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1.  Vitamin D Modulates Expression of the Airway Smooth Muscle Transcriptome in Fatal Asthma 
PLoS ONE  2015;10(7):e0134057.
Globally, asthma is a chronic inflammatory respiratory disease affecting over 300 million people. Some asthma patients remain poorly controlled by conventional therapies and experience more life-threatening exacerbations. Vitamin D, as an adjunct therapy, may improve disease control in severe asthma patients since vitamin D enhances glucocorticoid responsiveness and mitigates airway smooth muscle (ASM) hyperplasia. We sought to characterize differences in transcriptome responsiveness to vitamin D between fatal asthma- and non-asthma-derived ASM by using RNA-Seq to measure ASM transcript expression in five donors with fatal asthma and ten non-asthma-derived donors at baseline and with vitamin D treatment. Based on a Benjamini-Hochberg corrected p-value <0.05, 838 genes were differentially expressed in fatal asthma vs. non-asthma-derived ASM at baseline, and vitamin D treatment compared to baseline conditions induced differential expression of 711 and 867 genes in fatal asthma- and non-asthma-derived ASM, respectively. Functional gene categories that were highly represented in all groups included extracellular matrix, and responses to steroid hormone stimuli and wounding. Genes differentially expressed by vitamin D also included cytokine and chemokine activity categories. Follow-up qPCR and individual analyte ELISA experiments were conducted for four cytokines (i.e. CCL2, CCL13, CXCL12, IL8) to measure TNFα-induced changes by asthma status and vitamin D treatment. Vitamin D inhibited TNFα-induced IL8 protein secretion levels to a comparable degree in fatal asthma- and non-asthma-derived ASM even though IL8 had significantly higher baseline levels in fatal asthma-derived ASM. Our findings identify vitamin D-specific gene targets and provide transcriptomic data to explore differences in the ASM of fatal asthma- and non-asthma-derived donors.
PMCID: PMC4514847  PMID: 26207385
2.  Variants of Asthma and Chronic Obstructive Pulmonary Disease Genes and Lung Function Decline in Aging 
A substantial proportion of the general population has low lung function, and lung function is known to decrease as we age. Low lung function is a feature of several pulmonary disorders, such as uncontrolled asthma and chronic obstructive pulmonary disease. The objective of this study is to investigate the association of polymorphisms in asthma and chronic obstructive pulmonary disease candidate genes with rates of lung function decline in a general population sample of aging men.
We analyzed data from a cohort of 1,047 Caucasian men without known lung disease, who had a mean of 25 years of lung function data, and on whom DNA was available. The cohort was randomly divided into two groups, and we tested a total of 940 single-nucleotide polymorphisms in 44 asthma and chronic obstructive pulmonary disease candidate genes in the first group (testing cohort, n = 545) for association with change in forced expiratory volume in 1 second over time.
One hundred nineteen single-nucleotide polymorphisms that showed nominal associations in the testing cohort were then genotyped and tested in the second group (replication cohort, n = 502). Evidence for association from the testing and replication cohorts were combined, and after adjustment for multiple testing, seven variants of three genes (DPP10, NPSR1, and ADAM33) remained significantly associated with change in forced expiratory volume in 1 second over time.
Our findings that genetic variants of genes involved in asthma and chronic obstructive pulmonary disease are associated with lung function decline in normal aging participants suggest that similar genetic mechanisms may underlie lung function decline in both disease and normal aging processes.
PMCID: PMC4111635  PMID: 24253534
Genetics; Pulmonary; Normative aging; Successful aging.
3.  Genome-Wide Association Study Identifies Novel Pharmacogenomic Loci For Therapeutic Response to Montelukast in Asthma 
PLoS ONE  2015;10(6):e0129385.
Genome-wide association study (GWAS) is a powerful tool to identify novel pharmacogenetic single nucleotide polymorphisms (SNPs). Leukotriene receptor antagonists (LTRAs) are a major class of asthma medications, and genetic factors contribute to variable responses to these drugs. We used GWAS to identify novel SNPs associated with the response to the LTRA, montelukast, in asthmatics.
Using genome-wide genotype and phenotypic data available from American Lung Association - Asthma Clinical Research Center (ALA-ACRC) cohorts, we evaluated 8-week change in FEV1 related to montelukast administration in a discovery population of 133 asthmatics. The top 200 SNPs from the discovery GWAS were then tested in 184 additional samples from two independent cohorts.
Twenty-eight SNP associations from the discovery GWAS were replicated. Of these, rs6475448 achieved genome-wide significance (combined P = 1.97 x 10-09), and subjects from all four studies who were homozygous for rs6475448 showed increased ΔFEV1 from baseline in response to montelukast.
Through GWAS, we identified a novel pharmacogenomic locus related to improved montelukast response in asthmatics.
PMCID: PMC4470685  PMID: 26083242
4.  Association between prehospital vitamin D status and incident acute respiratory failure in critically ill patients: a retrospective cohort study 
BMJ Open Respiratory Research  2015;2(1):e000074.
We hypothesise that low 25-hydroxyvitamin D (25(OH)D) levels before hospitalisation are associated with increased risk of acute respiratory failure.
Retrospective cohort study.
Medical and Surgical Intensive care units of two Boston teaching hospitals.
1985 critically ill adults admitted between 1998 and 2011.
Measurements and main results
The exposure of interest was prehospital serum 25(OH)D categorised as ≤10 ng/mL, 11–19.9 ng/mL, 20–29.9 ng/mL and ≥30 ng/mL. The primary outcome was acute respiratory failure excluding congestive heart failure determined by International Classification of Diseases Ninth Edition (ICD-9) coding and validated against the Berlin Definition of acute respiratory sistress syndrome. Association between 25(OH)D and acute respiratory failure was assessed using logistic regression, while adjusting for age, race, sex, Deyo-Charlson Index and patient type (medical vs surgical).
In the cohort, the mean age was 63 years, 45% were male and 80% were white; 25(OH)D was ≤10 ng/mL in 8% of patients, 11–19.9 ng/mL in 24%, 20–29.9 ng/mL in 24% and ≥30 ng/mL in 44% of patients. Eighteen per cent (n=351) were diagnosed with acute respiratory failure. Compared to patients with 25(OH)D ≥30 ng/mL, patients with lower 25(OH)D levels had significantly higher adjusted odds of acute respiratory failure (≤10 ng/mL, OR=1.84 (95% CI 1.22 to 2.77); 11–19.9 ng/mL, OR=1.60 (95% CI 1.19 to 2.15); 20–29.9 ng/mL, OR=1.37 (95% CI 1.01 to 1.86)).
Prehospital 25(OH)D was associated with the risk of acute respiratory failure in our critically ill patient cohort.
PMCID: PMC4477148  PMID: 26113982
ARDS; Clinical Epidemiology
5.  Low serum 25-hydroxyvitamin D at critical care initiation is associated with increased mortality 
Critical care medicine  2012;40(1):63-72.
We hypothesized that deficiency in 25-hydroxy vitamin D at critical care initiation would be associated with all cause mortality.
Two-center observational study.
Two teaching hospitals in Boston, Massachusetts
1,325 patients, age ≥ 18 years, in whom 25-hydroxy vitamin D was measured 7 days prior to or after critical care initiation between 1998 and 2009.
25-hydroxy vitamin D was categorized as deficiency in 25-hydroxy vitamin D (≤15 ng/mL), insufficiency (16–29 ng/mL) and sufficiency (≥30 ng/mL). Logistic regression examined death by days 30, 90 and 365 post-critical care initiation and in hospital mortality. Adjusted odds ratios were estimated by multivariable logistic regression models.
Key Results
25-hydroxy vitamin D deficiency is predictive for short term and long term mortality. 30 days following critical care initiation, patients with 25-hydroxy vitamin D deficiency have an OR for mortality of 1.85 (95% CI, 1.15–2.98;P=0.01) relative to patients with 25-hydroxy vitamin D sufficiency. 25-hydroxy vitamin D deficiency remains a significant predictor of mortality at 30 days following critical care initiation following multivariable adjustment for age, gender, race, Deyo-Charlson index, sepsis, season, and surgical versus medical patient type (adjusted OR 1.94; 95% CI, 1.18–3.20;P=0.01). Results were similarly significant at 90 and 365 days following critical care initiation and for in hospital mortality. The association between vitamin D and mortality was not modified by sepsis, race, or Neighborhood poverty rate, a proxy for socioeconomic status.
Deficiency of 25-hydroxy vitamin D at the time of critical care initiation is a significant predictor of all cause patient mortality in a critically ill patient population.
PMCID: PMC4427350  PMID: 21926604
6.  Asthma, Allergy, and Responses to Methyl Donor Supplements and Nutrients 
After a brief period of stabilization, recent data has shown that the prevalence of asthma and allergic diseases continue to increase. Atopic diseases are major public health problems resulting in significant disability and resource utilization globally. While environmental factors influence the development of atopic disease, dietary changes may partially explain the high burden of atopic disease. One mechanism by which diet is suspected to impact asthma and allergy susceptibility is through epigenetic mechanisms including DNA methylation. Dietary methyl donors are important in the one-carbon metabolism pathway that is essential for DNA methylation. Findings from both observational studies and interventional trials of dietary methyl donor supplementation on the development and treatment of asthma and allergy have produced mixed results. While issues related to the differences in study design partially explain the heterogeneous results, two other issues have been largely overlooked in these studies. Firstly, these nutrients affect one of many pathways and occur in many of the same foods. Secondly, it is now becoming clear that the human intestinal microbiome is involved in the metabolism and production of the B vitamins and other methyl donor nutrients. Future studies will need to account for both the interrelationships between these nutrients and the effects of the microbiome.
PMCID: PMC4004707  PMID: 24360248
Allergy; Asthma; Betaine; Choline; Vitamin B2; Vitamin B6; Vitamin B12; Folate; Methyl Donor; Microbiome
7.  Peanut, milk, and wheat intake during pregnancy is associated with reduced allergy and asthma in children 
Maternal diet during pregnancy may influence childhood allergy and asthma.
To examine the associations between maternal intake of common childhood food allergens during early pregnancy and childhood allergy and asthma.
We studied 1277 mother-child pairs from a United States pre-birth cohort unselected for any disease. Using food frequency questionnaires administered during the first and second trimesters, we assessed maternal intake of common childhood food allergens during pregnancy. In mid-childhood (mean age 7.9 years), we assessed food allergy, asthma, allergic rhinitis, and atopic dermatitis by questionnaire and serum specific IgE levels. We examined the associations between maternal diet during pregnancy and childhood allergy and asthma. We also examined the cross-sectional associations between specific food allergies, asthma, and atopic conditions in mid-childhood.
Food allergy was common (5.6%) in mid-childhood, as was sensitization to at least one food allergen (28.0%). Higher maternal peanut intake (each additional z-score) during the first trimester was associated with 47% reduced odds of peanut allergic reaction (OR 0.53, 95%CI 0.30–0.94). Higher milk intake during the first trimester was associated with reduced asthma (OR 0.83, 95%CI 0.69–0.99) and allergic rhinitis (OR 0.85, 95%CI 0.74–0.97). Higher maternal wheat intake during the second trimester was associated with reduced atopic dermatitis (OR 0.64, 95%CI 0.46–0.90). Peanut, wheat, and soy allergy were each cross-sectionally associated with increased childhood asthma, atopic dermatitis, and allergic rhinitis (ORs 3.6 to 8.1).
Higher maternal intake of peanut, milk, and wheat during early pregnancy was associated with reduced odds of mid-childhood allergy and asthma.
PMCID: PMC4004710  PMID: 24522094
maternal diet; pregnancy; food allergy; sensitization; asthma; allergic rhinitis; peanut; milk; wheat; childhood
9.  Vitamin D Status and Hypertensive Disorders in Pregnancy 
Annals of epidemiology  2014;24(5):399-403.e1.
Several studies have reported increased risk of preeclampsia when 25-hyrdoxyvitamin D (25[OH]D) levels are low. The extent to which 25(OH)D may lower risk for hypertensive disorder during pregnancy remains unclear.
Among women enrolled in the Project Viva prenatal cohort in Massachusetts, we examined associations of 25(OH)D levels obtained at 16.4 –36.9 weeks of gestation (mean 27.9 weeks) with hypertensive disorders of pregnancy, including preeclampsia (56/1591, 3.5%) and gestational hypertension (109/1591, 6.9%).
We did not detect an association between plasma 25(OH)D concentration (mean 58, SD 22 nmol/L) and preeclampsia. For each 25 nmol/L increase in 25(OH)D, the adjusted odds ratio for preeclampsia was 1.14 (95% confidence interval: 0.77, 1.67). By contrast and contrary to hypothesis, higher 25(OH)D concentrations were associated with higher odds of gestational hypertension: adjusted odds ratio for gestational hypertension was 1.32 (95% confidence interval: 1.01, 1.72) per each 25nmol/L increment in 25(OH)D. Vitamin D intake patterns suggest this association was not because of reverse causation. While the elevated hypertension risk may be due to chance, randomized trials of vitamin D supplementation during pregnancy should monitor for gestational hypertension.
These data do not support the hypothesis that higher 25(OH)D levels lower the overall risk of hypertensive disorders of pregnancy.
PMCID: PMC4011985  PMID: 24631523
Pregnancy; Pre-Eclampsia; Hypertension; Pregnancy-Induced; Vitamin D; 25-hydroxyvitamin D
10.  The Vitamin D Antenatal Asthma Reduction Trial (VDAART): Rationale, design, and methods of a randomized, controlled trial of vitamin D supplementation in pregnancy for the primary prevention of asthma and allergies in children 
Contemporary clinical trials  2014;38(1):37-50.
There is intense interest in the role of vitamin D in the development of asthma and allergies. However, studies differ on whether a higher vitamin D intake or status in pregnancy or at birth is protective against asthma and allergies. To address this uncertainty, the Vitamin D Antenatal Asthma Reduction Trial (VDAART) was developed. VDAART is a randomized, double-blind, placebo-controlled trial of vitamin D supplementation in pregnant women to determine whether prenatal supplementation can prevent the development of asthma and allergies in the women’s offspring. A secondary aim is to determine whether vitamin D supplementation can prevent the development of pregnancy complications, such as preeclampsia, preterm birth, and gestational diabetes. Women were randomized to the treatment arm of 4,000 IU/day of vitamin D3 plus a daily multivitamin that contained 400 IU of vitamin D3 or the placebo arm of placebo plus a multivitamin that contained 400 IU daily of vitamin D3. Women who were between the gestational ages of 10–18 weeks were randomized from three clinical centers across the United States – Boston Medical Center, Washington University in St. Louis, and Kaiser Permanente Southern California Region (San Diego, CA). Supplementation took place throughout pregnancy. Monthly monitoring of urinary calcium to creatinine ratio was performed in addition to medical record review for adverse events. Offspring are being evaluated quarterly through questionnaires and yearly during in-person visits until the 3rd birthday of the child. Ancillary studies will investigate neonatal T-regulatory cell function, maternal vaginal flora, and maternal and child intestinal flora.
PMCID: PMC4086903  PMID: 24614387
Vitamin D; asthma; allergy; randomized controlled trial; Deveopmental Origins; prenatal
11.  Maternal intake of vitamin D during pregnancy and risk of recurrent wheeze in children at 3 y of age1–3 
Vitamin D deficiency and asthma are common at higher latitudes. Although vitamin D has important immunologic effects, its relation with asthma is unknown.
We hypothesized that a higher maternal intake of vitamin D during pregnancy is associated with a lower risk of recurrent wheeze in children at 3 y of age.
The participants were 1194 mother-child pairs in Project Viva—a prospective prebirth cohort study in Massachusetts. We assessed the maternal intake of vitamin D during pregnancy from a validated food-frequency questionnaire. The primary outcome was recurrent wheeze, ie, a positive asthma predictive index (≥2 wheezing attacks among children with a personal diagnosis of eczema or a parental history of asthma).
The mean (±SD) total vitamin D intake during pregnancy was 548 ± 167 IU/d. By age 3 y, 186 children (16%) had recurrent wheeze. Compared with mothers in the lowest quartile of daily intake (median: 356 IU), those in the highest quartile (724 IU) had a lower risk of having a child with recurrent wheeze [odds ratio (OR): 0.39; 95% CI: 0.25, 0.62; P for trend <0.001]. A 100-IU increase in vitamin D intake was associated with lower risk (OR: 0.81; 95% CI: 0.74, 0.89), regardless of whether vitamin D was from the diet (OR: 0.81; 95% CI: 0.69, 0.96) or supplements (OR: 0.82; 95% CI: 0.73, 0.92). Adjustment for 12 potential confounders, including maternal intake of other dietary factors, did not change the results.
In the northeastern United States, a higher maternal intake of vitamin D during pregnancy may decrease the risk of recurrent wheeze in early childhood.
PMCID: PMC4406411  PMID: 17344501
Vitamin D; pregnancy; dietary intake; childhood wheeze; asthma
12.  Vitamin D status among preterm and full-term infants at birth 
Pediatric research  2013;75(0):75-80.
Risk factors for maternal vitamin D deficiency and preterm birth overlap but the distribution of 25-hydroxyvitamin D (25(OH)D) levels among preterm infants is not known. We aimed to determine associations between 25(OH)D levels and gestational age.
We measured umbilical cord plasma levels of 25(OH)D from 471 infants born at Brigham and Women’s Hospital in Boston. We used generalized estimating equations to determine whether preterm (<37 weeks’ gestation) or very preterm (<32 weeks’ gestation) infants had greater odds of 25(OH)D levels < 20 ng/ml than more mature infants. We adjusted for potential confounding by season of birth, maternal age, race, marital status and singleton or multiple gestation.
Mean cord plasma 25(OH)D level was 34.0 ng/ml (range 4.1 to 95.3, and SD 14.1). Infants born before 32 weeks’ gestation had increased odds of 25(OH)D levels < 20 ng/ml in unadjusted (OR 2.2, 95% CI 1.1, 4.3) and adjusted models (OR 2.4, 95% CI 1.2, 5.3) compared to more mature infants.
Infants born < 32 weeks’ gestation are at higher risk than more mature infants for low 25(OH)D levels. Further investigation of the relationships between low 25(OH)D levels and preterm birth and its sequelae is thus warranted.
PMCID: PMC4349515  PMID: 24121425
13.  Inhaled corticosteroid treatment modulates ZNF432 gene variant's effect on bronchodilator response in asthmatics 
Single nucleotide polymorphisms (SNPs) influence a patient's response to inhaled corticosteroids and β2-agonists, and the effect of treatment with inhaled corticosteroids is synergistic with the effect of β2-agonists. We hypothesized that use of inhaled corticosteroids could influence the effect of SNPs associated with bronchodilator response.
To assess whether, among asthma subjects, the association of SNPs with bronchodilator response is different between those treated with inhaled corticosteroids vs. those on placebo.
A genome-wide association analysis was conducted using 581 white subjects from the Childhood Asthma Management Program (CAMP). Using data for 449,540 SNPs, we conducted a gene by environment analysis in PLINK with inhaled corticosteroid treatment as the environmental exposure and bronchodilator response as the outcome measure. We attempted to replicate the top 12 SNPs in the Leukotriene Modifier Or Corticosteroid or Corticosteroid-Salmeterol (LOCCS) Trial.
The combined P-value for the CAMP and LOCCS populations was 4.81E-08 for rs3752120, which is located in the zinc finger protein gene ZNF432, and has unknown function.
Inhaled corticosteroids appear to modulate the association of bronchodilator response with variant(s) in the ZNF432 gene among adults and children with asthma.
Clinical Implications
Clinicians who treat asthma patients with inhaled corticosteroids should be aware that the patient's genetic makeup likely influences response as measured in lung function.
Capsule Summary
Our study suggests that inhaled corticosteroids could influence the effect of multiple SNPs associated with bronchodilator response across the genome.
PMCID: PMC3943570  PMID: 24280104
asthma; bronchodilator response; lung function; inhaled corticosteroids; single nucleotide polymorphisms; zinc finger proteins; ZNF432
14.  Risk loci for chronic obstructive pulmonary disease: a genome-wide association study and meta-analysis 
The Lancet. Respiratory medicine  2014;2(3):214-225.
The genetic risk factors for susceptibility to chronic obstructive pulmonary disease (COPD) are still largely unknown. Additional genetic variants are likely to be identified by genome-wide association studies in larger cohorts or specific subgroups.
Genome-wide association analysis in COPDGene (non-Hispanic whites and African-Americans) was combined with existing data from the ECLIPSE, NETT/NAS, and GenKOLS (Norway) studies. Analyses were performed both using all moderate-to-severe cases and the subset of severe cases. Top loci not previously described as genome-wide significant were genotyped in the ICGN study, and results combined in a joint meta-analysis.
Analysis of a total of 6,633 moderate-to-severe cases and 5,704 controls confirmed association at three known loci: CHRNA3/CHRNA5/IREB2, FAM13A, and HHIP (10−12 < P < 10−14), and also showed significant evidence of association at a novel locus near RIN3 (overall P, including ICGN = 5•4×10−9). In the severe COPD analysis (n=3,497), the effects at two of three previously described loci were significantly stronger; we also identified two additional loci previously reported to affect gene expression of MMP12 and TGFB2 (overall P = 2•6x10−9 and 8•3×10−9). RIN3 and TGFB2 expression levels were reduced in a set of Lung Tissue Research Consortium COPD lung tissue samples compared with controls.
In a genome-wide study of COPD, we confirmed associations at three known loci and found additional genome-wide significant associations with moderate-to-severe COPD near RIN3 and with severe COPD near MMP12 and TGFB2. Genetic variants, apart from alpha-1 antitrypsin deficiency, increase the risk of COPD. Our analysis of severe COPD suggests additional genetic variants may be identified by focusing on this subgroup.
National Heart, Lung, and Blood Institute; the COPD Foundation through contributions from AstraZeneca, Boehringer Ingelheim, Novartis, and Sepracor; GlaxoSmithKline; Centers for Medicare and Medicaid Services; Agency for Healthcare Research and Quality; US Department of Veterans Affairs.
PMCID: PMC4176924  PMID: 24621683
16.  Diet and Asthma: Vitamins and Methyl Donors 
The lancet. Respiratory medicine  2013;1(10):813-822.
Dietary changes may partly explain the high burden of asthma in industrialized nations. Experimental studies have motivated a significant number of observational studies of the relation between vitamins (A, C, D, and E) or nutrients acting as methyl donors (folate, vitamin B12, and choline) and asthma. Because observational studies are susceptible to several sources of bias, well-conducted randomized controlled trials (RCTs) remain the “gold standard” to determine whether a vitamin or nutrient has an effect on asthma. Evidence from observational studies and/or relatively few RCTs most strongly justify ongoing and future RCTs of: 1) vitamin D to prevent or treat asthma, 2) choline supplementation as adjuvant treatment for asthma, and 3) vitamin E to prevent the detrimental effects of air pollution in subjects with asthma. At this time, there is insufficient evidence to recommend supplementation with any vitamin or nutrient acting as a methyl donor to prevent or treat asthma.
PMCID: PMC3904132  PMID: 24461761
17.  Diagnostic accuracy of the bronchodilator response in children 
The bronchodilator response (BDR) reflects the reversibility of airflow obstruction and is recommended as an adjunctive test to diagnose asthma. The validity of the commonly used definition of BDR, a 12% or greater change in FEV1 from baseline, has been questioned in childhood.
We sought to examine the diagnostic accuracy of the BDR test by using 3 large pediatric cohorts.
Cases include 1041 children with mild-to-moderate asthma from the Childhood Asthma Management Program.
Control subjects (nonasthmatic and nonwheezing) were chosen from Project Viva and Home Allergens, 2 population-based pediatric cohorts. Receiver operating characteristic curves were constructed, and areas under the curve were calculated for different BDR cutoffs.
A total of 1041 cases (59.7% male; mean age, 8.9 ± 2.1 years) and 250 control subjects (46.8% male; mean age, 8.7 ± 1.7 years) were analyzed, with mean BDRs of 10.7% ± 10.2% and 2.7% ± 8.4%, respectively. The BDR test differentiated asthmatic patients from nonasthmatic patients with a moderate accuracy (area under the curve, 73.3%).
Despite good specificity, a cutoff of 12% was associated with poor sensitivity (35.6%). A cutoff of less than 8% performed significantly better than a cutoff of 12% (P = .03, 8% vs 12%).
Our findings highlight the poor sensitivity associated with the commonly used 12% cutoff for BDR. Although our data show that a threshold of less than 8% performs better than 12%, given the variability of this test in children, we conclude that it might be not be appropriate to choose a specific BDR cutoff as a criterion for the diagnosis of asthma.
PMCID: PMC3759549  PMID: 23683464
Asthma; bronchodilator response; diagnosis
18.  A genome-wide association study of bronchodilator response in asthmatics 
The pharmacogenomics journal  2013;14(1):41-47.
Reversibility of airway obstruction in response to β2-agonists is highly variable among asthmatics, which is partially attributed to genetic factors. In a genome-wide association study of acute bronchodilator response (BDR) to inhaled albuterol, 534,290 single nucleotide polymorphisms (SNPs) were tested in 403 white trios from the Childhood Asthma Management Program using five statistical models to determine the most robust genetic associations. The primary replication phase included 1397 polymorphisms in three asthma trials (pooled n=764). The second replication phase tested 13 SNPs in three additional asthma populations (n=241, n=215, and n=592). An intergenic SNP on chromosome 10, rs11252394, proximal to several excellent biological candidates, significantly replicated (p=1.98×10−7) in the primary replication trials. An intronic SNP (rs6988229) in the collagen (COL22A1) locus also provided strong replication signals (p=8.51×10−6). This study applied a robust approach for testing the genetic basis of BDR and identified novel loci associated with this drug response in asthmatics.
PMCID: PMC3706515  PMID: 23508266
pharmacogenetics; asthma; bronchodilator response; genome-wide association study; albuterol
19.  Epigenetic Influences on Associations between Air Pollutants and Lung Function in Elderly Men: The Normative Aging Study 
Environmental Health Perspectives  2014;122(6):566-572.
Background: Few studies have been performed on pulmonary effects of air pollution in the elderly—a vulnerable population with low reserve capacity—and mechanisms and susceptibility factors for potential effects are unclear.
Objectives: We evaluated the lag structure of air pollutant associations with lung function and potential effect modification by DNA methylation (< or ≥ median) at 26 individual CpG sites in nine candidate genes in a well-characterized cohort of elderly men.
Methods: We measured forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), and blood DNA methylation one to four times between 1999 and 2009 in 776 men from the Normative Aging Study. Air pollution was measured at fixed monitors 4 hr to 28 days before lung function tests. We used linear mixed-effects models to estimate the main effects of air pollutants and effect modification by DNA methylation.
Results: An interquartile range (IQR) increase in subchronic exposure (3 to 28 days cumulated), but not in acute exposure (during the previous 4 hr, or the current or previous day), to black carbon, total and nontraffic particles with aerodynamic diameter ≤ 2.5 μm (PM2.5), carbon monoxide, and nitrogen dioxide was associated with a 1–5% decrease in FVC and FEV1 (p < 0.05). Slope estimates were greater for FVC than FEV1, and increased with cumulative exposure. The estimates slopes for air pollutants (28 days cumulated) were higher in participants with low (< median) methylation in TLR2 at position 2 and position 5 and high (≥ median) methylation in GCR.
Conclusions: Subchronic exposure to traffic-related pollutants was associated with significantly reduced lung function in the elderly; nontraffic pollutants (particles, ozone) had weaker associations. Epigenetic mechanisms related to inflammation and immunity may influence these associations.
Citation: Lepeule J, Bind MAC, Baccarelli AA, Koutrakis P, Tarantini L, Litonjua A, Sparrow D, Vokonas P, Schwartz JD. 2014. Epigenetic influences on associations between air pollutants and lung function in elderly men: the Normative Aging Study. Environ Health Perspect 122:566–572;
PMCID: PMC4050500  PMID: 24602767
20.  Dissecting direct and indirect genetic effects on chronic obstructive pulmonary disease (COPD) susceptibility 
Human genetics  2013;132(4):431-441.
Cigarette smoking is the major environmental risk factor for chronic obstructive pulmonary disease (COPD). Genome-wide association studies have provided compelling associations for three loci with COPD. In this study, we aimed to estimate direct, i.e., independent from smoking, and indirect effects of those loci on COPD development using mediation analysis. We included a total of 3,424 COPD cases and 1,872 unaffected controls with data on two smoking-related phenotypes: lifetime average smoking intensity and cumulative exposure to tobacco smoke (pack years). Our analysis revealed that effects of two linked variants (rs1051730 and rs8034191) in the AGPHD1/CHRNA3 cluster on COPD development are significantly, yet not entirely, mediated by the smoking-related phenotypes. Approximately 30 % of the total effect of variants in the AGPHD1/CHRNA3 cluster on COPD development was mediated by pack years. Simultaneous analysis of modestly (r2 = 0.21) linked markers in CHRNA3 and IREB2 revealed that an even larger (~42 %) proportion of the total effect of the CHRNA3 locus on COPD was mediated by pack years after adjustment for an IREB2 single nucleotide polymorphism. This study confirms the existence of direct effects of the AGPHD1/CHRNA3, IREB2, FAM13A and HHIP loci on COPD development. While the association of the AGPHD1/CHRNA3 locus with COPD is significantly mediated by smoking-related phenotypes, IREB2 appears to affect COPD independently of smoking.
PMCID: PMC3600068  PMID: 23299987
21.  Allergen sensitization is associated with increased DNA methylation in older men 
Variation in epigenetic modifications, arising from either environmental exposures or internal physiological changes, can influence gene expression, and may ultimately contribute to complex diseases such as asthma and allergies. We examined the association of asthma and allergic phenotypes with DNA methylation levels of retrotransposon-derived elements.
We used data from 704 men (mean age 73) in the longitudinal Normative Aging Study to assess the relationship between asthma, allergic phenotypes and DNA methylation levels of the retrotransposon derived elements Alu and LINE-1. Retrotransposons represent a large fraction of the genome (> 30%), and are heavily methylated to prevent expression. Percent methylation of Alu and LINE-1 elements in peripheral white blood cells was quantified using PCR pyrosequencing. Data on sensitization to common allergens by skin prick testing, asthma, and methacholine responsiveness was gathered approximately 8 years prior to DNA methylation analysis.
Prior allergen sensitization was associated with increased methylation of Alu (β=0.32 [sensitized vs. non-sensitized], p value 0.003), in models adjusted for pack-years, BMI, smoking, air pollutants, percent eosinophils, white blood cell count and age. Of the men interviewed, 5 % of subjects reported diagnosis of asthma. Neither Alu, nor LINE-1 methylation was associated with asthma.
These data suggest that increased DNA methylation of repetitive elements may be associated with allergen sensitization, but does not appear to be associated with asthma. Future work is needed to identify potential underlying mechanisms for these relationships.
PMCID: PMC3730837  PMID: 23257623
allergen sensitization; DNA methylation; Alu; and LINE-1
22.  Genome-Wide Association Analysis of Blood Biomarkers in Chronic Obstructive Pulmonary Disease 
Rationale: A genome-wide association study (GWAS) for circulating chronic obstructive pulmonary disease (COPD) biomarkers could identify genetic determinants of biomarker levels and COPD susceptibility.
Objectives: To identify genetic variants of circulating protein biomarkers and novel genetic determinants of COPD.
Methods: GWAS was performed for two pneumoproteins, Clara cell secretory protein (CC16) and surfactant protein D (SP-D), and five systemic inflammatory markers (C-reactive protein, fibrinogen, IL-6, IL-8, and tumor necrosis factor-α) in 1,951 subjects with COPD. For genome-wide significant single nucleotide polymorphisms (SNPs) (P < 1 × 10−8), association with COPD susceptibility was tested in 2,939 cases with COPD and 1,380 smoking control subjects. The association of candidate SNPs with mRNA expression in induced sputum was also elucidated.
Measurements and Main Results: Genome-wide significant susceptibility loci affecting biomarker levels were found only for the two pneumoproteins. Two discrete loci affecting CC16, one region near the CC16 coding gene (SCGB1A1) on chromosome 11 and another locus approximately 25 Mb away from SCGB1A1, were identified, whereas multiple SNPs on chromosomes 6 and 16, in addition to SNPs near SFTPD, had genome-wide significant associations with SP-D levels. Several SNPs affecting circulating CC16 levels were significantly associated with sputum mRNA expression of SCGB1A1 (P = 0.009–0.03). Several SNPs highly associated with CC16 or SP-D levels were nominally associated with COPD in a collaborative GWAS (P = 0.001–0.049), although these COPD associations were not replicated in two additional cohorts.
Conclusions: Distant genetic loci and biomarker-coding genes affect circulating levels of COPD-related pneumoproteins. A subset of these protein quantitative trait loci may influence their gene expression in the lung and/or COPD susceptibility.
Clinical trial registered with (NCT 00292552).
PMCID: PMC3622441  PMID: 23144326
biomarker; chronic obstructive pulmonary disease; genome-wide association study
23.  Nitric oxide synthase polymorphisms, gene expression and lung function in chronic obstructive pulmonary disease 
Due to the pleiotropic effects of nitric oxide (NO) within the lungs, it is likely that NO is a significant factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to test for association between single nucleotide polymorphisms (SNPs) in three NO synthase (NOS) genes and lung function, as well as to examine gene expression and protein levels in relation to the genetic variation.
One SNP in each NOS gene (neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3)) was genotyped in the Lung Health Study (LHS) and correlated with lung function. One SNP (rs1800779) was also analyzed for association with COPD and lung function in four COPD case–control populations. Lung tissue expression of NOS3 mRNA and protein was tested in individuals of known genotype for rs1800779. Immunohistochemistry of lung tissue was used to localize NOS3 expression.
For the NOS3 rs1800779 SNP, the baseline forced expiratory volume in one second in the LHS was significantly higher in the combined AG + GG genotypic groups compared with the AA genotypic group. Gene expression and protein levels in lung tissue were significantly lower in subjects with the AG + GG genotypes than in AA subjects. NOS3 protein was expressed in the airway epithelium and subjects with the AA genotype demonstrated higher NOS3 expression compared with AG and GG individuals. However, we were not able to replicate the associations with COPD or lung function in the other COPD study groups.
Variants in the NOS genes were not associated with lung function or COPD status. However, the G allele of rs1800779 resulted in a decrease of NOS3 gene expression and protein levels and this has implications for the numerous disease states that have been associated with this polymorphism.
PMCID: PMC3827989  PMID: 24192154
Chronic obstructive pulmonary disease; Nitric oxide synthase; Polymorphism; Gene expression
24.  A polymorphism in the thyroid hormone receptor gene is associated with bronchodilator response in asthmatics 
The pharmacogenomics journal  2012;13(2):130-136.
A pro-asthmatic culture milieu and β2-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing p values < 0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5’ of the thyroid hormone receptor beta gene was associated with BDR in the childhood population and two adult populations (p value = 0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.
PMCID: PMC3349771  PMID: 22212731
Bronchodilator response; transcription factor; association; thyroid hormone receptor β; asthma; pharmacogenetics
25.  Vitamin D Deficiency, Smoking, and Lung Function in the Normative Aging Study 
Rationale: Vitamin D has immunomodulatory and antiinflammatory effects that may be modified by cigarette smoke and may affect lung function.
Objectives: To examine the effect of vitamin D deficiency and smoking on lung function and lung function decline.
Methods: A total of 626 men from the Normative Aging Study had 25-hydroxyvitamin D levels measured at three different times between 1984 and 2003 with concurrent spirometry. Vitamin D deficiency was defined as serum level ≤ 20 ng/ml. Statistical analysis was performed using multivariable linear regression and mixed effects models.
Measurements and Main Results: In the overall cohort, there was no significant effect of vitamin D deficiency on lung function or on lung function decline. In both cross-sectional and longitudinal multivariable models, there was effect modification by vitamin D status on the association between smoking and lung function. Cross-sectional analysis revealed lower lung function in current smokers with vitamin D deficiency (FEV1, FVC, and FEV1/FVC; P ≤ 0.0002), and longitudinal analysis showed more rapid rates of decline in FEV1 (P = 0.023) per pack-year of smoking in subjects with vitamin D deficiency as compared with subjects who were vitamin D sufficient.
Conclusions: Vitamin D deficiency was associated with lower lung function and more rapid lung function decline in smokers over 20 years in this longitudinal cohort of elderly men. This suggests that vitamin D sufficiency may have a protective effect against the damaging effects of smoking on lung function. Future studies should seek to confirm this finding in the context of smoking and other exposures that affect lung function.
PMCID: PMC3480523  PMID: 22822023
vitamin D; vitamin D deficiency; lung function decline; smoking; effect modification

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