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 syndrome: a comprehensive open reading frame mutational analysis

Abstract

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 approximately 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.

Figure 1. Mutation clustering in the cardiac ryanodine receptor (RyR2)

Mutations are distributed in three “hot-spots” regions, called domains I (N-terminal), II (central) and III (channel region),. AA: amino-acid number estimated for each domain. Adapted from George CH, et.al., and Yano M, et. al.

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 on this study-cohort are shown in the approximate location. The number within the circle corresponds to the mutation # on Table 1.

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 side show the sensitivity in the 3 different subgroups of this cohort.

Figure 4. Possible tiered strategy for reflex genetic testing

Schematic representation of the 105 coding exons of the RYR2 gene. Boxes in colors: all the exon-containing mutations reported to date. Boxes in white: exons free of reported mutations. The tiered strategy was built based on the number of mutations containing in each exon as shown by three different colors. The 1^st tier included 16 exons, 2^nd tier 13 exons and 3^rd tier 16 exons. Exons containing control variants were not included.

Figure 5. Yield from RYR2 mutational analysis based on a tiered strategy

Retrospective analysis of the mutations detected in our cohort and in the world-wide compendium of mutations reported to date. The percentage of mutations that would be detected using the tired strategy is shown.