An Evidence-Based Evaluation of Gene Validity for Brugada Syndrome

10.1161/CIRCULATIONAHA.118.035070

Reappraisal of Reported Genes for Sudden Arrhythmic Death: An Evidence-Based Evaluation of Gene Validity for Brugada Syndrome

Running Title: Hosseini et al.; Gene Validity in Brugada Syndrome

S. Mohsen Hosseini, MD, PhD1*; Raymond Kim, MD, PhD1,3,4*; Sharmila Udupa, MD2*; Gregory Costain, MD, PhD3; Rebekah Jobling, MD1,3; Eriskay Liston, MSc1,3; Seema M. Jamal, MSc3; Marta Szybowska, MSc4; Chantal F. Morel, MD4; Sarah Bowdin, MD1,3; John Garcia, PhD5; Melanie Care, MSc6; Amy C. Sturm, MSc7; Valeria Novelli, PhD8; Michael J. Ackerman, MD, PhD9; James S. Ware, PhD, MRCP10; Ray E. Hershberger, MD11; Arthur A. M. Wilde, MD, PhD12; Michael H. Gollob, MD, (Chair)2,6,13; on behalf of the NIH-Clinical Genome Resource Consortium.

1Ted Rogers Cardiac Genome Clinic, The Hospital for Sick Children, Toronto, ON, Canada; 2Toronto General Hospital Research Institute, University of Toronto, Toronto, ON, Canada; Downloaded from 3Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada; 4Fred A. Litwin Family Center in Genetic Medicine, University Health Network, Toronto, ON, Canada; 5Invitae Corp, San Francisco, CA; 6Peter Munk Cardiac Centre, Department of Medicine, Toronto General Hospital, University of Toronto, Toronto, ON, http://circ.ahajournals.org/ Canada; 7Geisinger Health System Genomic Medicine Institute, Danville, PA; 8Centro Benito Stirpe per la Morte Improvvisa del Giovane Atleta,Fondazione Policlinico Universitario Agostino Gemelli, Catholic University of the Sacred Heart, Rome, Italy; 9Departments of Cardiovascular Diseases, Pediatrics, and Molecular Pharmacology & Experimental Therapeutics; Divisions of Heart Rhythm Services and Pediatric Cardiology; Windland Smith Rice Sudden Death Genomics Laboratory, Rochester, MN; 10National Heart and Lung Institute & MRC

by guest on July 2, 2018 London Institute of Medical Sciences, Imperial College London, and Royal Brompton & Harefield Hospitals, London UK; 11Division of Human Genetics & Cardiovascular Division, Department of Internal Medicine, Ohio State University, Columbus, OH; 12AMC Heart Center, Department of Clinical and Experimental Cardiology, Academic Medical Center, Amsterdam, the Netherlands & Princess Al-Jawhara Al-Brahim Centre of Excellence in Research of Hereditary Disorders, Jeddah, Kingdom of Saudi Arabia, and and Columbia University Irving Medical Centre, NewYork, NY; 13Department of Physiology and the Peter Munk Cardiovascular Molecular Medicine Laboratory, Toronto General Hospital, University of Toronto, Toronto, ON, Canada * The authors contributed equally to the manuscript.

Address for Correspondence: Michael H, Gollob, MD Peter Munk Cardiac Centre, Department of Medicine Toronto General Hospital, University Health Network Toronto, Ontario, Canada Tel: 416-340-4282 Fax: 416-340-3281 Email: [email protected]

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Abstract

Background—Implicit in the genetic evaluation of patients with suspected genetic diseases is the assumption that the genes evaluated are causative for the disease based on robust scientific and statistical evidence. However, in the past 20 years considerable variability has existed in the study design and quality of evidence supporting reported gene-disease associations raising concerns of the validity of many published disease-causing genes. Brugada syndrome (BrS) is an arrhythmia syndrome with a risk of sudden death. More than 20 genes have been reported to cause BrS and are assessed routinely on genetic testing panels in the absence of a systematic, evidence-based evaluation of the evidence supporting the causality of these genes. Methods—We evaluated the clinical validity of genes tested by diagnostic laboratories for BrS by assembling three gene curation teams. Using an evidence-based semi-quantitative scoring system of genetic and experimental evidence for gene-disease associations, curation teams independently classified genes as demonstrating Limited, Moderate, Strong or Definitive evidence for disease causation in BrS. The classification of curator teams was reviewed by a Downloaded from Clinical Domain Expert Panel who could modify the classifications based on their independent review and consensus. Results—Of 21 genes curated for clinical validity, biocurators classified only 1 gene (SCN5A) as Definitive evidence, while all other genes were classified as Limited evidence. Following http://circ.ahajournals.org/ comprehensive review by the Clinical Domain Expert Panel, all 20 genes classified as Limited evidence were re-classified as Disputed in regards to any assertions of disease causality for BrS. Conclusions—Our results contest the clinical validity of all but one gene clinically tested and reported to be associated with BrS. These findings warrant a systematic, evidence-based evaluation for reported gene-disease associations prior to use in patient care.

by guest on July 2, 2018 Key Words: Brugada syndrome; genetics; sudden death; ClinGen

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Clinical Perspective

What is new? • This evidence-based review of genes reported to cause Brugada Syndrome (BrS) and routinely clinically tested in patients indicates that 20 of 21 genes lack sufficient genetic evidence to support their causality for BrS. • Only the SCN5A gene is classified as having Definitive evidence as a cause for BrS.

What are the clinical implications? • Routine genetic evaluation of genes other than SCN5A is not currently warranted in the Downloaded from clinical care of BrS patients. • Genetic testing of genes without sufficient evidence supporting causality for BrS may lead to incorrect interpretation of rare variants in these genes and inappropriate diagnostic http://circ.ahajournals.org/ conclusions or interventions for patients and family members.

by guest on July 2, 2018

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Introduction

The Human Genome Project imbued science and medicine with the blueprint for human health

and disease.1 In the last 25 years, the evolution of this historic accomplishment has been

extraordinary, enabling the elucidation of the genetic and molecular underpinning of thousands

of human diseases. The impact on health care delivery has been extensive and has defined a new

era of Precision Medicine. Now, patients may have their clinical diagnosis genetically

‘confirmed’, in some cases specifically managed based on genotype, and may choose to share

their genetic information with at-risk family members to allow for presymptomatic genetic Downloaded from testing for disease risk. Provision of genetic testing services for clinical care is now widely

available and currently, 54,057 genetic tests offered by 503 labs are recorded by the Genetic http://circ.ahajournals.org/ Testing Registry of the National Center for Biotechnology Information

(https://www.ncbi.nlm.nih.gov/gtr/ queried 10-12-2017), examining 16,236 genes for 10,889

conditions. by guest on July 2, 2018 Successful implementation of Precision Medicine, at its foundation, necessitates that

reported gene-disease associations are reliably evidence-based to ensure the appropriate

application of genetic information in optimizing care while preventing inaccurate conclusions

that may cause harm. There remains considerable variability in the level of genetic and

experimental evidence of reported gene-disease associations, raising questions about the clinical

validity of some genes and potential concerns at their inclusion for clinical genetic testing.

Available gene-disease databases such as Online Mendelian Inheritance in Man (OMIM)2,

although valuable, lack the rigorous critical approach to examine the clinical validity of proposed

associations. The need for a systematic, evidence-based method for curating gene-disease

associations spurred the development of ClinGen (Clinical Genome Resource)3, an NIH-funded

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international consortium of geneticists, genomic scientists, and clinical domain experts, with the

common goal of defining standardized, evidence-based frameworks for assessment of reported

genetic associations for use in Precision Medicine.

Here, we report the first application of the ClinGen evidence-based gene curation

framework for sudden cardiac death (SCD)-predisposing, genetic heart rhythm diseases. Brugada

Syndrome (BrS) has an estimated prevalence of 1:2000.4,5 When familial, it follows an

autosomal dominant mode of inheritance.6 To date, over 20 genes have been reported to be

associated with BrS 6 and are routinely tested as single-gene causes for this condition on a Downloaded from variety of clinical genetic testing panels worldwide. As disease penetrance for BrS is incomplete

and age-related, genetic testing may be used for diagnostic purposes and for the screening of at- http://circ.ahajournals.org/ risk family members. In this context, genetic information may lead to disease-labeling in

individuals, influence physicians’ decision-making in guiding preventative treatments by means

of an implantable-cardioverter defibrillator, or lead to cascade screening of family members. In by guest on July 2, 2018 view of the significant impact a diagnosis of BrS (or any genetic disease) may have on an

individual and their family, it is critical that only genes with robust clinical validity be evaluated

in the care of patients to minimize the risk of incorrect interpretation of genetic information that

may ultimately cause undue harm or anxiety.

Methods

For purposes of replicating the results or process of this study, the analytic methods and study

materials for this study are described and referenced accordingly and all data is available to other

researchers in the Supplementary File (scores).

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Selection of Genes for Curation

Genes were selected for curation if they met all of the following criteria: i) ≥ 2 publications in

peer-reviewed medical literature suggesting single-gene causality for BrS, ii) reported literature

presenting both genetic and experimental data, and iii) present on ≥ 3 BrS clinical genetic testing

panels from accredited diagnostic laboratories. It should be noted that a number of the genes

evaluated for BrS have also been implicated in other diseases; however, this effort did not

evaluate the validity of any gene for disorders other than BrS.

Gene Curation Framework Downloaded from We formed three gene curation teams to independently curate each gene. Each curation team was

led by a board-certified medical geneticist and included two additional members. All gene http://circ.ahajournals.org/ curation team members were required to have graduate degrees in human genetics (three Masters

Degrees, three M.D./Ph.D. Scientists, three M.D. Clinical Geneticists). Gene curation teams

worked blinded to other curation teams and utilized the recently proposed ClinGen Gene by guest on July 2, 2018 Curation framework.7 Curation team members were required to review a Standard Operating

Procedure for gene curation using this framework (https://www.clinicalgenome.org/curation-

activities/gene-disease-validity/educational-and-training-materials/standard-operating-

procedures/) and received a webinar presentation illustrating the application of the analytic

process.

A detailed description of the ClinGen Gene-Disease Validity Classification Framework

has been published recently.7 This framework provides a systematic, evidence-based approach

for assessing reported gene-disease associations. Using a semi-quantitative scoring system, each

gene-disease relationship is categorized into a clinical validity classification level based on the

sum of its accompanying evidence. Clinical validity classification levels include the following:

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‘Definitive’ (12-18 points and replicated literature), ‘Strong’ (12-18 points), ‘Moderate’ (7-11

points), and ‘Limited’ (1-6 points).

Briefly, the evidence-based framework evaluates genetic and experimental data

separately and provides a scoring metric based on the level of evidence provided in the published

literature for the gene. Genetic evidence scores were weighted according to the design and

quality of the genetics study. For example, genes implicated in studies with familial data,

variant-disease segregation and significant LOD scores receive a greater assigned score than

genes implicated through ‘candidate gene’ approaches with small cohort sizes. Downloaded from Experimental evidence scores were based on the interpretation and phenotypic relevance

of in vitro assays assessing functional alterations of the disease-implicated gene variants, and http://circ.ahajournals.org/ model organism or rescue studies, as proposed by MacArthur et al.8

Details of this scoring matrix and a template spreadsheet can be accessed online 7:

https://www.clinicalgenome.org/working-groups/gene-curation/projects-initiatives/gene-disease- by guest on July 2, 2018 clinical-validity-scoring-matrix/ .

A Clinical Domain Expert Panel, consisting of 9 additional individuals with collectively

dozens of years of experience in the clinical care and/or research in the field of BrS, was tasked

with performing a final evaluation and classification on a gene by gene basis. This panel had the

option of modifying the classification of each gene (upgrade, no change, downgrade) based on

their collective experience and independent assessment of the medical literature and scientific

evidence following review of curator team summaries. Each individual on the panel

independently reviewed data for each gene, and together the panel discussed classifications for

each gene in teleconference and face-to-face meetings (Supplemental Figure 1). In the end, the

Chair of the panel requested each individual provide a confidential vote on a Final Classification

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for each gene.

Results

Identifying Brugada Genes for Curation

Using a PubMed search [Search terms: Gene (from Bookshelf) for PubMed (Search Brugada

syndrome gene)], we identified 23 genes reported to be associated with BrS (Table 1). As of

September 2017, we identified 30 accredited laboratories offering specific BrS multi-gene

panels, 15 in the USA, 15 internationally (NCBI Genetic Testing Registry: Downloaded from https://www.ncbi.nlm.nih.gov/gtr/ queried 2017-06-29; GeneTests website,

https://www.genetests.org/disorders/?disid=33991, accessed 2017-09-27). These panels varied in http://circ.ahajournals.org/ the number of genes tested (range 3-23 genes, median 11 genes per panel). Only four genes were

present on all testing panels (SCN5A, GPD1L, CACNA1C, and CACNB2). Of the 23 genes, 21

were reported in the literature in at least two peer-reviewed publications and were present on by guest on July 2, 2018 three or more BrS clinical genetic testing panels (range 3-29 panels, median 15 panels per gene)

(Figure 1). These 21 genes were selected for gene curation.

Gene Curation

Over 6-months, three gene curation teams independently reviewed the published literature for

each gene and applied the analytic framework spending a total of 318·5 hours (14·5 ± 5·9 hours

per gene, mean ± SD). A total of 130 publications were reviewed and logged by curation teams

(average 7 publications per gene, range 2-19). A complete list of publications reviewed for each

gene can be found in the online Supplementary File.

Figure 2 summarizes the clinical validity classifications and semi-quantitative scores for

genetic and experimental evidence from curation teams for each gene. There was complete

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concordance between the three curation teams in the clinical validity classifications of all 21

genes. SCN5A was the only gene that reached the Definitive evidence tier for BrS. All other

genes (20 of 21 genes) were classified as Limited evidence by the curator teams.

The Clinical Domain Expert Panel, although agreeing with the application of the scoring

template by curator teams, re-classified all 20 Limited evidence genes as Disputed, concluding

that currently published literature is not sufficient to assert causality for BrS for any of these 20

genes. Consensus of the Expert Panel was unanimous (voted 9-0) for re-classification based on

specific issues related to the methodology of genetic studies, the lack of supportive statistical Downloaded from evidence, the absence of genetically altered animal models recapitulating disease and the

uncertain interpretation of in vitro experimental data as related to the disease phenotype. These http://circ.ahajournals.org/ concepts are discussed in detail in the Discussion section. Summary tables on a gene by gene

basis are available in the Supplementary File (Scores).

Query of ClinVar submissions by guest on July 2, 2018 To evaluate the potential impact of available testing panels on gene variant interpretations, we

analyzed the number of ClinVar submissions for BrS by clinical testing laboratories. Our query

("Brugada syndrome" as [Disease/Phenotype] Filters: Clinical testing (queried 2017-11-01), i.e.

excluding “research” and “literature only” submissions] returned 1223 variations for BrS in 21

different genes, 33% in SCN5A (Figure 3A group(G) (G1, G2, G3).

Figure ). Of variants classified as pathogenic and likely pathogenic, 6% were in the 20 Disputed

evidence genes, while the remainder were in SCN5A. In total, Disputed evidence genes most

commonly had submitted variants classified as uncertain significance (VUS) or with conflicting

interpretations (56% or 420/747 of submitted variants) (Figure 3B).

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Discussion

In this study, we performed an evidence-based curation of 21 genes reported as single gene

causes for BrS that are routinely tested in patients by accredited laboratories’ clinical genetic

testing panels for this sudden arrhythmic death condition. Remarkably, only one (SCN5A) of 21

genes was classified as a ‘Definitive’ evidence gene for BrS. All other genes (20 of 21) received

a final classification of ‘Disputed’ in regards to assertions towards causality for BrS by a Clinical

Domain Expert Panel.

The findings of this study challenge the inclusion of 20 genes, out of 21, currently offered Downloaded from for clinical genetic testing for BrS. All three curator teams and the Clinical Domain Expert Panel

agreed only SCN5A has sufficient evidence for causality in BrS to warrant inclusion on clinical http://circ.ahajournals.org/ genetic testing panels and that current evidence does not support causality or clinical testing of

the 20 additional curated genes. The Expert Panel cited the following facts that alone or in

combination compelled the conclusion of Disputed gene classification: i) familial or segregation by guest on July 2, 2018 data of affected cases with rare variants was insufficient to support causality for the gene in most

studies; when sufficient data for familial linkage to a genomic region was present,

comprehensive sequencing data of all rare variants shared among affected individuals within the

shared genomic region was not provided; ii) reported genes were implied to have causality for

BrS on the basis of rare gene variants identified in BrS cohorts, an observation that is insufficient

to claim causality in light of the now known observation of frequent, benign rare genetic

variations in healthy human populations common to most genes; iii) the frequency of the

reported ‘rare’ gene variants in BrS cases leading to claims of causality were subsequently

determined to be grossly underestimated with observed frequencies in the general population that

are incompatible with disease causation based on disease prevalence; iv) supportive functional

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data was limited to in vitro experiments suggesting plausible molecular mechanism but without

phenotypic recapitulation, and in the absence of validated assays proven to distinguish disease-

causing rare genetic variants from benign rare genetic variants; v) existing data do not

demonstrate statistical evidence of an excess of rare genetic variants for the gene in BrS cases

versus healthy controls.

Of the 20 genes classified as Disputed, 19 of 20 were originally reported in pre-conceived

candidate gene studies on the basis of biological plausibility, in contrast to an a priori familial

linkage or other unbiased genome-wide methodology, approaches advocated for gene discovery.8 Downloaded from Seminal manuscripts for 13 of the 19 genes reported rare variants in only sporadic cases with no

segregation data or evidence of rare variant excess as compared to control subjects (ABCC9, http://circ.ahajournals.org/ ANK2, CACNA2D1, HCN4, KCND3, KCNH2, KCNJ8, RANGRF, SCN1B, SCN2B, SCN3B,

SLMAP, TRPM4). Three genes were reported with limited segregation in only two individuals in

two generations (CACNA1C, KCNE5, SCN10A). Only three genes (CACNB2, KCNE3, PKP2) by guest on July 2, 2018 were reported to have more than two segregations within a family.9-11 Although the original

description of a family harboring a CACNB2 variant identified via a candidate gene approach

indicated six segregations,9 our curators noted a reported affected individual who did not carry

the reported variant in CACNB2. Further independent reports were not identified with sufficient

genetic or statistical evidence to warrant classification beyond the Disputed evidence tier for this

gene. Similarly, four and three segregations were reported in original manuscripts using pre-

conceived candidate gene approaches implicating KCNE3 and PKP2, respectively, but without

sufficient statistical data, additional families or supporting evidence in the literature.10, 11

SCN5A gene was the only gene, among the 21 curated, to be classified as ‘Definitive’ by all three

curation teams. Interestingly, the original manuscript reporting this gene’s association with BrS

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was also a ‘candidate gene’ study in small pedigrees.12 However, subsequent papers reported

larger pedigrees with segregation and sufficient statistical evidence supporting gene causality.13,

14 In addition, curators and the Expert Panel cited the dense literature reporting protein-truncating

variants in this gene segregating with phenotype, and a published rare variant (MAF <0·001)

burden analysis of genes reported to be associated with BrS which identified a significant excess

of SCN5A variants in BrS cases compared to healthy controls (20·4% vs 2·4%, p < 1·4×10-7).15

In the same analysis, the authors evaluated 18 of the 20 Disputed evidence genes and did not find

any significant enrichment of rare variants in BrS cases.15 Downloaded from Interestingly, the GPD1L gene received Limited evidence classification by curator all 3

curator teams and a final classification of Disputed, despite its identification through the use of http://circ.ahajournals.org/ genetic linkage. Although genes identified with significant segregation and apparent linkage to

phenotype may receive higher scoring in the application of the evidence-based template, the

assigned scoring depends on whether comprehensive sequencing of all genes in the linked by guest on July 2, 2018 genomic region was performed versus selected screening of only specific genes. In the case of

GPD1L, curators and the expert panel noted the extensive size of the linked genomic region (at

least ~15 cM), the select sequencing of only a limited number of genes within this region

distanced by up to ten million base pairs with the lack of comprehensive sequencing of the

region to assess for alternative gene variants, the observation that the reported variant in this

gene is now recognized to be present in 1/5000 individuals in public databases

(http://gnomad.broadinstitute.org/gene/ENSG00000152642), and the absence of any subsequent

familial descriptions since the seminal publication in 2002.16, 17

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It is only recently appreciated that many benign variants can be extremely rare in a

population, and therefore finding of rare variation in a gene in patients with a genetic condition is

far from sufficient evidence to assert causality. Thirteen of the 20 Disputed evidence genes were

published earlier than 2013, prior to the availability of large public databases indicating gene

variant frequencies in thousands of individuals where there is no reason to anticipate over-

representation of this particular disorder with a 1 in 2000 prevalence. In the absence of large

databases to compare variant frequencies found in disease cases, early gene-disease reports

typically evaluated a small cohort of controls (100-1000 samples) to decide on variant rarity. Downloaded from Two curated genes in this study were originally implicated on the basis of variants suggested to

be ‘rare’ in the original studies, only to be subsequently noted to have heterozygosity frequency http://circ.ahajournals.org/ in public databases greater than or equal to the prevalence of BrS (KCNJ8 p.Ser422Leu, 1/250,

SCN3B p.Leu10Pro, p.Val110Ile, both 1/2500). This issue is further highlighted in a publication

from Risgaard et al assessing the prevalence of previously concluded ‘disease-causing by guest on July 2, 2018 mutations’ from original publications implicating 12 BrS genes. They found that of

approximately 4000 individual exomes made publicly available in 2012 from the NHLBI GO

Exome Sequencing Project, 1 in 23 individuals carried an originally reported putative BrS

“mutation”.18 Surprisingly, despite classification as a ‘Definitive’ evidence gene, an estimated

10-20% of early reported putative mutations in SCN5A may have been erroneously classified,

highlighting the challenges of variant interpretation for even definitive genes in the absence of

large databases.19

Lastly, the reported BrS genes curated in this study typically provided in vitro functional

data to suggest a plausible molecular mechanism for disease caused by rare variants. However,

an altered in vitro function demonstrated in non-cardiac, immortalized cell lines in the absence of

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phenotypic recapitulation in an intact animal is not synonymous with proof of disease causation.

Further, none of the experimental in vitro methodologies utilized for these 20 Disputed genes

have been validated to distinguish the function of benign rare variants known to exist in healthy

populations from the reported ‘disease-causing’ rare variants. To illustrate this issue, the reported

KCNJ8 variant p.Ser422Leu originally implicated as disease-causing for BrS was reported to

have a 2-fold gain-of-function of ion current in vitro as compared to wild-type, despite its

presence in 1/250 individuals in the general population, a frequency far too common to be a

cause of BrS.20, 21 Similarly, significant functional differences were reported for variants Downloaded from reported in SCN3B as a cause for BrS, despite the frequent presence of these same variants in

presumably healthy populations.22, 23 In the context of heart rhythm and electrocardiographic http://circ.ahajournals.org/ features in humans, it would be expected that both rare and common gene variants confer

functional differences explaining the large range of normal variation in heart rates, QRS and QT

parameters. Indeed, it is recognized that common gene variations (MAF > 0.5-20%) in disease- by guest on July 2, 2018 causing ‘channelopathy’ genes such as KCNH2 and SCN5A may provoke functional alterations

in vitro similar to putative mutations in those same genes yet not lead to an associated

phenotype.24, 25

Given the results of this gene curation effort, it is important to thoughtfully consider why

accredited laboratories include genes on BrS testing panels that do not have sufficient evidence

for disease causality. Foremost, accreditation bodies do not require laboratories to justify the

inclusion of genes on panels for clinical testing. It is also possible that the increasingly

competitive marketplace in laboratory genetics motivated a ‘more genes is better’ approach

leading to rapidly expanding gene panels. The practical implications of including these Disputed

evidence genes on testing panels in clinical care are potentially harmful. Physicians and genetic

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counsellors may trust that the inclusion of genes on disease panels by accredited laboratories

implies that they have valid associations with diseases. However, testing of genes with

insufficient evidence for causality creates unnecessary challenges for variant interpretation,

particularly for predicted loss of function variants that may be incorrectly assumed to be

pathogenic, and enhances the possibility of false-positive interpretations, a scenario that may

lead to inappropriate risk prediction in family members, unnecessary clinical testing,

prophylactic therapy and significant distress within a family. This is especially concerning for

age-related genetic diseases with incomplete penetrance, such as BrS, where genetic Downloaded from observations may be unduly persuasive towards diagnostic conclusions. These concerns are not

merely hypothetical. Our query of ClinVar indicated that of submitted variants classified as http://circ.ahajournals.org/ pathogenic or likely pathogenic for BrS, 6% represented variants in Disputed genes. In addition,

more than half of the variants submitted in these Disputed genes were classified as uncertain

significance or had conflicting interpretations. The ramifications of these interpretations for by guest on July 2, 2018 variants in genes with insufficient evidence to support causality for this sudden death condition

are concerning and may have led to inappropriate care in some families.

The conclusion of the Human Genome Project in 2003 was met with great enthusiasm in

all fields of medicine and led to the rapid reporting of thousands of genes over the last decade as

being disease-causing. Our study highlights the increasing threshold now necessary to conclude

gene-disease causality in light of the evolving knowledge of natural genetic variations in the

population, and the need for cautious interpretation of functional assays that do not recapitulate

the disease phenotype and are not validated to distinguish rare benign from rare disease-causing

variants. While the majority of reported gene-disease associations in our study currently lack

sufficient evidence to warrant their inclusion for clinical genetic testing to direct care for BrS

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patients and families, many of these genes should remain in the realm of research, and some of

these genes could ultimately gain clinical validity in future gene curation efforts. However,

further research aimed to promote Disputed genes or invoke novel single gene causes for disease

should provide genetic evidence using an unbiased analysis of genomic data with supportive

statistical evidence. These may include studies of large families or kindreds, which are

unfortunately uncommon in BrS. Alternatively, large case-control cohorts demonstrating a

statistically significant excess of rare variants in a gene of interest amongst cases could satisfy

strong genetic evidence. Importantly, in light of the common observation of sporadic, non- Downloaded from familial cases of BrS, oligogenic inheritance may play a significant role and create a more

challenging genetic landscape to study. http://circ.ahajournals.org/ Successful implementation of Precision Medicine requires that the inclusion of genes on

diagnostic testing panels accurately reflect clinically valid gene-disease associations. In the

absence of clinical validity of tested genes, unnecessary, costly and potentially harmful clinical by guest on July 2, 2018 tests or interventions may be ordered and used in the care and clinical decision making for a

patient or family. If Precision Medicine is to optimize the care of patients and families, it is

essential that practitioners, counselors and diagnostic laboratories come together to ensure the

most appropriate inclusion of genes for diagnostic testing and subsequent interpretation. Our

findings warrant a systematic, evidence-based approach to assess the validity of reported gene-

disease associations prior to use in patient care.

Contributors

MHG designed the study. SU and MHG did initial literature search and determined genes to be

curated. SMH, GC, RJ, EL, SMJ, MS, CFM, SB, and RHK performed the evidence-based gene

16 10.1161/CIRCULATIONAHA.118.035070

biocuration. SMH summarized the data, did the statistical analyses and generated figures. JG,

MC, ACS, VN, JSW, REH, MJA, AAMW, and MHG were the Clinical Domain Expert panel

and reviewed biocuration data and analyses. MHG wrote the manuscript. All authors reviewed

and edited the manuscript.

Disclosures

None

Downloaded from Acknowledgments

The authors would like to thank Jennifer Goldstein, Ph.D., C. Lisa Kurtz, Ph.D., and Roozbeh http://circ.ahajournals.org/ Manshaei, Ph.D. for their assistance. Dr. Ware is supported by Wellcome Trust (107469/Z15/Z)

and the British Heart Foundation. Dr. Wilde is supported by the Netherlands CardioVascular

Research Initiative (Predict). Dr. Gollob is supported by the Heart and Stroke Foundation Mid- by guest on July 2, 2018 Career Scientist Award (MC7449) and the Peter Munk Research Chair in Cardiovascular

Molecular Medicine (Toronto General Hospital, University of Toronto).

Sources of Funding

The US National Human Genome Research Institute partially funded this study. Funding agency

members had no role in the study design or collection, analysis, and interpretation of the data or

writing of the manuscript. The corresponding author had full access to all the data in the study

and had final responsibility for the decision to submit for publication. The Clinical Genome

Resource Consortium is funded by the National Human Genome Research Institute (U41

HG006834, U01 HG007436).

17 10.1161/CIRCULATIONAHA.118.035070

References

1. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann Y, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Downloaded from Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, http://circ.ahajournals.org/ Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Raymond C, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R,

by guest on July 2, 2018 Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowki J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ, Szustakowki J and International Human Genome Sequencing C. Initial sequencing and analysis of the human genome. Nature. 2001;409:860-921. 2. Amberger JS, Bocchini CA, Schiettecatte F, Scott AF and Hamosh A. OMIM.org: Online Mendelian Inheritance in Man (OMIM(R)), an online catalog of human genes and genetic disorders. Nucleic Acids Res. 2015;43:D789-798. 3. Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, Ledbetter DH, Maglott DR, Martin CL, Nussbaum RL, Plon SE, Ramos EM, Sherry ST, Watson MS and ClinGen. ClinGen--the Clinical Genome Resource. N Engl J Med. 2015;372:2235-2242. 4. Wilde AA, Antzelevitch C, Borggrefe M, Brugada J, Brugada R, Brugada P, Corrado D, Hauer RN, Kass RS, Nademanee K, Priori SG, Towbin JA and Study Group on the Molecular Basis of Arrhythmias of the European Society of C. Proposed diagnostic criteria for the Brugada syndrome: consensus report. Circulation. 2002;106:2514-2519.

18 10.1161/CIRCULATIONAHA.118.035070

5. Postema PG. About Brugada syndrome and its prevalence. Europace. 2012;14:925-928. 6. Watanabe H and Minamino T. Genetics of Brugada syndrome. J Hum Genet. 2016;61:57-60. 7. Strande NT, Riggs ER, Buchanan AH, Ceyhan-Birsoy O, DiStefano M, Dwight SS, Goldstein J, Ghosh R, Seifert BA, Sneddon TP, Wright MW, Milko LV, Cherry JM, Giovanni MA, Murray MF, O'Daniel JM, Ramos EM, Santani AB, Scott AF, Plon SE, Rehm HL, Martin CL and Berg JS. Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence- Based Framework Developed by the Clinical Genome Resource. Am J Hum Genet. 2017;100:895-906. 8. MacArthur DG, Manolio TA, Dimmock DP, Rehm HL, Shendure J, Abecasis GR, Adams DR, Altman RB, Antonarakis SE, Ashley EA, Barrett JC, Biesecker LG, Conrad DF, Cooper GM, Cox NJ, Daly MJ, Gerstein MB, Goldstein DB, Hirschhorn JN, Leal SM, Pennacchio LA, Stamatoyannopoulos JA, Sunyaev SR, Valle D, Voight BF, Winckler W and Gunter C. Guidelines for investigating causality of sequence variants in human disease. Nature. 2014;508:469-476. Downloaded from 9. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, Oliva A, Wollnik B, Gelber P, Bonaros EP, Jr., Burashnikov E, Wu Y, Sargent JD, Schickel S, Oberheiden R, Bhatia A, Hsu LF, Haissaguerre M, Schimpf R, Borggrefe M and Wolpert C. Loss-of-function mutations in the cardiac calcium channel underlie http://circ.ahajournals.org/ a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation. 2007;115:442-449. 10. Delpon E, Cordeiro JM, Nunez L, Thomsen PE, Guerchicoff A, Pollevick GD, Wu Y, Kanters JK, Larsen CT, Hofman-Bang J, Burashnikov E, Christiansen M and Antzelevitch C. Functional effects of KCNE3 mutation and its role in the development of Brugada syndrome. Circ Arrhythm Electrophysiol. 2008;1:209-218.

by guest on July 2, 2018 11. Cerrone M, Lin X, Zhang M, Agullo-Pascual E, Pfenniger A, Chkourko Gusky H, Novelli V, Kim C, Tirasawadichai T, Judge DP, Rothenberg E, Chen HS, Napolitano C, Priori SG and Delmar M. Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype. Circulation. 2014;129:1092-1103. 12. Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, Potenza D, Moya A, Borggrefe M, Breithardt G, Ortiz-Lopez R, Wang Z, Antzelevitch C, O'Brien RE, Schulze-Bahr E, Keating MT, Towbin JA and Wang Q. Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature. 1998;392:293-296. 13. Bezzina C, Veldkamp MW, van Den Berg MP, Postma AV, Rook MB, Viersma JW, van Langen IM, Tan-Sindhunata G, Bink-Boelkens MT, van Der Hout AH, Mannens MM and Wilde AA. A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res. 1999;85:1206-1213. 14. Hong K, Guerchicoff A, Pollevick GD, Oliva A, Dumaine R, de Zutter M, Burashnikov E, Wu YS, Brugada J, Brugada P and Brugada R. Cryptic 5' splice site activation in SCN5A associated with Brugada syndrome. J Mol Cell Cardiol. 2005;38:555-560. 15. Le Scouarnec S, Karakachoff M, Gourraud JB, Lindenbaum P, Bonnaud S, Portero V, Duboscq-Bidot L, Daumy X, Simonet F, Teusan R, Baron E, Violleau J, Persyn E, Bellanger L, Barc J, Chatel S, Martins R, Mabo P, Sacher F, Haissaguerre M, Kyndt F, Schmitt S, Bezieau S, Le Marec H, Dina C, Schott JJ, Probst V and Redon R. Testing the burden of rare variation in arrhythmia-susceptibility genes provides new insights into molecular diagnosis for Brugada syndrome. Hum Mol Genet. 2015;24:2757-2763.

19 10.1161/CIRCULATIONAHA.118.035070

16. Weiss R, Barmada MM, Nguyen T, Seibel JS, Cavlovich D, Kornblit CA, Angelilli A, Villanueva F, McNamara DM and London B. Clinical and molecular heterogeneity in the Brugada syndrome: a novel gene locus on chromosome 3. Circulation. 2002;105:707-713. 17. London B, Michalec M, Mehdi H, Zhu X, Kerchner L, Sanyal S, Viswanathan PC, Pfahnl AE, Shang LL, Madhusudanan M, Baty CJ, Lagana S, Aleong R, Gutmann R, Ackerman MJ, McNamara DM, Weiss R and Dudley SC, Jr. Mutation in glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) decreases cardiac Na+ current and causes inherited arrhythmias. Circulation. 2007;116:2260-2268. 18. Risgaard B, Jabbari R, Refsgaard L, Holst AG, Haunso S, Sadjadieh A, Winkel BG, Olesen MS and Tfelt-Hansen J. High prevalence of genetic variants previously associated with Brugada syndrome in new exome data. Clin Genet. 2013;84:489-495. 19. Kapplinger JD, Giudicessi JR, Ye D, Tester DJ, Callis TE, Valdivia CR, Makielski JC, Wilde AA and Ackerman MJ. Enhanced Classification of Brugada Syndrome-Associated and Long-QT Syndrome-Associated Genetic Variants in the SCN5A-Encoded Na(v)1.5 Cardiac Sodium Channel. Circ Cardiovasc Genet. 2015;8:582-595. Downloaded from 20. Barajas-Martinez H, Hu D, Ferrer T, Onetti CG, Wu Y, Burashnikov E, Boyle M, Surman T, Urrutia J, Veltmann C, Schimpf R, Borggrefe M, Wolpert C, Ibrahim BB, Sanchez- Chapula JA, Winters S, Haissaguerre M and Antzelevitch C. Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in http://circ.ahajournals.org/ KCNJ8. Heart Rhythm. 2012;9:548-555. 21. Medeiros-Domingo A, Tan BH, Crotti L, Tester DJ, Eckhardt L, Cuoretti A, Kroboth SL, Song C, Zhou Q, Kopp D, Schwartz PJ, Makielski JC and Ackerman MJ. Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes. Heart Rhythm. 2010;7:1466-1471. 22. Hu D, Barajas-Martinez H, Burashnikov E, Springer M, Wu Y, Varro A, Pfeiffer R,

by guest on July 2, 2018 Koopmann TT, Cordeiro JM, Guerchicoff A, Pollevick GD and Antzelevitch C. A mutation in the beta 3 subunit of the cardiac sodium channel associated with Brugada ECG phenotype. Circ Cardiovasc Genet. 2009;2:270-278. 23. Ishikawa T, Takahashi N, Ohno S, Sakurada H, Nakamura K, On YK, Park JE, Makiyama T, Horie M, Arimura T, Makita N and Kimura A. Novel SCN3B mutation associated with brugada syndrome affects intracellular trafficking and function of Nav1.5. Circ J. 2013;77:959-967. 24. Anson BD, Ackerman MJ, Tester DJ, Will ML, Delisle BP, Anderson CL and January CT. Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol Heart Circ Physiol. 2004;286:H2434-441. 25. Tan BH, Valdivia CR, Rok BA, Ye B, Ruwaldt KM, Tester DJ, Ackerman MJ and Makielski JC. Common human SCN5A polymorphisms have altered electrophysiology when expressed in Q1077 splice variants. Heart Rhythm. 2005;2:741-747.

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Table 1. List of the reported genes for Brugada syndrome

Downloaded from Number of core Gene Symbol Gene Name HGNC ID MIM phenotype record publications ABCC9 ATP binding cassette subfamily C member 9 60 - 1 ANK2 ankyrin 2 493 - 2

http://circ.ahajournals.org/ CACNA1C calcium voltage-gated channel subunit alpha1 C 1390 Brugada syndrome 3 - 611875 4 CACNA2D1 calcium voltage-gated channel auxiliary subunit alpha2delta 1 1399 - 2 CACNB2 calcium voltage-gated channel auxiliary subunit beta 2 1402 Brugada syndrome 4 - 611876 4 FGF12 fibroblast growth factor 12 3668 - 1 GPD1L glycerol-3-phosphate dehydrogenase 1 like 28956 Brugada syndrome 2 - 611777 2 HCN4 hyperpolarization activated cyclic nucleotide-gated potassium channel 4 16882 Brugada syndrome 8 - 613123 2 KCND3 potassium voltage-gated channel subfamily D member 3 6239 Brugada syndrome 9 - 616399 3 by guest on July 2, 2018 KCNE3 potassium voltage-gated channel subfamily E regulatory subunit 3 6243 Brugada syndrome 6 - 613119 2 KCNE5 potassium voltage-gated channel subfamily E regulatory subunit 5 6241 - 1 KCNH2 potassium voltage-gated channel subfamily H member 2 6251 - 2 KCNJ8 potassium voltage-gated channel subfamily J member 8 6269 - 4 RANGRF RAN guanine nucleotide release factor 17679 - 3 PKP2 plakophilin 2 9024 - 2 SCN10A sodium voltage-gated channel alpha subunit 10 10582 - 5 SCN1B sodium voltage-gated channel beta subunit 1 10586 Brugada syndrome 5 - 612838 9 SCN2B sodium voltage-gated channel beta subunit 2 10589 - 4 SCN3B sodium voltage-gated channel beta subunit 3 20665 Brugada syndrome 7 - 613120 4 SCN5A sodium voltage-gated channel alpha subunit 5 10593 Brugada syndrome 1 - 601144 7 SEMA3A semaphorin 3A 10723 - 2 SLMAP sarcolemma associated protein 16643 - 1 TRPM4 transient receptor potential cation channel subfamily M member 4 17993 - 2 HGNC ID: HUGO Gene Nomenclature Committee unique ID for the gene MIM phenotype record: Mendelian Inheritance in Men record number. Only genes with a record number have been curated as associated with Brugada syndrome by MIM. Number of core publications: Count of main publications establishing gene-BrS relationship

21 10.1161/CIRCULATIONAHA.118.035070

Figure Legends

Figure 1. Genes included on clinical gene test panels for Brugada syndrome.

30 multi-gene panels exclusively offered for Brugada syndrome were analyzed. The x-axis

shows the number of panels including each gene. Bars are color coded based on the labs offering

the test. List of labs is provided in the legend to the right of the graph.

*The two BrS panels testing for ANK3 and CACNA1D have marked them as “candidate genes

with no evidence, but likely to be related to the phenotype”. Downloaded from ƗLabPLUS offers a panel for BrS types 2,5,7 which doesn’t include SCN5A, CACNA1C,

CACNB2. http://circ.ahajournals.org/

Figure 2. Clinical validity classifications and matrix scores for Brugada Syndrome gene

associations

by guest on July 2, 2018 Of the 21 BrS-genes (y-axis) curated, only SCN5A reached Definitive classification and all other

genes were classified as Limited. Each bar represents scores from a single curator group(G) (G1,

G2, G3).

Figure 3. ClinVar variants for Brugada syndrome by gene and clinical interpretation.

A. All variants submitted to ClinVar (http://clinvar.com/) by clinical labs for Brugada syndrome

have been plotted (N = 1223, excluding 182 variants that were “literature only” or “research”).

Genes are listed along the y-axis while the x-axis shows the count of variants for each gene. Bars

are color coded based on the clinical classification of variants (see legend). B. The relative

proportion of submitted variant interpretation classifications for SCN5A and Limited evidence

genes.

22 Downloaded from http://circ.ahajournals.org/ by guest on July 2, 2018 Evidence Level Gene Expert Final Symbol LIMITED (0-6) MODERATE (7-11) STRONG (12-18) Classification

Disputed

Disputed Downloaded from Disputed http://circ.ahajournals.org/ Disputed

Disputed by guest on July 2, 2018

Disputed

Definitive

Disputed

Evidence type: Genetic Experimental A Downloaded from http://circ.ahajournals.org/ by guest on July 2, 2018

B Reappraisal of Reported Genes for Sudden Arrhythmic Death: An Evidence-Based Evaluation of Gene Validity for Brugada Syndrome S. Mohsen Hosseini, Raymond Kim, Sharmila Udupa, Gregory Costain, Rebekah Jobling, Eriskay Liston, Seema M. Jamal, Marta Szybowska, Chantal F. Morel, Sarah Bowdin, John Garcia, Melanie Care, Amy C. Sturm, Valeria Novelli, Michael J. Ackerman, James S. Ware, Ray E. Hershberger, Arthur A. M. Wilde and Michael H. Gollob on behalf of the NIH-Clinical Genome Resource Consortium Downloaded from

Circulation. published online June 29, 2018; Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2018 American Heart Association, Inc. All rights reserved. http://circ.ahajournals.org/ Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the

by guest on July 2, 2018 World Wide Web at: http://circ.ahajournals.org/content/early/2018/06/27/CIRCULATIONAHA.118.035070

Data Supplement (unedited) at: http://circ.ahajournals.org/content/suppl/2018/06/27/CIRCULATIONAHA.118.035070.DC1

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Supplemental Material

Supplement to:

Reappraisal of Reported Genes for Sudden Arrhythmic Death: An Evidence-Based Evaluation of Gene Validity for Brugada Syndrome.

Detailed gene by gene scoring files are available in an accompanying excel file to this manuscript.

NIH-Funded ClinGen Clinical Domain Bio-curators: Expert Panel: Michael H. Gollob, M.D. (Chair) Sarah Bowdin, M.D., M.Sc. Toronto General Hospital, University of Toronto, The Hospital for Sick Children, Toronto, Canada Toronto, Canada Gregory Costain, M.D., Ph.D. Michael J. Ackerman, M.D., Ph.D. The Hospital for Sick Children, Toronto, Canada Mayo Clinic, Rochester, USA S. Mohsen Hosseini, M.D., M.Sc., Ph.D. Melanie Care, M.Sc. The Hospital for Sick Children, Toronto, Canada Toronto General Hospital, University of Toronto, Seema M. Jamal, M.Sc. Toronto, Canada The Hospital for Sick Children, Toronto, Canada John Garcia, Ph.D. Rebekah Jobling, M.D. Invitae Corp, San Francisco, USA The Hospital for Sick Children, Toronto, Canada Ray E. Hershberger, M.D. Raymond H. Kim, M.D., Ph.D. Ohio State University, Columbus, USA The Fred A. Litwin Family Centre in Genetic Valeria Novelli, Ph.D. Medicine, University of Toronto, Toronto, Canada Catholic University of the Sacred Heart, Rome, Italy Eriskay Liston, M.Sc. Amy C. Sturm, M.Sc. The Hospital for Sick Children, Toronto, Canada Geisinger Health System Genomic Medicine Institute, Chantal F. Morel, M.D. Danville, USA The Fred A. Litwin Family Centre in Genetic James S. Ware, Ph.D. Medicine, University of Toronto, Toronto, Canada Imperial College London, London, UK Marta Szybowska, M.Sc. Arthur A. M. Wilde, M.D., Ph.D. The Fred A. Litwin Family Centre in Genetic Amsterdam Academic Medical Center, Amsterdam, Medicine, University of Toronto, Toronto, Canada The Netherlands

Figure 1 BrS Gene Curation Process

Formation of 3 Bio-curator teams (G1, G2, G3)

Review of NIH-ClinGen Gene Curation SOP and Web-Based demonstration of gene curation application of genetic and experimental scoring templates

Bio-curator team’s independent, gene curation

and semi-quantitative scoring of selected BrS genes

Clinical Domain Expert Group review and

discussion of bio-curator team’s scores and

consensus gene classification voting of 9

members on a gene by gene basis

Final Gene Classification

List of Publications Reviewed for Brugada Syndrome Gene Curation

Multiple Genes 1. Antzelevitch C, Pollevick GD, Cordeiro JM, Casis O, Sanguinetti MC, Aizawa Y, Guerchicoff A, Pfeiffer R, Oliva A, Wollnik B, Gelber P, Bonaros EP, Jr., Burashnikov E, Wu Y, Sargent JD, Schickel S, Oberheiden R, Bhatia A, Hsu LF, Haissaguerre M, Schimpf R, Borggrefe M and Wolpert C. Loss-of-function mutations in the cardiac calcium channel underlie a new clinical entity characterized by ST-segment elevation, short QT intervals, and sudden cardiac death. Circulation. 2007;115:442-449. 2. Koopmann TT, Beekman L, Alders M, Meregalli PG, Mannens MM, Moorman AF, Wilde AA and Bezzina CR. Exclusion of multiple candidate genes and large genomic rearrangements in SCN5A in a Dutch Brugada syndrome cohort. Heart Rhythm. 2007;4:752-755. 3. Burashnikov E, Pfeiffer R, Barajas-Martinez H, Delpon E, Hu D, Desai M, Borggrefe M, Haissaguerre M, Kanter R, Pollevick GD, Guerchicoff A, Laino R, Marieb M, Nademanee K, Nam GB, Robles R, Schimpf R, Stapleton DD, Viskin S, Winters S, Wolpert C, Zimmern S, Veltmann C and Antzelevitch C. Mutations in the cardiac L-type calcium channel associated with inherited J-wave syndromes and sudden cardiac death. Heart Rhythm. 2010;7:1872-1882. 4. Mishra S, Undrovinas NA, Maltsev VA, Reznikov V, Sabbah HN and Undrovinas A. Post-transcriptional silencing of SCN1B and SCN2B genes modulates late sodium current in cardiac myocytes from normal dogs and dogs with chronic heart failure. Am J Physiol Heart Circ Physiol. 2011;301:H1596-1605. 5. Crotti L, Marcou CA, Tester DJ, Castelletti S, Giudicessi JR, Torchio M, Medeiros-Domingo A, Simone S, Will ML, Dagradi F, Schwartz PJ and Ackerman MJ. Spectrum and prevalence of mutations involving BrS1- through BrS12-susceptibility genes in a cohort of unrelated patients referred for Brugada syndrome genetic testing: implications for genetic testing. J Am Coll Cardiol. 2012;60:1410-1418. 6. Duthoit G, Fressart V, Hidden-Lucet F, Simon F, Kattygnarath D, Charron P, Himbert C, Aouate P, Guicheney P, Lecarpentier Y, Frank R and Hebert JL. Brugada ECG pattern: a physiopathological prospective study based on clinical, electrophysiological, angiographic, and genetic findings. Front Physiol. 2012;3:1-16. 7. Holst AG, Saber S, Houshmand M, Zaklyazminskaya EV, Wang Y, Jensen HK, Refsgaard L, Haunso S, Svendsen JH, Olesen MS and Tfelt-Hansen J. Sodium current and potassium transient outward current genes in Brugada syndrome: screening and bioinformatics. Can J Cardiol. 2012;28:196-200. 8. Risgaard B, Jabbari R, Refsgaard L, Holst AG, Haunso S, Sadjadieh A, Winkel BG, Olesen MS and Tfelt- Hansen J. High prevalence of genetic variants previously associated with Brugada syndrome in new exome data. Clin Genet. 2013;84:489-495. 9. Allegue C, Coll M, Mates J, Campuzano O, Iglesias A, Sobrino B, Brion M, Amigo J, Carracedo A, Brugada P, Brugada J and Brugada R. Genetic Analysis of Arrhythmogenic Diseases in the Era of NGS: The Complexity of Clinical Decision-Making in Brugada Syndrome. PloS one. 2015;10:1-21. 10. Hasdemir C, Payzin S, Kocabas U, Sahin H, Yildirim N, Alp A, Aydin M, Pfeiffer R, Burashnikov E, Wu Y and Antzelevitch C. High prevalence of concealed Brugada syndrome in patients with atrioventricular nodal reentrant tachycardia. Heart Rhythm. 2015;12:1584-1594. 11. Le Scouarnec S, Karakachoff M, Gourraud JB, Lindenbaum P, Bonnaud S, Portero V, Duboscq-Bidot L, Daumy X, Simonet F, Teusan R, Baron E, Violleau J, Persyn E, Bellanger L, Barc J, Chatel S, Martins R, Mabo P, Sacher F, Haissaguerre M, Kyndt F, Schmitt S, Bezieau S, Le Marec H, Dina C, Schott JJ, Probst V and Redon R. Testing the burden of rare variation in arrhythmia-susceptibility genes provides new insights into molecular diagnosis for Brugada syndrome. Hum Mol Genet. 2015;24:2757-2763. 12. Peeters U, Scornik F, Riuro H, Perez G, Komurcu-Bayrak E, Van Malderen S, Pappaert G, Tarradas A, Pagans S, Daneels D, Breckpot K, Brugada P, Bonduelle M, Brugada R and Van Dooren S. Contribution of Cardiac Sodium Channel beta-Subunit Variants to Brugada Syndrome. Circ J. 2015;79:2118-2129. 13. Perez-Serra A, Mademont-Soler I, Riuro H, Pico F, Coll M, Iglesias A, Pagans S, Sarquella-Brugada G, Berne P, Benito B, Brugada J, Porres JM, Lopez Zea M, Castro-Urda V, Fernandez-Lozano I and Brugada R. Comprehensive Genetic Characterization of a Spanish Brugada Syndrome Cohort. PloS one. 2015;10:1-15.

14. Ghouse J, Have CT, Skov MW, Andreasen L, Ahlberg G, Nielsen JB, Skaaby T, Olesen SP, Grarup N, Linneberg A, Pedersen O, Vestergaard H, Haunso S, Svendsen JH, Hansen T, Kanters JK and Olesen MS. Numerous Brugada syndrome-associated genetic variants have no effect on J-point elevation, syncope susceptibility, malignant cardiac arrhythmia, and all-cause mortality. Genet Med. 2017;19:521-528.

ABCC9 15. Chutkow WA, Simon MC, Le Beau MM and Burant CF. Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels. Diabetes. 1996;45:1439-1445. 16. Bienengraeber M, Alekseev AE, Abraham MR, Carrasco AJ, Moreau C, Vivaudou M, Dzeja PP and Terzic A. ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex. FASEB J. 2000;14:1943-1952. 17. Chutkow WA, Pu J, Wheeler MT, Wada T, Makielski JC, Burant CF and McNally EM. Episodic coronary artery vasospasm and hypertension develop in the absence of Sur2 K(ATP) channels. J Clin Invest. 2002;110:203- 208. 18. Bienengraeber M, Olson TM, Selivanov VA, Kathmann EC, O'Cochlain F, Gao F, Karger AB, Ballew JD, Hodgson DM, Zingman LV, Pang YP, Alekseev AE and Terzic A. ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating. Nat Genet. 2004;36:382-387. 19. Beziau DM, Barc J, O'Hara T, Le Gloan L, Amarouch MY, Solnon A, Pavin D, Lecointe S, Bouillet P, Gourraud JB, Guicheney P, Denjoy I, Redon R, Mabo P, le Marec H, Loussouarn G, Kyndt F, Schott JJ, Probst V and Baro I. Complex Brugada syndrome inheritance in a family harbouring compound SCN5A and CACNA1C mutations. Basic Res Cardiol. 2014;109:446. 20. Hu D, Barajas-Martinez H, Terzic A, Park S, Pfeiffer R, Burashnikov E, Wu Y, Borggrefe M, Veltmann C, Schimpf R, Cai JJ, Nam GB, Deshmukh P, Scheinman M, Preminger M, Steinberg J, Lopez-Izquierdo A, Ponce- Balbuena D, Wolpert C, Haissaguerre M, Sanchez-Chapula JA and Antzelevitch C. ABCC9 is a novel Brugada and early repolarization syndrome susceptibility gene. Int J Cardiol. 2014;171:431-442.

ANK2 21. Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG and Bennett V. Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proc Natl Acad Sci. 2004;101:17533-17538. 22. Mohler PJ, Le Scouarnec S, Denjoy I, Lowe JS, Guicheney P, Caron L, Driskell IM, Schott JJ, Norris K, Leenhardt A, Kim RB, Escande D and Roden DM. Defining the cellular phenotype of "ankyrin-B syndrome" variants: human ANK2 variants associated with clinical phenotypes display a spectrum of activities in cardiomyocytes. Circulation. 2007;115:432-441. 23. Ichikawa M, Aiba T, Ohno S, Shigemizu D, Ozawa J, Sonoda K, Fukuyama M, Itoh H, Miyamoto Y, Tsunoda T, Makiyama T, Tanaka T, Shimizu W and Horie M. Phenotypic Variability of ANK2 Mutations in Patients With Inherited Primary Arrhythmia Syndromes. Circ J. 2016;80:2435-2442.

CACNA1C 24. Schultz D, Mikala G, Yatani A, Engle DB, Iles DE, Segers B, Sinke RJ, Weghuis DO, Klockner U, Wakamori M and et al. Cloning, chromosomal localization, and functional expression of the alpha 1 subunit of the L-type voltage-dependent calcium channel from normal human heart. Proc Natl Acad Sci. 1993;90:6228-6232. 25. Fukuyama M, Ohno S, Wang Q, Kimura H, Makiyama T, Itoh H, Ito M and Horie M. L-type calcium channel mutations in Japanese patients with inherited arrhythmias. Circ J. 2013;77:1799-1806.

26. Fukuyama M, Ohno S, Wang Q, Shirayama T, Itoh H and Horie M. Nonsense-mediated mRNA decay due to a CACNA1C splicing mutation in a patient with Brugada syndrome. Heart Rhythm. 2014;11:629-634. 27. Simms BA, Souza IA and Zamponi GW. Effect of the Brugada syndrome mutation A39V on calmodulin regulation of Cav1.2 channels. Mol Brain. 2014;7:34. 28. Canpolat U, Coteli C and Aytemir K. Brugada syndrome and calcium channel mutation in a patient with congenital deaf mutism. Indian Pacing Electrophysiol. 2017;17:16-17.

CACNA2D1 29. Fuller-Bicer GA, Varadi G, Koch SE, Ishii M, Bodi I, Kadeer N, Muth JN, Mikala G, Petrashevskaya NN, Jordan MA, Zhang SP, Qin N, Flores CM, Isaacsohn I, Varadi M, Mori Y, Jones WK and Schwartz A. Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit. Am J Physiol Heart Circ Physiol. 2009;297:H117-124. 30. Fagerberg L, Hallstrom BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, Habuka M, Tahmasebpoor S, Danielsson A, Edlund K, Asplund A, Sjostedt E, Lundberg E, Szigyarto CA, Skogs M, Takanen JO, Berling H, Tegel H, Mulder J, Nilsson P, Schwenk JM, Lindskog C, Danielsson F, Mardinoglu A, Sivertsson A, von Feilitzen K, Forsberg M, Zwahlen M, Olsson I, Navani S, Huss M, Nielsen J, Ponten F and Uhlen M. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. 2014;13:397-406. 31. Bourdin B, Shakeri B, Tetreault MP, Sauve R, Lesage S and Parent L. Functional characterization of CaValpha2delta mutations associated with sudden cardiac death. J Biol Chem. 2015;290:2854-2869.

CACNB2 32. Yamaguchi H, Okuda M, Mikala G, Fukasawa K and Varadi G. Cloning of the beta(2a) subunit of the voltage-dependent calcium channel from human heart: cooperative effect of alpha(2)/delta and beta(2a) on the membrane expression of the alpha(1C) subunit. Bioch Biophys Res Comm. 2000;267:156-163. 33. Cordeiro JM, Marieb M, Pfeiffer R, Calloe K, Burashnikov E and Antzelevitch C. Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome. Mol Cell Cardiol.. 2009;46:695-703. 34. Kanter RJ, Pfeiffer R, Hu D, Barajas-Martinez H, Carboni MP and Antzelevitch C. Brugada-like syndrome in infancy presenting with rapid ventricular tachycardia and intraventricular conduction delay. Circulation. 2012;125:14-22.

GPD1L 35. Weiss R, Barmada MM, Nguyen T, Seibel JS, Cavlovich D, Kornblit CA, Angelilli A, Villanueva F, McNamara DM and London B. Clinical and molecular heterogeneity in the Brugada syndrome: a novel gene locus on chromosome 3. Circulation. 2002;105:707-713. 36. London B, Michalec M, Mehdi H, Zhu X, Kerchner L, Sanyal S, Viswanathan PC, Pfahnl AE, Shang LL, Madhusudanan M, Baty CJ, Lagana S, Aleong R, Gutmann R, Ackerman MJ, McNamara DM, Weiss R and Dudley SC, Jr. Mutation in glycerol-3-phosphate dehydrogenase 1 like gene (GPD1-L) decreases cardiac Na+ current and causes inherited arrhythmias. Circulation. 2007;116:2260-2268. 37. Makiyama T, Akao M, Haruna Y, Tsuji K, Doi T, Ohno S, Nishio Y, Kita T and Horie M. Mutation analysis of the glycerol-3 phosphate dehydrogenase-1 like (GPD1L) gene in Japanese patients with Brugada syndrome. CircJ. 2008;72:1705-1706. 38. Valdivia CR, Ueda K, Ackerman MJ and Makielski JC. GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A. Am J Physiol Heart Circ Physiol. 2009;297:H1446-452.

HCN4 39. Ueda K, Nakamura K, Hayashi T, Inagaki N, Takahashi M, Arimura T, Morita H, Higashiuesato Y, Hirano Y, Yasunami M, Takishita S, Yamashina A, Ohe T, Sunamori M, Hiraoka M and Kimura A. Functional characterization of a trafficking-defective HCN4 mutation, D553N, associated with cardiac arrhythmia. J Biol Chem. 2004;279:27194-27198. 40. Ueda K, Hirano Y, Higashiuesato Y, Aizawa Y, Hayashi T, Inagaki N, Tana T, Ohya Y, Takishita S, Muratani H, Hiraoka M and Kimura A. Role of HCN4 channel in preventing ventricular arrhythmia. J Hum Genet. 2009;54:115-121. 41. Biel S, Aquila M, Hertel B, Berthold A, Neumann T, DiFrancesco D, Moroni A, Thiel G and Kauferstein S. Mutation in S6 domain of HCN4 channel in patient with suspected Brugada syndrome modifies channel function. Pflugers Arch. 2016;468:1663-1671.

KCND3 42. Dilks D, Ling HP, Cockett M, Sokol P and Numann R. Cloning and expression of the human kv4.3 potassium channel. J Neurophysiol. 1999;81:1974-1977. 43. Giudicessi JR, Ye D, Tester DJ, Crotti L, Mugione A, Nesterenko VV, Albertson RM, Antzelevitch C, Schwartz PJ and Ackerman MJ. Transient outward current (I(to)) gain-of-function mutations in the KCND3- encoded Kv4.3 potassium channel and Brugada syndrome. Heart Rhythm. 2011;8:1024-1032. 44. You T, Mao W, Cai B, Li F and Xu H. Two novel Brugada syndrome-associated mutations increase KV4.3 membrane expression and function. Int J Mol Med. 2015;36:309-315.

KCNE3 45. Lundquist AL, Manderfield LJ, Vanoye CG, Rogers CS, Donahue BS, Chang PA, Drinkwater DC, Murray KT and George AL, Jr. Expression of multiple KCNE genes in human heart may enable variable modulation of I(Ks). J Mol Cell Cardiol. 2005;38:277-287. 46. Delpon E, Cordeiro JM, Nunez L, Thomsen PE, Guerchicoff A, Pollevick GD, Wu Y, Kanters JK, Larsen CT, Hofman-Bang J, Burashnikov E, Christiansen M and Antzelevitch C. Functional effects of KCNE3 mutation and its role in the development of Brugada syndrome. Circ Arrhythm Eectrophysiol. 2008;1:209-218. 47. Nakajima T, Wu J, Kaneko Y, Ashihara T, Ohno S, Irie T, Ding WG, Matsuura H, Kurabayashi M and Horie M. KCNE3 T4A as the genetic basis of Brugada-pattern electrocardiogram. Circ J. 2012;76:2763-2772.

KCNE5 48. Piccini M, Vitelli F, Seri M, Galietta LJ, Moran O, Bulfone A, Banfi S, Pober B and Renieri A. KCNE1- like gene is deleted in AMME contiguous gene syndrome: identification and characterization of the human and mouse homologs. Genomics. 1999;60:251-257. 49. Ohno S, Zankov DP, Ding WG, Itoh H, Makiyama T, Doi T, Shizuta S, Hattori T, Miyamoto A, Naiki N, Hancox JC, Matsuura H and Horie M. KCNE5 (KCNE1L) variants are novel modulators of Brugada syndrome and idiopathic ventricular fibrillation. Circ Arrhythm Electrophysiol. 2011;4:352-361. 50. Abbott GW. KCNE4 and KCNE5: K(+) channel regulation and cardiac arrhythmogenesis. Gene. 2016;593:249-260.

KCNH2 51. Lees-Miller JP, Guo J, Somers JR, Roach DE, Sheldon RS, Rancourt DE and Duff HJ. Selective knockout of mouse ERG1 B potassium channel eliminates I(Kr) in adult ventricular myocytes and elicits episodes of abrupt sinus bradycardia. Mol Cell Biol. 2003;23:1856-1862. 52. Verkerk AO, Wilders R, Schulze-Bahr E, Beekman L, Bhuiyan ZA, Bertrand J, Eckardt L, Lin D, Borggrefe M, Breithardt G, Mannens MM, Tan HL, Wilde AA and Bezzina CR. Role of sequence variations in the human ether-a-go-go-related gene (HERG, KCNH2) in the Brugada syndrome. Cardiovasc Res. 2005;68:441-453. 53. Chung SK, MacCormick JM, McCulley CH, Crawford J, Eddy CA, Mitchell EA, Shelling AN, French JK, Skinner JR and Rees MI. Long QT and Brugada syndrome gene mutations in New Zealand. Heart Rhythm. 2007;4:1306-1314. 54. Itoh H, Sakaguchi T, Ashihara T, Ding WG, Nagaoka I, Oka Y, Nakazawa Y, Yao T, Jo H, Ito M, Nakamura K, Ohe T, Matsuura H and Horie M. A novel KCNH2 mutation as a modifier for short QT interval. Int J Cardiol. 2009;137:83-85. 55. Amin AS, Klemens CA, Verkerk AO, Meregalli PG, Asghari-Roodsari A, de Bakker JM, January CT, Wilde AA and Tan HL. Fever-triggered ventricular arrhythmias in Brugada syndrome and type 2 long-QT syndrome. Neth Heart J. 2010;18:165-169. 56. Wilders R and Verkerk AO. Role of the R1135H KCNH2 mutation in Brugada syndrome. Int J Cardiol. 2010;144:149-151. 57. Gianulis EC and Trudeau MC. Rescue of aberrant gating by a genetically encoded PAS (Per-Arnt-Sim) domain in several long QT syndrome mutant human ether-a-go-go-related gene potassium channels. J Biol Chem. 2011;286:22160-22169. 58. Wang Q, Ohno S, Ding WG, Fukuyama M, Miyamoto A, Itoh H, Makiyama T, Wu J, Bai J, Hasegawa K, Shinohara T, Takahashi N, Shimizu A, Matsuura H and Horie M. Gain-of-function KCNH2 mutations in patients with Brugada syndrome. J Cardiovasc Electrophysiol. 2014;25:522-530. 59. Saber S, Amarouch MY, Fazelifar AF, Haghjoo M, Emkanjoo Z, Alizadeh A, Houshmand M, Gavrilenko AV, Abriel H and Zaklyazminskaya EV. Complex genetic background in a large family with Brugada syndrome. Physiol Rep. 2015;2013. KCNJ8 60. Miki T, Suzuki M, Shibasaki T, Uemura H, Sato T, Yamaguchi K, Koseki H, Iwanaga T, Nakaya H and Seino S. Mouse model of Prinzmetal angina by disruption of the inward rectifier Kir6.1. Nat Med. 2002;8:466-472. 61. Haissaguerre M, Chatel S, Sacher F, Weerasooriya R, Probst V, Loussouarn G, Horlitz M, Liersch R, Schulze-Bahr E, Wilde A, Kaab S, Koster J, Rudy Y, Le Marec H and Schott JJ. Ventricular fibrillation with prominent early repolarization associated with a rare variant of KCNJ8/KATP channel. J Cardiovasc Electrophysiol. 2009;20:93-98. 62. Medeiros-Domingo A, Tan BH, Crotti L, Tester DJ, Eckhardt L, Cuoretti A, Kroboth SL, Song C, Zhou Q, Kopp D, Schwartz PJ, Makielski JC and Ackerman MJ. Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K(ATP) channel Kir6.1 as a pathogenic substrate for J-wave syndromes. Heart Rhythm. 2010;7:1466-1471. 63. Barajas-Martinez H, Hu D, Ferrer T, Onetti CG, Wu Y, Burashnikov E, Boyle M, Surman T, Urrutia J, Veltmann C, Schimpf R, Borggrefe M, Wolpert C, Ibrahim BB, Sanchez-Chapula JA, Winters S, Haissaguerre M and Antzelevitch C. Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8. Heart Rhythm. 2012;9:548-555. 64. Nakaya H. Role of ATP-sensitive K+ channels in cardiac arrhythmias. J Cardiovac Pharmacol Ther.. 2014;19:237-243.

PKP2 65. Mertens C, Kuhn C and Franke WW. Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque. J Cell Biol 1996;135:1009-1025. 66. Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW and Birchmeier W. Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation. J Cell Biol. 2004;167:149- 160. 67. Oxford EM, Musa H, Maass K, Coombs W, Taffet SM and Delmar M. Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells. Circ Res. 2007;101:703-711. 68. Sato PY, Musa H, Coombs W, Guerrero-Serna G, Patino GA, Taffet SM, Isom LL and Delmar M. Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes. Circ Res. 2009;105:523-526. 69. Cerrone M, Noorman M, Lin X, Chkourko H, Liang FX, van der Nagel R, Hund T, Birchmeier W, Mohler P, van Veen TA, van Rijen HV and Delmar M. Sodium current deficit and arrhythmogenesis in a murine model of plakophilin-2 haploinsufficiency. Cardiovasc Res. 2012;95:460-468. 70. Cerrone M, Lin X, Zhang M, Agullo-Pascual E, Pfenniger A, Chkourko Gusky H, Novelli V, Kim C, Tirasawadichai T, Judge DP, Rothenberg E, Chen HS, Napolitano C, Priori SG and Delmar M. Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype. Circulation. 2014;129:1092-1103. 71. Peters S. Arrhythmogenic cardiomyopathy and provocable Brugada ECG in a patient caused by missense mutation in plakophilin-2. Int J Cardiol. 2014;173:317-318. 72. Forkmann M, Tomala J, Huo Y, Mayer J, Christoph M, Wunderlich C, Salmas J, Gaspar T and Piorkowski C. Epicardial Ventricular Tachycardia Ablation in a Patient With Brugada ECG Pattern and Mutation of PKP2 and DSP Genes. Circ Arrhythm Electrophysiol. 2015;8:505-507.

RANGRF 73. Wu L, Yong SL, Fan C, Ni Y, Yoo S, Zhang T, Zhang X, Obejero-Paz CA, Rho HJ, Ke T, Szafranski P, Jones SW, Chen Q and Wang QK. Identification of a new co-factor, MOG1, required for the full function of cardiac sodium channel Nav 1.5. J Biol Chem. 2008;283:6968-6978. 74. Kattygnarath D, Maugenre S, Neyroud N, Balse E, Ichai C, Denjoy I, Dilanian G, Martins RP, Fressart V, Berthet M, Schott JJ, Leenhardt A, Probst V, Le Marec H, Hainque B, Coulombe A, Hatem SN and Guicheney P. MOG1: a new susceptibility gene for Brugada syndrome. Circ Cardiovasc Genet. 2011;4:261-268. 75. Olesen MS, Jensen NF, Holst AG, Nielsen JB, Tfelt-Hansen J, Jespersen T, Sajadieh A, Haunso S, Lund JT, Calloe K, Schmitt N and Svendsen JH. A novel nonsense variant in Nav1.5 cofactor MOG1 eliminates its sodium current increasing effect and may increase the risk of arrhythmias. Can J Cardiol. 2011;27:523.e17-23. 76. Chakrabarti S, Wu X, Yang Z, Wu L, Yong SL, Zhang C, Hu K, Wang QK and Chen Q. MOG1 rescues defective trafficking of Na(v)1.5 mutations in Brugada syndrome and sick sinus syndrome. Circ Arrhythm Electrophysiol. 2013;6:392-401. 77. Campuzano O, Berne P, Selga E, Allegue C, Iglesias A, Brugada J and Brugada R. Brugada syndrome and p.E61X_RANGRF. Cardiol J. 2014;21:121-127. 78. Zhou J, Wang L, Zuo M, Wang X, Ahmed AS, Chen Q and Wang QK. Cardiac sodium channel regulator MOG1 regulates cardiac morphogenesis and rhythm. Sci Rep. 2016;6:21538.

SCN10A 79. Rabert DK, Koch BD, Ilnicka M, Obernolte RA, Naylor SL, Herman RC, Eglen RM, Hunter JC and Sangameswaran L. A tetrodotoxin-resistant voltage-gated sodium channel from human dorsal root ganglia, hPN3/SCN10A. Pain. 1998;78:107-14.

80. Yang T, Atack TC, Stroud DM, Zhang W, Hall L and Roden DM. Blocking Scn10a channels in heart reduces late sodium current and is antiarrhythmic. Circ Res. 2012;111:322-332. 81. Hu D, Barajas-Martinez H, Pfeiffer R, Dezi F, Pfeiffer J, Buch T, Betzenhauser MJ, Belardinelli L, Kahlig KM, Rajamani S, DeAntonio HJ, Myerburg RJ, Ito H, Deshmukh P, Marieb M, Nam GB, Bhatia A, Hasdemir C, Haissaguerre M, Veltmann C, Schimpf R, Borggrefe M, Viskin S and Antzelevitch C. Mutations in SCN10A are responsible for a large fraction of cases of Brugada syndrome. J Am Coll Cardiol. 2014;64:66-79. 82. van den Boogaard M, Smemo S, Burnicka-Turek O, Arnolds DE, van de Werken HJ, Klous P, McKean D, Muehlschlegel JD, Moosmann J, Toka O, Yang XH, Koopmann TT, Adriaens ME, Bezzina CR, de Laat W, Seidman C, Seidman JG, Christoffels VM, Nobrega MA, Barnett P and Moskowitz IP. A common genetic variant within SCN10A modulates cardiac SCN5A expression. J Clin Invest. 2014;124:1844-1852. 83. Behr ER, Savio-Galimberti E, Barc J, Holst AG, Petropoulou E, Prins BP, Jabbari J, Torchio M, Berthet M, Mizusawa Y, Yang T, Nannenberg EA, Dagradi F, Weeke P, Bastiaenan R, Ackerman MJ, Haunso S, Leenhardt A, Kaab S, Probst V, Redon R, Sharma S, Wilde A, Tfelt-Hansen J, Schwartz P, Roden DM, Bezzina CR, Olesen M, Darbar D, Guicheney P, Crotti L and Jamshidi Y. Role of common and rare variants in SCN10A: results from the Brugada syndrome QRS locus gene discovery collaborative study. Cardiovasc Res. 2015;106:520-529. 84. Fukuyama M, Ohno S, Makiyama T and Horie M. Novel SCN10A variants associated with Brugada syndrome. Europace. 2016;18:905-911. 85. Stroud DM, Yang T, Bersell K, Kryshtal DO, Nagao S, Shaffer C, Short L, Hall L, Atack TC, Zhang W, Knollmann BC, Baudenbacher F and Roden DM. Contrasting Nav1.8 Activity in Scn10a-/- Ventricular Myocytes and the Intact Heart. J Am Heart Assoc. 2016;5. 86. Zhang L, Zhou F, Huang L, Wu Q, Zheng J, Wu Y, Yin K and Cheng J. Association of common and rare variants of SCN10A gene with sudden unexplained nocturnal death syndrome in Chinese Han population. .Int J Legal Med. 2017;131:53-60. SCN1B 87. Nuss HB, Chiamvimonvat N, Perez-Garcia MT, Tomaselli GF and Marban E. Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes. J Gen Physiol. 1995;106:1171-1191. 88. Qu Y, Isom LL, Westenbroek RE, Rogers JC, Tanada TN, McCormick KA, Scheuer T and Catterall WA. Modulation of cardiac Na+ channel expression in Xenopus oocytes by beta 1 subunits. J Biol Chem. 1995;270:25696-701. 89. Deschenes I, DiSilvestre D, Juang GJ, Wu RC, An WF and Tomaselli GF. Regulation of Kv4.3 current by KChIP2 splice variants: a component of native cardiac I(to)? Circulation. 2002;106:423-429. 90. Deschenes I and Tomaselli GF. Modulation of Kv4.3 current by accessory subunits. FEBS Lett. 2002;528:183-188. 91. Zimmer T, Bollensdorff C, Haufe V, Birch-Hirschfeld E and Benndorf K. Mouse heart Na+ channels: primary structure and function of two isoforms and alternatively spliced variants. Am J Physiol Heart Circ Physiol. 2002;282:H1007-1017. 92. Lopez-Santiago LF, Meadows LS, Ernst SJ, Chen C, Malhotra JD, McEwen DP, Speelman A, Noebels JL, Maier SK, Lopatin AN and Isom LL. Sodium channel Scn1b null mice exhibit prolonged QT and RR intervals. J Mol Cell Cardiol. 2007;43:636-647. 93. Watanabe H, Koopmann TT, Le Scouarnec S, Yang T, Ingram CR, Schott JJ, Demolombe S, Probst V, Anselme F, Escande D, Wiesfeld AC, Pfeufer A, Kaab S, Wichmann HE, Hasdemir C, Aizawa Y, Wilde AA, Roden DM and Bezzina CR. Sodium channel beta1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans. J Clin Invest. 2008;118:2260-2268. 94. Ogawa R, Kishi R, Takagi A, Sakaue I, Takahashi H, Matsumoto N, Masuhara K, Nakazawa K, Kobayashi S, Miyake F and Echizen H. A novel microsatellite polymorphism of sodium channel beta1-subunit gene (SCN1B) may underlie abnormal cardiac excitation manifested by coved-type ST-elevation compatible with Brugada syndrome in Japanese. Int J Clin Pharmacol Ther. 2010;48:109-119.

95. Hu D, Barajas-Martinez H, Medeiros-Domingo A, Crotti L, Veltmann C, Schimpf R, Urrutia J, Alday A, Casis O, Pfeiffer R, Burashnikov E, Caceres G, Tester DJ, Wolpert C, Borggrefe M, Schwartz P, Ackerman MJ and Antzelevitch C. A novel rare variant in SCN1Bb linked to Brugada syndrome and SIDS by combined modulation of Na(v)1.5 and K(v)4.3 channel currents. Heart Rhythm. 2012;9:760-769. 96. Olesen MS, Holst AG, Svendsen JH, Haunso S and Tfelt-Hansen J. SCN1Bb R214Q found in 3 patients: 1 with Brugada syndrome and 2 with lone atrial fibrillation. Heart Rhythm. 2012;9:770-773. 97. Ricci MT, Menegon S, Vatrano S, Mandrile G, Cerrato N, Carvalho P, De Marchi M, Gaita F, Giustetto C and Giachino DF. SCN1B gene variants in Brugada Syndrome: a study of 145 SCN5A-negative patients. Sci Rep. 2014;4:6470. 98. Lin X, O'Malley H, Chen C, Auerbach D, Foster M, Shekhar A, Zhang M, Coetzee W, Jalife J, Fishman GI, Isom L and Delmar M. Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current, altered intracellular calcium homeostasis and arrhythmias in murine hearts. J Physiol. 2015;593:1389-1407. 99. Aoki H, Nakamura Y, Ohno S, Makiyama T and Horie M. Cardiac conduction defects and Brugada syndrome: A family with overlap syndrome carrying a nonsense SCN5A mutation. J Arrhythm. 2017;33:35-39.

SCN2B 100. Watanabe H, Darbar D, Kaiser DW, Jiramongkolchai K, Chopra S, Donahue BS, Kannankeril PJ and Roden DM. Mutations in sodium channel beta1- and beta2-subunits associated with atrial fibrillation. Circ Arrhythm Electrophysiol. 2009;2:268-275. 101. Riuro H, Beltran-Alvarez P, Tarradas A, Selga E, Campuzano O, Verges M, Pagans S, Iglesias A, Brugada J, Brugada P, Vazquez FM, Perez GJ, Scornik FS and Brugada R. A missense mutation in the sodium channel beta2 subunit reveals SCN2B as a new candidate gene for Brugada syndrome. Hum Mut. 2013;34:961-966. 102. Bao Y, Willis BC, Frasier CR, Lopez-Santiago LF, Lin X, Ramos-Mondragon R, Auerbach DS, Chen C, Wang Z, Anumonwo J, Valdivia HH, Delmar M, Jalife J and Isom LL. Scn2b Deletion in Mice Results in Ventricular and Atrial Arrhythmias. Circ Arrhythm Electrophysiol. 2016;9:1-30.

SCN3B 103. Morgan K, Stevens EB, Shah B, Cox PJ, Dixon AK, Lee K, Pinnock RD, Hughes J, Richardson PJ, Mizuguchi K and Jackson AP. beta 3: an additional auxiliary subunit of the voltage-sensitive sodium channel that modulates channel gating with distinct kinetics. Proc Natl Acad Sci. 2000;97:2308-2313. 104. Fahmi AI, Patel M, Stevens EB, Fowden AL, John JE, 3rd, Lee K, Pinnock R, Morgan K, Jackson AP and Vandenberg JI. The sodium channel beta-subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart. J Physiol. 2001;537:693-700. 105. Maier SK, Westenbroek RE, McCormick KA, Curtis R, Scheuer T and Catterall WA. Distinct subcellular localization of different sodium channel alpha and beta subunits in single ventricular myocytes from mouse heart. Circulation. 2004;109:1421-1427. 106. Ko SH, Lenkowski PW, Lee HC, Mounsey JP and Patel MK. Modulation of Na(v)1.5 by beta1-- and beta3- subunit co-expression in mammalian cells. Pflugers Arch. 2005;449:403-412. 107. Hakim P, Gurung IS, Pedersen TH, Thresher R, Brice N, Lawrence J, Grace AA and Huang CL. Scn3b knockout mice exhibit abnormal ventricular electrophysiological properties. Prog Biophys Mol Biol. 2008;98:251- 266. 108. Hu D, Barajas-Martinez H, Burashnikov E, Springer M, Wu Y, Varro A, Pfeiffer R, Koopmann TT, Cordeiro JM, Guerchicoff A, Pollevick GD and Antzelevitch C. A mutation in the beta 3 subunit of the cardiac sodium channel associated with Brugada ECG phenotype. Circ Cardiovasc Genet. 2009;2:270-278. 109. Hakim P, Brice N, Thresher R, Lawrence J, Zhang Y, Jackson AP, Grace AA and Huang CL. Scn3b knockout mice exhibit abnormal sino-atrial and cardiac conduction properties. Acta Physiol. 2010;198:47-59.

110. Olesen MS, Jespersen T, Nielsen JB, Liang B, Moller DV, Hedley P, Christiansen M, Varro A, Olesen SP, Haunso S, Schmitt N and Svendsen JH. Mutations in sodium channel beta-subunit SCN3B are associated with early- onset lone atrial fibrillation. Cardiovasc Res. 2011;89:786-793. 111. Ishikawa T, Takahashi N, Ohno S, Sakurada H, Nakamura K, On YK, Park JE, Makiyama T, Horie M, Arimura T, Makita N and Kimura A. Novel SCN3B mutation associated with brugada syndrome affects intracellular trafficking and function of Nav1.5. Circ J. 2013;77:959-967. 112. Okata S, Yuasa S, Suzuki T, Ito S, Makita N, Yoshida T, Li M, Kurokawa J, Seki T, Egashira T, Aizawa Y, Kodaira M, Motoda C, Yozu G, Shimojima M, Hayashiji N, Hashimoto H, Kuroda Y, Tanaka A, Murata M, Aiba T, Shimizu W, Horie M, Kamiya K, Furukawa T and Fukuda K. Embryonic type Na+ channel beta-subunit, SCN3B masks the disease phenotype of Brugada syndrome. Sci Rep. 2016;6:34198.

SCN5A 113. Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, Potenza D, Moya A, Borggrefe M, Breithardt G, Ortiz-Lopez R, Wang Z, Antzelevitch C, O'Brien RE, Schulze-Bahr E, Keating MT, Towbin JA and Wang Q. Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature. 1998;392:293-296. 114. Bezzina C, Veldkamp MW, van Den Berg MP, Postma AV, Rook MB, Viersma JW, van Langen IM, Tan- Sindhunata G, Bink-Boelkens MT, van Der Hout AH, Mannens MM and Wilde AA. A single Na(+) channel mutation causing both long-QT and Brugada syndromes. Circ Res. 1999;85:1206-1213. 115. Rook MB, Bezzina Alshinawi C, Groenewegen WA, van Gelder IC, van Ginneken AC, Jongsma HJ, Mannens MM and Wilde AA. Human SCN5A gene mutations alter cardiac sodium channel kinetics and are associated with the Brugada syndrome. Cardiovasc Res. 1999;44:507-517. 116. Deschenes I, Baroudi G, Berthet M, Barde I, Chalvidan T, Denjoy I, Guicheney P and Chahine M. Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes. Cardiovasc Res. 2000;46:55-65. 117. Vatta M, Dumaine R, Antzelevitch C, Brugada R, Li H, Bowles NE, Nademanee K, Brugada J, Brugada P and Towbin JA. Novel mutations in domain I of SCN5A cause Brugada syndrome. Mol Genet Metab. 2002;75:317- 324. 118. Schulze-Bahr E, Eckardt L, Breithardt G, Seidl K, Wichter T, Wolpert C, Borggrefe M and Haverkamp W. Sodium channel gene (SCN5A) mutations in 44 index patients with Brugada syndrome: different incidences in familial and sporadic disease. Hum Mut. 2003;21:651-652. 119. Hong K, Guerchicoff A, Pollevick GD, Oliva A, Dumaine R, de Zutter M, Burashnikov E, Wu YS, Brugada J, Brugada P and Brugada R. Cryptic 5' splice site activation in SCN5A associated with Brugada syndrome. J Mol Cell Cardiol. 2005;38:555-560. 120. Kapplinger JD, Tester DJ, Alders M, Benito B, Berthet M, Brugada J, Brugada P, Fressart V, Guerchicoff A, Harris-Kerr C, Kamakura S, Kyndt F, Koopmann TT, Miyamoto Y, Pfeiffer R, Pollevick GD, Probst V, Zumhagen S, Vatta M, Towbin JA, Shimizu W, Schulze-Bahr E, Antzelevitch C, Salisbury BA, Guicheney P, Wilde AA, Brugada R, Schott JJ and Ackerman MJ. An international compendium of mutations in the SCN5A-encoded cardiac sodium channel in patients referred for Brugada syndrome genetic testing. Heart Rhythm. 2010;7:33-46.

SLMAP 121. Wigle JT, Demchyshyn L, Pratt MA, Staines WA, Salih M and Tuana BS. Molecular cloning, expression, and chromosomal assignment of sarcolemmal-associated proteins. A family of acidic amphipathic alpha-helical proteins associated with the membrane. J Biol Chem. 1997;272:32384-32394. 122. Guzzo RM, Salih M, Moore ED and Tuana BS. Molecular properties of cardiac tail-anchored membrane protein SLMAP are consistent with structural role in arrangement of excitation-contraction coupling apparatus. Am J Physiol Heart Circ Physiol. 2005;288:H1810-819.

123. Ishikawa T, Sato A, Marcou CA, Tester DJ, Ackerman MJ, Crotti L, Schwartz PJ, On YK, Park JE, Nakamura K, Hiraoka M, Nakazawa K, Sakurada H, Arimura T, Makita N and Kimura A. A novel disease gene for Brugada syndrome: sarcolemmal membrane-associated protein gene mutations impair intracellular trafficking of hNav1.5. Circ Arrhythm Electrophysiol. 2012;5:1098-1107. 124. Nader M, Westendorp B, Hawari O, Salih M, Stewart AF, Leenen FH and Tuana BS. Tail-anchored membrane protein SLMAP is a novel regulator of cardiac function at the sarcoplasmic reticulum. Am J Physiol Heart Circ Physiol. 2012;302:H1138-1145.

TRPM4 125. Stallmeyer B, Zumhagen S, Denjoy I, Duthoit G, Hebert JL, Ferrer X, Maugenre S, Schmitz W, Kirchhefer U, Schulze-Bahr E, Guicheney P and Schulze-Bahr E. Mutational spectrum in the Ca(2+)--activated cation channel gene TRPM4 in patients with cardiac conductance disturbances. Hum Mut. 2012;33:109-117. 126. Liu H, Chatel S, Simard C, Syam N, Salle L, Probst V, Morel J, Millat G, Lopez M, Abriel H, Schott JJ, Guinamard R and Bouvagnet P. Molecular genetics and functional anomalies in a series of 248 Brugada cases with 11 mutations in the TRPM4 channel. PloS one. 2013;8:e54131. 127. Demion M, Thireau J, Gueffier M, Finan A, Khoueiry Z, Cassan C, Serafini N, Aimond F, Granier M, Pasquie JL, Launay P and Richard S. Trpm4 gene invalidation leads to cardiac hypertrophy and electrophysiological alterations. PloS one. 2014;9:e115256. 128. Kruse M and Pongs O. TRPM4 channels in the cardiovascular system. Curr Opin Pharmacol. 2014;15:68- 73. 129. Mathar I, Kecskes M, Van der Mieren G, Jacobs G, Camacho Londono JE, Uhl S, Flockerzi V, Voets T, Freichel M, Nilius B, Herijgers P and Vennekens R. Increased beta-adrenergic inotropy in ventricular myocardium from Trpm4-/- mice. Circ Res. 2014;114:283-294. 130. Guinamard R, Bouvagnet P, Hof T, Liu H, Simard C and Salle L. TRPM4 in cardiac electrical activity. Cardiovasc Res. 2015;108:21-30. 131. Gualandi F, Zaraket F, Malagu M, Parmeggiani G, Trabanelli C, Fini S, Dang X, Wei X, Fang M, Bertini M, Ferrari R and Ferlini A. Mutation Load of Multiple Ion Channel Gene Mutations in Brugada Syndrome. Cardiol. 2017;137:256-260.