Luigi Ferrucci

Systematic identification of trans eQTLs as putative drivers of known disease associations

Identifying the downstream effects of disease-associated SNPs is challenging. To help overcome this problem, we performed expression quantitative trait locus (eQTL) meta-analysis in non-transformed peripheral blood samples from 5,311 individuals with replication in 2,775 individuals. We identified and replicated trans eQTLs for 233 SNPs (reflecting 103 independent loci) that were previously associated with complex traits at genome-wide significance. Some of these SNPs affect multiple genes in trans that are known to be altered in individuals with disease: rs4917014, previously associated with systemic lupus erythematosus (SLE), altered gene expression of C1QB and five type I interferon response genes, both hallmarks of SLE. DeepSAGE RNA sequencing showed that rs4917014 strongly alters the 3′ UTR levels of IKZF1 in cis, and chromatin immunoprecipitation and sequencing analysis of the trans-regulated genes implicated IKZF1 as the causal gene. Variants associated with cholesterol metabolism and type 1 diabetes showed similar phenomena, indicating that large-scale eQTL mapping provides insight into the downstream effects of many trait-associated variants.

Insulin-like Growth Factors, Their Binding Proteins, and Prostate Cancer Risk: Analysis of Individual Patient Data from 12 Prospective Studies

Context Insulin-like growth factors (IGFs) and IGF binding proteins may be associated with some cancers. Contribution This reanalysis of individual patient data from 12 studies of the association between IGFs and IGF binding proteins and prostate cancer suggests that higher levels of serum IGF-I are associated with higher risk for prostate cancer. Caution The 12 studies varied in the types of patients they studied and in how they measured IGFs. Implication High IGF-I levels seem to be a risk factor for prostate cancer. The Editors Prostate cancer is one of the most common types of cancer in men, yet few risk factors for the disease, other than age, race, and a family history, have been established (1, 2). Insulin-like growth factors (IGFs) and their associated binding proteins (IGFBPs) have been the subject of many epidemiologic investigations of prostate cancer because they are known to help regulate cell proliferation, differentiation, and apoptosis (3). Although results from some, but not all, studies suggest an association between IGFs and IGFBPs and prostate cancer risk, there has been much uncertainty about its consistency and magnitude. A previous meta-analysis that included only 3 prospective studies suggested that high levels could be associated with more than a 2-fold increase in risk (4), although recent studies have suggested the risk is lower. Furthermore, given that these peptides are correlated with each other, uncertainty remains about any observed relationships. The individual studies are rarely large enough to allow proper mutual adjustment for these correlated factors, and they are insufficiently powered to investigate the consistency of their findings in key subgroups (for example, stage and grade of disease). Such analyses are important because studies have suggested that IGF-I might be more associated with advanced than with localized disease (5, 6). The Endogenous Hormones and Prostate Cancer Collaborative Group was established to conduct collaborative reanalyses of individual data from prospective studies on the relationships between circulating levels of sex hormones and IGFs and subsequent prostate cancer risk. Results for the sex hormones have been reported elsewhere and show no statistically significant relation between androgen or estrogen levels in men and the subsequent risk for prostate cancer (7). We report results for concentrations of IGFs and IGFBPs. Methods Participants The Endogenous Hormones and Prostate Cancer Collaborative Group is described in detail elsewhere (7). In brief, the group invited principal investigators of all studies, found by searching PubMed, Web of Science, and CancerLit, that provided data on circulating concentrations of sex steroids, IGFs or IGFBPs, and prostate cancer risk by using prospectively collected blood samples to join the collaboration. Thirteen studies collected data on circulating IGF concentrations and the subsequent risk for prostate cancer (5, 6, 820), of which 1 contributed only data on sex hormones (20). Eleven of the studies used a matched casecontrol design nested within a prospective cohort study (5, 6, 812, 16, 19) or a randomized trial (1315, 17). One study used a casecohort design (18) and was converted into a matched casecontrol design by randomly matching up to 3 control participants to each case patient by age at recruitment, time between blood collection and diagnosis, time of blood draw, and race. (Table 1 provides a full description of the studies and matching criteria used.) Most of the prospective studies were population-based, with the exception of 1 based on health plan members (9), 1 that recruited male health professionals (16), and 1 that was a combination of an intervention study and a monitoring study for cardiovascular disease (6, 10). Two of the randomized trials did not have prostate cancer as a primary end point (5, 8, 15); the other 2 were based within a screening trial (13) or were about treatment of prostate-specific antigen (PSA)detected prostate cancer (14). Table 1. Study Characteristics Individual participant data were available for age; height; weight; smoking status; alcohol consumption; marital status; socioeconomic status (assessed by educational achievement); race; concentrations of IGFs, IGFBPs, and endogenous sex steroids; and PSA level. Information sought about prostate cancer included date of diagnosis, stage and grade of disease, and method of case patient ascertainment. Some studies (5, 6, 8, 10, 16) published more than 1 article or performed assays at different times on the association between IGFs and prostate cancer risk, sometimes with different matched casecontrol sets, laboratory measurements, and durations of follow-up. For each study, we created a single data set in which each participant appeared only once. In our analysis, we treated any participant who appeared in a study as both a control participant and a case patient as a case patient only. We removed matched set identifiers, and we generated a series of strata (equivalent to matched sets) in which participants in each study were grouped according to age at recruitment (2-year age bands) and date of recruitment (by year), because these matching criteria were common to most studies (Table 1). The number of strata used in the collaborative analysis was slightly less than that of matched sets used in the original analyses. To ensure that this process did not introduce any bias, we checked that the results for each study, using the original matched sets, were the same as those using the strata described above. Tumors were classified as advanced if the tumor was described as extending beyond the prostate capsule (T3/T4), and/or there was lymph node involvement (N1/N2/N3), and/or there were distant metastases (M1); tumors were classified as localized if they were T0/T1/T2 and N0/NX and M0. We classified tumors as high-grade if they had a Gleason score of 7 or more or were moderately poorly or poorly differentiated; otherwise, they were classified as low-grade. Statistical Analysis We calculated partial correlation coefficients between log-transformed IGF and IGFBP concentrations among control participants, adjusted for age at blood collection (<50, 50 to 59, 60 to 69, or 70 years) and study. For each IGF and IGFBP, we categorized men into quintiles of IGF and IGFBP serum concentrations, with cut-points defined by the study-specific quintiles of the distribution within control participants. For studies with more than 1 publication or in which the serum assays were done at different times, resulting in different absolute levels of IGFs (5, 6, 8, 10, 16), we calculated cut-points separately for each substudy. We used a conditional logistic regression stratified by study, age at recruitment (2-year age bands), and date of recruitment (single year) as our main method of analysis. To provide a summary measure of risk, we calculated a linear trend by scoring the quintiles of the serum IGF or IGFBP concentrations as 0, 0.25, 0.5, 0.75, and 1. Under the assumption of linearity, a unit change in this trend variable is equivalent to the odds ratio (OR) comparing the highest with the lowest quintile. All results are unadjusted for participant characteristics, except for those controlled by the stratification variables. We examined the possible influence of 5 participant characteristics by adjusting the relevant conditional logistic regression models for body mass index (BMI) (<22.5, 22.5 to 24.9, 25.0 to 27.4, 27.5 to 29.9, or >30 kg/m2), marital status (married or cohabiting, or not married or cohabiting), educational status (did not attend college or university, or attended college or university), smoking (never, previous, or current), and alcohol consumption (<10 or 10 g/d). We excluded participants from the analysis if they had a missing value for the characteristic under examination. We assessed heterogeneity in linear trends among studies by using a chi-square statistic to test whether the study-specific ORs were statistically different from the overall OR (21). Heterogeneity among studies was also quantified by calculating the H and I 2 statistics (22). To test whether the linear trend OR estimates for each IGF and IGFBP varied according to case patient characteristics, we estimated a series of subsets for each characteristic: stage at diagnosis (localized or advanced), grade at diagnosis (low or high), year of diagnosis (before 1990, 1990 to 1994, or 1995 onward; these year cutoffs were chosen to attempt to reflect differences in the use of the PSA test for cancer detection), age at diagnosis (<60, 60 to 69, or 70 years), and time between blood collection and diagnosis (<3, 3 to 6, or 7 years). We excluded case patients from the analyses of stage and grade at diagnosis if the relevant information was not available. For each of these case patient characteristics, we calculated a heterogeneity chi-square statistic to assess whether the estimated ORs statistically differed from each other (21). To assess whether the OR estimate of the linear trend for each IGF or IGFBP varied according to PSA level at recruitment (<2 g/L or 2 g/L), we entered an interaction term into the conditional logistic regression model for each IGF or IGFBP, and we tested the statistical significance of the interaction term with a likelihood ratio test. Statistical significance was set at the 5% level. All statistical tests were 2-sided. All statistical analyses were done with Stata, version 9.0 (StataCorp, College Station, Texas). Results Table 1 shows the characteristics of the studies. The 12 prospective studies included approximately 3700 case patients with prostate cancer and 5200 control participants. Insulin-like growth factor I and IGFBP-III measurements were available for all and 3600 case patients, respectively. However, IGF-II and IGFBP-II measurements were available for only 379 and 419 case patients, respectively (Table 2). Mean age at blood collection

Development and Validation of a Multidimensional Prognostic Index for One-Year Mortality from Comprehensive Geriatric Assessment in Hospitalized Older Patients

Our objective was to construct and validate a Multidimensional Prognostic Index (MPI) for 1-year mortality from a Comprehensive Geriatric Assessment (CGA) routinely carried out in elderly patients in a geriatric acute ward. The CGA included clinical, cognitive, functional, nutritional, and social parameters and was carried out using six standardized scales and information on medications and social support network, for a total of 63 items in eight domains. A MPI was developed from CGA data by aggregating the total scores of the eight domains and expressing it as a score from 0 to 1. Three grades of MPI were identified: low risk, 0.0-0.33; moderate risk, 0.34-0.66; and severe risk, 0.67-1.0. Using the proportional hazard models, we studied the predictive value of the MPI for all causes of mortality over a 12-month follow-up period. MPI was then validated in a different cohort of consecutively hospitalized patients. The development cohort included 838 and the validation cohort 857 elderly hospitalized patients. Of the patients in the two cohorts, 53.3 and 54.9% were classified in the low-risk group, respectively (MPI mean value, 0.18 +/- 0.09 and 0.18 +/- 0.09); 31.2 and 30.6% in the moderate-risk group (0.48 +/- 0.09 and 0.49 +/- 0.09); 15.4 and 14.2% in the severe-risk group (0.77 +/- 0.08 and 0.75 +/- 0.07). In both cohorts, higher MPI scores were significantly associated with older age (p = 0.0001), female sex (p = 0.0001), lower educational level (p = 0.0001), and higher mortality (p = 0.0001). In both cohorts, a close agreement was found between the estimated mortality and the observed mortality after both 6 months and 1 year of follow-up. The discrimination of the MPI was also good, with a ROC area of 0.751 (95%CI, 0.70-0.80) at 6 months and 0.751 (95%CI, 0.71-0.80) at 1 year of follow-up. We conclude that this MPI, calculated from information collected in a standardized CGA, accurately stratifies hospitalized elderly patients into groups at varying risk of mortality.

The ACTN3 R577X nonsense allele is under-represented in elite-level strength athletes

Previous reports have shown a lower proportion of the ACTN3 X/X genotype (R577X nonsense polymorphism) in sprint-related athletes compared to the general population, possibly attributed to impairment of muscle function related to α-actinin-3 deficiency. In the present study, we examined the frequency of the X/X genotype in both Black and White elite-level bodybuilders and strength athletes in comparison to the general population. A reference population of 668 Whites (363 men and 305 women) and 208 Blacks (98 men and 110 women) was genotyped for the ACTN3 R577X polymorphism. Strength athletes (52 white and 23 black; 4 women) consisting predominantly of world class and locally competitive bodybuilders, and elite powerlifters were recruited and similarly genotyped. Significantly lower X/X genotype frequencies were observed in the athletes (6.7%) vs controls (16.3%; P=0.005). The X/X genotype was significantly lower in White athletes (9.7%) vs controls (19.9%; P=0.018). No black athletes (0%) were observed with the X/X genotype, though this finding only approached statistical significance vs controls (4.8%; P=0.10). The results indicate that the ACTN3 R577X nonsense allele (X) is under-represented in elite strength athletes, consistent with previous reports indicating that α-actinin-3 deficiency appears to impair muscle performance.

Physical performance in peripheral arterial disease : A slower rate of decline in patients who walk more

Context Patients with lower-extremity peripheral arterial disease (PAD) benefit from supervised walking programs, but cost, travel, and other factors often limit participation. Contribution This prospective study shows that a self-directed program of walking at least 3 times per week for exercise is associated with a significantly reduced functional decline during the subsequent year in patients with PAD when compared with those who walk less frequently. Implications Self-directed walking for exercise may benefit a much larger proportion of patients with PAD than is currently being served by supervised rehabilitation programs. Cautions Observational studies, such as this one, cannot prove a causal relationship between walking frequency and functional decline. The Editors Peripheral arterial disease (PAD) of the lower extremities affects 20% to 30% of older patients in general medical practices (1, 2). Most patients with the disease do not have classical symptoms of intermittent claudication (1-3). Compared with those without PAD, persons with the disease have significantly greater functional impairment and more rapid functional decline (2-4). Exercise rehabilitation that includes supervised treadmill walking substantially improves treadmill walking performance in men and women with intermittent claudication (5). However, such barriers as cost, transportation, and program availability often limit access to exercise rehabilitation programs for patients with PAD (6, 7). Clinical guidelines for PAD recommend supervised walking exercise, but evidence for the benefits of unsupervised walking exercise is minimal to absent (8, 9). Specifically, it is unknown whether patients with PAD who engage in regular self-directed walking exercise have less functional decline than those who are sedentary. For persons with PAD, supervised treadmill walking exercise 3 or more times per week is more effective than less frequent supervised walking exercise (5). Therefore, we conducted a prospective observational study to examine whether patients with PAD who report that they walk for exercise 3 or more times per week have less functional decline than PAD participants who walk for exercise less frequently. Methods Methods for this longitudinal observational study of men and women with and without PAD have been described elsewhere (4). The protocol was approved by the institutional review boards of Northwestern University and Catholic Health Partners Hospital. Participants gave informed consent. Participant Eligibility Participants were 55 years of age and older at baseline. Potential participants were identified consecutively from patients who tested positive for PAD in 3 Chicago-area noninvasive vascular laboratories. A few participants were identified from lists of consecutive patients with recent appointments in our general internal medicine practice. This latter group of patients was screened for PAD by calculating their anklebrachial index at baseline; PAD was defined as an index of less than 0.90 (10-12). Baseline visits occurred between October 1998 and January 2000, and follow-up visits were scheduled annually. Exclusion Criteria Exclusion criteria have been previously reported (4). Patients with dementia, recent major surgery, or foot or leg amputations were excluded. We also excluded nursing home residents and wheelchair-bound patients. Patients who did not speak English were excluded because the investigators were not fluent in non-English languages. Participants who underwent leg revascularization were excluded from analyses after the procedure. AnkleBrachial Index Measurement In accordance with established methods, we used a hand-held Doppler probe (Nicolet Vascular Pocket Dop II, Golden, Colorado) to obtain systolic pressures in the right and left brachial, dorsalis pedis, and posterior tibial arteries (4, 13, 14). To maximize precision, each pressure was measured twice. We calculated the anklebrachial index in each leg by dividing the mean of all 4 dorsalis pedis and posterior tibial pressures by the mean of the 4 brachial pressures (13). When calculated by this method, the index correlates more closely with lower-extremity arterial function than when determined by alternative methods (13). We used the average brachial pressures in the arm with highest pressure when the measurement was higher in the same arm in both measurement sets and the 2 brachial pressures differed by 10 mm Hg or more in at least 1 measurement set; in such cases, subclavian stenosis was possible (13, 14). The lowest anklebrachial index was used in analyses. Leg Symptoms On the basis of a previous study (3, 4, 15), we used the San Diego Claudication Questionnaire to classify patients into 5 groups according to type of leg symptoms. Four groups had exertional leg symptoms as determined by an affirmative response to the question, Do you get pain in either leg or buttock on walking? These participants were further classified as having 1) intermittent claudication (n= 133), defined as exertional calf pain that does not begin at rest, causes the participant to stop walking, and resolves within 10 minutes of rest; 2) leg pain on exertion and rest (n= 76), defined as exertional leg pain that sometimes begins at rest; 3) atypical exertional leg pain/carry on (n= 39), defined as exertional leg symptoms that do not begin at rest and do not stop the individual while walking; and 4) atypical exertional leg pain/stop (n= 89), defined as exertional leg symptoms that do not begin at rest, stop the individual from walking, and do not involve the calves or resolve within 10 minutes of rest. A fifth group of patients was defined as asymptomatic (n= 80) because they reported no pain in either leg or buttock on walking. Comorbid Conditions We used algorithms developed for the Womens Health and Aging Study to document the following comorbid conditions: angina, diabetes mellitus, myocardial infarction, stroke, heart failure, pulmonary disease, spinal stenosis, disk disease, Parkinson disease, and hip fracture (16). American College of Rheumatology criteria were used to diagnose knee and hip osteoarthritis (17, 18). Functional Measures Rationale for use of the specific functional outcome measures used in the Walking and Leg Circulation Study has been described previously (19). Timed Walk Per a standardized protocol (20, 21), participants walked up and down a 100-foot hallway for 6 minutes after they were instructed to cover as much distance as possible during the allotted time. Repeated Chair Rises Participants sat in a straight-backed chair with their arms folded across their chest and rose to a standing position, repeating the exercise 5 consecutive times as quickly as possible. We measured the time each patient required to complete 5 chair rises. Standing Balance Participants were asked to hold 3 increasingly difficult standing positions for 10 seconds each: standing with feet together and parallel (side-by-side stand); standing with feet parallel, with the toes of 1 foot adjacent to and touching the heel of the opposite foot (semi-tandem stand); and standing with 1 foot directly in front of the other (tandem stand) (22, 23). Walking Velocity Walking velocity was measured with a 4-meter walk performed at usual and fastest pace. For the usual-paced walk, participants were instructed to walk at their usual pace, as if going down the street to the store. For the fastest-paced walk, participants were instructed to walk as fast as they could. Each walk was demonstrated by the research assistant. Participants were given the command ready, go; timing began on go. Each walk was performed twice, and the faster time in each pair was used in analyses (22, 23). Summary Performance Score The summary performance score combined data from the usual-paced 4-meter walking velocity, time to rise from a seated position 5 times, and standing balance. Individuals received a score of 0 for each task they were unable to complete. Scores ranging from 1 to 4 were assigned for all completed tasks; the scoring system was based on quartiles of performance for over 5000 participants in the Established Populations for the Epidemiologic Study of the Elderly (22, 23). Scores were then summed to obtain the summary performance score, which ranged from 0 to 12. Exercise During each study visit, participants were classified as current exercisers if they responded affirmatively to the question, During the past 2 weeks, have you gone walking for exercise? Frequency and duration of walking for exercise were also determined at each visit. A minimum frequency of 3 times per week and a minimum duration of 30 minutes per session are most optimal for supervised walking exercise programs in patients with PAD according to published literature (5). Therefore, we defined optimal walking frequency as 3 or more times per week and optimal walking duration as 90 minutes per week. Depressive Symptoms We measured depressive symptoms annually by using the Geriatric Depression Scale (short form), a 15-item questionnaire (24). Possible scores for the questionnaire ranged from 0 to 15; a score of 0 indicated no depressive symptoms and a score of 15 indicated that all depressive symptoms defined in the questionnaire were present. Other Measures Height and weight were measured at each visit. Body mass index (BMI) was calculated by dividing the patients body weight in kilograms by the square of his or her height in meters. Patients annually reported cigarette use (pack-years) and the number of blocks they walked during the past week. The principal investigator reviewed all medication use to identify patients who used aspirin, statins, and angiotensin-converting enzyme inhibitors. Physical Activity We measured each patients physical activity continuously over 7 days (beginning with the baseline visit) by using the Caltrac (Muscle Dynamics Fitness Network, Torrance, California) vertical accelerometer (25-29). This accelerometer

Age-Associated Declines in Complex Walking Task Performance: The Walking InCHIANTI Toolkit

OBJECTIVES: To describe a set of complex walking tasks (CWTs) that can be used to evaluate mobility and to characterize age‐ and sex‐specific performance on these tests.

Biomarkers of Inflammation and Thrombosis as Predictors of Near-Term Mortality in Patients with Peripheral Arterial Disease: A Cohort Study

Context Traditional cardiovascular disease risk factors predict distant but not near-term outcomes. Change in thrombogenic and inflammatory biomarkers might predict near-term events. Contribution The authors measured d-dimer, amyloid A protein, and C-reactive protein annually for 3.4 years in 377 patients with peripheral arterial disease. Elevated levelsand increases in levelswere associated with all-cause and cardiovascular diseaserelated deaths occurring 1 to 2 years after their measurement but not with deaths after 2 years. Caution The small number of deaths limits the power to detect patterns. Implication Increases in thrombogenic and inflammatory biomarker levels may signal near-termbut not distantevents. This observation requires confirmation in a larger sample. The Editors Atherosclerotic cardiovascular disease causes more than 19 million deaths per year worldwide and is a major cause of morbidity (1). Traditional atherosclerotic risk factors predict later-term cardiovascular events and mortality but are relatively poor predictors of near-term events (1, 2). Identifying biomarkers whose levels are elevated before an imminent clinical event could provide important prognostic information and elucidate mechanisms of acute events. Thrombogenic and inflammatory factors have been implicated in the pathogenesis of acute cardiovascular events. Elevated levels of inflammatory factors may encourage plaque instability and rupture (3, 4). Elevated d-dimer levels may reflect the degree of ongoing fibrin formation and degradation associated with unstable atherosclerotic plaque (57). The liver rapidly synthesizes the inflammatory markers serum amyloid A and C-reactive protein (CRP) in response to inflammatory stimuli (8). Elevated or increasing levels of these biomarkers may signal increased risk for acute arterial thrombosis or unstable plaque rupture. We studied associations between elevated levels of d-dimer, CRP, and serum amyloid A and near-term mortality versus long-term mortality in patients with lower-extremity peripheral arterial disease (PAD). We also studied associations of increases in these markers with death during the year after measurement. Persons with PAD have increased mortality and elevated levels of these biomarkers compared with persons without PAD (911). Therefore, persons with PAD are an important cohort in which to evaluate temporal associations between death and changes in levels of these biomarkers. We hypothesized that elevated biomarker levels would be more predictive of deaths occurring in the near term after biomarker measurement than of deaths occurring later after biomarker measurement. We also hypothesized that increases in biomarker levels would be associated with a higher risk for death during the first year after the biomarker increase. Methods Overview We designed an observational, prospective study of persons with PAD (12). Participants attended 1 baseline visit and 3 annual follow-up visits. We collected blood at each visit and followed participants for total and cardiovascular disease mortality. We studied the association between biomarker levels at each visit and death occurring during 3 intervals: the year immediately after biomarker measurement, 1 to 2 years after biomarker measurement, and 2 to 3 years after biomarker measurement. Yearly follow-up was defined according to the annual visit dates. We tested the hypothesis that associations of elevated biomarker levels with mortality were strongest for the shortest follow-up interval and weakest for the longest follow-up interval. We used time-dependent analyses for the biomarkers so that participants with blood levels obtained at multiple visits contributed data for each biomarker measurement. We also studied whether death during the first year after changes in biomarker levels was associated with the change in biomarker level between baseline and the 1-year follow-up visit, baseline and the 2-year follow-up visit, and baseline and the 3-year follow-up visit. Participants Participants were consecutive patients undergoing lower-extremity arterial testing in 3 Chicago-area noninvasive vascular laboratories (12). Some participants were identified among consecutive patients in a large, general internal medicine practice because they had a low anklebrachial index. The study protocol was approved by the institutional review boards of Northwestern Universitys Feinberg School of Medicine and Catholic Health Partners Hospitals (Chicago, Illinois). Participants gave written informed consent. Exclusion Criteria We defined PAD as an anklebrachial index less than 0.90 (13). Exclusion criteria are reported elsewhere (12). We excluded patients with dementia (because they could not answer questions accurately), nursing home residents, wheelchair-bound persons, persons with foot or leg amputations, and patients who had recently had major surgery. We also excluded nonEnglish-speaking patients because investigators were not fluent in non-English languages. AnkleBrachial Index Measurement After participants rested supine for 5 minutes, we used a hand-held Doppler probe (Pocket-Dop II, Nicolet Vascular, Golden, Colorado) to measure systolic pressures in the right brachial artery, right dorsalis pedis and posterior tibial arteries, left dorsalis pedis and posterior tibial arteries, and left brachial artery. Each pressure was measured twice (12). We calculated the anklebrachial index in each leg by dividing average pressures in each leg by the average of the 4 brachial pressures (14). We used average brachial pressures in the arm with highest pressure instead of the average of all 4 brachial pressures when 1 brachial pressure was higher than the other in both measurement sets, and the 2 brachial pressures differed by 10 mm Hg or more in at least 1 measurement set. These participants may have had subclavian stenosis (1416). The lowest leg anklebrachial index was used in analyses. Comorbid Conditions We used algorithms developed for the Womens Health and Aging Study to document comorbid conditions at baseline (17). These algorithms combine data from patient report, physical examination, medical record review, medications, laboratory values, and a primary care physician questionnaire. Blood Collection At each visit, we asked all participants to undergo blood sample collection and storage and to verify their assent by initialing the consent form. Phlebotomy was attempted for all consenting participants. The principal investigator certified health interviewers to do phlebotomy before beginning blood collection and again every 6 months to ensure continued adherence to protocol. A 10% randomly selected sample of participants underwent split sample testing for additional quality-control monitoring. Blood was collected into Vacutainer (Becton Dickenson, Franklin Lakes, New Jersey) tubes containing EDTA and sodium citrate and was put on ice immediately. Tubes were spun at 3000 rpm for 20 minutes at 4C in a refrigerated centrifuge. Blood was stored at 70C until analyses were completed, which was up to 3 years after blood collection. Serum Amyloid A and CRP Levels We measured serum amyloid A and CRP by using an immunotechnique on the Behring BN II analyzer (Dade Behring, Wilmington, Delaware). This method detects CRP concentrations as low as 0.15 mg/L (18). Coefficients of variability were 7.94% for serum amyloid A and 4.26% for CRP. d-Dimer Levels We used an Asserachrom D-Di kit (Diagnostica Stago, Asnires-sur-Seine, France) to measure d-dimer with an enzyme-linked immunosorbent assay procedure. The coefficient of variability was 9.4%. Total and High-Density Lipoprotein Cholesterol Levels Total cholesterol levels were measured by using enzymatic reaction with peroxidasephenol-4-aminophenazone indicator reaction (19). High-density lipoprotein cholesterol levels were measured by using direct enzymatic colorimetric assay (20). Cigarette Smoking We determined cigarette smoking history by patient report using a structured interview. Pack-years of smoking were calculated on the basis of the number of years smoked and the average number of packs smoked per day. Death We ascertained deaths from the Social Security Death Index. Survival status was available for all participants. We obtained death certificates from the state of Illinois or medical records. Cause of death was determined by a certified nosologist who had completed advanced training in identifying causes of death from death certificates. Cardiovascular deaths were those with International Statistical Classification of Diseases and Related Health Problems, 10th Revision, codes in the range I01.0 through I99.9, which includes deaths due to coronary heart disease, stroke, peripheral vascular disease, and other cardiovascular diseases. Statistical Analysis We compared baseline characteristics between decedents and survivors with PAD by using general linear models for continuous variables and chi-square tests for categorical variables. We used proportional hazards regression analyses to relate baseline biomarker levels to all-cause and cardiovascular mortality at 4-year follow-up,adjusting for age, sex, race, diabetes, number of cardiovascular diseases, smoking, and anklebrachial index. Analytic Approaches Used to Address Primary Study Aims We used 2 analytic approaches to model associations of biomarker levels with mortality. To assess the marginal associations of a given biomarker level with near-term versus later-term mortality, we used a 3-model approach in which we performed 3 proportional hazard analyses, each corresponding to 1 of 3 intervals: the first, second, and third years after the biomarker measurement. To assess longitudinal associations of changes in biomarker levels with mortality, we combined the data into 1 model (combined model approach). The Appendix presents detailed descriptions of these 2 analytic approaches. The 3-model approach provides more statistical power than the combined mode

Epigenetic Signatures of Cigarette Smoking

Background—DNA methylation leaves a long-term signature of smoking exposure and is one potential mechanism by which tobacco exposure predisposes to adverse health outcomes, such as cancers, osteoporosis, lung, and cardiovascular disorders. Methods and Results—To comprehensively determine the association between cigarette smoking and DNA methylation, we conducted a meta-analysis of genome-wide DNA methylation assessed using the Illumina BeadChip 450K array on 15u2009907 blood-derived DNA samples from participants in 16 cohorts (including 2433 current, 6518 former, and 6956 never smokers). Comparing current versus never smokers, 2623 cytosine–phosphate–guanine sites (CpGs), annotated to 1405 genes, were statistically significantly differentially methylated at Bonferroni threshold of P<1×10−7 (18u2009760 CpGs at false discovery rate <0.05). Genes annotated to these CpGs were enriched for associations with several smoking-related traits in genome-wide studies including pulmonary function, cancers, inflammatory diseases, and heart disease. Comparing former versus never smokers, 185 of the CpGs that differed between current and never smokers were significant P<1×10−7 (2623 CpGs at false discovery rate <0.05), indicating a pattern of persistent altered methylation, with attenuation, after smoking cessation. Transcriptomic integration identified effects on gene expression at many differentially methylated CpGs. Conclusions—Cigarette smoking has a broad impact on genome-wide methylation that, at many loci, persists many years after smoking cessation. Many of the differentially methylated genes were novel genes with respect to biological effects of smoking and might represent therapeutic targets for prevention or treatment of tobacco-related diseases. Methylation at these sites could also serve as sensitive and stable biomarkers of lifetime exposure to tobacco smoke.

ACTN3 genotype is associated with muscle phenotypes in women across the adult age span

The R577X polymorphism in the alpha-actinin-3 encoding gene (ACTN3) has been associated with elite athletic performance, and recently with differences in isometric and dynamic muscle strength and power in the general population. In this study we sought to determine the association of ACTN3 R577X genotype with muscle strength and mass phenotypes in men and women across the adult age span. Eight hundred forty-eight (n = 848) adult volunteers (454 men and 394 women) aged 22-90 yr were genotyped for ACTN3 R577X. Knee extensor (KE) shortening and lengthening peak torque values were determined using isokinetic dynamometry and fat-free mass (FFM) by dual-energy X-ray absorptiometry. Women deficient in alpha-actinin-3 (X/X; n = 53) displayed lower KE shortening peak torque (30 degrees /s: 89.5 +/- 3.5 vs. 99.3 +/- 1.4 N.m, P = 0.011; 180 degrees /s: 60.3 +/- 2.6 vs. 67.0 +/- 1.0 N.m, P = 0.019) and KE lengthening peak torque (30 degrees /s: 122.8 +/- 5.7 vs. 137.0 +/- 2.2 N.m, P = 0.022; 180 degrees /s: 121.8 +/- 5.8 vs. 138.5 +/- 2.2 N.m, P = 0.008) compared with R/X + R/R women (n = 341). Women X/X homozygotes also displayed lower levels of both total body FFM (38.9 +/- 0.5 vs. 40.1 +/- 0.2 kg, P = 0.040) and lower limb FFM (11.9 +/- 0.2 vs. 12.5 +/- 0.1 kg, P = 0.044) compared with R/X + R/R women. No genotype-related differences were observed in men. In conclusion, our results indicate that the absence of alpha-actinin-3 protein (i.e., ACTN3 X/X genotype) influences KE peak torque and FFM in women but not men.

Insulin resistance and executive dysfunction in older persons.

Objectives: To evaluate the association between insulin resistance (IR) and executive dysfunction in a large, population‐based study of older persons without diabetes mellitus (DM) or dementia.