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To determine the spectrum and prevalence of mutations in the RYR2-encoded the cardiac ryanodine receptor in cases with exertional syncope and normal QTc.
Mutations in the 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.
Mutational analysis of all RYR2 exons was performed using PCR, DHPLC, and DNA sequencing on 155 unrelated patients (49% females, 96% white, 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 (LQTS) but with QTc < 480 ms and a subsequent negative LQTS genetic test (n = 45).
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 one of 16 exons.
Possible CPVT1 mutations in RYR2 were identified in nearly 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.
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 30-50% by age 35 years, CPVT is one of the most malignant cardiac channelopathies expressed predominately in young patients with otherwise structurally normal hearts2. While the resting 12-lead electrocardiogram (ECG) is typically normal, the hallmark arrhythmia, bidirectional VT, is often 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 is 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 comprised of 4967 amino acids; localizes to the sarcoplasmic reticulum, and controls intracellular calcium release and cardiac contraction. Since the sentinel discovery of a CPVT-causing RYR2 mutation5, a cluster distribution involving three discrete protein regions has been reported. Based in a potential physiological role for these “hot-spots”, these regions have been termed “domains” I, II and III (Figure 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 disease8-10. However, since the majority of CPVT cases have not undergone the entire RYR2 scan, the prevalence of mutations residing outside these three canonical domains (i.e. ~61 exons that encode for 2570 amino-acids) remains unknown.
Currently, among research laboratories and clinical diagnostic laboratories, there is no consensus or clear definition of the “RYR2 targeted scan” resulting in 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.
We studied a cohort of 155 unrelated patients referred to either the Windland Smith Rice Sudden Death Genomics Laboratory at Mayo Clinic, Rochester, MN or the Department of Clinical Genetics, Academic Medical Center, University of Amsterdam, Netherlands for genetic testing between August 2001 and June 2008. A clinical diagnosis of CPVT was rendered in 110 patients by either one of the authors (MJA, AAMW) or the referring physician. Of these, 78 were classified as “strong CPVT phenotype” because of exertional syncope plus documentation of bidirectional or polymorphic ventricular tachycardia (BVT/PVT) while 32 were classified as “possible CPVT phenotype” based on the presence of exertional syncope and stress test induced ventricular ectopy but not BVT/PVT. In addition, 45 cases were referred as “possible/atypical long QT syndrome (LQTS)” because of exertional syncope and QTc values < 480 ms. All were genotype negative for the 12 known LQTS-susceptibility genes.
Following receipt of written consent for this Mayo Foundation Institutional Review Board and Amsterdam Academic Medical Center Medical Ethical Committee approved protocol, genomic DNA was extracted from peripheral blood lymphocytes using the Purgene DNA extraction kit (Gentra, Inc, Minneapolis, MN, USA). 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, USA).
Comprehensive open reading frame/splice site mutational analysis of all 105 RYR2 exons was performed using polymerase chain reaction (PCR), denaturing high performance liquid chromatography (DHPLC), and DNA sequencing as described previously.11 The flanking primers used for PCR were published previously or designed with Oligo software (Molecular Biology Insights, Inc., Cascade Colo.) and are available on request. We also searched for large genomic rearrangements affecting exon 3 as reported previously12.
All putative pathogenic variants must have been absent in 400 reference alleles (100 healthy white 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) in order to be considered as potentially disease-related.
We used the JMP Statistical Software (JMP 6.0, 2005; SAS Institute Inc, Cary, NC). 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.
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 yrs, and average QTc was 428 ± 29 ms. The mean age of onset of symptoms was significantly lower in RYR2 mutation positive subjects compared to those with a negative genetic test (16.7 ± 12.3 vs 23.8 ± 16.6 yrs respectively, p<0.004).
Overall, 77 (63 unique, 34 novel) putative disease causing mutations were identified in 73 cases (47%, Table 2, Figure 2). 41/73 mutation positive cases (56%) were females. Putative mutations were absent in 400 references alleles and most of the mutated residues exhibit highly 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 to 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, Figure 3). Four out of the 73 RYR2 mutation-positive cases hosted multiple mutations (5.5%). As expected, we observed a mutation clustering distribution across RYR2; nevertheless, ten mutations found in 11 cases resided outside the three canonical domains, specifically, between domain I and II; 8 of them exhibited a strong CPVT phenotype. Three large genomic rearrangements comprising exon 3 were detected in three unrelated cases involving a 3.6 kb deletion in one and a 1.1 kb deletion in two cases.
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 ECG; germline mosaicism was suspected clinically because more than one offspring was affected. Accordingly, Y4149S mosaicism was detected in her being highest in the hair-roots (~25%), less in leucocytes (~20%) and in fibroblasts and buccal epithelium (~15-18%).
Twelve non-synonymous single nucleotide polymorphisms (6 novel) were also identified, 7 of them were seen only in controls and 5 in cases and controls (Table 2). Four novel polymorphisms localize between domain I and II. The most common polymorphism was Q2958R with an 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% (3/100 Caucasians and 3/100 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; 127 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 while an additional 16 exons had, so far, only a single mutation reported (Figure 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 approximately $0.40 per coding nucleotide (http://pgxhealth.com, www.preventiongenetics.com), the estimated charge for the entire RYR2 coding region scan would be approximately $6000 per patient, meaning that the commercial charge to analyze this 155 patient cohort in its entirety would have approached $1 million US dollars. In comparison, the total charge to scan only the 45 mutation-hosting exons that have been reported to date exon-containing mutations reported to date would be about 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 $190,960.00 (~$1200 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 < $1000 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 US dlls ($795.00 US dlls/case, Figure 5).
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 mutation14. 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, isoproterenol infusion or by other forms of adrenergic stimulation15,16. The induction of polymorphic ventricular tachycardia or bidirectional VT, characterized by 180° alternating QRS axis on a beat-to-beat basis, sets CPVT apart from “concealed” or “borderline” LQTS.
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 function17-19 and a defective inter-molecular interaction may be crucial in disease phenotypes. Interestingly, in this study 11/64 (17%) of the putative mutations localize outside the considered canonical domains.
Based upon our results and after analyzing a large publicly available compendium of the 127 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 some exons (19) 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 3-28, 37-50, 75 and 83-105).
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 while 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 half (predicted $1232.00 US dlls/case for the first tier of 16 exons vs $3019.00 US dlls/case for the entire sequencing of exons-containing reported mutation).
In case of negative results, we suggest that the pseudo-comprehensive (64 exon) RYR2 scan mentioned previously (exons 3-28, 37-50, 75 and 83-105) 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 DHPLC or direct DNA sequencing. Validating this observation, exon 3 deletion was also reported recently in a different cohort where 2 unrelated cases (out of 33), hosted a 1.1kb deletion, including exon 321.
It has been considered that RYR2 is not a polymorphic gene. However, 15/142 (10.5%) missense variants reported to date were found in controls. We did not scan the entire RYR2 gene in control subjects. Instead, since we focused on the exon-containing mutations, the rate of non-synonymous 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 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 phenotype22,23; polymorphisms in RYR2 may have the same potential. RyR2-Q2958R is the most common RYR2 polymorphism; was described for the first time 9 years ago24 and is particularly common in 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 abolished25. The clinical consequences of this “RyR2 loss of function” in vitro phenotype is not clear, however, compound heterozygosity involving these two polymorphisms has been reported in right ventricular dysplasia26. 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 on this study have been functionally characterized to further bolster the contention of pathogenicity. However, less than 15% of the mutations reported to date in RYR2 have been studied in vitro, pathogenicity has been suspected based on co-segregation with the disease and absence in control subjects. Here, co-segregation with the disease data was not available for all cases. Instead, the prevalence of putative mutations amongst strong cases (~60%) was markedly higher than in controls (~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%).
This is the first report involving RYR2 mosaicism which was transmitted to descendants, presumably causing sudden death in two children and full blown CPVT in one child from the age of 9 years. RYR2 mutations, in many circumstances (~20% in our cohort) are de novo in origin, but it could also be present in a mosaic form in the asymptomatic parents, which requires attention during genetic counseling as well as during genetic screening.
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” region with important consequences in “gene negative” definition. The identification of novel common variants in control subjects will allow a better interpretation of the CPVT genetic test and the detection of RYR2 mosaicism and confirmation of exon 3 deletion in different patients-cohort, provide novel genetic possibilities in the pathogenesis of CPVT. Moreover, the possibility 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), two different diseases with a similar clinical presentation but different clinical outcomes and different responsiveness to pharmacotherapy.
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, though 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”.
Michael J. Ackerman and Argelia Medeiros-Domingo are supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death and Leducq Fondation programs. Arthur A. M. Wilde is supported by The Interuniversity Cardiology Institute of the Netherlands (ICIN) project 27 and by a Leducq Fondation program grant “05CVD01, Alliance Against Sudden Cardiac Death.”
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.
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