Genetic Testing in Inherited Arrhythmias: Approach, Limitations, and Challenges

Journal Pre-proof Genetic Testing in Inherited Arrhythmias: Approach, Limitations, and Challenges Christopher C. Cheung, MD FRCPC, Rafik Tadros, MD PhD, Brianna Davies, MSc CGC, Andrew D. Krahn, MD FRCPC FHRS PII:

S0828-282X(19)31222-X

DOI:

https://doi.org/10.1016/j.cjca.2019.08.041

Reference:

CJCA 3441

To appear in:

Canadian Journal of Cardiology

Received Date: 10 July 2019 Revised Date:

22 August 2019

Accepted Date: 23 August 2019

Please cite this article as: Cheung CC, Tadros R, Davies B, Krahn AD, Genetic Testing in Inherited Arrhythmias: Approach, Limitations, and Challenges, Canadian Journal of Cardiology (2019), doi: https:// doi.org/10.1016/j.cjca.2019.08.041. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the Canadian Cardiovascular Society.

Genetic Testing in Inherited Arrhythmias: Approach, Limitations, and Challenges

Authors: Christopher C. Cheung MD FRCPC1, Rafik Tadros MD PhD2, Brianna Davies MSc CGC1, Andrew D. Krahn MD FRCPC FHRS1

Affiliations: 1

Heart Rhythm Services, Division of Cardiology, University of British Columbia, Vancouver, BC.

2

Cardiovascular Genetics Center, Montreal Heart Institute, Montreal, QC

Corresponding Author: Dr. Andrew Krahn Heart Rhythm Vancouver 211-1033 Davie Street Vancouver, BC V6E 1M7 (e): [email protected] (p): (604) 682-2344, ext 63260 (f): (604) 806-8723

Journal: Canadian Journal of Cardiology Article Type: Training/Practice (CJC Training Program Initiative) Word Count: 1520 References: 5 Tables and Figures: 2 Figures (+1 Supplemental Table)

60-word Summary Genetic testing is becoming part of the “toolkit” for the cardiovascular clinician. In patients with inherited arrhythmias, genetic testing can establish or confirm a suspected diagnosis, and help facilitate cascade family screening. We present an approach to genetic testing for the clinician, provide an overview for history taking and pedigree construction, and highlight the challenges when interpreting genetic testing results.

Word Count: 60

Unstructured Abstract Genetic testing is playing an ever-expanding role in cardiovascular care and is becoming part of the “toolkit” for the cardiovascular clinician. In patients with inherited arrhythmias, genetic testing can confirm a suspected diagnosis, establish a diagnosis in unexplained cases, and help facilitate cascade family screening. Many inherited arrhythmia syndromes are monogenic diseases arising from a single pathogenic variant involved in the structure and function of cardiac ion channels or structural proteins. As such, “arrhythmia gene panels” will often cast a wide net for such heritable diseases. However, challenges may arise when genetic testing results are ambiguous, or when genetic testing results (genotype) and clinical phenotypes do not match.

In cases of “genotype-phenotype matching”, genetic results complement the clinical phenotype and genetic testing can be used in diagnosis, family screening, and occasionally prognostication. It becomes more challenging when genetic results are negative or non-contributory, and ‘contradict’ the clinical phenotype. “Genotype mismatches” can also occur when genotype-positive patients have no clinical phenotype, or when genetic testing results point towards a completely different disease than the clinical phenotype. We discuss an approach to genetic testing and review the challenges that may arise when interpreting genetic testing results.

Genetic testing has opened a wealth of opportunities in the diagnosis, management, and cascade screening of inherited arrhythmia syndromes, but has also opened a “Pandora’s box” of challenges. Genetic results should be interpreted with caution and in a multidisciplinary clinic, with support from genetic counsellors and an expert with a focused interest in cardiovascular genetics.

Word Count: 249

Background Genetic testing is playing an ever-expanding role in cardiovascular care and is becoming part of the “toolkit” for the cardiovascular clinician. In inherited arrhythmias, genetic testing can confirm a suspected diagnosis, establish a diagnosis in unexplained cases, and facilitate cascade family screening. Genetic “arrhythmia panels” cast a wide net for heritable diseases, ranging from common arrhythmia and cardiomyopathy genes, to obscure conditions. Direct-to-consumer technologies provide patients and the public with greater access to genetic information, but can easily mislead and cause anxiety. We discuss an approach to genetic testing for the cardiovascular clinician, and highlight challenges that may arise when interpreting genetic testing results.

Genetic History Taking and Pedigree Construction It is critical that the cardiovascular clinician takes and confirms a thorough family history prior to embarking on genetic testing. Pedigree construction can identify relatives at risk and obligate carriers, and help determine where to begin genetic testing. A fundamental principle is to begin testing on the proband or family member with the most severe/unequivocal phenotype, which may be an individual that died suddenly with banked tissue/DNA. Genetic testing in a relative with clinical findings of uncertain significance (e.g. borderline QT) can confuse family screening and should generally be avoided. Pedigrees with multiple affected individuals may include obligate carriers (Figure 1)- individuals that must carry the gene variant regardless of disease manifestation. Completed pedigrees can also identify unexplained deaths in the family, including sudden infant deaths, drownings, or motor vehicle accidents. In the case of multiple variants, segregation analysis amongst family members helps identify which gene variants are associated with the clinical phenotype and can, therefore, then be used for cascade screening.

Common Genes Associated with Inherited Arrhythmia Syndromes Many inherited arrhythmia syndromes are monogenic diseases arising from a single pathogenic variant involved in the assembly of the cardiac ion channels or structural cardiac proteins. In Supplemental Table

S1, we present a list of common genes involved in the inherited arrhythmia syndromes and arrhythmogenic cardiomyopathies, and the yield of genetic testing for each syndrome. In most monogenic inherited arrhythmia syndromes, the yield of genetic testing is modest (20-80%).

When to Consider Genetic Testing Genetic testing is typically arranged by a team with genetic counselling expertise in inherited arrhythmias. Monogenic conditions refer to genetic conditions where a single gene variant is sufficient to cause disease. Conversely, polygenic conditions have a multifactorial inheritance pattern where two or more genes may be involved in disease expression and penetrance. Mendelian inheritance dictates that genes segregate independently with dominant and/or recessive expression patterns, while non-Mendelian inheritance refers to inheritance patterns that do not follow these rules (i.e. co-dominance, gene conversion, imprinting, mitochondrial DNA inheritance). Genetic testing is most useful in the diagnosis, management, and cascade screening of monogenic conditions, and has been recommended in most inherited arrhythmia syndromes.(1) On the other hand, while genetic associations for polygenic conditions exist (i.e. idiopathic VF, familial atrial fibrillation), the yield of testing in these conditions is much lower.

Understanding Genetic Testing Complexity Genetic variation is very common and partly underlies variation of human traits such as height, skin color, cholesterol levels, blood cell count, electrocardiographic parameters, etc. Most genetic variants are functionally neutral or result in slight changes in protein function that are insufficient to cause disease. At the molecular level, genetic variation can either result from a DNA base pair substitution, insertion or deletion. Such changes in non-protein coding regions are generally not disease-causing, while changes that alter the protein amino acid sequence (coding regions) are more likely to be deleterious. According to the American College of Medical Genetics (ACMG), genetic variants are classified in one of five categories based on their likelihood of causing disease: pathogenic, likely pathogenic, variant of uncertain

significance (VUS), likely benign, or benign.(2) Benign and likely benign variants are frequently present in the general population and are typically not reported by genetics laboratories. In contrast, pathogenic and likely pathogenic variants are very rare in the general population and have a very high likelihood for pathogenicity based on the literature or public databases (e.g. ClinVar), predicted impact on protein function, and/or co-segregation with disease. Genetic variants are classified as VUS when there is insufficient data to classify them as benign or pathogenic. Common scenarios for VUS include rare or novel unpublished variants in a gene known to cause disease or a variant in a gene not clearly associated with disease. Although common in large genetic panels, VUS are generally not used in family screening, and are treated as non-contributory to the clinical picture.

Genotype Phenotype Match Patients harboring pathogenic or likely pathogenic variants are typically labeled as “genotype positive”. In cases of “genotype-phenotype matching”, genetic results complement the clinical phenotype allowing for diagnostic confirmation, family screening, and occasionally disease prognostication. For example, patients with Brugada syndrome and SCN5A mutations may have an increased risk of cardiac events compared to those without an identified pathogenic mutations.(3)

Genetic Testing Unhelpful It becomes more challenging in cases where genetic results are unhelpful or ‘contradict’ the clinical phenotype (Figure 2). This commonly occurs when patients have a clinical phenotype but negative genotype. In such cases, patients and families should be educated about the ongoing possibility of heritable syndromes despite negative genetic results. The field of medical genetics is sufficiently mature such that new undiscovered monogenic causes are unlikely; furthermore, recent studies have suggested that most inherited cardiac syndromes have a complex, and often polygenic, inheritance pattern.(4) As such, in families with ‘gene-elusive’ syndromes, cascade screening should occur on the basis of phenotype alone.

Genotype Mismatch “Genotype mismatch” can occur when genotype-positive patients have no clinical phenotype or when the clinical phenotype is different from that expected from genetic testing. Such cases may occur due to variable penetrance, leading to minimal or no disease manifestations in some individuals and manifest disease in others. For example, a grandparent may carry a pathogenic PKP2 variant and only manifest a mild ARVC phenotype- having survived to an older age, this patient has been protected by limited penetrance, and/or other genetic and epigenetic factors, often leading to a better prognosis than younger family members with manifest disease. Other examples of genotype mismatch occur when genetic results predict a completely different phenotype than what is expressed. Examples include individuals with SCN5A variants (typically associated with vagal-driven arrhythmias) manifesting with adrenergic arrhythmias, or vice versa; individuals with RYR2 variants (associated with adrenergic arrhythmias) with vagally-driven events. Given the complexity and differing types of genotype mismatch, such cases should be reviewed with expert consultation, integrating the clinical presentation, mechanism for pathogenesis, family expression of the variant, and response to therapy, rather than genotype alone.

Whole Exome or Whole Genome Sequencing There is growing excitement in the potential of whole exome or whole genome sequencing (WES/WGS), particularly in cases where current genetic testing is unhelpful (i.e. ‘gene-elusive’ conditions). Compared to conventional gene panels, WES/WGS casts a much wider net for gene variants, and may offer novel opportunities in gene discovery. Exploratory studies in WGS in cardiovascular disease have identified rare monogenic causes of disease, and derived polygenic risk scores to estimate the risk of cardiac events.(5) For example, genomic studies in atrial fibrillation have identified both rare variants in PITX2 and polygenic scores (incorporating up to 12 genetic risk markers) to predict subsequent risk of atrial fibrillation. However, the simultaneous challenges in genotype interpretation (i.e. frequent VUS, genotype mismatches) persist and are potentially magnified by the scope of WES/WGS.

Holistic Approach to Genetic Testing Before embarking on genetic testing, patient and family members must understand the potential implications of test results. Negative tests do not exclude disease, but reflect the absence of an explanatory variant known to be disease-causing. In patients and families with clinically manifest disease, a VUS, genotype mismatch, or negative genetic result may lead to confusion, uncertainty, or questions about relatives at risk. In contrast, the identification of pathogenic variants may provide closure following the loss of a loved one. Clinicians must be cautious when discussing results, as findings can also have long-lasting effects on family dynamics by assigning ‘genetic guilt’ to gene carriers. Notably, Canadian law (Bill S-201) forbids discrimination on the basis of genetic test results, prohibits companies and employers from requiring genetic testing or test results, and prohibits discrimination on life insurance based on genetic testing.

We strongly advocate for a multidisciplinary approach that integrates genetic expertise when pursuing genetic testing, involving genetic counselling, typically by a certified Genetic Counsellor with access to a medical geneticist or expert cardiogenetic expert. Families should be well-informed of the risks of positive and negative results, and should have the opportunity to consult with genetic expertise when receiving test results. A familiarity with commercially available gene panels and those offered by provincial labs will ensure appropriate selection and avoid inclusion of unrelated genes where test results may lead to confusion. Review of pathogenic variants and VUS may provide novel insights into genetic risk stratification and variant reclassification. Interval genetic testing, in the advent of large advances or gene discoveries, can also be considered following expert consultation.

Genetics and Precision Medicine Future advancements in genetics may have further broad ranging applications, particularly in the field of precision medicine. Advancements in WES/WGS and genomic editing can alter cell lines to facilitate

personalized pharmacological testing. Personalized drug susceptibilities may allow for targeted interventions, providing for novel treatments for inherited conditions. Furthermore, genomic editing using CRISPR-Cas technologies may also provide a unique opportunity to target actionable genetic variants themselves, potentially reducing or eliminating pathogenicity entirely. The CRISPR-Cas9 system, identified in bacteria, incorporates a DNA endonuclease and RNA guide sequence to deliver precise genome editing. Early experiments with animal models have suggested a potential to treat a wide variety of heritable diseases, including cardiovascular disease. This exciting capability raises the possibility of suppressing and even eliminating the effects of deleterious variants.

Conclusion Genetic testing has opened a wealth of opportunities in the diagnosis, management, and cascade screening of inherited arrhythmia syndromes, but has also opened a “Pandora’s box” of challenges. From VUS to genotype mismatches, genetic results should be interpreted with caution and in a multidisciplinary clinic, with support from genetic counsellors and an expert with a focused interest in cardiovascular genetics. Newer technologies in direct-to-consumer genetic testing and WES/WGS provide an escalating scale of genetic data at reasonable costs. However, interpretation of such large amounts of genetic data, and differentiating pathology from natural variance becomes increasingly difficult. The continued advancement of genetic technology will undoubtedly unlock further understanding into disease pathogenesis, but will simultaneously raise questions relating to its diagnostic certainty and clinical relevance.

References

1) Gollob MH, Blier L, Brugada R, Champagne J, Chauhan V, Connors S, et al. Recommendations for the use of genetic testing in the clinical evaluation of inherited cardiac arrhythmias associated with sudden cardiac death: Canadian Cardiovascular Society/Canadian Heart Rhythm Society joint position paper. Can J Cardiol. 2011 MarApr;27:232-45. 2) Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015 May;17:405-24. 3) Brugada J, Campuzano O, Arbelo E, Sarquella-Brugada G, Brugada R. Present Status of Brugada Syndrome: JACC State-of-the-Art Review. J Am Coll Cardiol. 2018 Aug 28;72:1046-1059. 4) Cerrone M, Remme CA, Tadros R, Bezzina CR, Delmar M. Beyond the One Gene-One Disease Paradigm: Complex Genetics and Pleiotropy in Inheritable Cardiac Disorders. Circulation. 2019 Aug 13;140:595-610. 5) Khera AV, Chaffin M, Zekavat SM, Collins RL, Roselli C, Natarajan P, et al. WholeGenome Sequencing to Characterize Monogenic and Polygenic Contributions in Patients Hospitalized With Early-Onset Myocardial Infarction. Circulation. 2019 Mar 26;139:1593-1602.

TABLES AND FIGURES

Figure 1. Example of Pedigree Diagram

Figure 2. Genotype-Phenotype Matching

Supplemental Table S1. Commons Genes and Associated Inherited Arrhythmia Syndromes

Figure 1 Legend. A three-generation pedigree is typically drawn in the evaluation for inherited cardiac conditions. Standard nomenclature include circles representing female, squares representing males, shaded symbols representing affected individuals, and crossed symbols representing deceased individuals.

Figure 2 Legend. Examples of genotype matching (middle), mismatch (left), or genotype unhelpful (right) and their clinical implications.

Figure 1. Example of Pedigree Diagram

Figure 1 Legend. A three-generation pedigree is typically drawn in the evaluation for inherited cardiac conditions. Standard nomenclature include circles representing female, squares representing males, shaded symbols representing affected individuals, and crossed symbols representing deceased individuals. In this pedigree, A and B refer to two different diseases, of which only one (A) segregates with the family tree. The +/- symbolize a heterozygous gene variant (i.e. autosomal dominant) segregating with disease.

Figure 2. Genotype-Phenotype Matching

Figure 2 Legend. Examples of genotype matching (middle), mismatch (left), or genotype unhelpful (right) and their clinical implications.

Supplemental Table S1. Inherited Arrhythmia Syndromes and Associated Genes Inherited Arrhythmia Syndrome

Yield of Testing

Associated Genes*

Channelopathies KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, KCNJ2, CACNA1C, CALM1, CALM2, CALM3, Catecholaminergic Polymorphic 50% RYR2, CASQ2, TRDN, CALM1, CALM2, Ventricular Tachycardia CALM3, TECRL Brugada Syndrome ~20% SCN5A Idiopathic Ventricular Fibrillation ? DPP6 Cardiomyopathies with Frequent Arrhythmic Presentations Arrhythmogenic Right Ventricular ~50% PKP2, DSP, TMEM43, SCN5A, DSG2, DSC2, Cardiomyopathy (ARVC) JUP ~50% MYBPC3, MYH7, TNNT2, TNNI3, MYL2, Hypertrophic Cardiomyopathy MYL3, TPM1 Other arrhythmogenic ? LMNA, PLN, DSP, FLNC, DES cardiomyopathies *The list includes genes unequivocally associated with the condition but is non-exclusive Long QT Syndrome

~80%