Denaturing HPLC-based approach for detecting RYR2 mutations involved in malignant arrhythmias.
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.
Bagattin
Alessia
A
Department of Biology, University of Padua, Padua, Italy.
Veronese
Caterina
C
Bauce
Barbara
B
Wuyts
Wim
W
Settimo
Luca
L
Nava
Andrea
A
Rampazzo
Alessandra
A
Danieli
Gian Antonio
GA
D000293:Adolescent; D000328:Adult; D001145:Arrhythmias, Cardiac / Q000235:genetics*; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008297:Male; D009154:Mutation; D020125:Mutation, Missense; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D012680:Sensitivity and Specificity
D019837:Ryanodine Receptor Calcium Release Channel