Replication studies of the Icelandic findings have yielded mixed results. shows the SNPs examined by all published studies of PDE4D and stroke, demonstrating that work has primarily focussed on the portion of the gene where deCODE associations were found.
Figure 5 A summary of PDE4D replication association studies in various populations. (A) The PDE4D gene structure. Exons are indicated by boxes. Differential promoters for PDE4D isoforms are indicated by horizontal arrows on the gene. The alternative splicing patterns (more ...)
Four European studies exclusively examined the strongest genotype and haplotype associations with all ischemic stroke or the combined atherosclerotic/cardioembolic group from the Iceland study and failed to replicate these findings (3
). A German study examined SNP 41(rs152312), SNP 45(rs12188950) and one microsatellite marker (AC008818-1), which had shown the most significant association with stroke in Iceland (9
). They further examined a set of haplotype tagging SNPs that distinguished 95% of all chromosomes within linkage disequilibrium blocks ‘B’ and ‘C’, which contained the haplotypes with the strongest association with stroke in Iceland. No positive associations with ischemic stroke were found.
An English study (6
) examined the positively associated Icelandic SNPs and haplotypes in relationship to carotid intima-media thickness and carotid plaque as well as risk of ischemic stroke and ischemic stroke subtypes. No SNPs or haplotyes were associated with all ischemic stroke or with the combined atherosclerotic/cardioembolic groups. Two SNPs, SNP 19(rs4133470) and SNP 87(rs2910829) were found to be associated with large vessel stroke after adjustment for other risk factors and one of these two, SNP 87(rs2910829), was associated with carotid intima-media thickness at the carotid bulb. Six deCODE SNPs [SNP2(rs152341), SNP13(rs26949), SNP 14(rs26950), SNP 15(rs35382), SNP 20(rs16878206), SNP 26(rs40512)] showed significant association with cardioembolic stroke after multivariate adjustment, and haplotypes derived from these SNPs were less strongly associated than the individual SNPs.
A Swedish study (3
) of predominantly ischemic strokes found no association with the deCODE-associated variants, the A allele of AC008818-1 marker and deCODE SNPs 45(rs12188950) and 41(rs152312) near PDE4D7 region. However, this study found associations with the B allele of AC008818-1 and an SNP near PDE4D3 region.
A study in a geographically isolated population in the Netherlands (7
) examined deCODE SNPs 39(rs3887175), 45(rs12188950), 83(rs966221) and their derived haplotypes. For the overall ischemic stroke group, no associations were found either in the total population or within inbred individuals. The subgroup of inbred individuals with small vessel disease did show an association with SNPs 39(rs3887175) and 45(rs12188950).
In contrast, recent American (4
) and Pakistani (8
) studies found significant associations between deCODE SNPs and all ischemic stroke. One American (4
) study, predominately consisting of Caucasians, found associations with two of four deCODE-associated variants, SNP 83(rs966221) and SNP 56(rs702553), with the most significant SNP 83(rs966221) having a stronger association with large artery atherosclerotic stroke. The other American (5
) study found SNP 87(rs2910829) to be associated with cardioembolic stroke among both whites and blacks and SNP 41(rs152312) to be associated with cardioembolic stroke among whites, all after multiple comparison corrections. The Pakistani study (8
) examined SNP 32(rs456009), 83(rs966221) and 87(rs2910829) and found an association with SNP 83(rs966221).
A recent Japanese (10
) study examined three microsatellites, one VNTR and 31 deCODE SNPs, finding no associations with any of the polymorphisms examined after correcting for multiple comparisons. However, haplotype analyses within two separate blocks revealed significant association with cerebral infarction. The first block, consistent in location with the deCODE findings, revealed a significant haplotype (P
= 0.002) comprising SNP83(rs966221), rs153031 and VNTR(AC008818). The second block in a different PDE4D
gene region revealed a significant haplotype (P
< 0.001) comprising of rs878567, rs3756739, rs6875372 and rs1423351.
Using existing information from databases, our SNP discovery efforts, as well as the Iceland results, we examined SNPs throughout the entire PDE4D
gene with greater emphasis on the regions around the first exons at the 5′ end of the gene. Among the deCODE stroke-associated variants evaluated in our study, several SNPs were found to be associated with ischemic stroke including SNP 83(rs966221) among Caucasians in an additive model, SNP 89(rs1396476) among African-Americans using an additive model and SNP 42(rs153031) in African-Americans using a dominant model. Additionally, rs918592, which was most associated with stroke in our study, was in linkage disequilibrium with rs152312(SNP 41), one of the polymorphisms more strongly associated with stroke in the Iceland population. Furthermore, the risk haplotype in the Iceland study contained rs152312 and had lower expression of the PDE4D
7 isoform (1
Although many of the studies of the PDE4D
gene focussed on replicating the Icelandic findings, the association between specific SNPs and stroke have been inconsistent. Only the Japanese study (10
) and our study have examined the PDE4D
gene more extensively, but neither study has comprehensively examined the genetic variation within the PDE4D
gene. The Japanese study identified two stroke-associated haplotype blocks. The more strongly associated haplotype was towards the 3′ end of the gene. The other associated haplotype was in the 5′ region of PDE4D
, near the first alternative exon for PDE4D
7, found to be associated with stroke in Iceland. Our strongest associated SNP was also in the 5′ region of PDE4D
. Thus, our study adds independent support to the findings of the Iceland investigators and extends these findings to early-onset stroke among African-Americans as well as Caucasians.
Our findings do not support the hypothesis that atherogenesis mediates the association. We have demonstrated an effect of the PDE4D
locus on stroke risk among young adults, a population with a very low prevalence of atherosclerotic disease. In contrast to the findings from Iceland (1
) and the recent American (4
) study, we found that our most strongly associated SNP, rs918592, was associated with multiple ischemic stroke subtypes. Significant findings were found not only for atherosclerotic stroke, but also for lacunar stroke and non-lacunar stroke of undetermined etiology. This later group included large artery strokes without evidence for a significant degree of proximal atherosclerosis. Although our findings differed from the Iceland results in that there was no association with cardiac embolism, the underlying cardiac conditions associated with stroke in a young population are quite different from those among the elderly.
Ours is the first study to examine the possibility of a PDE4D
gene–environment interaction with smoking. The risk genotype was not associated with stroke among never-smokers or former smokers, but highly associated with stroke among current smokers. Additionally, a strong dose–response relationship was seen among current smokers. Replication of these findings, including studies of males and older stroke patients, will be essential because of the small sample size, the number of risk factors tested for interaction and the lack of a similar interaction among the other stroke-associated PDE4D
SNPs. However, an interaction is biologically plausible. Cigarette smoking causes endothelial dysfunction (11
) and is known to modify the expression of many genes in endothelial cells, including cAMP response element binding protein (CREB) (12
). CREB is a transcription factor that induces the expression of a panel of genes with established roles in cell survival, metabolism and plasticity in the nervous system, including a potential role in ischemic preconditioning (13
). Thus, both PDE4D
and smoking may mediate effects via CREB providing a potential mechanism for the smoking interaction. An alternative mechanism could be that smoking may potentiate the effect of increased PDE4D
expression. In a mouse model, pre-natal exposure to cigarette smoking increased PDE4D
messenger RNA expression and decreased cAMP in the lung (14
In conclusion, we identified several PDE4D SNPs associated with stroke in both African-American and Caucasian females, providing support for association of this gene with stroke in non-Icelandic populations and among young adults. Our most significantly associated SNP was found to be in strong linkage disequilibrium with another previously associated with stroke in Iceland. The effect of the highly prevalent risk allele in this SNP was not subtype-specific but was specific to smokers. More comprehensive studies of the association between PDE4D genetic variation and both functional correlates and stroke risk in larger, ethnically diverse populations are needed.