Just Accepted by International Journal of Neuroscience
Association between PDE4D gene and ischemic stroke: Recent Advancements Satrupa Das, Sitara Roy, Anjana Munshi doi:10.3109/00207454.2015.1051621
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ABSTRACT Stroke is a severe complication and a leading cause of death worldwide and genetic studies among different ethnicities has provided the basis for involvement of Phosphodiesterase 4D (PDE4D) gene in cerebrovascular diseases. Recent advancements have evaluated the role of this gene in stroke and these studies have provided a stronger support for the involvement of this gene in stroke development and few studies also suggest that it may influence outcome. Further, case-control studies and meta-analysis studies have provided strong evidence for certain variants in PDE4D to predispose to stroke only among certain ethnicities. Thus, this review focuses on recent progress made in PDE4D gene research involving genetic, molecular and pharmacological aspect. A strong conclusion has emerged that clearly indicates a pivotal role played by this gene in ischemic stroke globally. Studies have also noticeably highlighted that PDE4D gene/pathway can be a suitable drug target for managing stroke however, a more comprehensive research is still required to understand the molecular and cellular intricacies this gene plays in stroke development, progression and its outcome.
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Publisher: Taylor & Francis
Journal: International Journal of Neuroscience
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DOI: http://dx.doi.org/10.3109/00207454.2015.1051621
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Satrupa Das, Sitara Roy, Anjana Munshi * Satrupa Das, Sitara Roy
Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad-500016, India.
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Dr. NTR University of Health Sciences,Vijayawada, Andhra Pradesh, India.
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Anjana Munshi *
Centre for Human Genetics, School of Health Sciences, Central University of Punjab, Bathinda, Punjab, India.
Corresponding author:
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*
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Association between PDE4D gene and ischemic stroke: Recent Advancements
Dr. Anjana Munshi Centre for Human Genetics School of Health Sciences Central university of Punjab, Bathinda Punjab, India. Email address: [email protected] Telephone No- +91-9872694373
ABSTRACT
Stroke is a severe complication and a leading cause of death worldwide and genetic studies among different ethnicities has provided the basis for 1
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KEYWORDS: Phosphodiesterase 4D gene, genetics, ischemic stroke, casecontrol study, meta-analysis study, expression study, Pharmacology study
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involvement of Phosphodiesterase 4D (PDE4D) gene in cerebrovascular diseases. Recent advancements have evaluated the role of this gene in stroke and these studies have provided a stronger support for the involvement of this gene in stroke development and few studies also suggest that it may influence outcome. Further, case-control studies and meta-analysis studies have provided strong evidence for certain variants in PDE4D to predispose to stroke only among certain ethnicities. Thus, this review focuses on recent progress made in PDE4D gene research involving genetic, molecular and pharmacological aspect. A strong conclusion has emerged that clearly indicates a pivotal role played by this gene in ischemic stroke globally. Studies have also noticeably highlighted that PDE4D gene/pathway can be a suitable drug target for managing stroke however, a more comprehensive research is still required to understand the molecular and cellular intricacies this gene plays in stroke development, progression and its outcome.
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Stroke is more of a clinical syndrome rather than a disease due to influence of complex family of traits such as genetic epidemiology, diabetes, obesity, hypertension, cardiovascular disease, schizophrenia, and autoimmune diseases [1]. It is of two types i.e ischemic stroke (IS) and hemorrhagic stroke, with an estimated incidence of 80% for IS. Various epidemiological studies in families and twins have revealed that genetic predisposition to stroke does occur. In recent years there have been several parallels of research to identify the functional variants of many candidate genes in association with stroke. One such widely studied plausible candidate gene is Phosphodiesterase 4D (PDE4D) which was identified via genome-wide association studies (GWAS) by the deCODE group and soon became a research hot-spot. This large gene has been mapped on to chromosome 5q12, spanning a region of >1.5mb; is known to have 22 exons, 8 splice variants and hundreds of gene variants [2].
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It belongs to a large superfamily of phosphodiesterases (PDEs) (PDE1PDE11) that encode for phosphodiesterase enzyme which regulates the cAMP levels in the body and is also the key signal transduction molecule in multiple cells including vascular smooth muscle cells (VSMCs) [3]. The four major families that play an important role in VSMCs are PDE1, PDE3, PDE4 and PDE5 of which PDE3 and PDE4 are most responsible for cAMP-hydrolyzing activity [4, 5]. PDE4 is further known to be consisting of four subfamilies (PDE4A, 4B, 4C and 4D) of which PDE4D expresses itself in different cell types, including arterial VSMCs where it degrades the secondary messenger molecule cAMP [6-9]. This decrease in cAMP level increases the proliferation and migration of VSMCs which is a central event in the development of atherosclerosis [10, 11]. The GWAS carried out by Gretarsdottir et al. (2003), examined 260 SNPs of which 6 were found to associate significantly with stroke after adjustment for multiple comparisons [2]. Many studies were subsequently undertaken in different populations of different ethnicities but with inconsistent results [12-19]. In our earlier review on PDE4D gene, we discussed about the work on different variants of this gene in association with IS evaluated in different population till 2007 [20]. Most of the studies then did not point towards any specific SNP to predispose to stroke, although the association was found to be more pronounced towards the cardioembolic IS subtype. Nevertheless, PDE4D gene continues to be a gene of prime importance in stroke research and therefore, this review was undertaken to highlight the
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INTRODUCTION
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discoveries/advancements made in PDE4D gene research from 2007 onwards to derive a better conclusion on the latest research and status of this gene among different populations.
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Case-control studies occupy the largest repository of data in genetic epidemiology. Such studies are helpful in deriving appropriate conclusions about the association of studied genes between cases (patients) and controls (healthy individuals). However, this kind of study is delicate and at times the most difficult, since proper age and sex matched control is needed for each case used in the study. From 2007 onwards, lot of such association studies have been published in different population groups (Table 1) and most of these have been carried out in Chinese population. Liu et al. (2013) investigated the linkage and association of SNP83 of PDE4D gene among IS patients and its related traits in 276 IS families from China and reported a probable association for allele ‘C’ of SNP83 with carotid intima media thickness (cIMT). Further, they also reported abnormal serum lipid, blood pressure and increasing cIMT to be associated with stroke [21]. Shao et al. (2013) evaluated the association of 3 SNPs (SNP83, SNP87 and SNP45) with IS among Chinese Han (Southeastern Chinese population) but could not establish an association [22]. He et al., (2013) on the other hand investigated the association of rs918592A/G and rs2910829C/T among young (G, however, analysis for this SNP did not report any significant association with IS [30, 13]. Kalita et al. (2011) on the other hand studied SNP32, 83 and 87 among North Indians and found frequency of CC genotype of SNP83 to be significantly more among IS patients. In case of SNP87 the frequency of TT genotype was found to be more among patients with abnormal intracranial MR angiography but its significance was lost after adjustment for risk factors. However, the genotypic and allelic frequency of SNP32 was not significantly related with stroke [31]. Milton et al. (2011) evaluated 6 SNPs (SNP13, 19, rs152312, 45, 83 and 87) in a well existing genetic database of Australian cohort and established the association with cardioembolic stroke for rs152312 and SNP45 [32]. Bondarenko et al. (2011, 2010) evaluated the role of SNP41 and 87 among Moldavians and population from Moscow and reported SNP41 to be associated with IS but not SNP87 in both the populations [33, 34]. A similar association of SNP41 but not SNP87 has been reported by Skvortsova et al. (2011) among IS subjects from Moscow [35]. Domingues-Montanari et al. (2010) evaluated the role of 5 SNPs (SNP41, 45, 56, 87 and 89) in two different subsets from Spain and Portugal but did not find association for any of the PDE4D variants [36]. Among the Tawainese, Liao et al. (2010) demonstrated a sex-differential effect of rs702553 among younger male stroke patients. This SNP was also found to influence cIMT, plaque index and stroke and thus highlighted the role of PDE4D in various aspects of atherogenesis [37]. Matsushita et al. (2009) studied the association of PDE4D among Japanese employing 3 methods- single marker, 5
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Meta-analysis studies
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Meta-analysis reports have been instrumental in collating data from all type of case-control studies involving different population groups, large and small sample sizes. However, with larger sample size meta-analysis has the potential to detect small effects in association studies [40]. A total of 6 meta-analysis reports have been documented since 2007 which has been summarised in Table 2. Yan et al. (2014) in their meta-analysis employed 25 studies to study the role of SNP83 among Chinese, Asians and Caucasians and reported a positive association for this variant among Chinese and Asians but not among Caucasians [41]. Liu et al. (2013) in their study for SNP83 variant among Chinese population employed 9 case-control studies and reported positive association for SNP83 with susceptibility to IS [42]. Yadav et al. (2013) on the other hand carried out a meta-analysis among South Asians and their study suggested SNP83 to be a significant risk factor for developing stroke [43]. However, Yoon et al. (2011) in their meta-analysis involving studies from different ethnic population reported only SNP56 to be associated with risk of IS and SNP83 to have a protective association with stroke [44]. Another study by Xu et al. (2010) too reported SNP83 to be an important marker for ischemic infarction among Asians [45]. In contrast to these findings, a met-analysis carried out by Bevan et al. (2008) involving 6 SNPs (SNP26, 45, 56, 83, 87 and 89) reported none of the genetic variants to have a reproducible association [46].
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haplotype and tag-single nucleotide polymorphism. In the single marker association test no significant association was found for 6 SNPs (SNP41, 45, 56, 83, 87 and 89) with stroke and its subtypes. Haplotype analysis also did not reveal any significant association and tag-SNP analysis found rs7730070, a SNP around 3’ end of PDE4D to be probably associated, but was reported to be nonsignificant on further testing [14]. A study among Koreans by Kim et al. (2008) involving SNP41, reported this SNP not to be a major risk factor for noncardiogenic IS [38]. Evaluation of four SNPs (SNP32, 45, 83 and 87) by Quarta et al. (2009) among Sardinians- a genetically homogenous population also did not reveal any significant association with any of the 4 variants [39].
Expression studies
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In the past decade there has been a surge in molecular genetics study in complex diseases and sophisticated techniques have been useful in deciphering the intricate role of genes and their expression, in understanding the molecular changes undergoing in such disease conditions. However, studies utilising molecular methods have been very few in cerebrovascular diseases including stroke. He et al. (2012) in their PDE4D study were the first to describe the changes in microvascular PDE4D expression since very little information is available on PDE4D function following cerebral ischemia. PDE4D is known to be expressed in cerebral microvessels and therefore, could be involved in regulating blood-brain barrier (BBB) permeability and to assess this, they measured the microvascular PDE4D expression, parenchymal albumin immunoreactivity and changes within the inside bore of brain microvasculature. This experiment was carried out on male Fischer young (4 months) and aged (24 months) rats subjected to sham surgery. Severe hippocampal CA1 region damage associated with significant increase in vascular PDE4D expression and parenchymal albumin immunoreactivities was seen among the younger animals. However, tissue density decrease in perimicrovascular space, increase in internal bore circumference and cross-sectional area of the hippocampal microvessels were reported among both the animals. Thus, this study reported increase in PDE4D expression following cerebral ischemia which can play a role in BBB permeability and thus affect ischemic outcome [47]. Yang et al. (2012) studied the in vitro effects of PDE inhibition and reported a potential relationship between PDE4 inhibition and endothelial tPA expression. This study demonstrated that rolipram (PDE4 inhibitor) decreased endothelial tPA expression and PDE4D siRNA reduced endothelial tPA mRNA to 40-50% in an in vitro ischemia model. Additionally, the study suggests a new role for Epac (cAMP effector) that regulates biological processes including endothelial barrier properties and vascular permeability. Activation of Epac mimicked the effects of rolipram while inhibiting it attenuated the effects of rolipram and PDE4D siRNA. Thus, it was found that the regulation of brain microvascular tPA expression by PDE4/PDE4D is mediated by Epac [48]. Further, a very recent study by the same group reported the role of PDE4 pathway in stroke pathogenesis independent of PDE4D isoform. The striking results of this investigation revealed a novel hypothesis that suggests a different isoform of PDE4 other than the much anticipated PDE4D to modulate stroke risk [49]. Another study by Liu et al. (2011) in the PDE4D gene confirmed that Plateletderived growth factor (PDGF) caused proliferation of VSMCs and that there 7
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was 30% reduction of PDGF-induced cell proliferation in PDE4D deficient VSMCs. Thus, in their study they infected VSMCs with lentivirus particles harbouring shRNA directed against PDE4D which significantly inhibited induced-PDGF, VSMC proliferation, and migration. Additionally they also found that the inhibitory effects were not associated with global intracellular cAMP levels. [50].
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This kind of study represents a different facet in stroke research and provides significant input for novel therapeutic strategy. Chen et al. (2011) in their study looked for solutions that could eliminate the risk factors that lead to stroke. Since uncontrolled PDE4D activities often lead to cAMP-induced stroke and cardiovascular disease they identified and analysed dual –targeting compounds that could reduce PDE4D and ALOX5AP (involved in inflammation pathway) activities from a traditional Chinese medicine (TCM) database. They used world’s largest TCM database for in silico drug identification and introduced a machine predictive model and pharmacophore model for characterizing drug like candidates. Myristic acid and pentadecanoic acid were identified and further analysis on molecular dynamics simulation determined the stable molecular interactions. The carboxyl group and hydrophobic regions were identified as the potent dual targets that inhibit PDE4D and ALOX5AP activities [51]. Another study by Chang et al. (2011) suggested that 3 compounds from ginger family were capable in inhibiting cAMP-binding and hydrolysis by PDE4D. In their pursuit of finding PDE4D inhibitors they screened compounds from TCM database followed by modelling techniques, molecular dynamics simulation and HypoGen validation. They also characterized the ligand binding properties that were associated with inhibition of PDE4D [52].
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Pharmacology studies
CONCLUSION Significant advances have been made in study on PDE4D gene from 2007 onwards and they have provided some fundamental insights into the pathogenesis and therapeutic interventions of stroke. Several case-control studies in different populations have been carried out that have provided genetic evidence for association of PDE4D gene with stroke. A number of SNPs in PDE4D gene have been studied as part of case-control approach of which many 8
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Rosand and co-workers published a paper in 2006 that provided a very serious critique of PDE4D as a stroke risk factor. The major critique was that the challenge for the study of complex disorders like stroke is that, given the possibility of large number of SNPs in the genome the probability of any one responsible for disease is highly unlikely. Additionally, such studies require confirmation in model systems and reproducible associations as demonstrated in other complex diseases like age-related macular degeneration and type2 diabetes where replication studies have confirmed one identical SNP in multiple population samples from different countries. We do not totally disagree with Rosand et al (2006) [54]. However, numerous studies published as a follow-up to the original article by deCODE group are divided between those that claim replication and those who did not. The discrepancies in the results might be on account of the ethnic differences and other factors. Therefore, investigations are warranted to evaluate possible ethnic specific effects. In spite of all these discrepancies the polymorphisms of PDE4D have emerged as significant risk factors for stroke among different ethnic groups. With a number of association studies with various sample size and variable differences the outcome of association results can lead to discrepancies and as such an investigation using a meta-analysis approach to study them is a better and a highly effective technique.
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associate it as a risk factor for stroke among different ethnicities. Nonetheless, false positive outcome as a result of selection bias in cases and controls and population admixture cannot be ruled out. Hence, in any genetic study a proper case-control design with sufficient sample size, proper variables (risk factors) and observed genotypes and alleles following Hardy-Weinberg equilibrium is a prerequisite for appropriate analysis. Further, genotyping error that can substantially contribute to wrong interpretation of statistical data should be paid utmost attention too [53].
Meta-analysis reports although useful in deriving a better conclusion in genetic epidemiology can nonetheless provide erroneous results if combined for different ethnic populations especially when a particular group has unique genetically homogenous structure [55]. Nevertheless, they may provide true association although the validity for individual candidate gene remains uncertain. In the current scenario for the role of PDE4D gene in the development of stroke, SNP 83 seems to be more reproducible in its association with stroke especially among the Chinese and Asians. Additionally, it has been 9
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On the other hand, investigations using molecular and expression studies involving in vitro system and animal models have provided the needed support for involvement of PDE4D in stroke. The results obtained clearly point towards a definite role played by this gene in stroke development, severity and its outcome but a recent report by Yang et al. (2014) suggests involvement of other PDE4 isoforms for stroke risk. Further, in silico pharmacology studies have evaluated natural compounds that are capable of inhibiting cAMP and hydrolysing PDE4D. These recent studies clearly present PDE4D as a potential drug target for stroke prevention. Nevertheless, future studies are needed that can focus on identifying the detailed molecular mechanisms for deducing the pathway in which PDE4D gene is involved in development of stroke.
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In conclusion, studies on different aspects of PDE4D gene and PDE4 pathway have provided a strong evidence for its role in stroke development. However, further advanced studies employing a new and comprehensive genomic, molecular and proteomic approach needs to be engaged that can revolutionize the understanding of PDE4 pathway/PDE4D gene in stroke pathophysiology in a better way. Such methods will also be instrumental in generating novel hypothesis and can possibly open options for new therapeutic targets.
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found that meta-analysis reports too can be compromised due to partial genotyping of overlapping marker sets and so to overcome this, a multi-locus Bayesian meta-analysis approach was proposed that revealed no association despite increase in statistical power [56]. This method provided the most thorough meta-analysis for PDE4D association study in stroke which was consistent with the results obtained by Bevan et al. (2008) [46].
Declaration of interest: The authors declare that they have no conflicts of interest.
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49. Yang F, Sumbria RK, Xue D, Yu C, He D, Liu S, Paganini-Hill A, Fisher M. Effects of PDE4 pathway inhibition in rat experimental stroke. J Pharm Pharm Sci. 2014;17:362-70. 50. Liu L, Xu X, Li J, Li X, Sheng W. Lentiviral-Mediated shRNA Silencing of PDE4D Gene Inhibits Platelet-Derived Growth Factor-Induced Proliferation and Migration of Rat Aortic Smooth Muscle Cells. Stroke Res Treat. 2011; 2011:534257. 51. Chen KC, Chang KW, Chen HY, Chen CY. Traditional Chinese medicine, a solution for reducing dual stroke risk factors at once? Mol Biosyst. 2011;7:271119.
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53. Kumar A, Sagar R, Kumar P, Sahu JK, Grover A, Srivastava AK, Vivekanandhan S, Prasad K. Identification of genetic contribution to ischemic stroke by screening of single nucleotide polymorphisms in stroke patients by using a case control study design. BMC Neurol. 2013;13:136-43.
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54. Rosand J, Bayley N, Rost N, de Bakker PI. Many hypotheses but no replication for the association between PDE4D and stroke.Nat Genet. 2006;38:1091-92. 55. Reich D, Thangaraj K, Patterson N, Price AL, Singh L. Reconstructing Indian population history. Nature 2009;461:489–94.
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56. Newcombe PJ, Verzilli C, Casas JP, Hingorani AD, Smeeth L, Whittaker JC. Multilocus Bayesian meta-analysis of gene-disease associations. Am J Hum Genet. 2009;84:567–80.
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52. Chang TT, Chen KC, Chang KW, Chen HY, Tsai FJ, Sun MF, Chen CY. In silico pharmacology suggests ginger extracts may reduce stroke risks. Mol Biosyst. 2011;7:2702-10.
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Reference
Population
SNP studied
1 2
Liu et al. (2013) Shao et al. (2013)
SNP83 SNPs83, 87 and 45
3
He et al. (2013)
Chinese South Eastern Han Chinese Chinese
4
He et al. (2012)
Henan Han
5
Zhao et al. (2012)
6 7
Li et al. (2010) Xue et al. (2009)
Northern Han Chinese Chinese Han Chinese Han
8
Lokvkist et al. (2012)
9
Munshi et al. (2012, 2009) Kalita et al. (2011) Milton et al. (2011)
12 13
AT haplotype of rs918592 and rs2910829 rs918592 and AT haplotype of rs2910829 rs918592 and rs2910829 SNPs83, 87, 219 AA haplotype of and 220 SNP219 and 220 SNP83 and 87 SNP83 SNPs32, 83 and 87 SNP83 and haplotype GCC SNP45 SNP45 – only in small population size SNPs41, 56, 83 and SNPs41, 56 and 83 87 SNPs32, 83 and 87 SNP83 SNPs13, 19, 45, SNP45 and 83, 87 and rs152312 rs152312 SNP41 and 87 SNP41
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rs918592 and rs2910829
C
AC
ST
10 11
Swedish
Risk associated SNP SNP83 None
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S.I No
South Indians North Indians Australians
Bondarenko et al. (2011, 2010) Skvortsova et al. (2011) DominguesMontanari et al. (2010) Lioa et al. (2010)
Moldavians and Moscow Moscow
SNP41 and 87
SNP41
Spanish and Portugals
SNPs41, 45, 56, 87 and 89
None
Tawainese
rs702553
Japanese
17
Matsushita et al. (2009) Kim et al. (2008)
SNPs41, 45, 56, 83, 87 and 89 SNP41
rs702553 – male patients None
18
Quarta et al. (2009)
Sardinians
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Table 1 Case-Control studies in PDE4D SNPs in different populations after 2007
15 16
Koreans
None
SNPs32, 45, 83 and None 87
15
Table 2 Meta-analysis reports involving PDE4D gene role in stroke among different
Reference
Population
SNP studied
1
Yan et al. (2014)
SNP83
2. 3.
Liu et al. (2013) Yadav et al. (2013)
Chinese, Asians and Caucasians Chinese South Asians
4.
Yoon et al. (2011)
Multiple ethnicities
5. 6.
Xu et al. (2010) Bevan et al. (2008)
Asians Multiple ethnicities
SNPs26, 45,56, 83, 87 and 89 SNP83 SNPs26, 45, 56, 83, 87 and 89
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SNP83 SNPs83, 32 and 87
C AC ST 16
Nature of risk association Positive among Chinese and Asians Positive Positive for SNP83
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S.I No
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populations after 2007
Positive for SNP56 Positive None found to be associated
Association between PDE4D gene and ischemic stroke: Recent Advancements Satrupa Das, Sitara Roy, Anjana Munshi doi:10.3109/00207454.2015.1051621
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ABSTRACT Stroke is a severe complication and a leading cause of death worldwide and genetic studies among different ethnicities has provided the basis for involvement of Phosphodiesterase 4D (PDE4D) gene in cerebrovascular diseases. Recent advancements have evaluated the role of this gene in stroke and these studies have provided a stronger support for the involvement of this gene in stroke development and few studies also suggest that it may influence outcome. Further, case-control studies and meta-analysis studies have provided strong evidence for certain variants in PDE4D to predispose to stroke only among certain ethnicities. Thus, this review focuses on recent progress made in PDE4D gene research involving genetic, molecular and pharmacological aspect. A strong conclusion has emerged that clearly indicates a pivotal role played by this gene in ischemic stroke globally. Studies have also noticeably highlighted that PDE4D gene/pathway can be a suitable drug target for managing stroke however, a more comprehensive research is still required to understand the molecular and cellular intricacies this gene plays in stroke development, progression and its outcome.
© 2015 Informa Healthcare USA, Inc. This provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. DISCLAIMER: The ideas and opinions expressed in the journal’s Just Accepted articles do not necessarily reflect those of Informa Healthcare (the Publisher), the Editors or the journal. The Publisher does not assume any responsibility for any injury and/or damage to persons or property arising from or related to any use of the material contained in these articles. The reader is advised to check the appropriate medical literature and the product information currently provided by the manufacturer of each drug to be administered to verify the dosages, the method and duration of administration, and contraindications. It is the responsibility of the treating physician or other health care professional, relying on his or her independent experience and knowledge of the patient, to determine drug dosages and the best treatment for the patient. Just Accepted have undergone full scientific review but none of the additional editorial preparation, such as copyediting, typesetting, and proofreading, as have articles published in the traditional manner. There may, therefore, be errors in Just Accepted articles that will be corrected in the final print and final online version of the article. Any use of the Just Accepted articles is subject to the express understanding that the papers have not yet gone through the full quality control process prior to publication.
Publisher: Taylor & Francis
Journal: International Journal of Neuroscience
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DOI: http://dx.doi.org/10.3109/00207454.2015.1051621
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Satrupa Das, Sitara Roy, Anjana Munshi * Satrupa Das, Sitara Roy
Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad-500016, India.
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Dr. NTR University of Health Sciences,Vijayawada, Andhra Pradesh, India.
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Anjana Munshi *
Centre for Human Genetics, School of Health Sciences, Central University of Punjab, Bathinda, Punjab, India.
Corresponding author:
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*
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Association between PDE4D gene and ischemic stroke: Recent Advancements
Dr. Anjana Munshi Centre for Human Genetics School of Health Sciences Central university of Punjab, Bathinda Punjab, India. Email address: [email protected] Telephone No- +91-9872694373
ABSTRACT
Stroke is a severe complication and a leading cause of death worldwide and genetic studies among different ethnicities has provided the basis for 1
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KEYWORDS: Phosphodiesterase 4D gene, genetics, ischemic stroke, casecontrol study, meta-analysis study, expression study, Pharmacology study
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involvement of Phosphodiesterase 4D (PDE4D) gene in cerebrovascular diseases. Recent advancements have evaluated the role of this gene in stroke and these studies have provided a stronger support for the involvement of this gene in stroke development and few studies also suggest that it may influence outcome. Further, case-control studies and meta-analysis studies have provided strong evidence for certain variants in PDE4D to predispose to stroke only among certain ethnicities. Thus, this review focuses on recent progress made in PDE4D gene research involving genetic, molecular and pharmacological aspect. A strong conclusion has emerged that clearly indicates a pivotal role played by this gene in ischemic stroke globally. Studies have also noticeably highlighted that PDE4D gene/pathway can be a suitable drug target for managing stroke however, a more comprehensive research is still required to understand the molecular and cellular intricacies this gene plays in stroke development, progression and its outcome.
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Stroke is more of a clinical syndrome rather than a disease due to influence of complex family of traits such as genetic epidemiology, diabetes, obesity, hypertension, cardiovascular disease, schizophrenia, and autoimmune diseases [1]. It is of two types i.e ischemic stroke (IS) and hemorrhagic stroke, with an estimated incidence of 80% for IS. Various epidemiological studies in families and twins have revealed that genetic predisposition to stroke does occur. In recent years there have been several parallels of research to identify the functional variants of many candidate genes in association with stroke. One such widely studied plausible candidate gene is Phosphodiesterase 4D (PDE4D) which was identified via genome-wide association studies (GWAS) by the deCODE group and soon became a research hot-spot. This large gene has been mapped on to chromosome 5q12, spanning a region of >1.5mb; is known to have 22 exons, 8 splice variants and hundreds of gene variants [2].
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It belongs to a large superfamily of phosphodiesterases (PDEs) (PDE1PDE11) that encode for phosphodiesterase enzyme which regulates the cAMP levels in the body and is also the key signal transduction molecule in multiple cells including vascular smooth muscle cells (VSMCs) [3]. The four major families that play an important role in VSMCs are PDE1, PDE3, PDE4 and PDE5 of which PDE3 and PDE4 are most responsible for cAMP-hydrolyzing activity [4, 5]. PDE4 is further known to be consisting of four subfamilies (PDE4A, 4B, 4C and 4D) of which PDE4D expresses itself in different cell types, including arterial VSMCs where it degrades the secondary messenger molecule cAMP [6-9]. This decrease in cAMP level increases the proliferation and migration of VSMCs which is a central event in the development of atherosclerosis [10, 11]. The GWAS carried out by Gretarsdottir et al. (2003), examined 260 SNPs of which 6 were found to associate significantly with stroke after adjustment for multiple comparisons [2]. Many studies were subsequently undertaken in different populations of different ethnicities but with inconsistent results [12-19]. In our earlier review on PDE4D gene, we discussed about the work on different variants of this gene in association with IS evaluated in different population till 2007 [20]. Most of the studies then did not point towards any specific SNP to predispose to stroke, although the association was found to be more pronounced towards the cardioembolic IS subtype. Nevertheless, PDE4D gene continues to be a gene of prime importance in stroke research and therefore, this review was undertaken to highlight the
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INTRODUCTION
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discoveries/advancements made in PDE4D gene research from 2007 onwards to derive a better conclusion on the latest research and status of this gene among different populations.
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Case-control studies occupy the largest repository of data in genetic epidemiology. Such studies are helpful in deriving appropriate conclusions about the association of studied genes between cases (patients) and controls (healthy individuals). However, this kind of study is delicate and at times the most difficult, since proper age and sex matched control is needed for each case used in the study. From 2007 onwards, lot of such association studies have been published in different population groups (Table 1) and most of these have been carried out in Chinese population. Liu et al. (2013) investigated the linkage and association of SNP83 of PDE4D gene among IS patients and its related traits in 276 IS families from China and reported a probable association for allele ‘C’ of SNP83 with carotid intima media thickness (cIMT). Further, they also reported abnormal serum lipid, blood pressure and increasing cIMT to be associated with stroke [21]. Shao et al. (2013) evaluated the association of 3 SNPs (SNP83, SNP87 and SNP45) with IS among Chinese Han (Southeastern Chinese population) but could not establish an association [22]. He et al., (2013) on the other hand investigated the association of rs918592A/G and rs2910829C/T among young (G, however, analysis for this SNP did not report any significant association with IS [30, 13]. Kalita et al. (2011) on the other hand studied SNP32, 83 and 87 among North Indians and found frequency of CC genotype of SNP83 to be significantly more among IS patients. In case of SNP87 the frequency of TT genotype was found to be more among patients with abnormal intracranial MR angiography but its significance was lost after adjustment for risk factors. However, the genotypic and allelic frequency of SNP32 was not significantly related with stroke [31]. Milton et al. (2011) evaluated 6 SNPs (SNP13, 19, rs152312, 45, 83 and 87) in a well existing genetic database of Australian cohort and established the association with cardioembolic stroke for rs152312 and SNP45 [32]. Bondarenko et al. (2011, 2010) evaluated the role of SNP41 and 87 among Moldavians and population from Moscow and reported SNP41 to be associated with IS but not SNP87 in both the populations [33, 34]. A similar association of SNP41 but not SNP87 has been reported by Skvortsova et al. (2011) among IS subjects from Moscow [35]. Domingues-Montanari et al. (2010) evaluated the role of 5 SNPs (SNP41, 45, 56, 87 and 89) in two different subsets from Spain and Portugal but did not find association for any of the PDE4D variants [36]. Among the Tawainese, Liao et al. (2010) demonstrated a sex-differential effect of rs702553 among younger male stroke patients. This SNP was also found to influence cIMT, plaque index and stroke and thus highlighted the role of PDE4D in various aspects of atherogenesis [37]. Matsushita et al. (2009) studied the association of PDE4D among Japanese employing 3 methods- single marker, 5
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Meta-analysis studies
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Meta-analysis reports have been instrumental in collating data from all type of case-control studies involving different population groups, large and small sample sizes. However, with larger sample size meta-analysis has the potential to detect small effects in association studies [40]. A total of 6 meta-analysis reports have been documented since 2007 which has been summarised in Table 2. Yan et al. (2014) in their meta-analysis employed 25 studies to study the role of SNP83 among Chinese, Asians and Caucasians and reported a positive association for this variant among Chinese and Asians but not among Caucasians [41]. Liu et al. (2013) in their study for SNP83 variant among Chinese population employed 9 case-control studies and reported positive association for SNP83 with susceptibility to IS [42]. Yadav et al. (2013) on the other hand carried out a meta-analysis among South Asians and their study suggested SNP83 to be a significant risk factor for developing stroke [43]. However, Yoon et al. (2011) in their meta-analysis involving studies from different ethnic population reported only SNP56 to be associated with risk of IS and SNP83 to have a protective association with stroke [44]. Another study by Xu et al. (2010) too reported SNP83 to be an important marker for ischemic infarction among Asians [45]. In contrast to these findings, a met-analysis carried out by Bevan et al. (2008) involving 6 SNPs (SNP26, 45, 56, 83, 87 and 89) reported none of the genetic variants to have a reproducible association [46].
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haplotype and tag-single nucleotide polymorphism. In the single marker association test no significant association was found for 6 SNPs (SNP41, 45, 56, 83, 87 and 89) with stroke and its subtypes. Haplotype analysis also did not reveal any significant association and tag-SNP analysis found rs7730070, a SNP around 3’ end of PDE4D to be probably associated, but was reported to be nonsignificant on further testing [14]. A study among Koreans by Kim et al. (2008) involving SNP41, reported this SNP not to be a major risk factor for noncardiogenic IS [38]. Evaluation of four SNPs (SNP32, 45, 83 and 87) by Quarta et al. (2009) among Sardinians- a genetically homogenous population also did not reveal any significant association with any of the 4 variants [39].
Expression studies
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In the past decade there has been a surge in molecular genetics study in complex diseases and sophisticated techniques have been useful in deciphering the intricate role of genes and their expression, in understanding the molecular changes undergoing in such disease conditions. However, studies utilising molecular methods have been very few in cerebrovascular diseases including stroke. He et al. (2012) in their PDE4D study were the first to describe the changes in microvascular PDE4D expression since very little information is available on PDE4D function following cerebral ischemia. PDE4D is known to be expressed in cerebral microvessels and therefore, could be involved in regulating blood-brain barrier (BBB) permeability and to assess this, they measured the microvascular PDE4D expression, parenchymal albumin immunoreactivity and changes within the inside bore of brain microvasculature. This experiment was carried out on male Fischer young (4 months) and aged (24 months) rats subjected to sham surgery. Severe hippocampal CA1 region damage associated with significant increase in vascular PDE4D expression and parenchymal albumin immunoreactivities was seen among the younger animals. However, tissue density decrease in perimicrovascular space, increase in internal bore circumference and cross-sectional area of the hippocampal microvessels were reported among both the animals. Thus, this study reported increase in PDE4D expression following cerebral ischemia which can play a role in BBB permeability and thus affect ischemic outcome [47]. Yang et al. (2012) studied the in vitro effects of PDE inhibition and reported a potential relationship between PDE4 inhibition and endothelial tPA expression. This study demonstrated that rolipram (PDE4 inhibitor) decreased endothelial tPA expression and PDE4D siRNA reduced endothelial tPA mRNA to 40-50% in an in vitro ischemia model. Additionally, the study suggests a new role for Epac (cAMP effector) that regulates biological processes including endothelial barrier properties and vascular permeability. Activation of Epac mimicked the effects of rolipram while inhibiting it attenuated the effects of rolipram and PDE4D siRNA. Thus, it was found that the regulation of brain microvascular tPA expression by PDE4/PDE4D is mediated by Epac [48]. Further, a very recent study by the same group reported the role of PDE4 pathway in stroke pathogenesis independent of PDE4D isoform. The striking results of this investigation revealed a novel hypothesis that suggests a different isoform of PDE4 other than the much anticipated PDE4D to modulate stroke risk [49]. Another study by Liu et al. (2011) in the PDE4D gene confirmed that Plateletderived growth factor (PDGF) caused proliferation of VSMCs and that there 7
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was 30% reduction of PDGF-induced cell proliferation in PDE4D deficient VSMCs. Thus, in their study they infected VSMCs with lentivirus particles harbouring shRNA directed against PDE4D which significantly inhibited induced-PDGF, VSMC proliferation, and migration. Additionally they also found that the inhibitory effects were not associated with global intracellular cAMP levels. [50].
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This kind of study represents a different facet in stroke research and provides significant input for novel therapeutic strategy. Chen et al. (2011) in their study looked for solutions that could eliminate the risk factors that lead to stroke. Since uncontrolled PDE4D activities often lead to cAMP-induced stroke and cardiovascular disease they identified and analysed dual –targeting compounds that could reduce PDE4D and ALOX5AP (involved in inflammation pathway) activities from a traditional Chinese medicine (TCM) database. They used world’s largest TCM database for in silico drug identification and introduced a machine predictive model and pharmacophore model for characterizing drug like candidates. Myristic acid and pentadecanoic acid were identified and further analysis on molecular dynamics simulation determined the stable molecular interactions. The carboxyl group and hydrophobic regions were identified as the potent dual targets that inhibit PDE4D and ALOX5AP activities [51]. Another study by Chang et al. (2011) suggested that 3 compounds from ginger family were capable in inhibiting cAMP-binding and hydrolysis by PDE4D. In their pursuit of finding PDE4D inhibitors they screened compounds from TCM database followed by modelling techniques, molecular dynamics simulation and HypoGen validation. They also characterized the ligand binding properties that were associated with inhibition of PDE4D [52].
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Pharmacology studies
CONCLUSION Significant advances have been made in study on PDE4D gene from 2007 onwards and they have provided some fundamental insights into the pathogenesis and therapeutic interventions of stroke. Several case-control studies in different populations have been carried out that have provided genetic evidence for association of PDE4D gene with stroke. A number of SNPs in PDE4D gene have been studied as part of case-control approach of which many 8
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Rosand and co-workers published a paper in 2006 that provided a very serious critique of PDE4D as a stroke risk factor. The major critique was that the challenge for the study of complex disorders like stroke is that, given the possibility of large number of SNPs in the genome the probability of any one responsible for disease is highly unlikely. Additionally, such studies require confirmation in model systems and reproducible associations as demonstrated in other complex diseases like age-related macular degeneration and type2 diabetes where replication studies have confirmed one identical SNP in multiple population samples from different countries. We do not totally disagree with Rosand et al (2006) [54]. However, numerous studies published as a follow-up to the original article by deCODE group are divided between those that claim replication and those who did not. The discrepancies in the results might be on account of the ethnic differences and other factors. Therefore, investigations are warranted to evaluate possible ethnic specific effects. In spite of all these discrepancies the polymorphisms of PDE4D have emerged as significant risk factors for stroke among different ethnic groups. With a number of association studies with various sample size and variable differences the outcome of association results can lead to discrepancies and as such an investigation using a meta-analysis approach to study them is a better and a highly effective technique.
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associate it as a risk factor for stroke among different ethnicities. Nonetheless, false positive outcome as a result of selection bias in cases and controls and population admixture cannot be ruled out. Hence, in any genetic study a proper case-control design with sufficient sample size, proper variables (risk factors) and observed genotypes and alleles following Hardy-Weinberg equilibrium is a prerequisite for appropriate analysis. Further, genotyping error that can substantially contribute to wrong interpretation of statistical data should be paid utmost attention too [53].
Meta-analysis reports although useful in deriving a better conclusion in genetic epidemiology can nonetheless provide erroneous results if combined for different ethnic populations especially when a particular group has unique genetically homogenous structure [55]. Nevertheless, they may provide true association although the validity for individual candidate gene remains uncertain. In the current scenario for the role of PDE4D gene in the development of stroke, SNP 83 seems to be more reproducible in its association with stroke especially among the Chinese and Asians. Additionally, it has been 9
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On the other hand, investigations using molecular and expression studies involving in vitro system and animal models have provided the needed support for involvement of PDE4D in stroke. The results obtained clearly point towards a definite role played by this gene in stroke development, severity and its outcome but a recent report by Yang et al. (2014) suggests involvement of other PDE4 isoforms for stroke risk. Further, in silico pharmacology studies have evaluated natural compounds that are capable of inhibiting cAMP and hydrolysing PDE4D. These recent studies clearly present PDE4D as a potential drug target for stroke prevention. Nevertheless, future studies are needed that can focus on identifying the detailed molecular mechanisms for deducing the pathway in which PDE4D gene is involved in development of stroke.
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In conclusion, studies on different aspects of PDE4D gene and PDE4 pathway have provided a strong evidence for its role in stroke development. However, further advanced studies employing a new and comprehensive genomic, molecular and proteomic approach needs to be engaged that can revolutionize the understanding of PDE4 pathway/PDE4D gene in stroke pathophysiology in a better way. Such methods will also be instrumental in generating novel hypothesis and can possibly open options for new therapeutic targets.
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found that meta-analysis reports too can be compromised due to partial genotyping of overlapping marker sets and so to overcome this, a multi-locus Bayesian meta-analysis approach was proposed that revealed no association despite increase in statistical power [56]. This method provided the most thorough meta-analysis for PDE4D association study in stroke which was consistent with the results obtained by Bevan et al. (2008) [46].
Declaration of interest: The authors declare that they have no conflicts of interest.
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1. Worrall BB, Mychaleckyj JC. PDE4D and Stroke: A Real Advance or a Case of the Emperor’s New Clothes? Stroke 2006;37:1955–57. 2. Gretarsdottir S, Thorleifsson G, Reynisdottir ST et al. The gene encoding phosphodiesterase 4D confers risk of ischemic stroke. Nature Genetics 2003; 35:131–38. 3. Essayan DM. Cyclic nucleotide phosphodiesterases. J Allergy Clin Immunol. 2001;108:671–80. 4. Kim D, Rybalkin SD, Pi X et al. Upregulation of phosphodiesterase 1A1 expression is associated with the development of nitrate tolerance. Circulation 2001;104:2338– 43. 5. Kim D, Aizawa T, Wei H et al. Angiotensin II increases phosphodiesterase 5A expression in vascular smooth muscle cells: a mechanism by which angiotensin II antagonizes cGMP signaling,. J Mol Cell Cardiol. 2005;38:175–84. 6. Muller T, Engels P, Fozard JR. Subtypes of the type 4 cAMP phosphodiesterases: structure, regulation and selective inhibition. Trends Pharmacol Sci. 1996;17:294– 98. 7. N´emoz G, Zhang R, Sette C, Conti M. Identification of cyclic AMPphosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells. FEBS Lett. 1996;384:97–102. 8. Peter D, Jin SLC, Conti M, Hatzelmann A, Zitt C. Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+ T cells: predominant role of PDE4D. J Immunol. 2007;178:4820–31. 9. Liu H and Maurice DH. Phosphorylation-mediated activation and translocation of the cyclic AMP- specific phosphodiesterase PDE4D3 by cyclic AMP-dependent protein kinase and mitogen-activated protein kinases: a potential mechanism allowing for the coordinated regulation of PDE4D activity and targeting. J Biol Chem. 1999;274:10557–65. 10. Cai X. Regulation of smooth muscle cells in development and vascular disease: current therapeutic strategies. Expert Rev Cardiovas Ther. 2006;4:789–800. 11. Koyama H, Bornfeldt KE, Fukumoto S, Nishizawa Y. Molecular pathways of cyclic nucleotide-induced inhibition of arterial smoothmuscle cell proliferation. J Cell Physiol. 2001;186:1–10. 12. Li N, He Z, Xu J, Liu F, Deng S, Zhang H. Association of PDE4D and IL-1 gene polymorphism with ischemic stroke in a Han Chinese population. Brain Res Bull. 2010;81:38–42. 13. Munshi A, Babu MS, Kaul S et al. Phosphodiesterase 4D (PDE4D) gene variants and the risk of ischemic stroke in a South Indian population. J Neurol Sci. 2009; 285:142–45. 14. Matsushita T, Kubo M, Yonemoto K et al. Lack of association between variations of PDE4D and ischemic stroke in the Japanese population. Stroke 2009;40:1245– 51. 15. Kostulas K, Gretarsdottir S, Kostulas V et al. PDE4D and ALOX5AP genetic variants and risk for Ischemic Cerebrovascular Disease in Sweden. J Neurol Sci. 2007; 263:113–17. 16. Song Q, Cole JW, O’Connell JR et al. Phosphodiesterase 4D polymorphisms and the risk of cerebral infarction in a biracial population: the Stroke Prevention in Young Women Study. Hum Mol Genet. 2006;15:2468–78.
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14
Reference
Population
SNP studied
1 2
Liu et al. (2013) Shao et al. (2013)
SNP83 SNPs83, 87 and 45
3
He et al. (2013)
Chinese South Eastern Han Chinese Chinese
4
He et al. (2012)
Henan Han
5
Zhao et al. (2012)
6 7
Li et al. (2010) Xue et al. (2009)
Northern Han Chinese Chinese Han Chinese Han
8
Lokvkist et al. (2012)
9
Munshi et al. (2012, 2009) Kalita et al. (2011) Milton et al. (2011)
12 13
AT haplotype of rs918592 and rs2910829 rs918592 and AT haplotype of rs2910829 rs918592 and rs2910829 SNPs83, 87, 219 AA haplotype of and 220 SNP219 and 220 SNP83 and 87 SNP83 SNPs32, 83 and 87 SNP83 and haplotype GCC SNP45 SNP45 – only in small population size SNPs41, 56, 83 and SNPs41, 56 and 83 87 SNPs32, 83 and 87 SNP83 SNPs13, 19, 45, SNP45 and 83, 87 and rs152312 rs152312 SNP41 and 87 SNP41
EP
rs918592 and rs2910829
C
AC
ST
10 11
Swedish
Risk associated SNP SNP83 None
TE D
S.I No
South Indians North Indians Australians
Bondarenko et al. (2011, 2010) Skvortsova et al. (2011) DominguesMontanari et al. (2010) Lioa et al. (2010)
Moldavians and Moscow Moscow
SNP41 and 87
SNP41
Spanish and Portugals
SNPs41, 45, 56, 87 and 89
None
Tawainese
rs702553
Japanese
17
Matsushita et al. (2009) Kim et al. (2008)
SNPs41, 45, 56, 83, 87 and 89 SNP41
rs702553 – male patients None
18
Quarta et al. (2009)
Sardinians
14
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Table 1 Case-Control studies in PDE4D SNPs in different populations after 2007
15 16
Koreans
None
SNPs32, 45, 83 and None 87
15
Table 2 Meta-analysis reports involving PDE4D gene role in stroke among different
Reference
Population
SNP studied
1
Yan et al. (2014)
SNP83
2. 3.
Liu et al. (2013) Yadav et al. (2013)
Chinese, Asians and Caucasians Chinese South Asians
4.
Yoon et al. (2011)
Multiple ethnicities
5. 6.
Xu et al. (2010) Bevan et al. (2008)
Asians Multiple ethnicities
SNPs26, 45,56, 83, 87 and 89 SNP83 SNPs26, 45, 56, 83, 87 and 89
EP
SNP83 SNPs83, 32 and 87
C AC ST 16
Nature of risk association Positive among Chinese and Asians Positive Positive for SNP83
TE D
S.I No
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populations after 2007
Positive for SNP56 Positive None found to be associated
