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Clinical Chemistry 50: 1148-1155, 2004. First published May 6, 2004; 10.1373/clinchem.2003.030734
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(Clinical Chemistry. 2004;50:1148-1155.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Denaturing HPLC-Based Approach for Detecting RYR2 Mutations Involved in Malignant Arrhythmias

Alessia Bagattin1, Caterina Veronese1, Barbara Bauce2, Wim Wuyts3, Luca Settimo4, Andrea Nava2, Alessandra Rampazzo1 and Gian Antonio Danieli1,a

Departments of1 Biology and 2 Cardiology, University of Padua, Padua, Italy. 3 Department of Medical Genetics, University of Antwerp, Antwerp, Belgium. 4 Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland.

aAddress correspondence to this author at: Department of Biology, University of Padua, via Ugo Bassi 58/B, 35131 Padua, Italy. Fax 39-049-8276209; e-mail danieli{at}bio.unipd.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Mutations in the RYR2 gene, which encodes the cardiac ryanodine receptor, have been reported in patients showing either arrhythmogenic right ventricular cardiomyopathy, type 2, or stress-induced polymorphic ventricular tachycardia. Both clinical phenotypes are characterized by a high risk of sudden death. Detection of RYR2 mutations is particularly important because beta-blocker treatment has been shown to be effective in preventing fatal arrhythmias in affected patients.

Methods: We used denaturing HPLC (DHPLC) to identify mutations in the human RYR2 gene. Fifty-three single exons, possibly targeted by mutations, were identified by comparison with the distribution of pathogenic mutations of the RYR1 gene, the skeletal muscle counterpart of RYR2. PCR primers for amplification of the entire coding sequence (116 amplicons, corresponding to 105 exons) were tested, and optimal DHPLC conditions were established. DHPLC analysis of critical exons was performed on 22 unrelated patients with effort-induced polymorphic ventricular arrhythmias but lacking a precise diagnosis.

Results: We identified four novel missense mutations among 22 patients. Their pathogenic role was related to present knowledge of the structure and function of RyR2 protein.

Conclusions: Under optimized conditions, DHPLC is a cost-effective, highly sensitive, rapid, and efficient method for mutation screenings. A four-step approach is proposed for mutation screening of the RYR2 gene: (a) DHPLC analysis of 48 critical exons (2–4, 6–15, 17–20, 39–49, 83, 84, 87–97, and 99–105); (b) DNA sequencing of 5 critical exons unsuitable for DHPLC; then, in case of negative results, (c) DHPLC analysis of the remaining 39 exons and (d) DNA sequencing of the last 13 amplicons unsuitable for DHPLC analysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ryanodine receptors are calcium-release channels located at the endoplasmic reticulum compartment. Three different isoforms of ryanodine receptors are known: ryanodine receptor type 1 (RyR1;1 OMIM 180901) and type 2 (RyR2; OMIM 180902) are produced in skeletal and cardiac muscle, respectively, whereas ryanodine receptor type 3 (RyR3; OMIM 180903) is produced in a wide range of tissue. In muscles, ryanodine receptors (RyR channels) play a fundamental role in excitation-contraction coupling.

RyRs share a common molecular morphology, with a "foot" structure bridging the junctional gap between sarcoplasmic reticulum and plasma membrane (1). RyR channels are homotetramers composed by four RyR polypeptides, associated with four FK506-binding proteins (FKBP12 for RyR1 and FKBP12.6 for RyR2), needed for normal gating (activation/inactivation) of the channel (2)(3).

RYR2 is one of the largest human genes, including 105 exons and encoding a mRNA of ~15 Kb (4). Mutations in the human RYR2 gene have been associated with two inherited cardiac diseases: arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2; OMIM 600996) and stress-induced polymorphic ventricular tachycardia [ventricular tachycardia, stress-induced, polymorphic (VTSIP); OMIM 604772] (5)(6)(7)(8)(9)(10). Both diseases are characterized by effort-induced polymorphic ventricular arrhythmias and a high risk of sudden death. Moreover, it was recently suggested that RYR2 mutations might be involved in atypical forms of long-QT syndrome (11). Detection of RYR2 mutations is particularly important because beta-blocker treatment has been shown to be beneficial in preventing fatal arrhythmias in asymptomatic carriers (6). We report here on a novel protocol for denaturing HPLC (DHPLC) mutation screening of the human RYR2 gene, based on clustering of known mutations along the RYR1 and RYR2 genes. DHPLC is a highly sensitive, specific, and automated method based on differences in retention times between hetero- and homoduplexes of mutated and wild-type DNA fragments in ion-pair reversed-phase chromatography under partially denaturing conditions (12)(13). The protocol described here allowed us to identify four novel RYR2 missense mutations in a set of 22 patients with effort-induced polymorphic ventricular arrhythmias.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
patients and controls
Index patients with effort-induced polymorphic ventricular arrhythmias were diagnosed at the Department of Cardiology of the University of Padua (Italy). An additional patient, diagnosed at the Department of Medical Genetics, University of Antwerp (Belgium), was also included in the present study. Blood samples were collected after informed consent. Genomic DNA was extracted from blood samples by a salting-out procedure. DNA samples from six patients with a previously identified RYR2 missense mutation were used as positive controls to verify the efficacy of the DHPLC mutation detection system. These mutations had been detected in our laboratory by single-strand conformation polymorphism analysis or direct sequencing (5)(6). PCR amplicons from DNA samples from healthy individuals were used to check the DHPLC working conditions.

pcr amplifications
PCR amplifications were performed in a final volume of 50 µL, containing 100 ng of genomic DNA, 1x PCR buffer II (Applied Biosystems), 1.5 mM MgCl2 (Applied Biosystems), 800 nM each primer (Sigma Genosys), 100 µM deoxynucleotide triphosphates (Invitrogen), and 1.25 U of Taq DNA polymerase (Promega).

To amplify exon 1, we added 1 µL (10% final concentration) of dimethyl sulfoxide to 10 µL of the reaction mixture. Cycling conditions (denaturation at 94 °C for 1 min, annealing at the working temperature for 1 min, and extension at 72 °C for 1 min) were repeated 35 times. All PCR reactions were performed on a PCR Express thermocycler (Hybaid).

dhplc analysis
DHPLC analysis was performed on a Transgenomic Wave DNA Fragment Analysis System (Model 3500HT; Transgenomic) with a DNASep column (Transgenomic). PCR fragments were denatured for 5 min at 95 °C and then left to reanneal slowly at room temperature to promote the formation of heteroduplexes.

Separation was performed at a flow rate of 0.9 mL/min, and the acetonitrile gradient was adjusted to elute the amplicon between 3 and 5 min. The gradient was obtained by mixing buffer A (0.1 mol/L triethylamine acetate, pH 7.0; Transgenomic) and buffer B (0.1 mol/L triethylamine acetate, pH 7.0, containing 250 mL/L acetonitrile; Transgenomic). The increase in buffer B was 2%/min. Column temperatures were calculated by NAVIGATOR software (Transgenomic). Whenever fragments showed distinct melting domains, additional analyzing temperatures were used. Heterozygous control probes (Sizing Standards; Transgenomic) were used to confirm system performance and the accuracy of the oven temperature. After each run, the column was cleaned with 750 mL/L acetonitrile and equilibrated for 0.1 min to restore optimal operating conditions.

dna sequencing
PCR amplicons were purified (PCR Product Pre-Sequencing Kit; USB) and sequenced by an ABI 3700 DNA sequencer (Applied Biosystems) with the BIG DYE dideoxy-terminator chemistry (Applied Biosystems). CHROMAS software (release 1.5; Technelsium) and the LASERGENE software package (DNASTAR) were used to edit, assemble, and translate sequences. Amplicons showing putative mutations were resequenced, with the product of an independent PCR reaction used as template.

controls
Whenever a putative mutation was detected, a search was performed on a single-nucleotide polymorphism (SNP) database (http://www.ncbi.nlm.nih.gov/SNP/snpblastByChr.html). In case of negative results, we performed DHPLC analysis on 120 individual samples of genomic DNA (240 chromosomes) from unrelated healthy control individuals to exclude DNA polymorphisms.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The PCR primers for amplification of the RYR2 coding regions have been published previously (5). Some amplicons, once tested for DHPLC by NAVIGATOR software, showed suboptimal melting profiles. Therefore, novel PCR primers were designed to amplify exons 3, 16, 37 (fragment 37a), 41, 53, 59, 63, 65, 67, 94, 96, and 99. The sequences of the PCR primers (116 pairs) designed for mutation screening of the entire coding sequence of RYR2 and the PCR and DHPLC conditions are given in Table 1 of the Data Supplement available with the online version of this article (http://www.clinchem.org/content/vol50/issue7/) and at http://telethon.bio.unipd.it/ARVDnet/molgen_arvd2.html.

As a first attempt, we performed DHPLC analyses on each RYR2 fragment at the melting temperatures suggested by NAVIGATOR. Only 19 amplicons (16%) gave a melting profile suitable for single-temperature analysis; most RYR2 PCR amplicons showed different melting domains within the same sequence. Thus, DHPLC conditions were set up for each amplicon by varying the temperature conditions (see Table 1 in the online Data Supplement). Eighteen fragments (1, 5, 16, 37d, 53, 54, 56, 64, 71, 73, 74, 76, 78, 80, 82, 85, 86, and 98, corresponding to 15% of the total amplicons) were found to be unsuitable for DHPLC analysis because of polyT stretches or domains with extremely different melting temperatures.

Once conditions for DHPLC analysis of 98 RYR2 fragments were established, we analyzed positive control amplicons carrying known RYR2 mutations. All such mutations produced abnormal DHPLC profiles.

Mutations associated with ARVD2 or VTSIP (5)(6)(7)(8)(9)(10) show clustering in three regions (Fig. 1 ), which roughly correspond to the RYR1 regions targeted by mutations causing malignant hyperthermia (MHS1; OMIM 145600) or central core disease (CCD; OMIM 117000) (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Therefore, a mutation screening aiming at the identification of pathogenic mutations associated with malignant arrhythmias should involve at least 53 exons (exons 2–20, 39–49, and 83–105). Because amplicons 5, 16, 85, 86, and 98 are unsuitable for DHPLC analysis, mutation screening of these five exons must be performed by direct DNA sequencing.



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  		Figure 1. Distribution of mutations along the RyR2 and RyR1 monomers.

The novel mutations reported in this study are indicated by arrows. The ARVD2 and VTSIP mutations of RyR2 cluster in three regions, where CCD and MH mutations of RyR1 are also clustered. The FKBP-interacting domains of RyRs include residues 305-1937 and 2361–2496 of RyR2 and residues 2407–2520 of RyR1. The RyR2 critical regions considered in this study are boxed. RyR1 and RyR2 monomers are displayed according to amino acid sequence alignment (NP_000531.1 for RyR1 and NP_001026.1 for RyR2).

To assess the sensitivity of DHPLC for detecting RYR2 mutations, we compared results from DHPLC analysis with direct sequencing of DNA samples from four patients with effort-induced polymorphic ventricular arrhythmias.

Mutation screening in such patients was performed on 54 amplicons, corresponding to RYR2 exons 2–4, 6–15, 17–20, 39–49, 83, 84, 87–97, and 99–105, which were selected based on being suitable for DHPLC analysis as well as "critical" (because of previously reported pathogenic mutations). Samples showing abnormal or extra peaks underwent DNA sequencing.

DHPLC analysis performed on 48 of the exons (for a total of 216 assays) detected 21 samples showing extra peaks, 4 of which were found to be false positives. No false negatives were detected. According to these data, the specificity of DHPLC was 98%, and the sensitivity was 100%. DNA sequencing revealed eight single-nucleotide substitutions, whereas DHPLC analysis produced six different elution profiles showing extra peaks. Every sequence alteration revealed by DNA sequencing was also detected by DHPLC. In a single case (amplicon 95), extra peaks revealed by DHPLC were found by DNA sequencing to correspond to three different SNPs in the same amplicon (see Table 1 ). Subsequently, we screened for RYR2 mutations (in the 48 critical exons) DNA samples from 18 patients for whom a precise diagnosis had not been obtained, but who had effort-induced polymorphic ventricular arrhythmias as a main symptom. The screening was performed by DHPLC, and 15 different abnormal DHPLC elution profiles were detected (for examples, see Fig. 2 ). Subsequent DNA sequencing confirmed the presence of single-nucleotide substitutions. In six cases, the three SNPs previously identified in amplicon 95 were detected again. Additional SNPs were detected in amplicon 92: SNPs 13476 + 16G>A and 13476 + 47G>A were found in five and two patients, respectively, whereas seven patients had both SNPs (see Table 1 ). Only four variants (Ala2387Pro, Met4504Ile, Ala4607Pro, and Val4880Ala) appeared to be novel missense mutations with a putative pathogenic effect and involved highly conserved amino acids. Mutation Ala2387Pro is located in the FKBP12.6-binding domain (30), whereas mutations Met4504Ile, Ala4607Pro, and Val4880Ala map to the C-terminal region of the RyR2 protein (Fig. 3 ) and play a role in the formation of the transmembrane domain (4). Because Ala4607 is located in an {alpha}-helix, as predicted by PSIPRED (31), mutation to Pro most likely destabilizes the {alpha}-helix.


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  		Table 1. Mutations and SNPs identified in the present study in 48 critical exons of the RYR2 gene (GenBank accession no. NM_001035.1).



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  		Figure 2. DHPLC elution profiles of some heterozygous sequence alterations in the RYR2 gene.

(A), different profiles of RYR2 amplicon 92, eluted at 56.3 °C with 54.7% buffer B. (B), different profiles of RYR2 amplicon 95, eluted at 55.5 °C with 55.9% buffer B. X axis, elution time of the DNA fragments; y axis, absorbance of DNA fragments at 260 nm



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  		Figure 3. Disease regions of RyR2.

(A), schematic representation of human cardiac RyR monomer sequence. (B), three-dimensional surface representation of RyR2 tetramer, top view. (C), three-dimensional surface representation of RyR2 tetramer, side view. D1, D2, and D3, highly variable regions among RyR1, RyR2, and RyR3. Segments I, II, and III, RyR2 regions corresponding to RyR1 regions where mutations are clustered (residues 35–614, 2117–2458, and 4200–4937). RyR2 segment I (residues 35–614 in RyR1) includes residues 176–433 in RyR2 (disease region I); RyR2 segment II (disease region II) includes part of region 2246–2504 and corresponds to residues 2117–2458 in RyR1. RyR2 segment III (disease region III) includes part of region 3778–4959 and corresponds to residues 4200–4937 in RyR1. I*, not yet verified experimentally; SR, sarcoplasmic reticulum. The drawing of RyR three-dimensional surface representation, originally referring to RyR1, was kindly provided by Prof. T. Wagenknecht and was adapted to RyR2 with his permission. RyR1, RyR2, and RyR3 are sufficiently similar to use the same structure for correlations between isoforms. The diagram in A is modified, with permission from Blackwell Publishing, from Fig. 3 in Stokes DL, Wagenknecht T. Calcium transport across the sarcoplasmic reticulum: structure and function of Ca2+-ATPase and the ryanodine receptor (Eur J Biochem 2000; 267:5274–9). The structure in B is modified, with permission from the American Society for Biochemistry and Molecular Biology, from Fig. 8A in Liu Z, Zhang J, Li P, Chen SR, Wagenknecht T. Three-dimensional reconstruction of the recombinant type 2 ryanodine receptor and localization of its divergent region 1 (J Biol Chem 277:46712–9).

Mutations Ala2387Pro and Met4504Ile were found in two young boys who showed effort-induced polymorphic ventricular arrhythmias during testing to obtain a sports certificate; currently, these patients are being treated successfully with beta-blockers. Screening of relatives revealed that the Ala2387Pro mutation is carried by the mother and sister of the one proband, whereas the Met4504Ile mutation was detected in the DNA of both the other proband and his father; the father, however, did not show polymorphic ventricular arrhythmia during a stress test.

The Ala4607Pro mutation was found in a patient who experienced a syncope during sports activity at the age of 15 years. At age 34 he showed effort-induced polymorphic ventricular arrhythmias during a stress test. He was therefore placed on beta-blocker therapy, which was effective. The Val4880Ala mutation was detected in a Belgian patient, who since childhood had repeatedly shown stress-induced syncope. At the age of 17, he started treatment with beta-blockers, which was effective. Segregation of the Ala4607Pro and Val4880Ala mutations with the disease could not be assessed in the families of these probands because DNA samples from the relatives were not available for study.

Exons 5, 16, 85, 86, and 98, which could not be analyzed by DHPLC, were screened by direct DNA sequencing. No putative pathogenic mutations were detected; only a SNP in amplicon 16 (1612 + 14T>C) was identified.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RYR2 mutations have been reported to be associated with effort-induced polymorphic ventricular arrhythmias, syncope, and sudden death (5)(6)(7)(8)(9)(10). Recently, putative pathogenic mutations in RYR2 have also been reported in 20 of 240 patients with long-QT syndrome (11).

All RyR2 mutations described to date show clustering in three specific domains: the N-terminal amino acid residues 176–433, the centrally located residues 2246–2504, and the C-terminal residues 3778–4959. The central portion of RyR2 (residues 2361–2496) has been reported to interact with the regulatory protein FKBP12.6 (30). Other studies have demonstrated that the RyR2-FKBP12.6 interaction is conformation-dependent and that a major binding site for FKBP12.6 is possibly located in the region between residues 305 and 1937 (32); therefore, mutations in this region would affect interaction of RyR2 with FKBP12.6, which stabilizes the RyR2 channel (33).

The last 500 amino acids (C-terminal region) correspond to a region including the transmembrane segments (4), which are important for the structure of the channel. Mutations in the C-terminal region would probably affect stability and gating of the channel.

Functional studies demonstrated that RyR2 mutations associated with catecholaminergic polymorphic ventricular tachycardia increase calcium release under exercise (34)(35). Moreover, it was suggested that the binding of RyR2 to its gating protein, FKBP12.6, could be differently affected by VTSIP- and ARVD2-associated point mutations (36).

For individuals carrying a pathogenic RYR2 mutation, sudden death is a real risk; fortunately, beta-blocker therapy is effective in most cases (6). Therefore, early identification of individuals carrying RYR2 mutations is extremely important.

The comparison between DNA sequencing and DHPLC analysis reported in the present study shows that DHPLC has 100% sensitivity and ~98% specificity. Unfortunately, DHPLC can be successfully applied to only 48 of 53 critical RYR2 exons because of unfavorable nucleotide composition in some amplicons. Nevertheless, DHPLC analysis of 48 critical exons appears to be cost-effective compared with DNA sequencing. Thus, although DHPLC-based analyses require a special and expensive instrument, the starting expense for such an instrument is rapidly balanced in diagnostic applications by a mutation-screening approach that is much faster and less expensive than direct DNA sequencing.

The data on PCR and DHPLC conditions reported here may be very helpful for other researchers using identical or similar DHPLC instrumentation, now available in many hospital laboratories. After the analytical conditions are established, rapid processing of numerous samples would be possible.

In the present study, performed on 22 isolated individuals with effort-induced polymorphic ventricular arrhythmias, we detected four putative pathogenic RYR2 mutations among 48 critical RYR2 exons suitable for DHPLC analysis. Such a low percentage of positives might be explained by genetic heterogeneity. Patients selected for the study had effort-induced polymorphic ventricular arrhythmias; this symptom was recently also found to be associated with mutations in the cardiac calsequestrin gene (CASQ2; OMIM 114251) (37). Therefore, the possibility that some patients may carry mutations in other genes involved in intracellular calcium homeostasis cannot be excluded. Moreover, the presence of phenocopies cannot be ruled out.

At present, no conclusive evidence on the physiologic role of the detected mutations is available because no functional studies for these specific variants have been reported to date. In two families, cosegregation with the disease could not be assessed because of a lack of available DNA samples. However, all four novel RYR2 mutations reported here involve highly conserved amino acids and map to functionally important regions of the channel, such as the FKBP12.6-binding domain and the transmembrane region. This strongly suggests their potential pathogenic effect.

Thirteen SNPs were detected by DHPLC in RYR2 amplicons from patients considered in this study. It is noteworthy that DHPLC was able to detect different nucleotide variations within the same fragment, as shown in Fig. 2Up . Hence, it may be possible to create a database of DHPLC profiles corresponding to known and frequent SNPs, facilitating the interpretation of abnormal DHPLC profiles and avoiding additional DNA sequencing.

As shown in Fig. 1Up , all ARVD2, VTSIP, MH, and CCD mutations cluster in similar regions of RyR1 and RyR2. The only mutation identified outside these regions to date is a missense mutation (Pro3527Ser) associated with a recessive form of CCD (19). This suggests that such "disease regions" may play a fundamental role in channel stability and function (Fig. 3Up ). In particular, disease regions I and II are likely to interact and undergo changes in juxtaposition during channel gating (38). Moreover, it has been suggested that mutations in disease region I of RyR1 may affect conformational changes associated with channel gating (39).

On the other hand, regions of RyR2 encoded by noncritical exons also include amino acid residues that are probably relevant for the properties of the channel, such as the N-terminal FKBP12.6-interacting domain (32), a calmodulin-binding site (40), ATP- and calcium-binding sites (4), binding domains for protein phosphatases PP1 and PP2A, and a site for protein kinase A phosphorylation (30). Therefore, in patients with effort-induced polymorphic ventricular arrhythmias, the entire RYR2 gene should be screened for mutations. DHPLC appears to be a cost-effective, highly sensitive, rapid, and efficient method for such screening. We propose a four-step approach for analysis of RYR2: (a) DHPLC analysis of the 48 critical exons (2–4, 6–15, 17–20, 39–49, 83, 84, 87–97, and 99–105); (b) DNA sequencing of critical exons 5, 16, 85, 86, and 98; then, in case of negative results, (c) DHPLC analysis of the remaining 39 exons and (d) DNA sequencing of the last 13 amplicons (1, 37d, 53, 54, 56, 64, 71, 73, 74, 76, 78, 80, and 82). In total, all individual DHPLC analyses, run at different temperatures, plus individual DNA sequencing reactions needed to screen the entire RYR2 coding sequence, would cost less than US $300, which appears to be a reasonable price for the analysis of such a large and relevant gene.


   Acknowledgments
 
We are indebted to Prof. T. Wagenknecht for comments and for permission to use the three-dimensional image of the RyR structure shown in Fig. 3Up . This publication is based on the project performed pursuant to Baylor College of Medicine Grant 1 U01 HL 65652 from the NIH. This work was also supported by TELETHON, Italy (Grant 1288 to G.A.D.), MURST 40%, ARVC EC Contract QLG1-CT-2000-01091, and Fondazione Cassa di Risparmio, Padova e Rovigo, Italy. A.B. and B.B. are recipients of a temporary position pursuant to ARVC EC Contract QLG1-CT-2000-01091.


   Footnotes
 
1 Nonstandard abbreviations: RyR1, RyR2, and RyR3, ryanodine receptor type 1, 2, and 3; FKBP12.6, FK506-binding protein 12.6; ARVD2, arrhythmogenic right ventricular cardiomyopathy, type 2; VTSIP, ventricular tachycardia, stress-induced, polymorphic; DHPLC, denaturing HPLC; SNP, single-nucleotide polymorphism; MH, malignant hyperthermia; and CCD, central core disease.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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