Exploration Of Association Between RYR2Gene Polymorphism At The G1885E And G1886S Sites With Unexplained Sudden Cardiac Death

Background: Ca²⁺plays an important role in the cardiac excitement-contraction coupling（ECC）which maintains a normal heart rhythm. Cardiac ryanodine receptors（RyR2）encoded by human RYR2gene（hRyR2） is an ion channel that allow the release of Ca²⁺from the sarcoplasmic reticulum，also，it’s the largest ion channel（²300kD） known todate and a major challenges for structural biology. When RyR2gene suffered from mutationas well as other abnormal regulation, RyR2located on the sarcoplasmic reticulum ofcardiac myocyte would have a spontaneous Ca²⁺leak which formed abnormal Ca²⁺homeostasis and arrhythmia. Extensive research has showed that mutations in RYR2genehad correlation with Arrhythmogenic right ventricular cardiomyopathy（ARVC） andCatecholaminergic polymorphic ventricular tachycardia（CPVT）. However, patients withCPVT or ARVC often had no obvious clinical signs and symptoms, with about50%patients developing syncope and sudden death as initial complaint. They are ease to causecivil disputes or misunderstandings, thus autopsy is often needed to confirm the cause ofdeath. But, in most cases, heart had no obvious morphological abnormalities, such caseswere often referred to as the unexplained sudden cardiac death （Sudden unexplained caidiacdeath, SUD）. SUD is one of the difficulties that plagued forensic pathology research.Therefore looking for a molecular pathology diagnostic methods to determine the cause of death of SUD will be not only important for the diagnosis and clinical prevention but alsoin the field of forensic pathology.Purpose: We examined two reported sites （G1885E and G1886S of RYR2gene） which hadan important regulatory role on the functional state of RyR2on single nucleotidepolymorphisms（SNPs） in unrelated healthy Chinese Han population（n=73） and in a cohortof29sudden unexplained cardiac death（SUD） victims, and analyzed the relationshipbetween the two sites and SUD. We did this in order to provide the basis for diagnosis ofthe cause of SUD cases at the molecular genetic level, to provide a reference for clinicalSUD prevention.Materials and methods: All samples were came from HuBei TongJi Forensicidentification center between the years of2005to2011, we selected healthy controlgroup（control group, n=78） and SUD group（experimental group, n=34） in strict accordancewith standard requirements, and then extracted genomic DNA of the control and SUDgroup by whole blood genomic DNA extraction kit and FFPE DNA kit respectively. Thisstudy selected G1885E and G1886S sites of RYR2gene as the research objects anddesigned specific primers which included these two sites by primer primer5.0software.PCR amplified the objective fragments of genomic DNA. Purified PCR products weresequenced, then the objective sites were genotyped and analyzed by NCBI-BLASTsoftware and the corresponded landing seauence in database. SPSS17.0software andHard-weinberg V2.0were adopted to analyze the statistics mentioned above.Results:（1）The unrelated healthy control group（n=78） and SUD group（n=34） weresequenced, each group having5samples with weak sequencing signals, i.e.73unrelatedhealthy control group samples and29SUD group samples were sequenced successfully.（2）Heterozygous mutation（AG） of G1886S site was found in both control group（n=1,1.37%） and SUD group（n=4,13.79%）, and there also had a homozygous mutation（AA） inSUD group（n=1,3.45%）. The wild genotype（GG） numbers for G1886S site of SUD andhealthy groups were24（13.79%） and72（98.63%） respectively. In SUD group, the WT “G” allele frequency for G1886S site was0.897, and the mutant “A” allele frequency was0.103.In healthy group, the WT “G” allele frequency for G1886S site was0.993, and the mutant“A” allele frequency was0.007. SPSS17.0software was adopted to analyze the statisticsabove-mentioned, it showed that regardless of the genotype（χ2=48.30, P<0.05） or genefrequencies（χ2=11.69, P<0.05）, the two groups had significant differences on G1886S site.（3） Conversely, DNA sequencing results showed that either heterozygous or homozygousmutation of G1885E site was found in both groups, i.e. every sample showed wild-type（WT）and no SNP was found at this site. In SUD group, the WT “G” allele frequency for G1885Esite was1, and the mutant “A” allele frequency was0. In healthy group, the WT “G” allelefrequency for G1885E site was1, and the mutant “A” allele frequency was0. Statisticalresults suggested that the two groups had no significant differences on G1885E site.Conclusion:（1） The results of this study indicated that polymorphism was found atG1886S site of RYR2gene in Chinese Han population, and the findings of our studysuggested that gene mutation of this site had a close relationship with SUD, as well as WT“G” allele may be a protective factor for SUD while the mutant “A” allele may be a riskfactor for SUD.（2）Contrary to the result of G1886S site, no polymorphism was found atthe G1885E site of RYR2gene in Chinese Han population. Simultaneously this experimentdid not find a correlation between this site and SUD.