Wilker et al. mercury sphygmomanometer with a 14-cm cuff. Systolic BP (SBP) and fifth-phase dia- stolic BP (DBP) were measured in each arm to the nearest 2 mmHg while the partici- pant was seated. The means of the right and left arm measurements were used as the BP measurement of each participant for analyti- cal purposes. Although there was no specific rest period prior to measurement of BP, SBP and DBP were measured immediately after a complete patient history was taken with the subject seated. SNP selection and genotyping. SNPs were selected based on previously published work investigating associations between genes involved in miRNA processing and disease (Horikawa et al. 2008; Yang et al. 2008). These SNPs were chosen because of overlap in path- ways involved in cancer processes related to autonomic function through cell signaling, Table 1. Descriptive statistics at baseline for par- ticipants included in analysis (n = 789).a Study characteristic BP measurements SBP (mmHg) DBP (mmHg) Clinical measures Age at visit (years) BMI (kg/m2 ) Pulse (beats/min) Hypertension diagnosis Hypertension medication use Two or more drinks per day Diabetesb Education (years) ≤ 12 13–16 > 16 Smoking status Never Current Former Value 132.0 ± 18.4 76.8 ± 10.9 72.3 ± 7.5 28.0 ± 4.1 71.1 ± 8.1 542 (81.8) 478 (72.1) 199 (25.2) 182 (23.1) 280 (35.5) 372 (47.2) 137 (17.3) 237 (30.0) 47 (6.0) 505 (64.0) Air pollution and weather data Seven-day moving average BC (µg/m3) 0.98 ± 0.415 Values are mean ± SD or no. (%). aA total of 2,349 observations (one to six study visits) from 789 NAS participants were included in these analy- ses. bDiabetes was classified as physician-diagnosed diabetes mellitus or fasting blood glucose > 126 mg/dL. Table 2. Linear mixed-effects models estimat- ing the change in BP associated with a 1-SD increasea in BC.b Model 1c Model 2d mmHg (95% CI) mmHg (95% CI) SBP 3.52 (2.77–4.26) 3.04 (2.29–3.79) DBP 2.72 (2.31–3.12) 2.28 (1.88–2.67) aCorresponding to a 0.415-µg/m3 increase in 7-day aver- age BC concentrations. bA total of 2,349 observations from 789 NAS participants were included in these analy- ses. cModel 1 was adjusted for age, BMI, smoking status, season, 7-day moving average of BC, and 7-day moving average of apparent temperature. dModel 2 was adjusted for education, blood urea nitrate, age, BMI, pulse, smok- ing status, pack-years, median income, apparent tem- perature, alpha blocker use, beta blocker use, calcium channel blocker use, angiotensin receptor blocker use, ACE inhibitor use, diuretics, season, two or more drinks per day, and diabetes diagnosis. 944 apoptosis, angiogenesis, and inflammation. Genotyping was performed using multiplex polymerase chain reaction assays designed with Sequenom SpectroDESIGNER software (Sequenom, Inc., San Diego, CA). The exten- sion product was then spotted onto a 384-well spectroCHIP before analysis in the MALDI- TOF mass spectrometer (Sequenom, Inc.). Duplication was performed on 5% of the sam- ples. The 24 SNPs analyzed for this study were all successfully genotyped. After genotyping, we excluded those SNPs for which fewer than 10 participants were homozygous variant carriers [rg1106042 in HIWI, rs3742330 in DICER, rs417309 in DiGeorge critical region-8 (DGCR8), rs636832 in Argonaute 1 (AGO1)] and also those with a Hardy–Weinberg p-value < 0.05 (rs10719 in DROSHA), leaving a total of 19 SNPs in 10 genes. BC and meteorologic measurements. Continuous BC was measured at a Harvard School of Public Health monitoring site located at Countway Library (10 Shattuck Street, Boston, MA, USA), 1 km from the clinical examination site, and was averaged by hour before BP measurement using an aethalometer (Magee Scientific, Berkeley, CA, USA). We obtained temperature and relative humidity measures from the Boston airport weather station. Statistical methods. Because repeated meas- ures of BP were available for many of these study participants with both BC measurements and genotyping, we were able to obtain greater power by using multiple measures. We evalu- ated SBP and DBP as dependent variables and analyzed their associations with BC in linear mixed-effects models. Previous studies have suggested that longer averaging times are more relevant to the associations between particles and BP (Zanobetti et al. 2004), and recent sen- sitivity analysis within the NAS found 7-day moving averages as being most strongly associ- ated with BP outcomes for short-term time windows (1 hr to 1 week) (Mordukhovich et al. 2009). Therefore, we used 7-day mov- ing averages of ambient BC concentrations matched on the time of BP measurement for each participant, and we evaluated SBP and DBP as dependent variables. We examined associations between BP and BC using two different approaches to address potential confounding. In model 1, we adjusted for age, BMI, BC, and apparent temperature (a marker of perceived tempera- ture) as continuous variables as well as smoking status (never, current, former) and season of clinical visit (spring: March–May; summer: June–August; fall: September–November; winter: December–February). In model 2, we adjusted for covariates included in model 1 as well as blood urea nitrate, pulse, and median income treated as continuous variables in addi- tion to education (≤ 12 years, 13–16 years, and > 16 years), alpha blocker, beta blocker, calcium channel blocker, angiotensin recep- tor blocker, angiotensin-converting enzyme (ACE) inhibitor, diuretic use, two or more alcoholic drinks per day, and diabetes diagno- sis. We chose to examine recessive models of inheritance only (rather than dominant and additive) to limit the number of associations tested. Covariates from model 2 were selected a priori to be included in the regression mod- els, regardless of statistical significance. We then examined the associations between the 19 SNPs in miRNA processing genes as well as SNP-by-BC cross-product terms to assess interactions. The default α level was defined as p = 0.05. We performed sensitivity analyses to determine whether our results were robust to changes in the covariates adjusted for in the models and also whether there were significant effects due to missing data. To address attri- tion in the study, we used inverse probability weighting to determine whether reweighting these observations with only one or two repeated measures significantly changed our results (Hernan et al. 2006). This was done by modeling the probability of having only one measurement or only two measurements using the covariates described in model 2 and treating those participants with three or more visits as the reference group. We then used the inverse of the predicted values as the weights. We also examined associations adjusting only for covariates selected in model 1 to determine whether the significance and or magnitude of our adjusted results changed. Linkage disequi- librium (LD) between SNPs in the same gene that met the significance criterion were assessed using the LDPlotter tool (Innate Immunity in Heart Lung and Blood Disease; http://www. pharmgat.org/IIPGA2/Bioinformatics/). Results Of the 2,280 men who originally entered the cohort in 1963, complete covariate data were available on participants who took part in one to six examinations during the study period. Because the NAS comprises over 95% white participants, we restricted our analy- sis to white individuals based on self-report. There were 942 participants with some or all microRNA-processing genotyping data and BP measurements. Of these, BC data were available for 799 participants who reside in Massachusetts. All visits occurred between 1995, when pollutant monitoring began, and 2008. Our full models (model 2) include data from the 789 participants with complete covariate data and some or all genotyping data. In this group, 645 (82%) participants had at least two study visits from 1995 to 2008; 475 (60%) had three or more visits. Our study population was composed entirely of males, most of whom were former cigarette ovlume 118 | number 7 | July 2010 • Environmental Health Perspectives