QUARTERLY FOCUS ISSUE: HEART RHYTHM DISORDERS
The RYR2-Encoded Ryanodine Receptor/Calcium Release Channel in Patients Diagnosed Previously With Either Catecholaminergic Polymorphic Ventricular Tachycardia or Genotype Negative, Exercise-Induced Long QT SyndromeA Comprehensive Open Reading Frame Mutational Analysis
Argelia Medeiros-Domingo, MD, PhD*,
Zahurul A. Bhuiyan, MD, PhD ,
David J. Tester, BS*,
Nynke Hofman, MSc,
Hennie Bikker, PhD,
J. Peter van Tintelen, MD, PhD¶,
Marcel M.A.M. Mannens, PhD,
Arthur A.M. Wilde, MD, PhD,|| and
Michael J. Ackerman, MD, PhD*, , ,*
* Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota
Department of Medicine, Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota
Department of Pediatrics, Division of Pediatric Cardiology, Mayo Clinic, Rochester, Minnesota
Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
|| Department of Cardiology and Heart Failure Research Centre, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
¶ Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands
Manuscript received May 5, 2009;
revised manuscript received August 28, 2009,
accepted August 30, 2009.
* Reprints requests and correspondence: Dr. Michael J. Ackerman, Director, Long QT Syndrome Clinic and the Mayo Clinic Windland Smith Rice Sudden Death Genomics Laboratory, Mayo Clinic, Guggenheim 501, 200 First Street SW, Rochester, Minnesota 55905 (Email: ackerman.michael{at}mayo.edu).
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Abstract
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Objectives: This study was undertaken to determine the spectrum and prevalence of mutations in the RYR2-encoded cardiac ryanodine receptor in cases with exertional syncope and normal corrected QT interval (QTc).
Background: Mutations in RYR2 cause type 1 catecholaminergic polymorphic ventricular tachycardia (CPVT1), a cardiac channelopathy with increased propensity for lethal ventricular dysrhythmias. Most RYR2 mutational analyses target 3 canonical domains encoded by <40% of the translated exons. The extent of CPVT1-associated mutations localizing outside of these domains remains unknown as RYR2 has not been examined comprehensively in most patient cohorts.
Methods: Mutational analysis of all RYR2 exons was performed using polymerase chain reaction, high-performance liquid chromatography, and deoxyribonucleic acid sequencing on 155 unrelated patients (49% females, 96% Caucasian, age at diagnosis 20 ± 15 years, mean QTc 428 ± 29 ms), with either clinical diagnosis of CPVT (n = 110) or an initial diagnosis of exercise-induced long QT syndrome but with QTc <480 ms and a subsequent negative long QT syndrome genetic test (n = 45).
Results: Sixty-three (34 novel) possible CPVT1-associated mutations, absent in 400 reference alleles, were detected in 73 unrelated patients (47%). Thirteen new mutation-containing exons were identified. Two-thirds of the CPVT1-positive patients had mutations that localized to 1 of 16 exons.
Conclusions: Possible CPVT1 mutations in RYR2 were identified in nearly one-half of this cohort; 45 of the 105 translated exons are now known to host possible mutations. Considering that  65% of CPVT1-positive cases would be discovered by selective analysis of 16 exons, a tiered targeting strategy for CPVT genetic testing should be considered.
Key Words: ryanodine receptor • catecholaminergic polymorphic ventricular tachycardia • sudden cardiac death • exertional syncope
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Abbreviations and Acronyms
| | CPVT = catecholaminergic polymorphic ventricular tachycardia | | DNA = deoxyribonucleic acid | | LQTS = long QT syndrome | | QTc = corrected QT interval | | RyR2 = cardiac ryanodine receptor |
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Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal, heritable arrhythmia syndrome often manifesting as exercise-induced ventricular arrhythmias, syncope, or sudden death (1). With mortality rates of 30% to 50% by age 35 years, CPVT is one of the most malignant cardiac channelopathies expressed predominately in young patients with otherwise structurally normal hearts (2). While the resting 12-lead electrocardiogram is typically normal, the hallmark arrhythmia, bidirectional VT, may be present during exercise stress testing and has been considered pathognomonic for CPVT (1,3).
CPVT stems from an alteration of intracellular calcium handling involving the critical calcium-induced calcium release mechanism of myocardial cells. At the molecular level, gain of function mutations in the cardiac ryanodine receptor encoded by RYR2 account for at least 50% of CPVT cases and are annotated as type 1 CPVT (CPVT1) (3). Mutations in CASQ2-encoded calsequestrin are responsible for the very rare, autosomal recessive form known as type 2 CPVT (CPVT2) (2,4).
The cardiac ryanodine receptor (RyR2), encoded by the 105-exon–containing RYR2 gene, is one of the largest ion channel proteins, comprising 4,967 amino acids; it localizes to the sarcoplasmic reticulum, and controls intracellular calcium release and cardiac contraction. Since the sentinel discovery of a CPVT-causing RYR2 mutation (5), a cluster distribution involving 3 discrete protein regions has been reported. On the basis of a potential physiological role for these "hot spots," these regions have been termed "domains" I, II, and III (Fig. 1) (6,7). Similar mutation clustering is observed in the RYR1 gene, which encodes the skeletal muscle RyR1 and is linked to malignant hyperthermia and central core disease (8–10). However, because the majority of CPVT cases have not undergone the entire RYR2 scan, the prevalence of mutations residing outside these 3 canonical domains (i.e., 61 exons that encode for 2,570 amino acids) remains unknown.
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Figure 1 Mutation Clustering in the RyR2
Mutations clustered in the cardiac ryanodine receptor (RyR2) are distributed in 3 "hot-spot" regions, called domains I (N-terminal), II (central), and III (channel region). AA = amino acid number estimated for each domain. Adapted from George et al. (7) and Yano et al. (6).
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Currently, among research laboratories and clinical diagnostic laboratories, there is no consensus or clear definition of the "RYR2 targeted scan," resulting in an enormous discrepancy in the number of exons studied by each research group or commercial company. This situation has an important impact in "gene-negative" definition, genotype-phenotype correlation, and patient quality of care. In the present study, we sought to determine the prevalence of mutations throughout RYR2's entire open reading frame in a large cohort of unrelated cases referred to 2 different institutions for exertional syncope and, using a combined analysis of the previous reported mutations and the novel mutations found in this cohort, we propose a novel, targeted "genetic approach" for CPVT1 genetic testing.
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Methods
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Study participants.
We studied a cohort of 155 unrelated patients referred to either the Windland Smith Rice Sudden Death Genomics Laboratory at Mayo Clinic, Rochester, Minnesota, or the Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, the Netherlands, for genetic testing between August 2001 and June 2008. A clinical diagnosis of CPVT was rendered in 110 patients by either the authors (M.J.A., A.A.M.W.) or the referring physician. Of these patients, 78 were classified as "strong CPVT phenotype" because of exertional syncope plus documentation of bidirectional or polymorphic ventricular tachycardia, and 32 were classified as "possible CPVT phenotype" on the basis of presence of exertional syncope and stress test induced ventricular ectopy but not bidirectional or polymorphic ventricular tachycardia. In addition, 45 cases were referred as "possible/atypical long QT syndrome" (LQTS) because of exertional syncope and corrected QT interval (QTc) values <480 ms. All 45 were genotype negative for the 12 known LQTS-susceptibility genes.
After receipt of written consent for this protocol approved by the Mayo Foundation Institutional Review Board and Amsterdam Academic Medical Center Medical Ethical Committee, genomic deoxyribonucleic acid (DNA) was extracted from peripheral blood lymphocytes using the Purgene DNA extraction kit (Gentra, Minneapolis, Minnesota). In cases with suspected mosaicism, additional DNA from saliva was isolated using the ORAgene kit (DNA Genotek, Ottawa, Ontario, Canada), and DNA from skin fibroblasts and hair-roots was isolated using the QIAamp DNA minikit (Qiagen, Valencia, California).
Mutational analysis.
Comprehensive open reading frame/splice site mutational analysis of all 105 RYR2 exons was performed using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing as described previously (11). The flanking primers used for polymerase chain reaction were published previously or designed with Oligo software (Molecular Biology Insights, Cascade, Colorado) and are available on request. We also searched for large genomic rearrangements affecting exon 3, as reported previously (12).
All putative pathogenic variants must have been absent in 400 reference alleles (100 healthy Caucasian and 100 healthy black) obtained from the Human Genetic Cell Repository sponsored by the National Institute of General Medical Sciences and the Coriell Institute for Medical Research (Camden, New Jersey) to be considered as potentially disease related.
Statistical analysis.
We used the JMP Statistical Software (version 6.0, SAS Institute, Cary, North Carolina). All continuous variables are reported as mean ± SD. Differences between continuous variables were evaluated using unpaired Student t tests, and nominal variables were analyzed using chi-square analysis. Statistical significance was considered at p < 0.05.
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Results
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The demographic characteristics of the 155 unrelated patients are shown in Table 1; 96% were Caucasians, 49% were females, age at symptoms was 20 ± 15 years, and average QTc was 428 ± 29 ms. The mean age of onset of symptoms was significantly lower in RYR2 mutation-positive subjects compared with subjects who had a negative genetic test (16.7 ± 12.3 years vs. 23.8 ± 16.6 years, respectively; p < 0.004).
Overall, 77 (63 unique, 34 novel) putative disease-causing mutations were identified in 73 cases (47%) (Table 2, Fig. 2) (1–3,5,6,12,14,24,26–49). Forty-one of 73 (56%) mutation-positive cases were females. Putative mutations were absent in 400 reference alleles, and most of the mutated residues exhibit high conservation across species (Supplemental Table). The yield of the genetic test was significantly higher among the 78 cases classified clinically as "strong CPVT phenotype" compared with the 32 cases diagnosed as "possible CPVT phenotype" (60% vs. 37.5%, p < 0.04). Notably, nearly one-third of the 45 "gene negative LQTS" cases had a rare missense mutation in RYR2 (Table 1, Fig. 3). Four of the 73 RYR2 mutation-positive cases hosted multiple mutations (5.5%). As expected, we observed a mutation clustering distribution across RYR2; nevertheless, 10 mutations found in 11 cases resided outside the 3 canonical domains, specifically, between domains I and II; 8 of them exhibited a strong CPVT phenotype. Three large genomic rearrangements comprising exon 3 were detected in 3 unrelated cases involving a 3.6 kb deletion in 1 and a 1.1 kb deletion in 2 cases.
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Figure 2 RyR2 Channel Topology and Localization of Mutations and Polymorphisms
Linear topology of the cardiac ryanodine receptor (RyR2); putative pathogenic mutations (yellow circles) and polymorphisms (blue circles) found in this study cohort are shown in the approximate location. The number within the circle corresponds to the mutation number on Table 2. SR = sarcoplasmic reticulum.
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Figure 3 Prevalence of RYR2 Mutations by Subgroups
The yield from the entire RYR2 scan on this cohort is shown on the left. Bars on the right show the yield in the 3 different subgroups of this cohort. CPVT = catecholaminergic polymorphic ventricular tachycardia; LQTS = long QT syndrome.
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One proband had a maternally inherited Y4149S (tyrosine, Y, at position 4149 mutated to serine, S) missense mutation. Although the proband's mother was asymptomatic and had an unremarkable exercise electrocardiogram, germline mosaicism was suspected clinically because more than 1 offspring was affected. Accordingly, Y4149S mosaicism was detected in her, being highest in the hair-roots (25%), and less in leukocytes (20%) and in fibroblasts and buccal epithelium (15% to 18%).
Twelve nonsynonymous single nucleotide polymorphisms (6 novel) were also identified; 7 of them were seen only in controls, and 5 were seen in cases and controls (Table 2). Four novel polymorphisms localized between domains I and II. The most common polymorphism was Q2958R with a heterozygous prevalence of 34% in Caucasians and 10% in African Americans, followed by G1886S with a prevalence of 20% (African Americans) and 9% (Caucasians). V377M was found only in African Americans with a prevalence of 3%. Finally, Y2156C, E2183V, M2389L, V4010M, A4282V, and G4315E are rare variants observed only once in different control subjects. Thus, within the exons hosting putative CPVT1-associated mutations, the background prevalence of rare amino acid substitutions among the 200 apparently healthy volunteers was 3% (3 of 100 Caucasians and 3 of 100 African Americans) (Table 2).
We evaluated the number of mutations in each exon reported to date in the literature (Table 2), excluding exons containing only polymorphisms. As such, 128 unique mutations were analyzed, including those found within this cohort. Sixteen exons hosted  3 distinct CPVT1-associated mutations, 13 exons had at least 2 mutations reported, and an additional 16 exons have had, so far, only a single mutation reported (Fig. 4). This mutation clustering phenomena might facilitate a tiered strategy that may yield a more cost-effective approach for CPVT genetic testing. If we consider that the average charge for the current RYR2 commercial tests available on the market is $0.40 per coding nucleotide (50,51), the estimated charge for the entire RYR2 coding region scan would be $6,000 per patient, meaning that the commercial charge to analyze this 155-patient cohort in its entirety would have approached U.S. $1 million. In comparison, the total charge to scan only the 45 mutation-hosting exons that have been reported to date would be 50% less. Further, a reflex tiered strategy would reduce the cost significantly. As modeled here, using a 3-tiered reflex genetic test strategy, based on Figure 4, the genetic scan of the first tier of exons in our cohort would cost $190,960 ($1,200 per case) and would detect nearly two-thirds of those CPVT cases that are due to mutations in RYR2. The charge to reflex to the second tier genetic scan would add <$1,000 per case and combined, nearly 90% of the RYR2-mutation positive cases (CPVT1) would be identified. Reflexing to the third tier would capture the remaining RYR2-positive cases and the charge to do so would be $123,225 ($795 per case) (Fig. 5).
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Figure 4 Possible Tiered Strategy for RYR2 Genetic Testing
Schematic representation of the 105 coding exons of the RYR2 gene. Colored boxes represent all of the exon-containing mutations reported to date; white boxes represent exons free of reported mutations. The tiered strategy was built on the basis of the number of mutations contained in each exon as shown by 3 different colors. The first tier included 16 exons; the second tier, 13 exons; and the third tier, 16 exons. Exons containing control variants were not included.
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Figure 5 Yield From RYR2 Mutational Analysis on the Basis of a Tiered Strategy
Retrospective analysis of the mutations detected in our cohort (purple bars) and in the world-wide compendium (orange bars) of mutations reported to date. The percentage of mutations that would be detected using the tiered strategy is shown.
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Discussion
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Exertional syncope: LQTS or CPVT?.
It has been reported that nearly 30% of CPVT cases have been misdiagnosed as "LQTS with normal QT intervals" or "concealed LQTS" (13). Recently, we demonstrated that nearly 6% of 269 LQTS genotype-negative patients hosted a putative CPVT1-causing RYR2 mutation (14). Here, we included only referral cases of "atypical/possible LQTS" with a phenotype of exertional syncope and QTc <480 ms. Herein, the yield of RYR2 mutations for these 45 cases was 31%, indicating the critical importance of properly distinguishing between CPVT and LQTS. CPVT-related arrhythmias can be easily reproduced during an exercise stress test, by isoproterenol infusion, or by other forms of adrenergic stimulation (15,16). The induction of polymorphic ventricular tachycardia or bidirectional VT, characterized by a 180° alternating QRS axis on a beat-to-beat basis, sets CPVT apart from "concealed" or "borderline" LQTS.
RYR2 genetic approach: targeted scan and tiered strategy.
Our results confirm that mutation clustering exists. The functional significance of mutation clustering remains unclear. It has been suggested, however, that a domain-domain interaction is crucial for channel function (17–19), and a defective intermolecular interaction may be crucial in disease phenotypes. Interestingly, in this study, 11 of 64 (17%) putative mutations localized outside the considered canonical domains.
On the basis of our results and after analyzing a large publicly available compendium of the 128 RYR2 putative mutations known to date (Table 2), we propose an expanded genetic approach for research/investigational laboratories. A reasonable RYR2 scan will include the analysis of at least 45 exons in total known to host all published mutations reported to date. Since 19 exons imbibed in the hot-spot region remain free of mutations so far, a more ambitious and "comprehensive" RYR2 genetic test would include these exons as well, resulting in a 64-exon scan (exons 3 to 28, 37 to 50, 75, and 83 to 105).
The mutation clustering phenomena might facilitate a tiered strategy that may yield a more cost-effective approach for CPVT genetic testing. Figure 4 summarizes this proposed tiered strategy. The approach was developed considering the number of mutations in each exon reported to date in the literature. The first tier comprises those exons (n = 16) now known to host 3 unique CPVT-associated mutations. The second tier includes 13 exons with at least 2 mutations reported, and the third tier consists of the final 16 exons where, so far, only a single mutation within that exon has been reported. Considering that 65% of the RYR2 mutation-positive cases might have a mutation in the first tier of 16 RYR2 exons, the charge of the genetic analysis in this group could be reduced by approximately one-half (predicted U.S. $1,232 per case for the first tier of 16 exons vs. $3,019 per case for the entire sequencing of exons containing reported mutations).
In case of negative results, we suggest that the pseudocomprehensive (64-exon) RYR2 scan mentioned previously (exons 3 to 28, 37 to 50, 75, and 83 to 105) be performed. Additional "rare" although documented causes of CPVT should also be considered, like large RYR2 genomic rearrangements involving exon 3 and mutations in calsequestrin 2 (CASQ2) and Kir2.1 (KCNJ2) (20). The area surrounding exon 3 is highly susceptible to large Alu-repeat–mediated genomic rearrangements; we documented 3 unrelated cases hosting large heterozygous deletions involving exon 3 that could not be detected by regular genetic screening using high-performance liquid chromatography or direct DNA sequencing. Validating this observation, exon 3 deletion was also reported recently in a different cohort, in which 2 unrelated cases (of 33) hosted a 1.1kb deletion, including exon 3 (21).
Polymorphisms in RYR2, not that rare and with potential functional effect.
It has been considered that RYR2 is not a polymorphic gene. However, 15 of 142 (10.5%) missense variants reported to date were found in control subjects. We did not scan the entire RYR2 gene in control subjects. Instead, because we focused on the exon-containing mutations, the rate of nonsynonymous genetic variation throughout all of RYR2 may be higher. Importantly however, among the exons now known to host possible CPVT1-associated missense mutations, similarly rare amino acid substitutions were found in only 6 of the 200 control subjects (3%) examined in this study. Although not a true case-control genetic epidemiologic study, if validated, this would suggest that among cases where CPVT is strongly suspected, there would be a 95% estimated probability that the identification of a rare missense mutation would likely represent the pathogenic basis for the patient's CPVT, rather than merely being only a rare amino acid substitution.
We have learned that common polymorphisms in other ion channels have the potential to modify the clinical phenotype (22,23); polymorphisms in RYR2 may have the same potential. The most common RYR2 polymorphism is RyR2-Q2958R, described for the first time 9 years ago (24), and it is particularly common among Caucasians (34%). The second most common polymorphism in RYR2 is G1886S (20% African Americans, 9% Caucasians), followed by G1885E (6% Caucasians). Interestingly, in vitro studies in heterologous systems have demonstrated that both G1885E and G1886S polymorphisms caused a significant increase in the cellular Ca(2+) oscillation activity compared with RyR2 wild-type channels. Further, when both polymorphisms were introduced in the same RyR2 subunit, the store-overload-induced calcium release activity was nearly completely abolished (25). The clinical consequences of this "RyR2 loss of function" in vitro phenotype is not clear; however, compound heterozygosity involving these 2 polymorphisms has been reported in right ventricular dysplasia (26). The potential functional effects of the 6 novel polymorphisms identified in this study are unknown.
It is important to remark that none of the novel mutations detected in this study have been functionally characterized to further bolster the contention of pathogenicity. However, <15% of the mutations reported to date in RYR2 have been studied in vitro; pathogenicity has been suspected based on cosegregation with the disease and absence in control subjects. Here, cosegregation with the disease data was not available for all cases. Instead, the prevalence of rare mutations among strong cases (60%) was markedly higher than in control cases (3%), and all putative mutations were absent in 400 reference alleles. Thus, although the precise contribution of each discrete mutation to the phenotype remains to be determined, statistically, the estimated probability for pathogenicity for RYR2 mutations found in strong cases is quite high (95%).
Mosaicism in RYR2.
This is the first report involving RYR2 mosaicism that was transmitted to descendants, presumably causing sudden death in 2 children and full-blown CPVT in 1 child from the age of 9 years. The RYR2 mutations, in many circumstances (20% in our cohort) are de novo in origin, but they could also be present in a mosaic form in the asymptomatic parents, and that requires attention during genetic counseling as well as during genetic screening.
Clinical significance.
This study represents the first analysis of RYR2 mutation distribution in a large cohort of cases. Our results contribute to a better delineation of the "hot spot" regions with important consequences in "gene negative" definition. The identification of novel common variants in control subjects facilitates better interpretation of the CPVT genetic test. Detection of RYR2 mosaicism, and confirmation of exon 3 deletion in different patient-cohorts, provides novel genetic possibilities for the pathogenesis of CPVT. Moreover, the development of a tiered strategy for RYR2 genetic scan may enable a more cost-effective genetic approach to analyzing one of the largest genes in the human genome. Finally, we emphasize the critical importance of properly distinguishing between CPVT and LQTS (including Andersen-Tawil syndrome), 2 different diseases with a similar clinical presentation but different clinical outcomes and different responsiveness to pharmacotherapy.
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Conclusions
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Although intimidating, as one of the largest genes in the human genome, results from this comprehensive open reading frame analysis involving one of the largest cohorts of unrelated patients examined, combined with a detailed analysis of all published CPVT1-associated mutations indicate that to date, only 45 of RYR2's 105 translated exons host a putative CPVT1-associated mutation thus far. Moreover, an initial targeting of only 16 exons would allow the identification of putative mutations in 65% of the RYR2-mutation positive cases, although compound heterozygosity may be missed. Finally, given the present estimate of 3% frequency for rare missense mutations among controls, one must be cognizant of the possibility of a "false positive" especially as the pre-test probability of a CPVT diagnosis decreases. The 33% yield that was observed among the "possible" cases of CPVT indicates that perhaps 90% of the mutations, identified among cases labeled as "possible CPVT" or so-called "atypical LQTS" with exercise-induced syncope and QTc <480 ms, are pathogenic, whereas 10% of those mutations may represent false positives.
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Appendix
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For a supplementary table listing conservation across species of the novel RYR2 variants, please see the online version of this article.
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Footnotes
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This work was supported (in part) by National Institutes of Health grant P01 HL094291 (to Drs. Ackerman and Moss). Dr. Ackerman is a consultant for PGxHealth and chairs their FAMILION Medical/Scientific Advisory Board (approved by Mayo Clinic's Medical-Industry Relations Office and Conflict of Interests Review Board). In addition, a license agreement pertaining to mutations in the ryanodine receptor 2 gene and heart disease, resulting in consideration and royalty payments, was established between PGxHealth and Mayo Clinic Health Solutions in 2007. Dr. Tester receives moderate royalties from PGxHealth Inc. Drs. Ackerman and Medeiros-Domingo are supported by a grant from the Fondation Leducq (08CVD01) and by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. Dr. Wilde is supported by the Interuniversity Cardiology Institute of the Netherlands (ICIN) Project 27 and by Leducq Fondation Program Grant 05CVD01, Alliance Against Sudden Cardiac Death. Drs. Medeiros-Domingo and Bhuiyan contributed equally to this work.
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