Genetic variants of phosphodiesterase 4D gene are associated with an enhanced risk for ischemic stroke in young Chinese population
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.108131
Source of Support: None, Conflict of Interest: None
Background: Previous studies have shown that the phosphodiesterase 4D (PDE4D) gene is a susceptibility gene for ischemic stroke (IS) primarily in elder populations. However, few studies have reported the role of the PDE4D gene polymorphisms in a young cohort. Aims: To investigate the association between the PDE4D gene polymorphisms and young-onset IS in Chinese population. Materials and Methods: A total of 186 young patients (18-45 years) with IS and 232 matched control subjects were recruited. Two SNPs (rs918592 and rs2910829) in PDE4D gene were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio and 95% confidence intervals (95% CI) were calculated to test the association between the genetic factors and IS. Results: The rs918592A/A genotype frequency and A allele frequency, rs2910829 CT/TT genotype frequency and T allele frequency of young IS group were significantly higher than those of the control group ( P < 0.05). Besides, the frequency of Hap (A-T) was remarkably higher in the young patients than that in the controls (OR =4.047, 95% CI: 3.521-4.652). Hap (A-C) and Hap (G-C) were associated with decreased risk of IS (OR =0.640, 95% CI: 0.452-0.906; OR =0.675, 95% CI: 0.466-0.978, respectively). Conclusions: Our findings suggest that the rs918592 and rs2910829 polymorphisms and haplotypes of PDE4D gene are significantly associated with IS in Chinese young population.
Keywords: Haplotype, ischemic stroke, PDE4D, polymorphism
Ischemic stroke (IS) is a leading cause of death worldwide, and its prevalence among young adults (aged 15-45 years) ranges from 3% to 5%. , Although acute IS is more common among elderly persons, 10% of all cerebral infarctions occur among patients aged 45 years or younger.  Familial aggregation and twin studies provide evidence for a strong genetic component for IS risk, particularly for young-onset stroke. ,,,, Despite extensive investigations, the etiology of this potentially devastating condition remains unknown in approximately one-third of young adults. ,
A genome-wide linkage analysis conducted by the deCODE group in an Icelandic population suggested a possible role of PDE4D gene in the risk of stroke. PDE4D is a member of the superfamily of cyclic nucleotide phosphodiesterases and has been implicated in the etiology of stroke based on the hypothesis that PDE4D selectively degrades cAMP, which exerts protean effects on the vasculature and nervous system.  Extension of the findings to non-Icelandic populations subsequently led to controversial and conflicting results. [12-15] Furthermore, few studies have reported the role of the PDE4D polymorphisms in early-onset IS. Genetic effects may be more prominent in the young, because cumulative environmental factors have not had enough time to modify phenotype substantially.  The aim of this study was to assess the association of rs918592 and rs2910829 polymorphisms of PDE4D gene with IS in young (<45 years) Chinese population.
A total of 186 young patients (18-45 years) with IS (males: females =122:64) were recruited from the First Affiliated Hospital of Zhengzhou University (Henan, China) between January 2008 and June 2010. IS was defined by a loss of global or focal cerebral function persisting for >24 hours, with corresponding infarction on brain imaging and a probable vascular cause.  Neurologist classified IS subtypes based on the Trial of Org10172 in Acute Stroke Treatment (TOAST) classification.  All patients underwent a detailed clinical scrutiny, including medical history, family history, general physical, and neurological examinations, standardized blood tests, brain computed tomography (CT) and/or magnetic resonance imaging (MRI) examinations within 2 days after the onset of the symptoms. Patients with arterial fibrillation, cerebral hemorrhage, peripheral vascular diseases, and kidney diseases were excluded from this study. Hypertension was defined as systolic blood pressure of ≥140 mmHg or diastolic blood pressure of ≥90 mmHg or current treatment with hypertensive medication. Diabetes was diagnosed if a fasting glucose level was >7.8 mmol/L or a glucose level of >11.1 mmol/L at 2 h after oral glucose challenge or both. Hypercholesterolemia was defined as a total fasting plasma cholesterol >5.2 mmol/L. If a subject had smoked at least 1 cigarette per day for at least 1 year and had not quitted smoking by the time of study, he/she was defined as a current smoker.
The control group included 232 individuals matched for age and gender (males:females =135:97). All controls were unrelated Henan Han nationality and free of cerebrovascular and cardiovascular diseases, cancer, hepatic, or renal diseases. Signed consent form was obtained from each participant. The study protocols were approved by the Ethics Committee on Human Research of Zhengzhou University.
PDE4D gene genotyping
Genomic DNA was extracted from peripheral white blood cells using the phenol-chloroform method. Determinations of rs918592 and rs2910829 gene polymorphisms were done by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The sequences of rs918592 primer pair were: 5′-GCC AGT GCT CAC GTT TAA CA-3′ (forward) and 5′-CAA TTG CTG AAG CTG CAA AG-3'(reverse). The rs2910829 primers were: 5'-AGG TAT GAA GAC ACC TGA AAG ATC-3'(forward) and 5'-GCA GTA TGT TTA AAG ATG AGG AAG-3'(reverse) (Sangon, China). PCRs (PE9600, Applied Biosystem, USA) were performed using a mixture containing genomic DNA 2.0 uL, 2-CAA TTG CTG AAG CTG CAA AG-3′ (reverse). The rs2910829 primers were: 5′-AGG TAT GAA GAC ACC TGA AAG ATC-3,(forward) and 5,-GCA GTA TGT TTA AAG ATG AGG AAG'3-(reverse) (Sangon, China). PCRs (PE9600, Applied Biosystem, USA) were performed using a mixture containing genomic DNA 2.0 uL, 2-AGG TAT GAA GAC ACC TGA AAG ATC-3′ (forward) and 5′-GCA GTA TGT TTA AAG ATG AGG AAG'3-(reverse) (Sangon, China). PCRs (PE9600, Applied Biosystem, USA) were performed using a mixture containing genomic DNA 2.0 uL, 2-GCA GTA TGT TTA AAG ATG AGG AAG-3′ (reverse) (Sangon, China). PCRs (PE9600, Applied Biosystem, USA) were performed using a mixture containing genomic DNA 2.0 uL, 2× PCR Master Mix 8.0 uL, 2.0 uL each primer and 11 uL ddH 2 O in a final volume of 25 uL. The conditions for PCR amplifications were initial denaturation at 95°C for 5 min, 38 cycles of denaturation at 94°C for 60 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 60 seconds, with a final extension at 72°C for 5 min.
The PCR products of rs918592 were digested with restriction endonuclease ApaLI (Sangon, China) and the fragments were separated on 3% agarose gel electrophoresis. The AA genotype were detected as fragments of 255 bp, and AG genotype were detected as fragments of 255, 205, and 50 bp, whereas GG genotype were detected as fragments of 205 and 50 bp. The amplified products of rs2910829 were digested with restriction endonuclease SspI (Sangon, China). After digestion, fragment sizes for carriers of the polymorphic allele decreased from 211 bp (wild-type) to 211, 159, and 52 bp (heterozygous) and to 159, 52 bp (variant).
Eight randomly chosen samples with positive genotyping results were confirmed further by direct sequencing of the PCR products with ABI3730xl (ABI, USA). All genotypes were identical with those obtained from the first round of genotyping.
All statistical tests were performed using SPSS (version 16.0) software for Windows (SPSS Inc, Chicago, USA). The quantitative variables were compared between the two groups by u test.  Differences in the allele and genotype frequencies of rs918592 and rs2910829 between IS patients and controls were calculated using the Chi-square test. Testing for deviation of genotype distribution from Hardy-Weinberg equilibrium and haplotype-based case-control study were performed using the SHEsis software (http://analysis.bio-x.cn).  The comparison associations were expressed in terms of odds ratio (OR) adjusted for age and gender, with the corresponding 95% confidence interval (CI). Statistical significance was defined as P < 0.05.
Clinical characteristics of subjects
The clinical characteristics of the controls and the IS patients are shown in [Table 1]. The mean age of the IS and control subjects was 36.5 and 36.8 years, respectively. The conventional risk factors including hypertension, diabetes, cigarette smoking, drinking, and cholesterol were more prevalent among young IS patients than controls (P < 0.05).
The genotyping results of PDE4Drs918592 are shown in [Figure 1]. The genotyping results of PDE4D rs2910829 are shown in [Figure 2]. In order to confirm the veracity of the PCR-RFLP results, DNA sequencing was performed. The results are shown in [Figure 3] and [Figure 4].
Association with the PDE4D genetic variants
The distributions of rs918592 and rs2910829 genotype frequencies both in the IS group and in the control group were in accord with the Hardy-Weinberg equilibrium (P > 0.05). The rs918592A/A genotype frequencies (33.3%) and A allele frequencies (58.6%) of the young IS group were significantly higher than those of control group (P < 0.05). CT and TT genotype frequencies (44.1%, 10.7%) of rs2910829 in the IS group were significantly higher than that in the control group (25.0%, 2.6%; P =0.000). T allele frequencies (32.8%, 15.1%) in the young IS group was also significantly higher than that in the control group (OR =2.747; 95% CI, 1.967-3.836; P =0.000). The results are shown in [Table 2].
Linkage disequilibrium test and haplotype analysis
The extent of pair-wise linkage disequilibrium of rs918592 and rs2910829 in the present population was calculated (D' =0.491, r 2 =0.061). Then, haplotypes were analyzed based on the observed genotypes. Four common haplotypes in the patients and controls are presented in [Table 3]. The frequency of Hap (A-T) was significantly higher in the young patients than in the controls (P =0.000). While the frequencies of Hap (A-C) and Hap (G-C) in the IS group were significantly lower than that in the controls (P =0.012; P =0.037, respectively) [Table 3].
In the present study, we focused on young subjects. First, young people' exposure to environmental risk factors is less than that of elder subjects. Therefore, the contribution of genetic determinants may be more easily detectable. Second, the occurrence of IS in young people brings heavy social and economical burden. The prevention of IS in this age group has important public health implications.
Premature atherosclerosis is considered the important risk factor for young-onset IS.  Gretarsdottir et al. found that phosphodiesterase 4D (PDE4D) was highly associated with the combination of two forms of stroke: Cardioembolic and atherosclerothic stroke; they proposed that PDE4D gene was involved in the pathogenesis of stroke through atherosclerosis.  The human PDE4D gene spans a 1.6-Mb region on chromosome 5q12 and contains 24 exons (OMIM 600129).  Since the initial identification of PDE4D as a risk gene for IS, several analyses were subsequently carried out in different populations through the case-control approach. No association with PDE4D SNPs and the risk of stroke was found in several European populations ,, and Pakistanian.  Evidence of a significant positive association was reported in studies conducted in North America, Australia, and Asia. ,, Any association that may exist was likely to be potentially restricted to specific populations.
Our findings revealed that the CT/TT genotypes and T allele frequencies of rs2910829 had increased the risk of IS in young Chinese cohort (OR =2.828, OR =6.667, and OR =2.747, respectively). While in our previous study, no significant association of rs2910829 with IS was discovered in elder Chinese cohort.  This association was consistent with an English study, in which SNP 87 (rs2910829) was associated with carotid intima-media thickness at the carotid bulb.  The American study also found SNP 87 (rs2910829) to be associated with cardioembolic stroke among both white people and black people after multiple comparison corrections.  As for rs918592, we also found that the AA genotype and A allele frequencies of rs918592 were associated with an increased risk of young IS (OR =2.186, OR =1.453, respectively). To our knowledge, there was only one study investigating the SNP rs918592 of PDE4D in the young population. Our association result supported Song et al., who showed that rs918592 was strongly associated with early onset stroke among African-Americans as well as Caucasians. 
Haplotype analysis is thought to be more powerful than single SNP analysis in searching for genetic determinants of complex diseases or human traits.  In our study, the Hap (A-T) was strongly associated with an increased risk of IS (OR =4.047, 95% CI: 3.521-4.652) . Hap (A-T) was the combination of risk A allele of rs918592 and risk T allele of rs2910829. May be this additive effect made a combined impact and produced an obvious phenotype. Therefore, the Hap (A-T) was probably the risk haplotype in young Chinese population. While the frequencies of Hap (A-C) and Hap (G-C) in the IS group were significantly lower than in controls (P =0.012; P =0.037, respectively). Hap (A-C) and Hap (G-C) were associated with decreased risk of IS (OR =0.640, 95% CI : 0.452-0.906; OR =0.675, 95% CI: 0.466-0.978, respectively). Our results indicated that Hap (A-C) and Hap (G-C) were protective factors in young Chinese cohort.
In conclusion, rs918592 and rs2910829 of PDE4D were associated with IS in Chinese young population, which provided support for association of PDE4D with IS in non-Icelandic populations and among young adults. Haplotype (A-T) was strongly associated with an increased risk of young-onset stroke and Hap (A-C) and Hap (G-C) were protective factors among young Chinese in this study. More comprehensive studies of the association between PDE4D genetic variation and both functional correlates and stroke risk in larger, ethnically diverse populations are needed.
We acknowledge the technical assistance of staff members of the Neurology Department of First Affiliated Hospital of Zhengzhou University. We also wish to thank all the patients and healthy controls for providing blood samples.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3]