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1.  Common Genetic Polymorphisms Influence Blood Biomarker Measurements in COPD 
PLoS Genetics  2016;12(8):e1006011.
Implementing precision medicine for complex diseases such as chronic obstructive lung disease (COPD) will require extensive use of biomarkers and an in-depth understanding of how genetic, epigenetic, and environmental variations contribute to phenotypic diversity and disease progression. A meta-analysis from two large cohorts of current and former smokers with and without COPD [SPIROMICS (N = 750); COPDGene (N = 590)] was used to identify single nucleotide polymorphisms (SNPs) associated with measurement of 88 blood proteins (protein quantitative trait loci; pQTLs). PQTLs consistently replicated between the two cohorts. Features of pQTLs were compared to previously reported expression QTLs (eQTLs). Inference of causal relations of pQTL genotypes, biomarker measurements, and four clinical COPD phenotypes (airflow obstruction, emphysema, exacerbation history, and chronic bronchitis) were explored using conditional independence tests. We identified 527 highly significant (p < 8 X 10−10) pQTLs in 38 (43%) of blood proteins tested. Most pQTL SNPs were novel with low overlap to eQTL SNPs. The pQTL SNPs explained >10% of measured variation in 13 protein biomarkers, with a single SNP (rs7041; p = 10−392) explaining 71%-75% of the measured variation in vitamin D binding protein (gene = GC). Some of these pQTLs [e.g., pQTLs for VDBP, sRAGE (gene = AGER), surfactant protein D (gene = SFTPD), and TNFRSF10C] have been previously associated with COPD phenotypes. Most pQTLs were local (cis), but distant (trans) pQTL SNPs in the ABO blood group locus were the top pQTL SNPs for five proteins. The inclusion of pQTL SNPs improved the clinical predictive value for the established association of sRAGE and emphysema, and the explanation of variance (R2) for emphysema improved from 0.3 to 0.4 when the pQTL SNP was included in the model along with clinical covariates. Causal modeling provided insight into specific pQTL-disease relationships for airflow obstruction and emphysema. In conclusion, given the frequency of highly significant local pQTLs, the large amount of variance potentially explained by pQTL, and the differences observed between pQTLs and eQTLs SNPs, we recommend that protein biomarker-disease association studies take into account the potential effect of common local SNPs and that pQTLs be integrated along with eQTLs to uncover disease mechanisms. Large-scale blood biomarker studies would also benefit from close attention to the ABO blood group.
Author Summary
Precision medicine is an emerging approach that takes into account variability in genes, gene and protein expression, environment and lifestyle. Recent advances in high-throughput genome-wide genotyping, genomics, and proteomics coupled with the creation of large, highly-phenotyped clinical cohorts now allows for integration of these molecular data sets at the individual level. Here we use genome-wide genotyping and blood measurements of 88 biomarkers in 1,340 subjects from two large NIH-supported clinical cohorts of smokers (SPIROMICS and COPDGene) to identify more than 300 novel DNA variants that influence measurement of blood protein levels (pQTLs). We find that many DNA variants explain a large portion of the variability of measured protein expression in blood. Furthermore, we show that integration of DNA variants with blood biomarker levels can improve the ability of predictive models to reflect the relationship between biomarker and disease features (e.g., emphysema) within chronic obstructive pulmonary disease (COPD).
PMCID: PMC4988780  PMID: 27532455
2.  Effect of Naturally Occurring Ozone Air Pollution Episodes on Pulmonary Oxidative Stress and Inflammation 
This study aimed to determine if naturally occurring episodes of ozone air pollution in the Salt Lake Valley in Utah, USA, during the summer are associated with increased pulmonary inflammation and oxidative stress, increased respiratory symptoms, and decreased lung function in individuals with chronic obstructive pulmonary disease (COPD) compared to controls. We measured biomarkers (nitrite/nitrate (NOx), 8-isoprostane) in exhaled breath condensate (EBC), spirometry, and respiratory symptoms in 11 former smokers with moderate-to-severe COPD and nine former smokers without airflow obstruction during periods of low and high ozone air pollution. High ozone levels were associated with increased NOx in EBC in both COPD (8.7 (±8.5) vs. 28.6 (±17.6) μmol/L on clean air vs. pollution days, respectively, p < 0.01) and control participants (7.6 (±16.5) vs. 28.5 (±15.6) μmol/L on clean air vs. pollution days, respectively, p = 0.02). There was no difference in pollution effect between COPD and control groups, and no difference in EBC 8-isoprostane, pulmonary function, or respiratory symptoms between clean air and pollution days in either group. Former smokers both with and without airflow obstruction developed airway oxidative stress and inflammation in association with ozone air pollution episodes.
PMCID: PMC4454954  PMID: 25985308
air pollution; ozone; chronic obstructive pulmonary disease; exhaled breath condensate; oxidative stress; airway inflammation
3.  Polymorphisms in key pulmonary inflammatory pathways and the development of acute respiratory distress syndrome 
Experimental lung research  2014;41(3):155-162.
Acute Respiratory Distress Syndrome (ARDS) is an important clinical and public health problem. Why some at-risk individuals develop ARDS and others do not is unclear but may be related to differences in inflammatory and cell signaling systems. The Receptor for Advanced Glycation Endproducts (RAGE) and Granulocyte-Monocyte Stimulating Factor (GM-CSF) pathways have recently been implicated in pulmonary pathophysiology; whether genetic variation within these pathways contributes to ARDS risk or outcome is unknown.
Materials and Methods
We studied 842 patients from three centers in Utah and 14 non-Utah ARDS Network centers. We studied patients at risk for ARDS and patients with ARDS to determine whether Single Nucleotide Polymorphisms (SNPs) in the RAGE and GM-CSF pathways were associated with development of ARDS. We studied 29 SNPs in 5 genes within the two pathways and controlled for age, sepsis as ARDS risk factor, and severity of illness, while targeting a false discovery rate of ≤5%. In a secondary analysis we evaluated associations with mortality.
Of 842 patients, 690 had ARDS, and 152 were at-risk. Sepsis was the risk factor for ARDS in 250 (30%) patients. When controlling for age, APACHE III score, sepsis as risk factor, and multiple comparisons, no SNPs were significantly associated with ARDS. In a secondary analysis, only rs743564 in CSF2 approached significance with regard to mortality (OR 2.17, unadjusted p = 0.005, adjusted p = 0.15).
Candidate SNPs within 5 genes in the RAGE and GM-CSF pathways were not significantly associated with development of ARDS in this multi-centric cohort.
PMCID: PMC4406221  PMID: 25513711
acute respiratory distress syndrome; GM-CSF; genetics; RAGE
4.  Severity of cough in idiopathic pulmonary fibrosis is associated with MUC5 B genotype 
A polymorphism (rs35705950) in the promoter region of the mucin MUC5B is associated with both familial and sporadic forms of idiopathic pulmonary fibrosis. (IPF) We hypothesize that this common MUC5B variant will impact the expression of cough, a frequent disabling symptom seen in subjects with IPF.
We genotyped 136 subjects with IPF. All living subjects were provided with a Leicester Cough Questionnaire (LCQ) to measure cough severity. We assessed allele effects of the MUC5B polymorphism on the LCQ scores using SAS General Linear Models (GLM) in the patients with IPF.
In the 68 of the total 136 IPF patients who returned the LCQ, MUC5B minor allele frequency (T) is consistent with prior published studies (31%). We found a significant independent effect of the T allele on the LCQ score (p = 0.002 for subjects with IPF). This effect is independent of other common causes of cough, including gastroesophogeal reflux disease and upper airway cough syndrome.
Cough severity, a common disabling phenotypic component of IPF, is significantly associated with the presence of the minor allele of a MUC5B promoter polymorphism. This study highlights a possible genetic mechanism for phenotypic heterogeneity in pulmonary fibrosis.
PMCID: PMC4038402  PMID: 24667072
5.  Comparison of serum, EDTA plasma and P100 plasma for luminex-based biomarker multiplex assays in patients with chronic obstructive pulmonary disease in the SPIROMICS study 
As a part of the longitudinal Chronic Obstructive Pulmonary Disease (COPD) study, Subpopulations and Intermediate Outcome Measures in COPD study (SPIROMICS), blood samples are being collected from 3200 subjects with the goal of identifying blood biomarkers for sub-phenotyping patients and predicting disease progression. To determine the most reliable sample type for measuring specific blood analytes in the cohort, a pilot study was performed from a subset of 24 subjects comparing serum, Ethylenediaminetetraacetic acid (EDTA) plasma, and EDTA plasma with proteinase inhibitors (P100™).
105 analytes, chosen for potential relevance to COPD, arranged in 12 multiplex and one simplex platform (Myriad-RBM) were evaluated in duplicate from the three sample types from 24 subjects. The reliability coefficient and the coefficient of variation (CV) were calculated. The performance of each analyte and mean analyte levels were evaluated across sample types.
20% of analytes were not consistently detectable in any sample type. Higher reliability and/or smaller CV were determined for 12 analytes in EDTA plasma compared to serum, and for 11 analytes in serum compared to EDTA plasma. While reliability measures were similar for EDTA plasma and P100 plasma for a majority of analytes, CV was modestly increased in P100 plasma for eight analytes. Each analyte within a multiplex produced independent measurement characteristics, complicating selection of sample type for individual multiplexes.
There were notable detectability and measurability differences between serum and plasma. Multiplexing may not be ideal if large reliability differences exist across analytes measured within the multiplex, especially if values differ based on sample type. For some analytes, the large CV should be considered during experimental design, and the use of duplicate and/or triplicate samples may be necessary. These results should prove useful for studies evaluating selection of samples for evaluation of potential blood biomarkers.
PMCID: PMC3928911  PMID: 24397870
Chronic obstructive pulmonary disease; COPD; SPIROMICS; Biomarkers; Blood analytes; Multiplex assays; P100 plasma; Serum; EDTA plasma; Pilot study
6.  A Semiautomated Framework for Integrating Expert Knowledge into Disease Marker Identification 
Disease markers  2013;35(5):513-523.
Background. The availability of large complex data sets generated by high throughput technologies has enabled the recent proliferation of disease biomarker studies. However, a recurring problem in deriving biological information from large data sets is how to best incorporate expert knowledge into the biomarker selection process. Objective. To develop a generalizable framework that can incorporate expert knowledge into data-driven processes in a semiautomated way while providing a metric for optimization in a biomarker selection scheme. Methods. The framework was implemented as a pipeline consisting of five components for the identification of signatures from integrated clustering (ISIC). Expert knowledge was integrated into the biomarker identification process using the combination of two distinct approaches; a distance-based clustering approach and an expert knowledge-driven functional selection. Results. The utility of the developed framework ISIC was demonstrated on proteomics data from a study of chronic obstructive pulmonary disease (COPD). Biomarker candidates were identified in a mouse model using ISIC and validated in a study of a human cohort. Conclusions. Expert knowledge can be introduced into a biomarker discovery process in different ways to enhance the robustness of selected marker candidates. Developing strategies for extracting orthogonal and robust features from large data sets increases the chances of success in biomarker identification.
PMCID: PMC3809975  PMID: 24223463
7.  Airway PI3K Pathway Activation Is an Early and Reversible Event in Lung Cancer Development 
Science translational medicine  2010;2(26):26ra25.
Although only a subset of smokers develop lung cancer, we cannot determine which smokers are at highest risk for cancer development, nor do we know the signaling pathways altered early in the process of tumorigenesis in these individuals. On the basis of the concept that cigarette smoke creates a molecular field of injury throughout the respiratory tract, this study explores oncogenic pathway deregulation in cytologically normal proximal airway epithelial cells of smokers at risk for lung cancer. We observed a significant increase in a genomic signature of phosphatidylinositol 3-kinase (PI3K) pathway activation in the cytologically normal bronchial airway of smokers with lung cancer and smokers with dysplastic lesions, suggesting that PI3K is activated in the proximal airway before tumorigenesis. Further, PI3K activity is decreased in the airway of high-risk smokers who had significant regression of dysplasia after treatment with the chemopreventive agent myo-inositol, and myo-inositol inhibits the PI3K pathway in vitro. These results suggest that deregulation of the PI3K pathway in the bronchial airway epithelium of smokers is an early, measurable, and reversible event in the development of lung cancer and that genomic profiling of these relatively accessible airway cells may enable personalized approaches to chemoprevention and therapy. Our work further suggests that additional lung cancer chemoprevention trials either targeting the PI3K pathway or measuring airway PI3K activation as an intermediate endpoint are warranted.
PMCID: PMC3694402  PMID: 20375364
8.  Low-Dose Naltrexone for Pruritus in Systemic Sclerosis 
Pruritus is a common symptom in systemic sclerosis (SSc), an autoimmune disease which causes fibrosis and vasculopathy in skin, lung, and gastrointestinal tract (GIT). Unfortunately, pruritus has limited treatment options in this disease. Pilot trials of low-dose naltrexone hydrochloride (LDN) for pruritus, pain, and quality of life (QOL) in other GIT diseases have been successful. In this case series we report three patients that had significant improvement in pruritus and total GIT symptoms as measured by the 10-point faces scale and the University of California Los Angeles Scleroderma Clinical Trials Consortium Gastrointestinal Tract 2.0 (UCLA SCTC GIT 2.0) questionnaire. This small case series suggests LDN may be an effective, highly tolerable, and inexpensive treatment for pruritus and GIT symptoms in SSc.
PMCID: PMC3171757  PMID: 21918649
9.  Multistudy Fine Mapping of Chromosome 2q Identifies XRCC5 as a Chronic Obstructive Pulmonary Disease Susceptibility Gene 
Rationale: Several family-based studies have identified genetic linkage for lung function and airflow obstruction to chromosome 2q.
Objectives: We hypothesized that merging results of high-resolution single nucleotide polymorphism (SNP) mapping in four separate populations would lead to the identification of chronic obstructive pulmonary disease (COPD) susceptibility genes on chromosome 2q.
Methods: Within the chromosome 2q linkage region, 2,843 SNPs were genotyped in 806 COPD cases and 779 control subjects from Norway, and 2,484 SNPs were genotyped in 309 patients with severe COPD from the National Emphysema Treatment Trial and 330 community control subjects. Significant associations from the combined results across the two case-control studies were followed up in 1,839 individuals from 603 families from the International COPD Genetics Network (ICGN) and in 949 individuals from 127 families in the Boston Early-Onset COPD Study.
Measurements and Main Results: Merging the results of the two case-control analyses, 14 of the 790 overlapping SNPs had a combined P < 0.01. Two of these 14 SNPs were consistently associated with COPD in the ICGN families. The association with one SNP, located in the gene XRCC5, was replicated in the Boston Early-Onset COPD Study, with a combined P = 2.51 × 10−5 across the four studies, which remains significant when adjusted for multiple testing (P = 0.02). Genotype imputation confirmed the association with SNPs in XRCC5.
Conclusions: By combining data from COPD genetic association studies conducted in four independent patient samples, we have identified XRCC5, an ATP-dependent DNA helicase, as a potential COPD susceptibility gene.
PMCID: PMC2937234  PMID: 20463177
emphysema; genetic linkage; metaanalysis; single nucleotide polymorphism
10.  Smoking, COPD, and 3-Nitrotyrosine Levels of Plasma Proteins 
Environmental Health Perspectives  2011;119(9):1314-1320.
Background: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress.
Objective: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation.
Methods: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed.
Results: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests.
Conclusions: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.
PMCID: PMC3230408  PMID: 21652289
cigarette smoke; COPD; ELISA; eNOS; nitrotyrosine; posttranslational modification
11.  The Vascular Microenvironment and Systemic Sclerosis 
The role of the vascular microenvironment in the pathogenesis Systemic Sclerosis (SSc) is appreciated clinically as Raynaud's syndrome with capillary nail bed change. This manifestation of vasculopathy is used diagnostically in both limited and diffuse cutaneous subsets of SSc, and is thought to precede fibrosis. The degree of subsequent fibrosis may also be determined by the vascular microenvironment. This paper describes why the vascular microenvironment might determine the degree of end-organ damage that occurs in SSc, with a focus on vascular cell senescence, endothelial progenitor cells (EPC) including multipotential mesenchymal stem cells (MSC), pericytes, and angiogenic monocytes. An explanation of the role of EPC, pericytes, and angiogenic monocytes is important to an understanding of SSc pathogenesis. An evolving understanding of the vascular microenvironment in SSc may allow directed treatment.
PMCID: PMC2931393  PMID: 20814552
12.  High-Sensitivity NanoLC—MS/MS Analysis of Urinary Desmosine and Isodesmosine 
Analytical chemistry  2009;81(5):1881-1887.
Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium- based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D4-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 µL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 ± 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 ± 3.1 ng/mg crea), (c) healthy smokers (13.2 ± 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 ± 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.
PMCID: PMC2787797  PMID: 19178285
13.  A Candidate Gene Approach Identifies the CHRNA5-A3-B4 Region as a Risk Factor for Age-Dependent Nicotine Addiction 
PLoS Genetics  2008;4(7):e1000125.
People who begin daily smoking at an early age are at greater risk of long-term nicotine addiction. We tested the hypothesis that associations between nicotinic acetylcholine receptor (nAChR) genetic variants and nicotine dependence assessed in adulthood will be stronger among smokers who began daily nicotine exposure during adolescence. We compared nicotine addiction—measured by the Fagerstrom Test of Nicotine Dependence—in three cohorts of long-term smokers recruited in Utah, Wisconsin, and by the NHLBI Lung Health Study, using a candidate-gene approach with the neuronal nAChR subunit genes. This SNP panel included common coding variants and haplotypes detected in eight α and three β nAChR subunit genes found in European American populations. In the 2,827 long-term smokers examined, common susceptibility and protective haplotypes at the CHRNA5-A3-B4 locus were associated with nicotine dependence severity (p = 2.0×10−5; odds ratio = 1.82; 95% confidence interval 1.39–2.39) in subjects who began daily smoking at or before the age of 16, an exposure period that results in a more severe form of adult nicotine dependence. A substantial shift in susceptibility versus protective diplotype frequency (AA versus BC = 17%, AA versus CC = 27%) was observed in the group that began smoking by age 16. This genetic effect was not observed in subjects who began daily nicotine use after the age of 16. These results establish a strong mechanistic link among early nicotine exposure, common CHRNA5-A3-B4 haplotypes, and adult nicotine addiction in three independent populations of European origins. The identification of an age-dependent susceptibility haplotype reinforces the importance of preventing early exposure to tobacco through public health policies.
Author Summary
Tobacco use is a global health care problem, and persistent smoking takes an enormous toll on individual health. The onset of daily smoking in adolescence is related to chronic use and severe nicotine dependence in adulthood. Since nicotine is the key addictive chemical in tobacco, we tested the hypothesis that genetic variants within nicotinic acetylcholine receptors will influence the severity of addiction measured in adulthood. Using genomic resequencing to define the patterns of variation found in these candidate genes, we observed that common haplotypes in the CHRNA5-A3-B4 gene cluster are associated with adult nicotine addiction, specifically among those who began daily smoking before age 17. We show that in populations of European origins, one haplotype is a risk factor for dependence, one is protective, and one is neutral. These observations suggest that genetic determinants expressed during human adolescence contribute to the risk of lifetime addiction severity produced from early onset of cigarette use. Because disease risk from the adverse health effects of tobacco smoke is related to lifetime tobacco exposure, the finding that an age-dependent effect of these haplotypes has a strong influence on lifetime smoking behavior reinforces the public health significance of delaying smoking onset.
PMCID: PMC2442220  PMID: 18618000

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