotype analysis has been extended including, and brief description of criteria for patients distribution among groups of analysis.

Thankfully, conclusions are based on the numbers presented in the manuscript text and this confusion with presentation of numbers in the table does not change the conclusions and key messages from our study.

Submitted filename: Reviewer_FB-JJ.docx

Click here for additional data file.

25 Mar 2020

Prognostic impact of misdiagnosis of cardiac channelopathies as epilepsy

PONE-D-19-31902R1

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31 Mar 2020

PONE-D-19-31902R1

Prognostic impact of misdiagnosis of cardiac channelopathies as epilepsy

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