Forensic Science International 235 (2014) 14–18
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Postmortem genetic screening of SNPs in RyR2 gene in sudden unexplained nocturnal death syndrome in the southern Chinese Han population Lei Huang a,1, Chao Liu b,1, Shuangbo Tang a,1, Terry Su a, Jianding Cheng a,* a b
Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Guangzhou Institute of Forensic Science, Guangzhou 510030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2013 Received in revised form 3 October 2013 Accepted 6 December 2013 Available online 16 December 2013
To investigate the genetic variants of the RyR2 gene in sudden unexplained nocturnal death syndrome (SUNDS) in the southern Chinese Han population, we genetically screened 29 of the 105 coding exons of the RyR2 gene associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC) in sporadic SUNDS victims using polymerase chain reaction (PCR) and direct sequencing methods. Genomic DNA was extracted from blood samples of 127 SUNDS cases and 165 healthy unrelated controls. None of the published or novel RyR2 missense mutations were found in 127 SUNDS cases. A total of sixteen genetic variants of the RyR2 gene were identified, comprised of: one novel synonymous coding mutation (c.13710C>A), one novel synonymous rare polymorphism (c.14871C>T), and fourteen previously reported polymorphisms. The genotype and allele frequency of previously reported missense polymorphism c.5656G>A (G1886S) was of no statistical difference between SUNDS cases and controls (x2 = 0.390, P > 0.05; x2 = 0.271, P > 0.05). This is the first report of genetic phenotype of RyR2 gene of SUNDS in the southern Chinese Han population. Previously reported plausible pathogenic missense polymorphism G1886S may not be an independent predisposition factor of SUNDS in the southern Chinese Han population. The association of genetic variants of the RyR2 gene with SUNDS needs further elucidation. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: The ryanodine receptor 2 (RyR2) gene Calcium release channel Sudden unexplained nocturnal death syndrome Single nucleotide polymorphism Cardiac arrhythmia
1. Introduction Sudden unexplained nocturnal death syndrome (SUNDS), also called sudden unexplained death during sleep (SUDS), predominantly prevails in Southeast Asia [1] and has different countryspecific synonyms, such as ‘‘bangungut’’ (Philippines) [2], ‘‘lai-tai’’ (Thailand) [1], ‘‘pokkuri’’ (Japan) [3], and ‘‘sudden manhood death syndrome’’ (China) [4]. The annual incidence of SUNDS has been reported to be as high as 43 per 100,000 people aged 20–40 years in the Philippines [5] and 38 per 100,000 people aged 20–49 years in Thailand [6]. In southern China, the annual incidence is about 1 per 100,000 people [4]. These syndromes have some common phenotypes: (1) the majority of deceased were apparently healthy young males; (2) death occurred at night during sleep with symptoms of moaning, tachypnea, and abrupt tic of limbs; and (3)
* Corresponding author at: Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, No. 74, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. Tel.: +86 20 87330704; fax: +86 20 87334353. E-mail address: [email protected] (J. Cheng). 1 These authors contributed equally to this work. 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.12.007
standard forensic autopsy, histopathology, and toxicology showed no morphological changes to reveal the cause of death. Potentially lethal and inheritable arrhythmias, such as congenital Long-QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular cardiomyopathy (ARVC) were considered to play an important role in the pathogenesis of sudden cardiac death in the young [7]. Most recently, we have already performed postmortem molecular autopsy on the potentially lethal and inherited cardiac arrhythmia-associated SCN5A, KCNQ1, KCNH2, KCNE1, and KCNE2 genes in sporadic cases of SUNDS in the southern Chinese Han population and have revealed that part SUNDS cases may be caused by inherited cardiac arrhythmia, such as LQTS and BrS [4,8]. The cardiac sarcoplasmic reticulum (SR) calcium release channel, ryanodine receptor 2 (RyR2), is extremely vital to myocardial excitation–contraction coupling (ECC) [9]. The RyR2 is located in the membrane of the SR and is responsible for the regulation of calcium release in the SR. Its function is regulated by several modulatory proteins such as triadin, junction, PKA, CASQ2, and FK506-binding-protein (FKBP12.6) [10]. Under normal conditions, depolarization triggers a minute amount of calcium influx
L. Huang et al. / Forensic Science International 235 (2014) 14–18
through the L-type calcium channels. This activates the RyR2 in the membrane of the SR, and results in a large amount of calcium release from the SR to the cytosol, through the RyR2, to initiate myocardial contraction. This process is known as calcium-induced calcium release (CICR) [9]. In addition to CICR, calcium can spontaneously be released from the SR to the cytosol, through the RyR2, under calcium overload conditions. This process is termed store-overload-induced calcium release (SOICR) [11]. It is of no doubt that dysregulation of the RyR2 can lead to severe and potentially lethal cardiac arrhythmias. Indeed, mutations in the RyR2 gene have previously been reported to be associated with CPVT [12] and ARVC [13]. Both CPVT and ARVC are severe cardiac arrhythmias that can result in sudden death [14,15]. In this study, we tested the hypothesis that genetic variants in the RyR2 gene may contribute to some SUNDS victims, in a case–control study. 2. Materials and methods 2.1. Study population In this study, 127 sporadic SUNDS cases (ages from 18 to 52 years) were diagnosed or collected by the Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, from 2005 to 2012. The inclusion criteria for SUNDS were as follows [4]: (1) a southern Chinese Han male greater than or equal to 18 years of age that was; (2) previously healthy without any significant disease; (3) prior to experiencing a sudden unexpected death during sleep; (4) and had a negative standard forensic autopsy, toxicology, histopathology, and death-scene investigation resulting in an unexplained death. As a control, blood samples collected from 165 unrelated apparently healthy southern Chinese Han males (ages from 20 to 57 years) were provided by the First Affiliated Hospital of Sun Yatsen University. None of the control subjects had a history of syncope or cardiovascular disease. All participants or agents gave informed consent and the principles outlined in the Declaration of Helsinki were followed. The project was approved for human study by the ethics committee of Sun Yat-sen University. 2.2. Genomic DNA extraction For the SUNDS cases and healthy unrelated controls, genomic DNA was extracted from blood samples using Maxwell1 16 System DNA Purification Kits (Promega Corporation, Madison, WI, USA). 2.3. PCR and direct sequencing We genetically screened 29 of the 105 coding exons of the RyR2 gene previously reported to be associated with CPVT and ARVC in 127 sporadic cases of SUNDS. The 29 selected coding exons are comprised of exons 3, 8, 10, 12, 14, 15, 37, 41, 44–47, 49, 50, 83, 88– 90, 93–97, 99–103, and 105. These 29 coding exons were selected according to the ‘‘tiered strategy for RyR2 genetic testing’’ [16]. The primer sequences and conditions for polymerase chain reaction
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(PCR) refer to Tiso et al. [13] and Larsen et al. [17]. The coding regions, exon–intron boundaries, and 30 UTR of the RyR2 gene were amplified by PCR and then run on 3730 Genetic Analyzers (Applied Biosystems, Foster City, CA, USA) using BigDye1 Terminator Cycle Sequencing Kits version 3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The primers for direct sequencing were equal to the primers for PCR. 2.4. Bioinformatics analyses The data obtained from direct sequencing were compared with the corresponding reference cDNA sequence of the RyR2 gene (NM_001035.2) using SeqManTMII sequence analysis software (DNASTAR, Inc., Madison, WI, USA). All suspicious variants were sequenced again using the opposite primers. To assess the genotype and allele frequency of G1886S polymorphism between SUNDS cases and controls, 165 unrelated ethnically matched healthy controls were also sequenced. A standard nomenclature to describe sequence variants has been agreed on http://www.hgvs.org/mutnomen/. To determine whether or not the identified genetic variants were novel mutations, the identified genetic variants were also compared with published data including the inherited arrhythmias database (http://www.fsm.it/cardmoc/), the 1000 Genomes Project (http://www.1000genomes.org/ensembl-browser), the NHLBI Go Exome Sequencing Project (ESP) (http://snp.gs.washington.edu/ EVS), and the database of single nucleotide polymorphism (http:// www.ncbi.nlm.nih.gov/snp/). 2.5. Statistical analysis Statistical analysis was performed by IBM SPSS Statistics 19.0 (IBM Corporation, Armonk, New York, USA) and a P value less than 0.05 was considered to be of statistical significance. Genotype frequencies were calculated for each SNP site by the genotype counting method. Genotype frequencies of G1886S polymorphism in both of the groups were tested for deviation from the Hardy–Weinberg equilibrium, using a chi-square test. Differences in genotype and allele frequencies between SUNDS cases and healthy controls were also analyzed by chi-square test or Fisher’s exact probability test. The association between G1886S polymorphism and SUNDS risk were estimated by calculating odds ratio (OR) and 95% confidence interval (95% CI) using chi-square test analysis. 3. Results In sporadic cases of SUNDS, sixteen genetic variants in total were identified. Two novel synonymous variants located in exons 94 and 105 were identified (Table 1). The c.13710 C>A was located in exon 94 of the RyR2 mRNA encoding the transmembrane domain of RyR2 and results in the synonymous exchange of a proline residue to a proline residue at amino acid position 4570. The c.14871 C>T was located in exon 105 of the RyR2 mRNA encoding the C terminal of RyR2 and results in the synonymous exchange of a cysteine residue to a cysteine residue at amino acid position 4957. The c.13710 C>A was absent in all of the 165 health controls
Table 1 Novel rare variants of RyR2 gene in SUNDS. Exon
Locia
Cases hosting variant/sample sizeb
Identity in dbSNPc
Amino acid change
Type of variant
Locationd
94 105
c.13710C>A c.14871C>T
1/123 1/124
Novel Novel
p.Pro4570Pro p.Cys4957Cys
Synonymous Synonymous
TMD CT
a b c d
Nucleotide number relative to the translation start site. Due to the quality and quantity limit of the samples, the sample size of each site that can be successfully detected are different. Database of single nucleotide polymorphism. TMD, transmembrane domain; CT, C terminal.
L. Huang et al. / Forensic Science International 235 (2014) 14–18
16 Table 2 Polymorphisms of RyR2 gene in SUNDS. Region
Locia
Homo/Heter/cases hosting variant/sample sizeb
Identity in dbSNPc
Amino acid change
Genotype freq.
Allele freq.
8 15 37 37 45 46 83 90 90 95 95 99 103 105
c.4648A>C c.1359C>T c.5400A>G c.5656G>A c.6906T>C c.7115+58T>C c.1132623C>T c.1196311T>C c.13260+48G>A c.137836A>G c.13913+12A>C c.14298+55A>G c.14756+12C>T c.1480920C>T
15/64/79/113 66/52/118/123 1/16/17/111 1/16/17/111 123/0/123/123 123/0/123/123 87/33/120/125 62/35/97/115 0/5/5/101 111/14/125/125 110/15/125/125 36/60/96/124 0/12/12/122 0/2/2/124
rs10925391 rs3765097 rs3820216 rs3766871 rs707189 rs530109 rs2253831 rs790889 rs77082151 rs790901 rs790900 rs790879 rs2275693 rs14111926
– p.Ser453Ser p.Lys1800Lys p.Gly1886Ser p.Leu2302Leu – – – – – – – – –
AA/AC/CC0.301/0.566/0.133 CC/CT/TT0.041/0.423/0.536 AA/AG/GG0.847/0.144/0.009 GG/GA/AA0.847/0.144/0.009 CC1.000 CC1.000 CC/CT/TT0.040/0.264/0.696 TT/TC/CC0.157/0.304/0.539 GG/GA0.950/0.050 AG/GG0.112/0.888 AC/CC0.120/0.880 AA/AG/GG0.226/0.484/0.290 CC/CT0.902/0.098 CC/CT0.984/0.016
A/C0.584/0.416 C/T0.252/0.748 A/G0.919/0.081 G/A0.919/0.081 C1.000 C1.000 C/T0.172/0.828 T/C0.309/0.691 G/A0.975/0.025 A/G0.056/0.944 A/C0.060/0.940 A/G0.468/0.532 C/T0.951/0.049 C/T0.992/0.008
a
Nucleotide number relative to the translation start site. Homo, homozygote; Heter, heterozygote. Due to the quality and quantity limit of the blood samples, the sample size of each site that can be successfully detected are different. c Database of single nucleotide polymorphism. b
(330 reference alleles). The c.14871 C>T was detected in one of the 165 health controls (330 reference alleles). Fourteen reported polymorphisms were also identified, including ten polymorphisms in intron region, one missense polymorphism, and three synonymous polymorphisms in exon region. The genotype and allele frequencies of all 14 SNPs are as follows (Table 2). The previously published potentially functional variant c.5656G>A (G1886S) was located in exon 37 of the RyR2 mRNA encoding the divergent region 3 (DR3) domain of RyR2 and results in the missense exchange of a glycine residue to a serine residue at amino acid position 1886. The G1886S was present in both SUNDS cases and controls. The genotype distributions of the G1886S and controls were all in the Hardy–Weinberg equilibrium (x2 = 0.119, P value = 0.731; x2 = 1.994, P value = 0.158). The genotype and allele frequency of G1886S was of no statistical difference between SUNDS cases and controls (x2 = 0.390, P value > 0.05; x2 = 0.271, P value > 0.05) (Table 3). 4. Discussion The RyR2 gene is comprised of 105 exons and is one of the largest human genes, encoding an mRNA of approximately 15 kb. Mutations in the RyR2 gene have previously been reported to be associated with CPVT [12] and ARVC [13]. We performed the initial postmortem genetic screening in RyR2 gene in SUNDS in southern Chinese Han population and there were neither published nor novel non-synonymous plausible pathogenic RyR2 mutations found in this cohort of 127 sporadic SUNDS cases. In this study, the c.14871 C>T was detected in one of the 124 SUNDS cases and in one of the 165 health controls (Table 1). Considering the low variant incidence (minor allele frequency, Table 3 The genotype and allele frequencies of G1886S polymorphism. c.5656G>A (G1886S)
SUNDS (n = 111)
Controls (n = 165)
GG GA AA P (x2) G A P (x2) OR (95% CI)
94 (0.847) 16 (0.144) 1 (0.009) 0.859 (0.390) 204 (0.919) 18 (0.081) 0.649 (0.271) 0.851 (0.4641.562)
137 (0.830) 25 (0.152) 3 (0.018) 299 (0.906) 31 (0.094)
OR, odds ratio and CI, confidence interval. Due to the quality and quantity limit of the blood samples, the sample size of each site that can be successfully detected are different.
MAF < 0.01) in SUNDS cases and controls, the c.14871 C>T was regarded as a rare synonymous polymorphism. In addition, the c.13710 C>A was detected in one of the 123 SUNDS cases and absent in the 165 health controls regarding as a synonymous mutation (Table 1). To our knowledge, synonymous mutations do not change the sequence and structure of protein, but may affect mRNA stability and synthesis of the receptor [18]. Thus, the correlation between synonymous mutation and SUNDS cases cannot be ruled out. The c.13710 C>A (Pro4570Pro) may play a critical role in the development of SUNDS by affecting mRNA stability and synthesis of RyR2. This hypothesis requires further elucidation. The previously published genetic variants of G1885E and G1886S were located in exon 37 of the RyR2 mRNA encoding the DR3 domain of RyR2. The DR3 domain includes residues 1852– 1890 of RyR2 that has been reported to be involved in myocardial excitation–contraction coupling (ECC) and in channel regulation by Ca2+, Mg2+, or FK506-binding-protein (FKBP12.6) [19]. Milting et al. [20] identified composite heterozygous polymorphisms G1885E/G1886S, which are associated with ARVC. The G1885E/ G1886S in the DR3 domain cause a modified channel gating with highly increased channel activity at diastolic free calcium concentrations, leading to SR Ca2+ leak. Diastolic Ca2+ release can result in delayed afterdepolarizations (DADs), which can trigger fatal cardiac arrhythmias [21]. The c.5656G>A leading to the amino acid substitution G1886S may create a putative protein kinase C (PKC) phosphorylation site in the human RyR2 [20]. Dysfunction of Ca2+ regulating protein due to an altered phosphorylation contributes to important cardiac diseases such as heart failure [22]. However, Koop et al. [23] found that the phosphorylation site at the position 1886 of RyR2 is unlikely to be a target of any of the tested kinases including PKA, PKC, PKG, and CaMKII. Furthermore, expression of each amino acid substitution G1886S or G1885E caused a significant increase in the cellular Ca2+ oscillation activity compared with that of wild-type RyR2. But, when G1885E/G1886S were introduced in the same RyR2 subunit, the SOICR activity was strongly inhibited. These results indicate that the glycine residues 1885 and 1886 are important for the functional properties of RyR2. Recently, Ran et al. [24] found that the A allele of c.5656G>A leading to the amino acid substitution G1886S in RyR2, not only associates with an increased risk of ventricular arrhythmias in patients with chronic heart failure, but also serves as an independent predictor of sudden cardiac death. In this study, the previously published potentially functional common polymorphism c.5656G>A (G1886S) was present in both SUNDS cases and controls. Meanwhile, the c.5654G>A (G1885E)
L. Huang et al. / Forensic Science International 235 (2014) 14–18
was found neither in SUNDS cases nor in controls. The G1885E/ G1886S genotype was not found, suggesting that composite polymorphisms may play an important role in the development of ARVC [20], but not in SUNDS. The genotype and allele frequency of G1886S was of no statistical difference between SUNDS cases and controls (Table 3), revealing that the G1886S genotype may not be a genetic susceptibility factor in SUNDS of the southern Chinese Han population. In addition to G1886S, thirteen reported polymorphisms were also detected (Table 2). None of these reported polymorphisms resulted in amino acid change and ten of thirteen reported polymorphisms were located in introns. Many transcription factor binding sites are located in the promoter or intron regions of the gene and thus intron variation may affect the level of gene expression and protein translation [25,26]. Thus, the variants in the promoter or intron regions may alter the transcription factor binding sites and affect both RyR2 gene expression and RyR2 formation. This hypothesis also requires further elucidation. According to a recently published study, Tester et al. [27] performed a mutational analysis of 18 exons of the RyR2 gene in 49 cases of sudden unexplained death (SUD) found 7 mutations, revealing the prevalence of mutations was 14%. Furthermore, Larsen et al. [17] genetically screened 29 exons of the RyR2 gene in 74 cases of SUD identified 7 rare variants, revealing the prevalence of rare variants was 9.4%. In our study, the prevalence of rare variants in 29 exons of the RyR2 gene was present in approximately 1% of the examined cases of SUNDS. These results strongly indicated that RyR2 maybe not the key susceptible gene for Chinese SUNDS. Since the discovery of CPVT-causing mutations in the RyR2 gene, a cluster distribution involving three discrete protein regions has been reported [12,16]. On the basis of a potential physiological function, these ‘‘hot-spots’’ regions have been termed domains I, II and III [28,29]. However, because the examined cases of SUNDS have not undergone a scan of the entire RyR2 gene, the prevalence of rare variants residing outside these three domains remains unknown [16]. In addition, the human species may be one of the reasons for the difference of the prevalence of rare variants. Thus, the entire coding exons, promoter, and transcription factor binding sites of the RyR2 gene should be investigated for further comprehensive information of pathogenic mutations. 5. Conclusions This is the first report of genetic phenotype of RyR2 gene of SUNDS in the southern Chinese Han population. Multiple genetic variants of the RyR2 gene were found in SUNDS cases. Association of the multiple genetic variants with SUNDS needs further elucidation. In this study, we genetically screened 29 of the 105 coding exons of the RyR2 gene. For further comprehensive information of pathogenic mutations: (1) the open reading frame (ORF), promoter, and transcription factor binding sites should be investigated; (2) the sample size of SUNDS cases and controls should be enlarged; (3) more candidate genes should be tested, such as Plakophilins2 (PkP2), Connexin43 (Cx43), and CASQ2. For the comprehensive evaluation of SUNDS, clinical information, forensic autopsy records, and molecular autopsy data of SUNDS case should be detailed. Conflict of interest None. Funding This work was supported by the National Natural Science Foundation of China (81172901, 81373238), the Fundamental
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Research Funds for the Central Universities (11ykpy04), the Natural Science Foundation of Guangdong (S2012010009045), and the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAK02B02). Acknowledgment We thank Mr. Danny Andersen for reviewing this manuscript. References [1] K. Nademanee, G. Veerakul, S. Nimmannit, V. Chaowakul, K. Bhuripanyo, K. Likittanasombat, K. Tunsanga, S. Kuasirikul, P. Malasit, S. Tansupasawadikul, P. Tatsanavivat, Arrhythmogenic marker for the sudden unexplained death syndrome in Thai men, Circulation 96 (1997) 2595–2600. [2] A.C. Gaw, B. Lee, G. Gervacio-Domingo, C. Antzelevitch, R. Divinagracia, F.J. Jocano, Unraveling the enigma of Bangungut: is sudden unexplained nocturnal death syndrome (SUNDS) in the Philippines a disease allelic to the Brugada syndrome? Philipp. J. Intern. Med. 49 (2011) 165–176. [3] K. Nakajima, S. Takeichi, Y. Nakajima, M.Q. 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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Postmortem genetic screening of SNPs in RyR2 gene in sudden unexplained nocturnal death syndrome in the southern Chinese Han population Lei Huang a,1, Chao Liu b,1, Shuangbo Tang a,1, Terry Su a, Jianding Cheng a,* a b
Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China Guangzhou Institute of Forensic Science, Guangzhou 510030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 August 2013 Received in revised form 3 October 2013 Accepted 6 December 2013 Available online 16 December 2013
To investigate the genetic variants of the RyR2 gene in sudden unexplained nocturnal death syndrome (SUNDS) in the southern Chinese Han population, we genetically screened 29 of the 105 coding exons of the RyR2 gene associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy (ARVC) in sporadic SUNDS victims using polymerase chain reaction (PCR) and direct sequencing methods. Genomic DNA was extracted from blood samples of 127 SUNDS cases and 165 healthy unrelated controls. None of the published or novel RyR2 missense mutations were found in 127 SUNDS cases. A total of sixteen genetic variants of the RyR2 gene were identified, comprised of: one novel synonymous coding mutation (c.13710C>A), one novel synonymous rare polymorphism (c.14871C>T), and fourteen previously reported polymorphisms. The genotype and allele frequency of previously reported missense polymorphism c.5656G>A (G1886S) was of no statistical difference between SUNDS cases and controls (x2 = 0.390, P > 0.05; x2 = 0.271, P > 0.05). This is the first report of genetic phenotype of RyR2 gene of SUNDS in the southern Chinese Han population. Previously reported plausible pathogenic missense polymorphism G1886S may not be an independent predisposition factor of SUNDS in the southern Chinese Han population. The association of genetic variants of the RyR2 gene with SUNDS needs further elucidation. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: The ryanodine receptor 2 (RyR2) gene Calcium release channel Sudden unexplained nocturnal death syndrome Single nucleotide polymorphism Cardiac arrhythmia
1. Introduction Sudden unexplained nocturnal death syndrome (SUNDS), also called sudden unexplained death during sleep (SUDS), predominantly prevails in Southeast Asia [1] and has different countryspecific synonyms, such as ‘‘bangungut’’ (Philippines) [2], ‘‘lai-tai’’ (Thailand) [1], ‘‘pokkuri’’ (Japan) [3], and ‘‘sudden manhood death syndrome’’ (China) [4]. The annual incidence of SUNDS has been reported to be as high as 43 per 100,000 people aged 20–40 years in the Philippines [5] and 38 per 100,000 people aged 20–49 years in Thailand [6]. In southern China, the annual incidence is about 1 per 100,000 people [4]. These syndromes have some common phenotypes: (1) the majority of deceased were apparently healthy young males; (2) death occurred at night during sleep with symptoms of moaning, tachypnea, and abrupt tic of limbs; and (3)
* Corresponding author at: Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, No. 74, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. Tel.: +86 20 87330704; fax: +86 20 87334353. E-mail address: [email protected] (J. Cheng). 1 These authors contributed equally to this work. 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.12.007
standard forensic autopsy, histopathology, and toxicology showed no morphological changes to reveal the cause of death. Potentially lethal and inheritable arrhythmias, such as congenital Long-QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular cardiomyopathy (ARVC) were considered to play an important role in the pathogenesis of sudden cardiac death in the young [7]. Most recently, we have already performed postmortem molecular autopsy on the potentially lethal and inherited cardiac arrhythmia-associated SCN5A, KCNQ1, KCNH2, KCNE1, and KCNE2 genes in sporadic cases of SUNDS in the southern Chinese Han population and have revealed that part SUNDS cases may be caused by inherited cardiac arrhythmia, such as LQTS and BrS [4,8]. The cardiac sarcoplasmic reticulum (SR) calcium release channel, ryanodine receptor 2 (RyR2), is extremely vital to myocardial excitation–contraction coupling (ECC) [9]. The RyR2 is located in the membrane of the SR and is responsible for the regulation of calcium release in the SR. Its function is regulated by several modulatory proteins such as triadin, junction, PKA, CASQ2, and FK506-binding-protein (FKBP12.6) [10]. Under normal conditions, depolarization triggers a minute amount of calcium influx
L. Huang et al. / Forensic Science International 235 (2014) 14–18
through the L-type calcium channels. This activates the RyR2 in the membrane of the SR, and results in a large amount of calcium release from the SR to the cytosol, through the RyR2, to initiate myocardial contraction. This process is known as calcium-induced calcium release (CICR) [9]. In addition to CICR, calcium can spontaneously be released from the SR to the cytosol, through the RyR2, under calcium overload conditions. This process is termed store-overload-induced calcium release (SOICR) [11]. It is of no doubt that dysregulation of the RyR2 can lead to severe and potentially lethal cardiac arrhythmias. Indeed, mutations in the RyR2 gene have previously been reported to be associated with CPVT [12] and ARVC [13]. Both CPVT and ARVC are severe cardiac arrhythmias that can result in sudden death [14,15]. In this study, we tested the hypothesis that genetic variants in the RyR2 gene may contribute to some SUNDS victims, in a case–control study. 2. Materials and methods 2.1. Study population In this study, 127 sporadic SUNDS cases (ages from 18 to 52 years) were diagnosed or collected by the Department of Forensic Pathology, Zhongshan School of Medicine, Sun Yat-sen University, from 2005 to 2012. The inclusion criteria for SUNDS were as follows [4]: (1) a southern Chinese Han male greater than or equal to 18 years of age that was; (2) previously healthy without any significant disease; (3) prior to experiencing a sudden unexpected death during sleep; (4) and had a negative standard forensic autopsy, toxicology, histopathology, and death-scene investigation resulting in an unexplained death. As a control, blood samples collected from 165 unrelated apparently healthy southern Chinese Han males (ages from 20 to 57 years) were provided by the First Affiliated Hospital of Sun Yatsen University. None of the control subjects had a history of syncope or cardiovascular disease. All participants or agents gave informed consent and the principles outlined in the Declaration of Helsinki were followed. The project was approved for human study by the ethics committee of Sun Yat-sen University. 2.2. Genomic DNA extraction For the SUNDS cases and healthy unrelated controls, genomic DNA was extracted from blood samples using Maxwell1 16 System DNA Purification Kits (Promega Corporation, Madison, WI, USA). 2.3. PCR and direct sequencing We genetically screened 29 of the 105 coding exons of the RyR2 gene previously reported to be associated with CPVT and ARVC in 127 sporadic cases of SUNDS. The 29 selected coding exons are comprised of exons 3, 8, 10, 12, 14, 15, 37, 41, 44–47, 49, 50, 83, 88– 90, 93–97, 99–103, and 105. These 29 coding exons were selected according to the ‘‘tiered strategy for RyR2 genetic testing’’ [16]. The primer sequences and conditions for polymerase chain reaction
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(PCR) refer to Tiso et al. [13] and Larsen et al. [17]. The coding regions, exon–intron boundaries, and 30 UTR of the RyR2 gene were amplified by PCR and then run on 3730 Genetic Analyzers (Applied Biosystems, Foster City, CA, USA) using BigDye1 Terminator Cycle Sequencing Kits version 3.1 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The primers for direct sequencing were equal to the primers for PCR. 2.4. Bioinformatics analyses The data obtained from direct sequencing were compared with the corresponding reference cDNA sequence of the RyR2 gene (NM_001035.2) using SeqManTMII sequence analysis software (DNASTAR, Inc., Madison, WI, USA). All suspicious variants were sequenced again using the opposite primers. To assess the genotype and allele frequency of G1886S polymorphism between SUNDS cases and controls, 165 unrelated ethnically matched healthy controls were also sequenced. A standard nomenclature to describe sequence variants has been agreed on http://www.hgvs.org/mutnomen/. To determine whether or not the identified genetic variants were novel mutations, the identified genetic variants were also compared with published data including the inherited arrhythmias database (http://www.fsm.it/cardmoc/), the 1000 Genomes Project (http://www.1000genomes.org/ensembl-browser), the NHLBI Go Exome Sequencing Project (ESP) (http://snp.gs.washington.edu/ EVS), and the database of single nucleotide polymorphism (http:// www.ncbi.nlm.nih.gov/snp/). 2.5. Statistical analysis Statistical analysis was performed by IBM SPSS Statistics 19.0 (IBM Corporation, Armonk, New York, USA) and a P value less than 0.05 was considered to be of statistical significance. Genotype frequencies were calculated for each SNP site by the genotype counting method. Genotype frequencies of G1886S polymorphism in both of the groups were tested for deviation from the Hardy–Weinberg equilibrium, using a chi-square test. Differences in genotype and allele frequencies between SUNDS cases and healthy controls were also analyzed by chi-square test or Fisher’s exact probability test. The association between G1886S polymorphism and SUNDS risk were estimated by calculating odds ratio (OR) and 95% confidence interval (95% CI) using chi-square test analysis. 3. Results In sporadic cases of SUNDS, sixteen genetic variants in total were identified. Two novel synonymous variants located in exons 94 and 105 were identified (Table 1). The c.13710 C>A was located in exon 94 of the RyR2 mRNA encoding the transmembrane domain of RyR2 and results in the synonymous exchange of a proline residue to a proline residue at amino acid position 4570. The c.14871 C>T was located in exon 105 of the RyR2 mRNA encoding the C terminal of RyR2 and results in the synonymous exchange of a cysteine residue to a cysteine residue at amino acid position 4957. The c.13710 C>A was absent in all of the 165 health controls
Table 1 Novel rare variants of RyR2 gene in SUNDS. Exon
Locia
Cases hosting variant/sample sizeb
Identity in dbSNPc
Amino acid change
Type of variant
Locationd
94 105
c.13710C>A c.14871C>T
1/123 1/124
Novel Novel
p.Pro4570Pro p.Cys4957Cys
Synonymous Synonymous
TMD CT
a b c d
Nucleotide number relative to the translation start site. Due to the quality and quantity limit of the samples, the sample size of each site that can be successfully detected are different. Database of single nucleotide polymorphism. TMD, transmembrane domain; CT, C terminal.
L. Huang et al. / Forensic Science International 235 (2014) 14–18
16 Table 2 Polymorphisms of RyR2 gene in SUNDS. Region
Locia
Homo/Heter/cases hosting variant/sample sizeb
Identity in dbSNPc
Amino acid change
Genotype freq.
Allele freq.
8 15 37 37 45 46 83 90 90 95 95 99 103 105
c.4648A>C c.1359C>T c.5400A>G c.5656G>A c.6906T>C c.7115+58T>C c.1132623C>T c.1196311T>C c.13260+48G>A c.137836A>G c.13913+12A>C c.14298+55A>G c.14756+12C>T c.1480920C>T
15/64/79/113 66/52/118/123 1/16/17/111 1/16/17/111 123/0/123/123 123/0/123/123 87/33/120/125 62/35/97/115 0/5/5/101 111/14/125/125 110/15/125/125 36/60/96/124 0/12/12/122 0/2/2/124
rs10925391 rs3765097 rs3820216 rs3766871 rs707189 rs530109 rs2253831 rs790889 rs77082151 rs790901 rs790900 rs790879 rs2275693 rs14111926
– p.Ser453Ser p.Lys1800Lys p.Gly1886Ser p.Leu2302Leu – – – – – – – – –
AA/AC/CC0.301/0.566/0.133 CC/CT/TT0.041/0.423/0.536 AA/AG/GG0.847/0.144/0.009 GG/GA/AA0.847/0.144/0.009 CC1.000 CC1.000 CC/CT/TT0.040/0.264/0.696 TT/TC/CC0.157/0.304/0.539 GG/GA0.950/0.050 AG/GG0.112/0.888 AC/CC0.120/0.880 AA/AG/GG0.226/0.484/0.290 CC/CT0.902/0.098 CC/CT0.984/0.016
A/C0.584/0.416 C/T0.252/0.748 A/G0.919/0.081 G/A0.919/0.081 C1.000 C1.000 C/T0.172/0.828 T/C0.309/0.691 G/A0.975/0.025 A/G0.056/0.944 A/C0.060/0.940 A/G0.468/0.532 C/T0.951/0.049 C/T0.992/0.008
a
Nucleotide number relative to the translation start site. Homo, homozygote; Heter, heterozygote. Due to the quality and quantity limit of the blood samples, the sample size of each site that can be successfully detected are different. c Database of single nucleotide polymorphism. b
(330 reference alleles). The c.14871 C>T was detected in one of the 165 health controls (330 reference alleles). Fourteen reported polymorphisms were also identified, including ten polymorphisms in intron region, one missense polymorphism, and three synonymous polymorphisms in exon region. The genotype and allele frequencies of all 14 SNPs are as follows (Table 2). The previously published potentially functional variant c.5656G>A (G1886S) was located in exon 37 of the RyR2 mRNA encoding the divergent region 3 (DR3) domain of RyR2 and results in the missense exchange of a glycine residue to a serine residue at amino acid position 1886. The G1886S was present in both SUNDS cases and controls. The genotype distributions of the G1886S and controls were all in the Hardy–Weinberg equilibrium (x2 = 0.119, P value = 0.731; x2 = 1.994, P value = 0.158). The genotype and allele frequency of G1886S was of no statistical difference between SUNDS cases and controls (x2 = 0.390, P value > 0.05; x2 = 0.271, P value > 0.05) (Table 3). 4. Discussion The RyR2 gene is comprised of 105 exons and is one of the largest human genes, encoding an mRNA of approximately 15 kb. Mutations in the RyR2 gene have previously been reported to be associated with CPVT [12] and ARVC [13]. We performed the initial postmortem genetic screening in RyR2 gene in SUNDS in southern Chinese Han population and there were neither published nor novel non-synonymous plausible pathogenic RyR2 mutations found in this cohort of 127 sporadic SUNDS cases. In this study, the c.14871 C>T was detected in one of the 124 SUNDS cases and in one of the 165 health controls (Table 1). Considering the low variant incidence (minor allele frequency, Table 3 The genotype and allele frequencies of G1886S polymorphism. c.5656G>A (G1886S)
SUNDS (n = 111)
Controls (n = 165)
GG GA AA P (x2) G A P (x2) OR (95% CI)
94 (0.847) 16 (0.144) 1 (0.009) 0.859 (0.390) 204 (0.919) 18 (0.081) 0.649 (0.271) 0.851 (0.4641.562)
137 (0.830) 25 (0.152) 3 (0.018) 299 (0.906) 31 (0.094)
OR, odds ratio and CI, confidence interval. Due to the quality and quantity limit of the blood samples, the sample size of each site that can be successfully detected are different.
MAF < 0.01) in SUNDS cases and controls, the c.14871 C>T was regarded as a rare synonymous polymorphism. In addition, the c.13710 C>A was detected in one of the 123 SUNDS cases and absent in the 165 health controls regarding as a synonymous mutation (Table 1). To our knowledge, synonymous mutations do not change the sequence and structure of protein, but may affect mRNA stability and synthesis of the receptor [18]. Thus, the correlation between synonymous mutation and SUNDS cases cannot be ruled out. The c.13710 C>A (Pro4570Pro) may play a critical role in the development of SUNDS by affecting mRNA stability and synthesis of RyR2. This hypothesis requires further elucidation. The previously published genetic variants of G1885E and G1886S were located in exon 37 of the RyR2 mRNA encoding the DR3 domain of RyR2. The DR3 domain includes residues 1852– 1890 of RyR2 that has been reported to be involved in myocardial excitation–contraction coupling (ECC) and in channel regulation by Ca2+, Mg2+, or FK506-binding-protein (FKBP12.6) [19]. Milting et al. [20] identified composite heterozygous polymorphisms G1885E/G1886S, which are associated with ARVC. The G1885E/ G1886S in the DR3 domain cause a modified channel gating with highly increased channel activity at diastolic free calcium concentrations, leading to SR Ca2+ leak. Diastolic Ca2+ release can result in delayed afterdepolarizations (DADs), which can trigger fatal cardiac arrhythmias [21]. The c.5656G>A leading to the amino acid substitution G1886S may create a putative protein kinase C (PKC) phosphorylation site in the human RyR2 [20]. Dysfunction of Ca2+ regulating protein due to an altered phosphorylation contributes to important cardiac diseases such as heart failure [22]. However, Koop et al. [23] found that the phosphorylation site at the position 1886 of RyR2 is unlikely to be a target of any of the tested kinases including PKA, PKC, PKG, and CaMKII. Furthermore, expression of each amino acid substitution G1886S or G1885E caused a significant increase in the cellular Ca2+ oscillation activity compared with that of wild-type RyR2. But, when G1885E/G1886S were introduced in the same RyR2 subunit, the SOICR activity was strongly inhibited. These results indicate that the glycine residues 1885 and 1886 are important for the functional properties of RyR2. Recently, Ran et al. [24] found that the A allele of c.5656G>A leading to the amino acid substitution G1886S in RyR2, not only associates with an increased risk of ventricular arrhythmias in patients with chronic heart failure, but also serves as an independent predictor of sudden cardiac death. In this study, the previously published potentially functional common polymorphism c.5656G>A (G1886S) was present in both SUNDS cases and controls. Meanwhile, the c.5654G>A (G1885E)
L. Huang et al. / Forensic Science International 235 (2014) 14–18
was found neither in SUNDS cases nor in controls. The G1885E/ G1886S genotype was not found, suggesting that composite polymorphisms may play an important role in the development of ARVC [20], but not in SUNDS. The genotype and allele frequency of G1886S was of no statistical difference between SUNDS cases and controls (Table 3), revealing that the G1886S genotype may not be a genetic susceptibility factor in SUNDS of the southern Chinese Han population. In addition to G1886S, thirteen reported polymorphisms were also detected (Table 2). None of these reported polymorphisms resulted in amino acid change and ten of thirteen reported polymorphisms were located in introns. Many transcription factor binding sites are located in the promoter or intron regions of the gene and thus intron variation may affect the level of gene expression and protein translation [25,26]. Thus, the variants in the promoter or intron regions may alter the transcription factor binding sites and affect both RyR2 gene expression and RyR2 formation. This hypothesis also requires further elucidation. According to a recently published study, Tester et al. [27] performed a mutational analysis of 18 exons of the RyR2 gene in 49 cases of sudden unexplained death (SUD) found 7 mutations, revealing the prevalence of mutations was 14%. Furthermore, Larsen et al. [17] genetically screened 29 exons of the RyR2 gene in 74 cases of SUD identified 7 rare variants, revealing the prevalence of rare variants was 9.4%. In our study, the prevalence of rare variants in 29 exons of the RyR2 gene was present in approximately 1% of the examined cases of SUNDS. These results strongly indicated that RyR2 maybe not the key susceptible gene for Chinese SUNDS. Since the discovery of CPVT-causing mutations in the RyR2 gene, a cluster distribution involving three discrete protein regions has been reported [12,16]. On the basis of a potential physiological function, these ‘‘hot-spots’’ regions have been termed domains I, II and III [28,29]. However, because the examined cases of SUNDS have not undergone a scan of the entire RyR2 gene, the prevalence of rare variants residing outside these three domains remains unknown [16]. In addition, the human species may be one of the reasons for the difference of the prevalence of rare variants. Thus, the entire coding exons, promoter, and transcription factor binding sites of the RyR2 gene should be investigated for further comprehensive information of pathogenic mutations. 5. Conclusions This is the first report of genetic phenotype of RyR2 gene of SUNDS in the southern Chinese Han population. Multiple genetic variants of the RyR2 gene were found in SUNDS cases. Association of the multiple genetic variants with SUNDS needs further elucidation. In this study, we genetically screened 29 of the 105 coding exons of the RyR2 gene. For further comprehensive information of pathogenic mutations: (1) the open reading frame (ORF), promoter, and transcription factor binding sites should be investigated; (2) the sample size of SUNDS cases and controls should be enlarged; (3) more candidate genes should be tested, such as Plakophilins2 (PkP2), Connexin43 (Cx43), and CASQ2. For the comprehensive evaluation of SUNDS, clinical information, forensic autopsy records, and molecular autopsy data of SUNDS case should be detailed. Conflict of interest None. Funding This work was supported by the National Natural Science Foundation of China (81172901, 81373238), the Fundamental
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Research Funds for the Central Universities (11ykpy04), the Natural Science Foundation of Guangdong (S2012010009045), and the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2012BAK02B02). Acknowledgment We thank Mr. Danny Andersen for reviewing this manuscript. References [1] K. Nademanee, G. Veerakul, S. Nimmannit, V. Chaowakul, K. Bhuripanyo, K. Likittanasombat, K. Tunsanga, S. Kuasirikul, P. Malasit, S. Tansupasawadikul, P. Tatsanavivat, Arrhythmogenic marker for the sudden unexplained death syndrome in Thai men, Circulation 96 (1997) 2595–2600. [2] A.C. Gaw, B. Lee, G. Gervacio-Domingo, C. Antzelevitch, R. Divinagracia, F.J. Jocano, Unraveling the enigma of Bangungut: is sudden unexplained nocturnal death syndrome (SUNDS) in the Philippines a disease allelic to the Brugada syndrome? Philipp. J. Intern. Med. 49 (2011) 165–176. [3] K. Nakajima, S. Takeichi, Y. Nakajima, M.Q. 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