Phosphodiesterase 4D (PDE4D) gene variants and the risk of ischemic stroke in a South Indian population

Journal of the Neurological Sciences 285 (2009) 142–145

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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s

Phosphodiesterase 4D (PDE4D) gene variants and the risk of ischemic stroke in a South Indian population Anjana Munshi a,⁎, M Sai Babu a, Subhash Kaul b, Gowhar Shafi a, A.N. Anila a, Suvarna Alladi b, A. Jyothy a a b

Institute of Genetics and Hospital for Genetic Diseases, Begumpet, Hyderabad-500016, India Nizams Institute of Medical Sciences, Punjagutta, Hyderabad-500082, India

a r t i c l e

i n f o

Article history: Received 5 March 2009 Received in revised form 12 June 2009 Accepted 12 June 2009 Available online 15 July 2009 Keywords: Ischemic stroke Phosphodiesterase 4D gene Intracranial large artery atherosclerosis Small artery occlusion

a b s t r a c t Stroke is the third largest cause of death and a major cause of adult disability and mortality worldwide. Experimental evidence suggests that genetic determinants do contribute a large part to stroke risk. The identification of phosphodiesterase 4D gene as a risk factor for stroke caused a great deal of interest in stroke genetics. Many of the studies of PDE4D gene have focused on the original Icelandic findings but the association between specific SNPs and haplotypes has been inconsistent. The aim of the present study was to investigate the association of three SNPs 32 (rs 456009), 83 (rs 966221) and 87 (rs 2910829), originally described by deCODE group; with stroke in a South Indian population from Andhra Pradesh. Two hundred and fifty ischemic stroke patients and two hundred and fifty controls were included in the study. The stroke patients were sub typed according to TOAST classification. SNP 83 showed significant association with stroke in the population under study while SNPs 87 and 32 were monomorphic. Further SNP 83 was found to be significantly associated with two stroke subtypes, intracranial large artery atherosclerosis (the most frequent subtype in the population) and small artery occlusion. The association with other subtypes was found to be insignificant. Further, SNP 83 was found to be associated significantly with some conventional stroke risk factors like diabetes and smoking. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Stroke or brain attack is the third largest killer in the world after heart attack and cancer [1–3]. Studies involving twins, siblings and families suggest that stroke has a strong genetic component [4,5]. Genetic causes of stroke range from classic Mendelian to complex. In recent years several parallels of research have been conducted to establish the relationship between the functional variants of a variety of genes and the risk of stroke. These include the angiotensin converting enzyme gene [6,7], the endothelial nitric oxide synthase gene [8,9], genes associated with inflammation [10], thrombosis and coagulation [11], lipid metabolism and apolipoprotein E [12]. Most genetic determinants of stroke modulate the effects of known risk factors such as hypertension, diabetes etc. A more recent and exciting discovery in this field, is the identification of phosphodiesterase 4D (PDE4D) gene which seems to contribute to the risk of stroke in carriers independent of other genetically based risk factors [13]. PDE4D is a large gene, which spans a 1.6 Mb region on 5q12 and has 24 exons [14,15].The gene can express nine different functional protein isoforms through alternative splicing or the use of differential ⁎ Corresponding author. Department of Molecular Biology, Institute of Genetics and Hospital for Genetic Diseases, Begumpet, Hyderabad-500016, India. Tel.: +91 40 27762776. E-mail address: [email protected] (A. Munshi). 0022-510X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2009.06.024

promoters. The different PDE4D variants are expressed in various tissues including brain, lungs, kidneys, monocytes, B and T lymphocytes and vascular smooth muscles [16]. After Gretarsdottir et al. identified PDE4D gene as a susceptibility gene for stroke, attempts have been made to confirm whether this gene was also a susceptibility gene in other cohorts and populations [13]. The association of PDE4D with stroke has not been investigated in any South Indian population. The aim of the present study was to evaluate the association of three single nucleotide polymorphisms (SNPs) SNP 32, 83 and 87 with ischemic stroke in a South Indian population from Andhra Pradesh. 2. Materials and methods 2.1. Subjects Two hundred and fifty ischemic stroke patients (males:females = 188:62) presenting with new or recurrent stroke evaluated in the stroke clinic of Nizams Institute of Medical Sciences, Hyderabad (A.P, India) between July 2007 and February 2009 were included in the study. Informed consent was obtained from all the subjects included in the study. The ethics committee of the study hospital approved the study. All the patients were examined by a qualified stroke neurologist and ischemic strokes were differentiated by computed tomography scans and magnetic resonance imaging. The ischemic

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stroke was classified into subtypes according to the TOAST classification [17]. As a control group 250 healthy individuals matched for sex and age were recruited from the same demographic area. Information on demographic characteristics and risk factors were collected by using a structured questionnaire. Hypertension, alcoholism, diabetes and smoking were defined as reported previously [7]. 2.2. DNA isolation and genotyping A total of 5 ml of venous blood was collected in EDTA tubes. Genomic DNA was extracted using phenol-chloroform method. The specific regions containing these SNPs (32, 83 and 87) were amplified by polymerase chain reaction [18]. The amplified PCR products were digested with restriction endonucleases and the digested fragments were analyzed by gel electrophoresis [18]. The polymorphism in SNP 83 was confirmed by sequencing the PCR products. The sequencing was done by Chromus Biotech (Hyderabad, India). SNP 32 (go.59832491 GNC), SNP 83 (go.59538277 TNC) and SNP 87 (go.59505656 CNT) were digested with MSPA1I (New England Biolabs), TaiI fast digest ( Fermentas Life Sciences ) and SSP1 (New England Biolabs) respectively [18]. 2.3. Statistical analysis Hardy Weinberg equilibrium was first tested for PDE4D gene polymorphism. Association between genotypes and stroke was examined by using odds ratio (or) with 95% confidence interval (CI) and chi square analysis using EPI6 software (EPI info 6 CDC). All the statistical tests were two sided, and were considered significant at P b 0.05. Allelic frequencies were calculated according to the number of different alleles observed and the total number of alleles examined. Bonferroni correction was done for multiple comparisons. 3. Results During the study period, 250 ischemic stroke patients and 250 controls were included in the study. The demographic features of the population under study have been listed in Table 1. When subjected to PCR analysis and restriction digestion none of the subjects was either heterozygous or homozygous for the SNPs 32 and 87.However, SNP 83 polymorphism (T/C) was found to be significantly associated with the stroke while SNPs 32 and 87 were monomorphic in the population under study. To confirm the polymorphism, the PCR reaction products were sequenced (Figs. 1–3). Gretasdottir et al. have described the respective positions of these three SNPs in the PDE4D gene [13]. The genotypic distribution of SNP 83 (rs 996621) and allelic frequency for T and C alleles in patients and healthy controls have been given in Table 2. Statistically significant difference in the genotypic distribution and allelic frequency between the patients and healthy controls was observed (x2 = 25.06, df = 2; p b 0.0001 for genotypes; x2 = 21.33, l df = 1; p b 0.0001 for allelic frequency). The odds ratio for CC genotype and CT genotype as independent risk factors for ischemic stroke was 7.54 (95% CI; 2.53–23.23 p b 0.001) and 1.83 (CI 95%: 1.23–

Fig. 1. Showing the amplification of region bearing SNP 83.

2.64 p = 0.006) respectively. The odds ratio for C allele was 1.93 (95% CI; 1.44–2.57). In a further analysis of the association of SNP 83 with stroke sub types classified according to the TOAST classification, we found a statistically significant difference in the allelic frequency between patients having large artery intracranial atherosclerosis and small artery occlusion (Tables 3 and 4). As far as other subtypes are concerned, the association was found to be insignificant. The association of C allele with conventional risk factors for stroke was analyzed. The hypertensives, diabetics, smokers and alcohol users showed a higher frequency of C allele in comparison with normotensives, non-diabetics, non-smokers and non-alcoholics among the patient population. Bonferroni correction was done for these multiple comparisons. Although all the parameters survived this correction, the significance was high for diabetes (p = 7.379− 08(P)) and smoking (p = 1.988− 06 (P)) and borderline for hypertension and alcoholism (p = 0.019 and 0.0125 respectively). 4. Discussion PDE4D is a large gene which spans a 1.6 Mb region on 5q12 and bears 24 exons [14,15]. It belongs to a super family of phosphodiesterases (PDE4 family) involved in the degradation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Before the report by deCODE Genetics, the PDE4 gene family was never tested as candidate genes associated with any disease or

Table 1 Demographic features of the study population.

Age (mean ± SD) Male:female Hypertension Diabetes Smokers Alcoholics Family history of stoke

Patients (n = 250)

Controls (n = 250)

48.53 (16.34) 188:62 70.63% 44% 45% 35% 26%

47.01 (17.78) 185:65 51% 30% 40% 30% 12%

Fig. 2. PCR products (SNP 83) digested with Tai I, Lanes 1, 6 and 7 represent CT heterozygotes. Lanes 2, 5 and 8 represent TT homozygotes. Lane 3 represents CC homozygote and lane 4 represents 100 bp ladder.

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A. Munshi et al. / Journal of the Neurological Sciences 285 (2009) 142–145 Table 3 SNP 83 genotypic and allelic frequencies in patients classified according to TOAST classification. TOAST classification

No. of PDE4D genotype (%) patients TT CT CC

1. Large artery 111 atherosclerosis a. Intracranial large 92 artery atherosclerosis 19 b. Extracranial large artery atherosclerosis 2. Small artery 40 occlusion 3. Cardioembolism 29 4. Other determined 5 etiology 5. Undetermined 65 etiology

Fig. 3. Sequenced PCR products of the region bearing SNP 83 showing TT homozygote, CC homozygote and CT heterozygote respectively.

phenotype [19]. PDE4D gene codes for cAMP-specific 3′ 5′-cyclic phosphodiesterase 4D, which seems to be a secondary signal pathway regulator of phenotype, with indirect effects on cardiovascular or stroke biomarkers [20]. PDE4D degrades second-messenger cAMP which might be playing an important role in the proliferation of smooth muscle cells and macrophages, and therefore possibly in atherosclerosis and plaque stability [21–25]. After Gretarsdottir et al. identified PDE4D gene as a susceptibility gene for stroke, more than a dozen follow up studies have been taken up to study the single nucleotide polymorphism across the PDE4D gene for association with the disease. Cohorts of different ethnicities have been studied. A detailed account of all these studies has been already published [5]. Recently Hsieh et al. have studied two SNPs

Study group Patients n (%) Control n (%)

PDE4D genotype

Allelic frequency

TT

CT

CC

Total

T

C

Total

100 (40) 145 (58)

124 (50) 100 (40)

26 (10) 5 (2)

250 250

324 (0.64) 390 (0.78)

176 (0.35) 110 (0.22)

500 500

χ2 = 25.06 (2 df), p b 0.0001 for genotypes; χ2 = 21.33 (1 df), p b 0.0001 for allelic frequency. Odds ratio for C allele, CC and CT genotype is 1.93 (1.44–2.57), 7.54 (2.63–23.23) and 1.80 (1.23–2.64) respectively.

16 (84) 11 (27)

3 (16) – 21 (53)

101 (0.54) 83 (0.45) 35 (0.92)

8 (20)

C

3 (0.08)

43 (0.53) 37 (0.46)

17 (57) 12 (41) – 1 (20) 4 (80) –

46 (0.79) 12 (0.20) 6 (0.6) 4 (0.4)

30 (46) 33 (50)

93 (0.7)

2 (3)

35 (0.3)

i.e. 87 and 41 from the gene in a Taiwanese population [26]. No significant association with ischemic stroke was found. A metaanalysis was carried out on 5200 cases and 6600 controls by Bevan et al. [27]. They did not find any genetic variant examined in PDE4D, showing a robust and reproducible association to stroke. According to them any association that may exist seems to be weak and restricted to specific populations. In the present study we evaluated the association of three SNPs 83 (rs 966221), 32 (rs 456009) and 87 (rs 2910829) with ischemic stroke in an Andhra Pradesh population. To the best of our knowledge this is the first study to investigate the association of PDE4D genetic variants with ischemic stroke in a South Indian population. All the three SNPs were found to be associated with stroke in an Icelandic population [13]. As far as SNPs 32 and 87 are concerned, we did not find any mutation in the subjects. These seem to be either monomorphic or the mutations are very rare in the population under study. However, SNP 83 was found to be significantly associated with the disease (p b 0.0001). Further, examining the association of this SNP with stroke subtypes, we found a significant association with intracranial large artery atherosclerosis and small artery occlusion (p b 0.0001 in each case).A Pakistani study also found SNP 83 to have a strong association with ischemic stroke [18]. SNP 83 has been found to be associated with cardioembolic stroke in an American population [28]. Staton et al. have found a significant association of SNP 83 with ischemic stroke in an Australian population [29]. A recent study from North India also found SNP 83 to be significantly associated with stroke in the North Indian population [10]. In addition we found a strong association of C allele with diabetes and smoking and a marginal significance with hypertension and alcoholism. Therefore diabetics and smokers bearing C allele in a high frequency are more predisposed to stroke.

Table 4 The odds ratio and p values in different stroke subtypes. TOAST classification

Table 2 Distribution of SNP 83 genotypes and allelic frequencies of the study population.

25 (27) 51 (55) 16 (17)

Allelic frequency T

Genotype

Allelic frequency

χ2 (2 df) p-value

χ2 (1 df) Odds ratio (95% CI) p-value

Large artery atherosclerosis a) Intracranial large 42.43 artery atherosclerosis b) Extracranial large 5.11 artery atherosclerosis Small artery occlusion 32.18 Cardioembolism 0.59 Other determined 3.26 etiology Undetermined etiology 2.99

b0.0001 35.46

2.92 (2.0–4.24)

b0.0001

4.23

0.30 (0.07–1.06)

0.039

b0.0001 21.43 0.743 0.05 0.195 1.83

3.05 (1.82–5.11) 0.92 (0.45–1.88) 2.36 (0.55–9.65)

b0.0001 0.819 0.176

1.33 (0.84–2.12)

0.200

0.077

0.224

1.64

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The identification of candidate genes such as PDE4D associated with stroke gives some hope that genetic studies might have a direct impact on the treatment of the disease. Moreover PDE4D and members of PDE4 family are characterized by selective inhibition by Rolipram [30]. The selective inhibition of PDE4D using drugs might ultimately lead to the prevention of a stroke. Further studies will hopefully tell us how far the genetic information will assist to tailor clinical and therapeutic decision to an individual's genotype. In conclusion our results suggest that SNP 83 of PDE4D gene is significantly associated with ischemic stroke in the South Indian population from Andhra Pradesh. Moreover, significant association is found with intracranial large artery atherosclerosis which is the most frequent subtype in this region [31] and small artery occlusion. This might be because the frequency of other subtypes is low in the study population. This is similar to previous report from the study hospital [31]. Most notable differences of the stroke registry of Nizams Institute of Medical Sciences were the predominance of intracranial rather than extracranial atherosclerosis and also the fact that other stroke subtypes which are quite frequent in western registries, are less frequent in the study population [31]. References [1] del Zoppo GJ, Hallenbeck JM. Advances in vascular pathophysiology of ischemic stroke. Thromb Res 2000;98(3):73–81. [2] Cacas JP, Bautista LE, Smeeth L, Sharma P, Hingorani AD. Homocysteine and stroke; evidence on a causal link from Mendelian randomization. Lancet 2005;365 (9455):224–32. [3] Manica G. Prevention and treatment of stroke in patients with hypertension. Clin Ther 2004;26:631–48. [4] Bak S, Gaist D, Sindrup SH, Skytthe Ma, Christensen K. Genetic liability in stroke; a long term follow-up study of Danish twins. Stroke 2002;33:769–74. [5] Munshi A, Kaul S. Stroke genetics—focus on PDE4D gene. Int Jour of Stroke 2008;3 (3):188–92. [6] Kostulas K, Huang WX, Crisby M, et al. An angiotensin converting enzyme gene polymorphism suggests a genetic distinction between ischemic stroke and carotid stenosis. Eur J Clin Invest 1999;29:478–83. [7] Munshi A, Shehnaz S, Kaul S, Pullareddy B, Alladi S, Jyothy A. Angiotensin converting enzyme insertion/deletion polymorphism and the risk of ischemic stroke in a South Indian population. J Neurol Sci 2008;272:132–5. [8] Markus HS, Ruigork Y, Ali N, Powell JF. Endothelial nitric oxide synthase exon 7 polymorphism, ischemic cerebrovascular disease, and carotid atheroma. Stroke 1998;29:1908–11. [9] Congning S, Kang X, Wang Y, Zhou Y. The coagulation factor V leiden, MTHFR C677T variant and Enos 4ab polymorphism in young Chinese population with ischemic stroke. Clinica Chim Acta 2008;396:7–9.

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