ISSN 10227954, Russian Journal of Genetics, 2010, Vol. 46, No. 6, pp. 765–768. © Pleiades Publishing, Inc., 2010. Original Russian Text © E.A. Bondarenko, T.V. Tupitsina, P.A. Slominsky, I.M. Shetova, N.A. Shamalov, A.Yu. Botsina, V.I. Skvortsova, S.A. Limborska, 2010, published in Genetika, 2010, Vol. 46, No. 6, pp. 861–864.
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Phosphodiesterase 4D (PDE4D) Gene Polymorphism in Patients with Acute Stroke from Moscow E. A. Bondarenkoa, T. V. Tupitsinaa, P. A. Slominskya, I. M. Shetovab, N. A. Shamalovb, A. Yu. Botsinab, V. I. Skvortsovab, and S. A. Limborskaa a
Institute of Molecular Genetics, Russian Academy of Medical Sciences, Moscow, 12318 Russia; email:
[email protected] b Research Institute of Cerebral Vascular Pathology and Stroke, Russian State Medical University, Moscow, 117997 Russia Received September 29, 2009
Abstract—Two PDE4D gene polymorphisms [SNP41 (rs152312 and SNP87 (rs2910829)] were studied in patients with acute stroke (n = 577) and in control sample (n = 270). Significant differences in the genotype and allele frequency distribution were found between these samples for polymorphism SNP41. We showed that the AA and AG genotypes of SNP41 polymorphism were associated with higher risk of acute stroke devel opment in the Moscow population (OR = 1.6). No association of SNP87 polymorphism with the disease was observed. DOI: 10.1134/S1022795410060189
Vascular pathology of the brain is the most impor tant contributor to disability and mortality in the developed countries. According to the World Develop ment Report, stroke is the second cause of mortality after cardiovascular pathology in Russia. Over 6 mil lion individuals in the world and 450000 in Russia are annually subjected to stroke. Moreover, this disease is the leading cause of disability. A trend for younger age of people suffering with stroke is observed, as it is spreading among the people of the working age. Thus, stroke is a problem of extreme medical and social importance. In 2002, the DeCode group has published the results of genomewide linkage analysis for predispo sition to stroke. The authors has mapped the STRK1 locus associated with stroke to the 5q12 chromosome [1]. A more detailed analysis of this of chromosome region revealed a strong association of the PDE4D gene located in the examined locus with two forms of stroke related to atherosclerosis: cardioembolic and atherosclerotic [2]. The PDE4D gene encodes phosphodiesterase 4D, which belongs to PDE4 of the phosphodiesterase superfamily and is involved in the selective cAMP deg radation. A decrease in the cAMP concentration results in an increase of proliferation and migration of vascular smoothmuscle cells in vitro. It is known that such processes produce fibrous plaques [3–6]. In addition, animal models have shown that application of PDE4 antagonists inhibit proliferation of these cells [7, 8]. The PDE4D gene is also expressed in the acti vated macrophages and thus might produce inflam mation in the atherosclerotic plaque. Thus, phos phodiesterase 4D could be involved either in the
atherogenesis, or in instability of atherosclerotic plaques, or in both processes [9–11]. The 1600kb PDE4D gene composed of 22 exons and has at least seven promoters [2]. Through alterna tive splicing or the use of differential promoters, this gene can express at least eight functionally variable isoforms of the protein: two short (PDE4D1 and PDE4D2) and six long forms (PDE4D3, 4, 5, 7, 8, and 9) [12]. All the isoforms have similar Cterminal cata lytic domain and differ in the Nterminal domain structure [2]. It is likely that these domains could be involved in the phosphodiesterase activity regulation [13]. The different PDE4D variants are expressed in many types of cells and tissues including brain, lungs, kidney, monocytes, B and Tlymphocytes as well as vascular smooth muscle cells [14, 15]. Using EBVtransformed B cell lines it was shown that stroke patients have a significantly reduced level of total PDE4D mRNA compared to the control group. This difference resulted from lower expression of PDE4D1, PDE4D2, and PDE4D5 isoform mRNAs [2]. It is not clear at present how the lower level of definite phosphodiesterase 4D isoforms is involved in stroke pathogenesis. According to one hypothesis, the decrease in the expression of one or several phosphodiesterase 4D isoforms or the imbal ance produced by the disturbance in their expression or splicing increases the risk of ischemic stroke devel opment [16]. 260 single nucleotide polymorphisms (SNPs) were found at the PDE4D gene. Gretarsdottir et al. [2] determined three haploblocks A, B, and C, which involve the first three exons of PDE4D gene and the adjacent regions: haploblock A (exons D73 and D7
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Comparative genotype and allele analysis of polymorphisms SNP87 and SNP41 in PDE4D gene in acute stroke patients and in the control sample Genotype, allele SNP 87 C/C C/T T/T C/T + T/T C T SNP 41 G/G G/A A/A G/A + A/A G A Notes:
Acute stroke N
Control
Frequency, %
N
Frequency, %
p
115 309 153 462 539 615
19.9 53.6 26.5 80.1 46.7 53.3
51 143 76 219 245 295
18.9 53.3 28.1 81.4 45.4 54.6
0.84* 0.84* 0.84* 0.79** 0.86*** 0.86***
449 119 9 128 1017 137
77.8 20.6 1.6 22.2 88.1 11.9
230 38 2 40 498 42
85.2 14.1 0.7 14.8 92.2 7.8
0.04* 0.04* 0.04* 0.02** 0.014*** 0.014***
* Calculated using the GraphPad InStat Software for the 2 × 3 contingency table. **, *** This was calculated using the GraphPad InStat Software for the 2 × 2 contingency table.
2, 300 kb, contains 19 SNP with MAF > 20%), hap loblock B (the first exon D71, 200 kb, contains 22 SNP with MAF > 20%), and haploblock C (adjacent to haploblock B, 60 kb, contains 25 SNP with MAF > 20%). A positive association between PDE4D SNPs and stroke has been reported in some cohorts in different nonEuropean countries such as United States (for SNP56, haploblock B), Pakistan (for SNP83, haplob lock A), Japan (for SNP83), and Australia (for SNP83, SNP87, and SNP89, haploblock A). These results were not reproduced in European populations, where only van Rijn et al. [17] have reported a positive association in a small population from the Nether lands (for SNP39 and SNP45, haploblock B). Gretar sdottir et al. [2] showed that SNP41 (haploblock B) and SNP87 (haploblock A) were associated with car dioembolic and atherosclerotic stroke in the Iceland population. No association was observed between SNP of haploblock C with the risk of stroke develop ment. Thus, the data on association of PDE4D gene with the risk of this disease development are contro versial. So, we conducted analysis of two single nucleotide polymorphisms: SNP41 (rs152312) and SNP87 (rs2910829), which belong to various haploblocks (haploblock A and haploblock B, respectively), in the patients with acute stroke (n = 577, average age 67 ± 12.6 years) from Moscow and a control sample (n = 270, average age 51.7 ± 9.1) matched by sex and eth nicity. Patient selection and their clinical investigation was carried out in the Clinical City Hospital no. 31 affiliated to the Department of Basic and Clinical Neurology, Russian State Medical University. Blood
withdrawal has been performed on day 2 or day 3 after stroke. Molecular genetic studies have been conducted using DNA specimens obtained from 3 ml of venous blood. DNA was isolated using AxyPrepTM Blood Genomic DNA Midiprep kit (Axygen Biosciences, United States). Polymorphic alleles of PDE4D gene were examined using realtime polymerase chain reaction (TaqMan technology). PCR was conducted using a Mx3000P thermal cycler (Stratagene). The amplification reaction mixture in 25 μl contained 2.5 μl 10× PCR buffer (Syntol, Russia); 2.5 μl 2.5 mM dNTPs solution (dATP, dCTP, dGTP, dTTP); 10 pM of each primer; 4 pM of each probe; 1.25 units HotTaq polymerase (Syntol, Russia); 0.1–0.2 μg of genomic DNA and deionized water up to 25 μl. The amplifica tion procedure involved decontamination at 50°С for 3 min, denaturation at 95°С for 10 min, followed by 40 cycles at 95°С for 15 s, 58°С (in case SNP87) or 56°С (in case of SNP41) for 50 s. The sequences of primers and probes are presented in [18]. Statistical analysis was performed using GraphPad InStat Software (United States) (http:// www.graph pad.com) to examine HardyWeinberg equilibrium as well as the odds ratio (OR) value. Significance of dif ferences in allele and genotype frequencies at the poly morphic loci in the compared groups was determined using χ2 test. Comparison of the genotype frequencies was conducted using contingency 2 × 3 tables while for the alleles and joint genotypes, using contingency 2 × 2 tables. P values less than 0.05 were considered signif icant. The results of SNP87(C T) and SNP41 (G A) polymorphism genotyping are presented in Table 1.
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The distribution of genotype and allele frequencies in the control sample in case of both SNP is in accor dance with Hardy–Weinberg expectations (χ2 = 0.73, 0.00; p = 0.69, 1.00, respectively). Table 1 shows the lack of significant differences between genotype and allele frequency distribution between examined groups in case of SNP87 polymor phism. The CC genotype frequencies in the group of acute stroke patients and in the control sample were 19.9% and 18.9%, respectively, for CT genotype, 53.6% and 53.3%, respectively, and for TT genotype, 26.5% and 28.1%, respectively. Thus, the frequency of the minor C allele was 46.7% and 45.4% in the group of acute stroke patients and the control sample, respectively. The GG genotype frequencies in SNP41 polymor phism were 77.8% and 85.2%, respectively, in the group of acute stroke patients and control sample (p = 0.04). Therefore, in the control sample the GG geno type frequency was 7.4% higher than in the group of acute stroke patients. The frequency of the AA geno type was 2.3fold higher in the sample of acute stroke patients. The G allele frequency was also 4.1% higher in the control group (p = 0.014). In the sample of acute stroke patients, the A allele carriers (individuals with genotypes GA and AA) were observed with frequency 22.2%, while in the control group, with frequency 14.8% (p = 0.02). Our results suggest that allele A of SNP41 and the genotypes containing this allele are associated with increased risk of acute stroke development in the Mos cow population. The relative risk of stroke develop ment in the individuals carrying genotypes AA and GA is 1.6fold higher (OR = 1.639; 95% CI from 1.1 to 2.4). Thus, SNP41 polymorphism in the PDE4D gene, which is caused by G A substitution, is associated with the risk of acute stroke development in the Rus sian population. SNP41 is located in the noncoding region of the PDE4D gene, which flanks D71 exon. This SNP has no functional importance and its contribution to the increased risk of acute stroke development is not clear. It is likely that SNP41 alone does not contribute to the increased stroke incidence. However, it is in linkage disequilibrium with the risk allele. Therefore, the lev els of linkage disequilibrium in these loci could vary between populations. Phosphodiestease 4D comprises 80% of the activity of all the other phosphodiesterases in the cell that are involved in inflammation [14, 19]. The PDE4D enzyme activity is important for the risk of stroke development as it is involved in the inflammation, generation of the physiologic response to vascular dis turbance, angiogenesis, etc. [17, 20]. It is likely that PDE4D is involved in the regulation of biologic activ ity in the tissues where it is expressed. SNP41 is located in the noncoding region flanking D71 exon of the PDE4D gene. Although the functional significance RUSSIAN JOURNAL OF GENETICS
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of the PDE4D gene SNP is not clear so far, we suggest that it could affect enzyme activity due to modulation of the definite enzyme isoform expression. This is sup ported by the data of the DeCode research group and other authors. It is possible that other genetic disturbances in the PGEsignaling pathway or supplementary signaling pathways regulated by phosphodiesterases could be involved in the predisposition to stroke. A search for candidate genes associated with stroke could enable development of the pharmacological approaches for the disease treatment. Taking into consideration that phosphodiesterases are well characterized we suggest the likelihood for their pharmacologic correction. For example, isoformspecific, clinically important mod ulators could be manufactured. It is well established that PDE4D is selectively inhibited by Rolipram [21]. This permits to anticipate the development in future drugs for stroke development prevention in the risk groups. ACKNOWLEDGMENTS This study was supported by the State Contract (project no. 02.522.11.2018), the Russian Foundation for Basic Research (project no. 070401511 and 07 0400027), and the Programs of the Russian Academy of Sciences “Molecular and Cellular Biology, ” “Innovation and Development Support. ” REFERENCES 1. Gretarsdottir, S., Sveinbjörnsdottir, S., Jonsson, H.H., et al., Localization of a Susceptibility Gene for Com mon Forms of Stroke to 5q12, Am. J. Hum. Genet., 2002, vol. 70, no. 3, pp. 593–603. 2. Gretarsdottir, S., Thorleifsson, G., Reynisdottir, S.T., et al., The Gene Encoding Phosphodiesterase 4D Con fers Risk of Ischemic Stroke, Nat. Genet., 2003, vol. 35, no. 2, pp. 131–138. 3. Fukumoto, S., Koyama, H., Hosoi, M., et al., Distinct Role of cAMP and cGMP in the Cell Cycle Control of Vascular Smooth Muscle Cells: cGMP Delays Cell Cycle Transition through Suppression of Cyclin D1 and CyclinDependent Kinase 4 Activation, Circ. Res., 1999, vol. 85, no. 11, pp. 985–991. 4. Houslay, M.D. and Adams, D.R., PDE4 cAMP Phos phodiesterases: Modular Enzymes That Orchestrate Signalling CrossTalk, Desensitization and Compart mentalization, Biochem. J., 2003, vol. 370, pp. 1–18. 5. Pan, X., Arauz, E., Krzanowski, J.J., et al., Synergistic Interactions between Selective Pharmacological Inhib itors of Phosphodiesterase Isozyme Families PDE III and PDE IV to Attenuate Proliferation of Rat Vascular Smooth Muscle Cells, Biochem. Pharmacol., 1994, vol. 48, no. 4, pp. 827–835. 6. Palmer, D., Tsoi, K., and Maurice, D.H., Synergistic Inhibition of Vascular Smooth Muscle Cell Migration by Phosphodiesterase 3 and Phosphodiesterase 4 Inhib itors, Circ. Res., 1998, vol. 82, no. 8, pp. 852–861. 2010
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