Wilker et al. BP in carriers of the homozygous variant (95% CI, –1.65 to 1.91) and 2.32 (95% CI, 1.91–2.72) in others. Under a reces- sive model, homozygous carriers of the vari- ant alleles of rs7813 and rs910925, both in GEMIN4, were both associated with lower BP in response to a 1-SD increase in BC com- pared with others. Both these SNPs produced very similar magnitudes of effect and were found to be in high LD (r2 = 0.9). As a sen- sitivity analysis, we also investigated whether there were significant associations in models adjusted only for model 1 variables, and the same SNPs achieved significant results. We also tested our associations using inverse prob- ability weighting (IPW) to address attrition and found that our most significant results for both SBP and DBP became more significant, although changes associated with the IPW analysis were modest. We report the model 1 p-values and IPW p-values for sensitivity addi- tionally in Table 4. Discussion We observed that BC concentration averaged over the 7 days preceding each study center visit was positively associated with SBP and DBP in a cohort of elderly men. Furthermore, SNPs in miRNA processing genes modified the BC associations with BP that we observed. Our analysis focused on potentially functional SNPs located in genes involved in the processing of miRNAs. To our knowledge, this is the first study to investigate the role of polymorphisms in miRNA processing genes to be identified as effect modifiers of an association between air pollution and cardiovascular response. We selected these SNPs because of their involvement in the biogenesis and processing of miRNA. The transcription and process- ing of miRNAs has been described in detail elsewhere (Bartel 2004). Briefly, miRNAs are processed in a multistep pathway (Figure 1). First, long primary transcripts coded within the introns of protein-coding genes are tran- scribed into primary miRNAs (pri-miRNAs) approximately 100 nucleotides (nt) in length. Through a series of steps mediated by DROSHA and DGCR8, the pre-miRNA stem-loop (approximately 70–100 nt) is formed. After export from the nucleus via the RAN-GTP complex and XPO5, these pre- miRNAs undergo modification by DICER within the cytoplasm to generate an ~ 22-nt duplex from the loop complex, which com- prises miRNA and its complement. AGO2, GEMIN3, and GEMIN4 then interact with miRNA to form a ribonucleoprotein, which guides the miRNA into the RNA-induced silencing complex (RISC), where the miRNA mRNA RISC TRBP AGO Double stranded DNA Ran-GTP XPO5 Pre-miRNA GEMIN4 GEMIN3 DICER mRNA degradation Translational repression DROSHA DGCR8 Pri-miRNA Transcription mRNA degradation. 946 Figure 1. Major steps in miRNA processing. Long primary transcripts coded within the introns of protein- coding genes are transcribed into primary miRNAs (pri-miRNAs). Then DROSHA and the DGCR8 complex mediate the formation of the pre-miRNA stem-loop. After export from the nucleus via the RAN-GTP com- plex and Exportin5, these pre-miRNAs undergo modification by DICER within the cytoplasm to generate an ~ 22-nt duplex from the loop complex, which comprises the miRNA and its complement. AGO2, GEMIN3, and GEMIN4 then interact with miRNA to form a ribonucleoprotein that guides the miRNA into the RISC, where the miRNA strand anneals to the 3´ UTRs of target mRNAs, promoting translational repression or strand anneals to the 3´ untranslated regions (UTRs) of target mRNAs, promoting transla- tional repression or mRNA degradation. More than 700 human miRNA have been identi- fied in the miRBase Registry (http://microrna. sanger.ac.uk/) (Griffiths-Jones et al. 2008). It has been shown that miRNAs, which are generally associated with silencing or sup- pression of target gene transcripts (negative regulation), may become activators of gene expression during stress (Rocha 2007; Yang and Paschen 2008). Animal studies have also shown that miRNA expression is down- regulated in lungs of rats exposed to cigarette smoke, which is consistent with the hypothesis that alterations of the miRNA control mecha- nism can occur even after relatively short peri- ods of exposure (Izzotti et al. 2009). Other forms of epigenetic regulation, such as DNA methylation, have been associated with expo- sure to traffic particles (Baccarelli et al. 2009). Recently, miRNA processing genes, such as DICER, have been associated with endothelial function and angiogenesis (Suarez et al. 2007). None of the interactions identified were significantly predictive of both SBP and DBP. Although BC interactions with SNPs in DGCR8, DICER, and GEMIN4 were observed in models of DBP, only interactions with sin- gle SNPs in GEMIN3 and GEMIN4 were predictive of SBP. The DICER and DGCR8 SNPs for which we observed interactions pre- dicting DBP are both located within 3´ UTRs. These regions are not translated directly into the protein; however, they are thought to play a role in mRNA stability. Additionally, these polymorphisms may affect miRNA binding to the 3´ UTR of target mRNAs. Previously, DICER knockdown increased activation of the endothelial nitric oxide synthase pathway (Suarez et al. 2007). DICER is thought to be more generally involved in miRNA process- ing, whereas DGCR8 is thought to be more specific to particular miRNAs (Han et al. 2006). DGCR8 is required for the maturation of miRNA primary transcripts at the initial stages. A recent study found that deletion of DGCR8 in cardiomyocytes leads to left ven- tricular malfunction progressing to a dilated cardiomyopathy and premature lethality (Rao et al. 2009), which suggests a central role of this gene in cardiovascular function. Lack of DICER activity in knockout studies has been suggested to be associated with defective blood vessel formation (Bernstein et al. 2003; Yang et al. 2005). However, no associations predict- ing SBP have been observed for the particular SNPs that we observed interacting with BC. In our analyses, we found that BC interac- tions with SNPs in GEMIN4 were associated with SBP and DBP. Interactions with one SNP in GEMIN3, rs197414, was also associ- ated with SBP. These genes code for proteins associated with the survival motor neuron ovlume 118 | number 7 | July 2010 • Environmental Health Perspectives