Association of the RYR3 gene polymorphisms with atherosclerosis in elderly Japanese population
© Zhao et al.; licensee BioMed Central Ltd. 2014
Received: 5 August 2013
Accepted: 31 December 2013
Published: 14 January 2014
The Ryanodine receptor 3 gene (RYR3) encodes an intracellular calcium channel that mediates the efflux of Ca2+ from intracellular stores. Two single-nucleotide polymorphisms (SNPs) in the RYR3 gene have been shown to associate with stroke (rs877087) and carotid intima-media thickness (rs2229116) in two independent genome-wide association studies (GWAS) in Caucasian. We investigated the effect of these two SNPs as well as the 31.1 kilobases spanning region on atherosclerosis in Japanese population.
Atherosclerotic severity was assessed by carotid artery (n = 1374) and pathological atherosclerosis index (PAI) (n = 1262), which is a macroscopic examination of the luminal surfaces of 8 systemic arteries in consecutive autopsy samples. 4 tag SNPs in the 31.1 Kb region, rs877087, rs2132207, rs658750 and rs2229116, were genotyped and haplotypes were inferred to study the association with atherosclerotic indices.
rs877087 and rs2229116 were associated with PAI (OR = 2.07 [1.04-4.12] (95% CI), p = 0.038; and OR = 1.38 [1.02-1.86], p = 0.035, respectively). rs2229116 was also associated with common carotid atherosclerosis (OR = 1.45 [1.13-1.86], p = 0.003). The risk allele of rs2229116 was opposite from the original report. The haplotype block of this 31.1 Kb region was different between Caucasian and Japanese. Haplotype analysis revealed that only TAGG haplotype was associated with PAI (OR = 0.67 [0.48-0.94], p = 0.020) and atherosclerosis of common carotid artery (OR = 0.75 [0.58-0.98], p = 0.034).
rs877087 and rs2229116 of RYR3 gene are associated with atherosclerosis severity in Japanese. The functional difference caused by rs2229116 needs to be investigated.
KeywordsAtherosclerosis Polymorphism Ryanodine receptor 3 Japanese
Atherosclerosis is currently thought to be an inflammatory disease with deposits of cholesterol and inflammatory cells in the arterial wall [1–3]. In the later stages, thrombosis forms, and this leads to fatal events, like myocardial and cerebral infarctions [4, 5]. The etiology of atherosclerosis is multi-factorial, in that it includes various genetic and environmental factors [6, 7]. It is known that genetic factors play a pivotal role in atherosclerosis, but the influence of specific genes on susceptibility is not fully understood [8–10]. The discovery of novel risk factors for atherosclerosis may lead to efficient prevention and new therapeutic targets for this fatal disease.
Recently, two independent genome-wide association studies (GWAS) of atherosclerotic diseases in Caucasians pointed to two SNPs, closely resided in the 31.1 Kb (intron 14 ~ exon 19) region of the RYR3 gene [11, 12]. RYR3 gene is located on chromosome 15q14-q15, and encode a large intracellular homotetrameric protein (> 2MDa) that comprises 4780 amino acids [13–15]; RYR3 protein resides on the sarcoplasmic reticulum membrane and releases Ca2+ from intracellular stores to regulates intracellular calcium concentration [16, 17]. The RyR3-deficient mice lack calcium regulation properties in arterial smooth muscle cells, which lead to dysregulation of arterial tone . In human arterial endothelial cells, Ca2+ release mediated by RYR3, but not by the RYR1 and RYR2, plays a role in endothelial vasodilation . Thus, it is conceivable that RYR3 is a good candidate for atherosclerosis susceptible gene. The rs877087 polymorphism, which is an intron SNP, was associated with stroke in a Caucasian population from 4 large cohorts (n = 19,602) . The rs2229116, which is an Ile to Val non-synonymous SNP, was associated with common carotid intima-media thickness (cIMT), a clinical parameter for subclinical atherosclerosis . This was found in a specific group of Caucasian males infected with HIV and were treated with highly active antiretroviral therapy (n = 177) . Since rs877087 and rs2229116 on RYR3 gene is separated by 31.1 Kb, we hypothesize that this region might confer susceptibility to atherosclerosis, and thus studied this region in detail using tag SNPs and haplotypes.
A total of 1536 consecutive autopsy cases of aged patients registered in the JG-SNP database were examined (http://www.tmghig.jp/jg-snp/english/E_top.html) which contains abundant pathological information on atherosclerosis . The autopsies were performed from 1995 to 2004 at the Tokyo Metropolitan Geriatric Hospital in Tokyo, Japan. The distribution of age at death is 65 ~ 102 years old, and the mean is 80.21 ± 8.90 (mean ± SD). The major clinical diagnosis and direct cause of death in this Japanese population was described elsewhere . The study was approved by the ethical committees of the Tokyo Metropolitan Geriatric Hospital and the Tokyo Medical and Dental University.
The methods for the pathological macroscopic evaluation of atherosclerosis severity are published previously [22, 23]. Briefly, a total of eight arteries were examined, including the common carotid artery, subclavian artery, aorta, splenic artery, superior mesenteric artery, common iliac artery, external iliac artery, and femoral artery. Assessments were performed by macroscopic examinations of the luminal surface in formalin-fixed arteries. The degree of atherosclerosis had been scored according to the ratio of the atheroma area to the entire intimal area. The scale ranged from 0–8, where 0 = absent, or a ratio less than 1/20; 2 = minimal, or a ratio of 1/20 ~ 1/6; 4 = mild, or a ratio of 1/6 ~ 1/3; 6 = moderate, or a ratio of 1/3 ~ 2/3; and 8 = severe, or a ratio of 2/3 ~ 1. The pathological atherosclerotic index (PAI) was defined as the average degree of atherosclerotic severity in all eight arteries . Since not all eight arteries were evaluated in some samples, the number of PAI records (n = 1262) is less than the number of common carotid artery records (n = 1374).
The RYR3 polymorphism (rs877087, rs2132207, rs658750 and rs2229116) was analyzed by the Taqman assay (Applied BioSystems) using specific primers and probes on a Light-Cycler 480 (Roche), according to previously described protocols . The pathological assessments and genotyping were performed in different institutions in a double-blind fashion.
Statistical analyses were performed with the IBM SPSS 19.0.0 statistical software. The associations of genotypes with atherosclerosis severity were tested by multiple logistic regression analyses with adjustment for conventional risk factors. All continuous and discrete variables of atherosclerotic severity were categorized by the cut-point of 75% severity, and divided subjects into Ath(+) group with top 25% severity, and Ath(−) group with severity lower than that, as we did previously . The conventional risk factors were also categorized as follows: gender (male vs. female), hypertension, diabetes mellitus, hyperlipidemia, and history of smoking or drinking (absent vs. present were classified as 0 vs. 1, respectively). P-values less than 0.05 were considered statistically significant, and correction for multiple testing was not considered in this study. Haplotype frequency estimation and haplotype-based associations were performed using PLINK software (http://pngu.mgh.harvard.edu/purcell/plink/) . Hardy–Weinberg equilibrium (HWE) was determined with Fisher’s exact test. Linkage disequilibrium among SNPs was assessed using Haploview software .
Genetic structure data
Genotype and haplotype distribution of the SNPs
P-value for HWE test
Estimated frequencies and number of samples a
0.504, n = 681
0.158, n = 214
0.121, n = 164
0.102, n = 138
0.057, n = 77
Relationship between RYR3 polymorphisms and atherosclerosis severity
Relationship between RYR3 polymorphisms and atherosclerosis severity >75%
rs877087 genotypes, n(%)
rs2229116 genotypes, n(%)
TT vs. CT + CC
AA vs. AG + GG
Atherosclerosis degrees in each rs2229116genotype
Means of atherosclerosis degrees in rs2229116 genotype groups
rs2229116 genotypes, mean(n)
Relationships between RYR3 haplotypes and atherosclerosis severity
Haplotype-based association test for linear and logistic regression
The previous GWAS showed that the RYR3 gene polymorphisms rs877087 and rs2229116 was associated with stroke and cIMT, respectively [11, 12]. In this study, we investigated these two SNPs on RYR3 gene and the 31.1 Kb region in between employing elderly Japanese. We observed that both rs877087 and rs2229116 was associated with PAI. Our study suggested that the atherosclerosis risk allele is T for rs877087, as the same risk allele from the GWAS of stroke. It should be noted that the minor allele frequency of rs877087 is 0.035 for C allele in Japanese, and 0.42 for T allele in Caucasian according to the HapMap data. rs2229116 was associated with PAI and atherosclerosis severity in common carotid after adjusted for conventional risk factors (Table 2). However, our risk allele was opposite from the original paper , in that, the GWAS for cIMT the risk allele was G, but in our study it was A (Table 3). The LD patterns between rs877087 and rs2229116 from HapMap data showed great differences between CEU and JPT (Figure 1 and Additional file 3: Figure S1 and Additional file 4: Figure S2). As reference to rs2229116, the LD values of two populations showed large difference (ΔD’ > 0.5) in several SNP sites, including the 2 tag SNPs (rs2132207 and rs658750), suggesting that the haplotypes within this region may be different between CEU and JPT (Figure 1). Therefore, different haplotypes might account for the different observation between Japanese and European. In order to further investigate the region between the two reported SNPs, we further selected 2 tag SNPs (rs2132207 and rs658750) and build haplotypes (Table 1). Haplotype linear regression analyses revealed that the carriers of TAGG haplotype had a decrease trend of atherosclerosis severity in both common carotid arteries and PAI. Also, an increase trend was observed in TGGA haplotype in common carotid arteries and TAAA haplotypes in PAI, respectively (Table 4). In haplotype logistic regression model for atherosclerosis severity trait, we observed TAGG haplotype had a preventive effect in both common carotid arteries and PAI (Table 4). Although we tried to explain the opposite effect in Japanese and Caucasian, using trans-ethnic fine mapping, and selected these two tag SNPs which gave a significant difference in LD value between two ethnics, we could not explain the association with either of the SNP. Nevertheless, taken together the results of haplotype analysis, it is likely that the non-synonymous polymorphism rs2229116 (Val/Ile) itself may be the primary determinant for the observed associations (Table 4).
The rs2229116 SNP causes amino acid substitution (Ile to Val) at the 731st amino acid site of RYR3. However, the functional consequence has not yet been investigated. RYR3 have a major role in calcium signaling in the vasculation and thus is a good candidate gene in atherosclerosis pathogenesis.
Along with the function of RYR3 in vascular cells, recent studies have found that RYR3 have function in T cells, which is involved in the inflammation process of atherosclerotic lesions [27, 28]. RYR3 appear to regulate Ca2+ signaling in T lymphocytes , in which, calcium signaling is essential for its activation and differentiation . The association of T cell activation markers and carotid arterial stiffness was also reported in a group of HIV-infected women . These lines of evidence suggest that, intracellular calcium mobilization mediated by RYR3 may be involved in atherosclerosis process through its common participants, such as arterial smooth muscle cells, endothelial cells and T lymphocytes.
Our study has some limitations. The subjects of the present study were consecutive autopsy cases of patients in a geriatric hospital. Therefore, the selection bias and whether the subjects can represent the demographics of Japan should be evaluated. The autopsy rate of the hospital was kept around 40% and those who died suddenly outside the hospital and medicolegal cases were not autopsied for this study. The leading mortality probability at the age of 75 years according the census data in ‘Abridged Life Tables For Japan 2010’ by the Ministry of Health, Labor and Welfare of Japan (http://www.mhlw.go.jp/english/database/db-hw/lifetb10/index.html) were as follows; 25.9% men vs. 16.5% women by malignant neoplasms, 15.2% men vs. 20.3% women by heart diseases, 10.4% men vs. 12.1% women by cerebrovascular diseases, and 15.3% men vs. 12.4% women by pneumonia. Almost all death rates for major diseases at 75 years of age in Tokyo were consistent with our autopsy data, except for the frequency of malignant neoplasms is a little higher and that of cerebrovascular diseases is lower in our autopsy subjects. Since the national health insurance covers the whole population of Japan and in Japan most death occurred in hospitals, the selection bias from socioeconomic difference of the subjects seems minimal. Altogether, the subjects in our study did not differ greatly from the standard elderly residents in Tokyo, and may represent the general population, including young residents, in Japan rather than a selected hospitalized population.
RYR3 gene rs877087 and rs2229116 polymorphisms are associated with atherosclerosis in elderly Japanese. Functional studies are still needed to identify the causal variation.
Ryanodine receptor 3 gene
Single nucleotide polymorphism
Genome-wide association studies
Pathological atherosclerosis index
Common carotid intima-media thickness
This study was supported by the Joint Usage/Research Program of the Medical Research Institute, Tokyo Medical and Dental University. The authors express their gratitude for the financial support for the study.
- Ross R: Atherosclerosis–an inflammatory disease. N Engl J Med. 1999, 340 (2): 115-126. 10.1056/NEJM199901143400207.View ArticlePubMedGoogle Scholar
- Hansson GK: Atherosclerosis–an immune disease: the anitschkov lecture 2007. Atherosclerosis. 2009, 202 (1): 2-10. 10.1016/j.atherosclerosis.2008.08.039.View ArticlePubMedGoogle Scholar
- Libby P: Inflammation in atherosclerosis. Nature. 2002, 420 (6917): 868-874. 10.1038/nature01323.View ArticlePubMedGoogle Scholar
- Falk E: Plaque rupture with severe pre-existing stenosis precipitating coronary thrombosis. Characteristics of coronary atherosclerotic plaques underlying fatal occlusive thrombi. Br Heart J. 1983, 50 (2): 127-134. 10.1136/hrt.50.2.127.View ArticlePubMedPubMed CentralGoogle Scholar
- Jackson SP: Arterial thrombosis--insidious, unpredictable and deadly. Nat Med. 2011, 7;17 (11): 1423-1436.View ArticleGoogle Scholar
- Singh RB, Mengi SA, Xu YJ, Arneja AS, Dhalla NS: Pathogenesis of atherosclerosis: a multifactorial process. Exp Clin Cardiol. 2002, 7 (1): 40-53.PubMedPubMed CentralGoogle Scholar
- Fox CS, Polak JF, Chazaro I, Cupples A, Wolf PA, D’Agostino RA, O’Donnell CJ: Genetic and environmental contributions to atherosclerosis phenotypes in men and women: heritability of carotid intima-media thickness in the Framingham Heart Study. Stoke. 2003, 34: 397-401.Google Scholar
- Blanco-Colio LM, Martín-Ventura JL, Vivanco F, Michel JB, Meilhac O, Egido J: Biology of atherosclerotic plaques: what we are learning from proteomic analysis. Cardiovasc Res. 2006, 1;72 (1): 18-29.View ArticleGoogle Scholar
- Stylianou IM, Bauer RC, Reilly MP, Rader DJ: Genetic basis of atherosclerosis: insights from mice and humans. Circ Res. 2012, 20;110 (2): 337-355.View ArticleGoogle Scholar
- Maouche S, Schunkert H: Strategies beyond genome-wide association studies for atherosclerosis. Arterioscler Thromb Vasc Biol. 2012, 32 (2): 170-181. 10.1161/ATVBAHA.111.232652.View ArticlePubMedGoogle Scholar
- Ikram MA, Seshadri S, Bis JC, Fornage M, DeStefano AL, Aulchenko YS, Debette S, Lumley T, Folsom AR, van den Herik EG, Bos MJ, Beiser A, Cushman M, Launer LJ, Shahar E, Struchalin M, Du Y, Glazer NL, Rosamond WD, Rivadeneira F, Kelly-Hayes M, Lopez OL, Coresh J, Hofman A, DeCarli C, Heckbert SR, Koudstaal PJ, Yang Q, Smith NL, Kase CS, et al: Genomewide association studies of stroke. N Engl J Med. 2009, 23;360 (17): 1718-1728.View ArticleGoogle Scholar
- Shrestha S, Irvin MR, Taylor KD, Wiener HW, Pajewski NM, Haritunians T, Delaney JA, Schambelan M, Polak JF, Arnett DK, Chen YD, Grunfeld C: A genome-wide association study of carotid atherosclerosis in HIV-infected men. AIDS. 2010, 24 (4): 583-592. 10.1097/QAD.0b013e3283353c9e.View ArticlePubMedPubMed CentralGoogle Scholar
- Sorrentino V, Giannini G, Malzac P, Mattei MG: Localization of a novel ryanodine receptor gene (RYR3) to human chromosome 15q14-q15 by in situ hybridization. Genomics. 1993, 18 (1): 163-165. 10.1006/geno.1993.1446.View ArticlePubMedGoogle Scholar
- Nakashima Y, Nishimura S, Maeda A, Barsoumian EL, Hakamata Y, Nakai J, Allen PD, Imoto K, Kita T: Molecular cloning and characterization of a human brain ryanodine receptor. FEBS Lett. 1997, 417: 157-162. 10.1016/S0014-5793(97)01275-1.View ArticlePubMedGoogle Scholar
- Leeb T, Brenig B: cDNA cloning and sequencing of the human ryanodine receptor type 3 (RYR3) reveals a novel alternative splice site in the RYR3 gene. FEBS Lett. 1998, 423 (3): 367-370. 10.1016/S0014-5793(98)00124-0.View ArticlePubMedGoogle Scholar
- Jeyakumar LH, Copello JA, O’Malley AM, Wu GM, Grassucci R, Wagenknecht T, Fleischer S: Purification and characterization of ryanodine receptor 3 from mammalian tissue. J Biol Chem. 1998, 273 (26): 16011-16020. 10.1074/jbc.273.26.16011.View ArticlePubMedGoogle Scholar
- Lanner JT, Georgiou DK, Joshi AD, Hamilton SL: Ryanodine receptors: structure, expression, molecular details, and function in calcium release. Cold Spring Harb Perspect Biol. 2010, 2 (11): a003996-View ArticlePubMedPubMed CentralGoogle Scholar
- Löhn M, Jessner W, Fürstenau M, Wellner M, Sorrentino V, Haller H, Luft FC, Gollasch M: Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells. Circ Res. 2001, 89 (11): 1051-1057. 10.1161/hh2301.100250.View ArticlePubMedGoogle Scholar
- Köhler R, Brakemeier S, Kühn M, Degenhardt C, Buhr H, Pries A, Hoyer J: Expression of ryanodine receptor type 3 and TRP channels in endothelial cells: comparison of in situ and cultured human endothelial cells. Cardiovasc Res. 2001, 51 (1): 160-168. 10.1016/S0008-6363(01)00281-4.View ArticlePubMedGoogle Scholar
- Finn AV, Kolodgie FD, Virmani R: Correlation between carotid intimal/medial thickness and atherosclerosis: a point of view from pathology. Arterioscler Thromb Vasc Biol. 2010, 30 (2): 177-181. 10.1161/ATVBAHA.108.173609.View ArticlePubMedGoogle Scholar
- Sawabe M, Arai T, Kasahara I, Esaki Y, Nakahara K, Hosoi T, Orimo H, Takubo K, Murayama S, Tanaka N: Developments of geriatric autopsy database and Internet-based database of Japanese single nucleotide polymorphisms for geriatric research (JG-SNP). Mech Ageing Dev. 2004, 125 (8): 547-552. 10.1016/j.mad.2004.06.005.View ArticlePubMedGoogle Scholar
- Oda K, Tanaka N, Arai T, Araki J, Song Y, Zhang L, Kuchiba A, Hosoi T, Shirasawa T, Muramatsu M, Sawabe M: Polymorphisms in pro- and anti-inflammatory cytokine genes and susceptibility to atherosclerosis: a pathological study of 1503 consecutive autopsy cases. Hum Mol Genet. 2007, 16 (6): 592-599.View ArticlePubMedGoogle Scholar
- Sawabe M, Arai T, Kasahara I, Hamamatsu A, Esaki Y, Nakahara K, Harada K, Chida K, Yamanouchi H, Ozawa T, Takubo K, Murayama S, Tanaka N: Sustained progression and loss of the gender-related difference in atherosclerosis in the very old: a pathological study of 1074 consecutive autopsy cases. Atherosclerosis. 2006, 186 (2): 374-379. 10.1016/j.atherosclerosis.2005.07.023.View ArticlePubMedGoogle Scholar
- Song Y, Araki J, Zhang L, Froehlich T, Sawabe M, Arai T, Shirasawa T, Muramatsu M: Haplotyping of TNFalpha gene promoter using melting temperature analysis: detection of a novel −856(G/A) mutation. Tissue Antigens. 2005, 66 (4): 284-290. 10.1111/j.1399-0039.2005.00464.x.View ArticlePubMedGoogle Scholar
- Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Maller J, Sklar P, de Bakker PIW, Daly MJ, Sham PC: PLINK: a toolset for whole-genome association and population-based linkage analysis. Am J Hum Genet. 2007, 81 (3): 559-575. 10.1086/519795.View ArticlePubMedPubMed CentralGoogle Scholar
- Barrett JC, Fry B, Maller J, Daly MJ: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005, 21 (2): 263-265. 10.1093/bioinformatics/bth457.View ArticlePubMedGoogle Scholar
- Emeson EE, Robertson AL: T lymphocytes in aortic and coronary intimas. Their potential role in atherogenesis. Am J Pathol. 1988, 130 (2): 369-376.PubMedPubMed CentralGoogle Scholar
- Kleindienst R, Xu Q, Willeit J, Waldenberger FR, Weimann S, Wick G: Immunology of atherosclerosis. Demonstration of heat shock protein 60 expression and T lymphocytes bearing alpha/beta or gamma/delta receptor in human atherosclerotic lesions. Am J Pathol. 1993, 142 (6): 1927-1937.PubMedPubMed CentralGoogle Scholar
- Schwarzmann N, Kunerth S, Weber K, Mayr GW, Guse AH: Knock-down of the type 3 ryanodine receptor impairs sustained Ca2+ signaling via the T cell receptor/CD3 complex. J Biol Chem. 2002, 277 (52): 50636-50642. 10.1074/jbc.M209061200.View ArticlePubMedGoogle Scholar
- Boubali S, Liopeta K, Virgilio L, Thyphronitis G, Mavrothalassitis G, Dimitracopoulos G, Paliogianni F: Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: a distinct target in the calcium dependent pathway. Mol Immunol. 2012, 52 (2): 51-60. 10.1016/j.molimm.2012.04.008.View ArticlePubMedGoogle Scholar
- Kaplan RC, Sinclair E, Landay AL, Lurain N, Sharrett AR, Gange SJ, Xue X, Parrinello CM, Hunt P, Deeks SG, Hodis HN: T cell activation predicts carotid artery stiffness among HIV-infected women. Atherosclerosis. 2011, 217 (1): 207-213. 10.1016/j.atherosclerosis.2011.03.011.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/14/6/prepub
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.