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1.  Mapping Splicing Quantitative Trait Loci in RNA-Seq 
Cancer Informatics  2014;13(Suppl 4):35-43.
One of the major mechanisms of generating mRNA diversity is alternative splicing, a regulated process that allows for the flexibility of producing functionally different proteins from the same genomic sequences. This process is often altered in cancer cells to produce aberrant proteins that drive the progression of cancer. A better understanding of the misregulation of alternative splicing will shed light on the development of novel targets for pharmacological interventions of cancer.
In this study, we evaluated three statistical methods, random effects meta-regression, beta regression, and generalized linear mixed effects model, for the analysis of splicing quantitative trait loci (sQTL) using RNA-Seq data. All the three methods use exon-inclusion levels estimated by the PennSeq algorithm, a statistical method that utilizes paired-end reads and accounts for non-uniform sequencing coverage.
Using both simulated and real RNA-Seq datasets, we compared these three methods with GLiMMPS, a recently developed method for sQTL analysis. Our results indicate that the most reliable and powerful method was the random effects meta-regression approach, which identified sQTLs at low false discovery rates but higher power when compared to GLiMMPS.
We have evaluated three statistical methods for the analysis of sQTLs in RNA-Seq. Results from our study will be instructive for researchers in selecting the appropriate statistical methods for sQTL analysis.
PMCID: PMC4218654  PMID: 25452687
alternative splicing; quantitative trait loci; RNA-Seq
2.  Candidate Gene Association Study of Coronary Artery Calcification in Chronic Kidney Disease: Findings from the Chronic Renal Insufficiency Cohort Study 
To identify loci for coronary artery calcification (CAC) in patients with chronic kidney disease (CKD).
CKD is associated with increased CAC and subsequent coronary heart disease (CHD) but the mechanisms remain poorly defined. Genetic studies of CAC in CKD may provide a useful strategy for identifying novel pathways in CHD.
We performed a candidate gene study (~2,100 genes; ~50,000 SNPs) of CAC within the Chronic Renal Insufficiency Cohort (CRIC) Study (n=1,509; 57% European, 43% African ancestry). SNPs with preliminary evidence of association with CAC in CRIC were examined for association with CAC in PennCAC (n=2,560) and Amish Family Calcification Study (AFCS; n=784) samples. SNPs with suggestive replication were further analyzed for association with myocardial infarction (MI) in the Pakistan Risk of Myocardial Infarction study (PROMIS) (n=14,885).
Of 268 SNPs reaching P <5×10−4 for CAC in CRIC, 28 SNPs in 23 loci had nominal support (P <0.05 and in same direction) for CAC in PennCAC or AFCS. Besides chr9p21 and COL4A1, known loci for CHD, these included SNPs having reported GWAS association with hypertension (e.g., ATP2B1). In PROMIS, four of the 23 suggestive CAC loci (chr9p21, COL4A1, ATP2B1 and ABCA4) had significant associations with MI consistent with their direction of effect on CAC.
We identified several loci associated with CAC in CKD that also relate to MI in a general population sample. CKD imparts a high risk of CHD and may provide a useful setting for discovery of novel CHD genes and pathways.
PMCID: PMC3953823  PMID: 23727086
Coronary artery calcification (CAC); chronic kidney disease (CKD); Chronic Renal Insufficiency Cohort Study (CRIC); myocardial infarction (MI); risk factors; candidate genes; single nucleotide polymorphisms (SNPs)
3.  The Value of Statistical or Bioinformatics Annotation for Rare Variant Association with Quantitative Trait 
Genetic epidemiology  2013;37(7):666-674.
In the past few years, a plethora of methods for rare variant association with phenotype have been proposed. These methods aggregate information from multiple rare variants across genomic region(s), but there is little consensus as to which method is most effective. The weighting scheme adopted when aggregating information across variants is one of the primary determinants of effectiveness. Here we present a systematic evaluation of multiple weighting schemes through a series of simulations intended to mimic large sequencing studies of a quantitative trait. We evaluate existing phenotype-independent and -dependent methods, as well as weights estimated by penalized regression approaches including Lasso, Elastic Net and SCAD. We find that the difference in power between phenotype-dependent schemes is negligible when high quality functional annotations are available. When functional annotations are unavailable or incomplete, all methods suffer from power loss; however, the variable selection methods outperform the others at the cost of increased computational time. Therefore, in the absence of good annotation, we recommend variable selection methods (which can be viewed as “statistical annotation”) on top regions implicated by a phenotype independent weighting scheme. Further, once a region is implicated, variable selection can help to identify potential causal SNPs for biological validation. These findings are supported by an analysis of a high coverage targeted sequencing study of 1898 individuals.
PMCID: PMC4083762  PMID: 23836599
rare variants; association; weighting; variable selection; variant annotation
4.  A Hierarchical Bayesian Model for Estimating and Inferring Differential Isoform Expression for Multi-Sample RNA-Seq Data 
Statistics in biosciences  2011;5(1):119-137.
RNA-Seq has drastically changed our ways of studying transcrip-tomes in providing more precise estimates of gene expression, including isoform-specific expression. Most of the available methods for RNA-Seq data focus on one sample at a time. We present in this paper a Poisson-Gamma hierarchical model for multi-sample RNA-Seq data analysis in order to simultaneously estimate isoform-specific expression and to identify differentially expressed iso-forms. Our model has the advantage of borrowing information across all samples in estimating expression levels, which can improve the estimates drastically, particularly for low abundance isoforms. Furthermore, our hierarchical model has the ability to account for overdispersion in the data and also can incorporate sample-specific covariates in the underlying model, which facilitates the isoform-specific differential expression analysis. Simulation studies demonstrated that this Bayesian multi-sample approach can lead to more precise estimates of isoform-specific expression and higher power to detect differential expression by borrowing information across all samples than single sample analysis, especially for isoforms of low abundance. We further illustrated our methods using the RNA-Seq data of 10 Yoruban and 10 Caucasian individuals.
PMCID: PMC3669631  PMID: 23737925
Mixture of Poisson-Gamma model; Markov Chain Monte Carlo Sampling Next Generation Sequencing
5.  IL1RN Coding Variant Is Associated with Lower Risk of Acute Respiratory Distress Syndrome and Increased Plasma IL-1 Receptor Antagonist 
Rationale: Acute respiratory distress syndrome (ARDS) behaves as a complex genetic trait, yet knowledge of genetic susceptibility factors remains incomplete.
Objectives: To identify genetic risk variants for ARDS using large scale genotyping.
Methods: A multistage genetic association study was conducted of three critically ill populations phenotyped for ARDS. Stage I, a trauma cohort study (n = 224), was genotyped with a 50K gene-centric single-nucleotide polymorphism (SNP) array. We tested SNPs associated with ARDS at P < 5 × 10−4 for replication in stage II, a trauma case–control population (n = 778). SNPs replicating their association in stage II (P < 0.005) were tested in a stage III nested case–control population of mixed subjects in the intensive care unit (n = 2,063). Logistic regression was used to adjust for potential clinical confounders. We performed ELISA to test for an association between ARDS-associated genotype and plasma protein levels.
Measurements and Main Results: A total of 12 SNPs met the stage I threshold for an association with ARDS. rs315952 in the IL1RN gene encoding IL-1 receptor antagonist (IL1RA) replicated its association with reduced ARDS risk in stages II (P < 0.004) and III (P < 0.02), and was robust to clinical adjustment (combined odds ratio = 0.81; P = 4.2 × 10−5). Plasma IL1RA level was associated with rs315952C in a subset of critically ill subjects. The effect of rs315952 was independent from the tandem repeat variant in IL1RN.
Conclusions: The IL1RN SNP rs315952C is associated with decreased risk of ARDS in three populations with heterogeneous ARDS risk factors, and with increased plasma IL1RA response. IL1RA may attenuate ARDS risk.
PMCID: PMC3707367  PMID: 23449693
functional genetic polymorphism; acute lung injury; acute respiratory distress syndrome; IL-1 receptor antagonist; replication
6.  Transferability and Fine Mapping of Type 2 Diabetes Loci in African Americans 
Diabetes  2013;62(3):965-976.
Type 2 diabetes (T2D) disproportionally affects African Americans (AfA) but, to date, genetic variants identified from genome-wide association studies (GWAS) are primarily from European and Asian populations. We examined the single nucleotide polymorphism (SNP) and locus transferability of 40 reported T2D loci in six AfA GWAS consisting of 2,806 T2D case subjects with or without end-stage renal disease and 4,265 control subjects from the Candidate Gene Association Resource Plus Study. Our results revealed that seven index SNPs at the TCF7L2, KLF14, KCNQ1, ADCY5, CDKAL1, JAZF1, and GCKR loci were significantly associated with T2D (P < 0.05). The strongest association was observed at TCF7L2 rs7903146 (odds ratio [OR] 1.30; P = 6.86 × 10−8). Locus-wide analysis demonstrated significant associations (Pemp < 0.05) at regional best SNPs in the TCF7L2, KLF14, and HMGA2 loci as well as suggestive signals in KCNQ1 after correction for the effective number of SNPs at each locus. Of these loci, the regional best SNPs were in differential linkage disequilibrium (LD) with the index and adjacent SNPs. Our findings suggest that some loci discovered in prior reports affect T2D susceptibility in AfA with similar effect sizes. The reduced and differential LD pattern in AfA compared with European and Asian populations may facilitate fine mapping of causal variants at loci shared across populations.
PMCID: PMC3581206  PMID: 23193183
7.  Testing Genetic Association With Rare Variants in Admixed Populations 
Genetic epidemiology  2012;37(1):38-47.
Recent studies suggest that rare variants play an important role in the etiology of many traits. Although a number of methods have been developed for genetic association analysis of rare variants, they all assume a relatively homogeneous population under study. Such an assumption may not be valid for samples collected from admixed populations such as African Americans and Hispanic Americans as there is a great extent of local variation in ancestry in these populations. To ensure valid and more powerful rare variant association tests performed in admixed populations, we have developed a local ancestry-based weighted dosage test, which is able to take into account local ancestry of rare alleles, uncertainties in rare variant imputation when imputed data are included, and the direction of effect that rare variants exert on phenotypic outcome. We used simulated sequence data to show that our proposed test has controlled type I error rates, whereas naïve application of existing rare variants tests and tests that adjust for global ancestry lead to inflated type I error rates. We showed that our test has higher power than tests without proper adjustment of ancestry. We also applied the proposed method to a candidate gene study on low-density lipoprotein cholesterol. Our results suggest that it is important to appropriately control for potential population stratification induced by local ancestry difference in the analysis of rare variants in admixed populations.
PMCID: PMC3524352  PMID: 23032398
admixed population; rare variants; population stratification
8.  MaCH-Admix: Genotype Imputation for Admixed Populations 
Genetic epidemiology  2012;37(1):25-37.
Imputation in admixed populations is an important problem but challenging due to the complex linkage disequilibrium (LD) pattern. The emergence of large reference panels such as that from the 1,000 Genomes Project enables more accurate imputation in general, and in particular for admixed populations and for uncommon variants. To efficiently benefit from these large reference panels, one key issue to consider in modern genotype imputation framework is the selection of effective reference panels. In this work, we consider a number of methods for effective reference panel construction inside a hidden Markov model and specific to each target individual. These methods fall into two categories: identity-by-state (IBS) based and ancestry-weighted approach. We evaluated the performance on individuals from recently admixed populations. Our target samples include 8,421 African Americans and 3,587 Hispanic Americans from the Women’s Health Initiative, which allow assessment of imputation quality for uncommon variants. Our experiments include both large and small reference panels; large, medium, and small target samples; and in genome regions of varying levels of LD. We also include BEAGLE and IMPUTE2 for comparison. Experiment results with large reference panel suggest that our novel piecewise IBS method yields consistently higher imputation quality than other methods/software. The advantage is particularly noteworthy among uncommon variants where we observe up to 5.1% information gain with the difference being highly significant (Wilcoxon signed rank test P-value < 0.0001). Our work is the first that considers various sensible approaches for imputation in admixed populations and presents a comprehensive comparison.
PMCID: PMC3524415  PMID: 23074066
genotype imputation; admixed populations; large reference panel; uncommon variants; MaCH-Admix
9.  PennSeq: accurate isoform-specific gene expression quantification in RNA-Seq by modeling non-uniform read distribution 
Nucleic Acids Research  2013;42(3):e20.
Correctly estimating isoform-specific gene expression is important for understanding complicated biological mechanisms and for mapping disease susceptibility genes. However, estimating isoform-specific gene expression is challenging because various biases present in RNA-Seq (RNA sequencing) data complicate the analysis, and if not appropriately corrected, can affect isoform expression estimation and downstream analysis. In this article, we present PennSeq, a statistical method that allows each isoform to have its own non-uniform read distribution. Instead of making parametric assumptions, we give adequate weight to the underlying data by the use of a non-parametric approach. Our rationale is that regardless what factors lead to non-uniformity, whether it is due to hexamer priming bias, local sequence bias, positional bias, RNA degradation, mapping bias or other unknown reasons, the probability that a fragment is sampled from a particular region will be reflected in the aligned data. This empirical approach thus maximally reflects the true underlying non-uniform read distribution. We evaluate the performance of PennSeq using both simulated data with known ground truth, and using two real Illumina RNA-Seq data sets including one with quantitative real time polymerase chain reaction measurements. Our results indicate superior performance of PennSeq over existing methods, particularly for isoforms demonstrating severe non-uniformity. PennSeq is freely available for download at
PMCID: PMC3919567  PMID: 24362841
10.  Development of a Genotyping Microarray for Studying the Role of Gene-Environment Interactions in Risk for Lung Cancer 
A microarray (LungCaGxE), based on Illumina BeadChip technology, was developed for high-resolution genotyping of genes that are candidates for involvement in environmentally driven aspects of lung cancer oncogenesis and/or tumor growth. The iterative array design process illustrates techniques for managing large panels of candidate genes and optimizing marker selection, aided by a new bioinformatics pipeline component, Tagger Batch Assistant. The LungCaGxE platform targets 298 genes and the proximal genetic regions in which they are located, using ∼13,000 DNA single nucleotide polymorphisms (SNPs), which include haplotype linkage markers with a minimum allele frequency of 1% and additional specifically targeted SNPs, for which published reports have indicated functional consequences or associations with lung cancer or other smoking-related diseases. The overall assay conversion rate was 98.9%; 99.0% of markers with a minimum Illumina design score of 0.6 successfully generated allele calls using genomic DNA from a study population of 1873 lung-cancer patients and controls.
PMCID: PMC3792704  PMID: 24294113
genetic association; environmental exposures; Tagger Batch Assistant; LungCaGxE
11.  Secretory Phospholipase A2-IIA and Cardiovascular Disease 
Holmes, Michael V. | Simon, Tabassome | Exeter, Holly J. | Folkersen, Lasse | Asselbergs, Folkert W. | Guardiola, Montse | Cooper, Jackie A. | Palmen, Jutta | Hubacek, Jaroslav A. | Carruthers, Kathryn F. | Horne, Benjamin D. | Brunisholz, Kimberly D. | Mega, Jessica L. | van Iperen, Erik P.A. | Li, Mingyao | Leusink, Maarten | Trompet, Stella | Verschuren, Jeffrey J.W. | Hovingh, G. Kees | Dehghan, Abbas | Nelson, Christopher P. | Kotti, Salma | Danchin, Nicolas | Scholz, Markus | Haase, Christiane L. | Rothenbacher, Dietrich | Swerdlow, Daniel I. | Kuchenbaecker, Karoline B. | Staines-Urias, Eleonora | Goel, Anuj | van 't Hooft, Ferdinand | Gertow, Karl | de Faire, Ulf | Panayiotou, Andrie G. | Tremoli, Elena | Baldassarre, Damiano | Veglia, Fabrizio | Holdt, Lesca M. | Beutner, Frank | Gansevoort, Ron T. | Navis, Gerjan J. | Mateo Leach, Irene | Breitling, Lutz P. | Brenner, Hermann | Thiery, Joachim | Dallmeier, Dhayana | Franco-Cereceda, Anders | Boer, Jolanda M.A. | Stephens, Jeffrey W. | Hofker, Marten H. | Tedgui, Alain | Hofman, Albert | Uitterlinden, André G. | Adamkova, Vera | Pitha, Jan | Onland-Moret, N. Charlotte | Cramer, Maarten J. | Nathoe, Hendrik M. | Spiering, Wilko | Klungel, Olaf H. | Kumari, Meena | Whincup, Peter H. | Morrow, David A. | Braund, Peter S. | Hall, Alistair S. | Olsson, Anders G. | Doevendans, Pieter A. | Trip, Mieke D. | Tobin, Martin D. | Hamsten, Anders | Watkins, Hugh | Koenig, Wolfgang | Nicolaides, Andrew N. | Teupser, Daniel | Day, Ian N.M. | Carlquist, John F. | Gaunt, Tom R. | Ford, Ian | Sattar, Naveed | Tsimikas, Sotirios | Schwartz, Gregory G. | Lawlor, Debbie A. | Morris, Richard W. | Sandhu, Manjinder S. | Poledne, Rudolf | Maitland-van der Zee, Anke H. | Khaw, Kay-Tee | Keating, Brendan J. | van der Harst, Pim | Price, Jackie F. | Mehta, Shamir R. | Yusuf, Salim | Witteman, Jaqueline C.M. | Franco, Oscar H. | Jukema, J. Wouter | de Knijff, Peter | Tybjaerg-Hansen, Anne | Rader, Daniel J. | Farrall, Martin | Samani, Nilesh J. | Kivimaki, Mika | Fox, Keith A.A. | Humphries, Steve E. | Anderson, Jeffrey L. | Boekholdt, S. Matthijs | Palmer, Tom M. | Eriksson, Per | Paré, Guillaume | Hingorani, Aroon D. | Sabatine, Marc S. | Mallat, Ziad | Casas, Juan P. | Talmud, Philippa J.
This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.
Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy.
We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable.
PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE.
Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.
PMCID: PMC3826105  PMID: 23916927
cardiovascular diseases; drug development; epidemiology; genetics; Mendelian randomization; ACS, acute coronary syndrome(s); CI, confidence interval; LDL-C, low-density lipoprotein cholesterol; MI, myocardial infarction; MVE, major vascular events; OR, odds ratio; RCT, randomized clinical trial; SNP, single-nucleotide polymorphism; sPLA2, secretory phospholipase A2
12.  Development of a Genotyping Microarray for Studying the Role of Gene-Environment Interactions in Risk for Lung Cancer 
A microarray (LungCaGxE), based on Illumina BeadChip technology, was developed for high-resolution genotyping of genes that are candidates for involvement in environmentally driven aspects of lung cancer oncogenesis and/or tumor growth. The iterative array design process illustrates techniques for managing large panels of candidate genes and optimizing marker selection, aided by a new bioinformatics pipeline component, Tagger Batch Assistant. The LungCaGxE platform targets 298 genes and the proximal genetic regions in which they are located, using ∼13,000 DNA single nucleotide polymorphisms (SNPs), which include haplotype linkage markers with a minimum allele frequency of 1% and additional specifically targeted SNPs, for which published reports have indicated functional consequences or associations with lung cancer or other smoking-related diseases. The overall assay conversion rate was 98.9%; 99.0% of markers with a minimum Illumina design score of 0.6 successfully generated allele calls using genomic DNA from a study population of 1873 lung-cancer patients and controls.
PMCID: PMC3792704  PMID: 24294113
genetic association; environmental exposures; Tagger Batch Assistant; LungCaGxE
13.  Genetics of coronary artery calcification among African Americans, a meta-analysis 
BMC Medical Genetics  2013;14:75.
Coronary heart disease (CHD) is the major cause of death in the United States. Coronary artery calcification (CAC) scores are independent predictors of CHD. African Americans (AA) have higher rates of CHD but are less well-studied in genomic studies. We assembled the largest AA data resource currently available with measured CAC to identify associated genetic variants.
We analyzed log transformed CAC quantity (ln(CAC + 1)), for association with ~2.5 million single nucleotide polymorphisms (SNPs) and performed an inverse-variance weighted meta-analysis on results for 5,823 AA from 8 studies. Heritability was calculated using family studies. The most significant SNPs among AAs were evaluated in European Ancestry (EA) CAC data; conversely, the significance of published SNPs for CAC/CHD in EA was queried within our AA meta-analysis.
Heritability of CAC was lower in AA (~30%) than previously reported for EA (~50%). No SNP reached genome wide significance (p < 5E-08). Of 67 SNPs with p < 1E-05 in AA there was no evidence of association in EA CAC data. Four SNPs in regions previously implicated in CAC/CHD (at 9p21 and PHACTR1) in EA reached nominal significance for CAC in AA, with concordant direction. Among AA, rs16905644 (p = 4.08E-05) had the strongest association in the 9p21 region.
While we observed substantial heritability for CAC in AA, we failed to identify loci for CAC at genome-wide significant levels despite having adequate power to detect alleles with moderate to large effects. Although suggestive signals in AA were apparent at 9p21 and additional CAC and CAD EA loci, overall the data suggest that even larger samples and an ethnic specific focus will be required for GWAS discoveries for CAC in AA populations.
PMCID: PMC3733595  PMID: 23870195
Atherosclerosis; Coronary artery calcium; Genetics; Meta-analysis; African-American
14.  A Gaussian copula approach for the analysis of secondary phenotypes in case–control genetic association studies 
Biostatistics (Oxford, England)  2011;13(3):497-508.
In many case–control genetic association studies, a set of correlated secondary phenotypes that may share common genetic factors with disease status are collected. Examination of these secondary phenotypes can yield valuable insights about the disease etiology and supplement the main studies. However, due to unequal sampling probabilities between cases and controls, standard regression analysis that assesses the effect of SNPs (single nucleotide polymorphisms) on secondary phenotypes using cases only, controls only, or combined samples of cases and controls can yield inflated type I error rates when the test SNP is associated with the disease. To solve this issue, we propose a Gaussian copula-based approach that efficiently models the dependence between disease status and secondary phenotypes. Through simulations, we show that our method yields correct type I error rates for the analysis of secondary phenotypes under a wide range of situations. To illustrate the effectiveness of our method in the analysis of real data, we applied our method to a genome-wide association study on high-density lipoprotein cholesterol (HDL-C), where “cases” are defined as individuals with extremely high HDL-C level and “controls” are defined as those with low HDL-C level. We treated 4 quantitative traits with varying degrees of correlation with HDL-C as secondary phenotypes and tested for association with SNPs in LIPG, a gene that is well known to be associated with HDL-C. We show that when the correlation between the primary and secondary phenotypes is >0.2, the P values from case–control combined unadjusted analysis are much more significant than methods that aim to correct for ascertainment bias. Our results suggest that to avoid false-positive associations, it is important to appropriately model secondary phenotypes in case–control genetic association studies.
PMCID: PMC3372941  PMID: 21933777
Case–control studies; Statistical genetics; Statistical methods in Epidemiology
15.  Evaluating the Impact of Sequencing Depth on Transcriptome Profiling in Human Adipose 
PLoS ONE  2013;8(6):e66883.
Recent advances in RNA sequencing (RNA-Seq) have enabled the discovery of novel transcriptomic variations that are not possible with traditional microarray-based methods. Tissue and cell specific transcriptome changes during pathophysiological stress in disease cases versus controls and in response to therapies are of particular interest to investigators studying cardiometabolic diseases. Thus, knowledge on the relationships between sequencing depth and detection of transcriptomic variation is needed for designing RNA-Seq experiments and for interpreting results of analyses. Using deeply sequenced Illumina HiSeq 2000 101 bp paired-end RNA-Seq data derived from adipose of a healthy individual before and after systemic administration of endotoxin (LPS), we investigated the sequencing depths needed for studies of gene expression and alternative splicing (AS). In order to detect expressed genes and AS events, we found that ∼100 to 150 million (M) filtered reads were needed. However, the requirement on sequencing depth for the detection of LPS modulated differential expression (DE) and differential alternative splicing (DAS) was much higher. To detect 80% of events, ∼300 M filtered reads were needed for DE analysis whereas at least 400 M filtered reads were necessary for detecting DAS. Although the majority of expressed genes and AS events can be detected with modest sequencing depths (∼100 M filtered reads), the estimated gene expression levels and exon/intron inclusion levels were less accurate. We report the first study that evaluates the relationship between RNA-Seq depth and the ability to detect DE and DAS in human adipose. Our results suggest that a much higher sequencing depth is needed to reliably identify DAS events than for DE genes.
PMCID: PMC3691247  PMID: 23826166
16.  Genetic determinants of the ankle-brachial index: A meta-analysis of a cardiovascular candidate gene 50K SNP panel in the candidate gene association resource (CARe) consortium 
Atherosclerosis  2012;222(1):138-147.
Candidate gene association studies for peripheral artery disease (PAD), including subclinical disease assessed with the ankle-brachial index (ABI), have been limited by the modest number of genes examined. We conducted a two stage meta-analysis of ~50,000 SNPs across ~2100 candidate genes to identify genetic variants for ABI.
Methods and results
We studied subjects of European ancestry from 8 studies (n = 21,547, 55% women, mean age 44–73 years) and African American ancestry from 5 studies (n = 7267, 60% women, mean age 41–73 years) involved in the candidate gene association resource (CARe) consortium. In each ethnic group, additive genetic models were used (with each additional copy of the minor allele corresponding to the given beta) to test each SNP for association with continuous ABI (excluding ABI > 1.40) and PAD (defined as ABI < 0.90) using linear or logistic regression with adjustment for known PAD risk factors and population stratification. We then conducted a fixed-effects inverse-variance weighted meta-analyses considering a p < 2 × 10−6 to denote statistical significance.
In the European ancestry discovery meta-analyses, rs2171209 in SYTL3 (β = −0.007, p = 6.02 × 10−7) and rs290481 in TCF7L2 (β = −0.008, p = 7.01 × 10−7) were significantly associated with ABI. None of the SNP associations for PAD were significant, though a SNP in CYP2B6 (p = 4.99 × 10−5) was among the strongest associations. These 3 genes are linked to key PAD risk factors (lipoprotein(a), type 2 diabetes, and smoking behavior, respectively). We sought replication in 6 population-based and 3 clinical samples (n = 15,440) for rs290481 and rs2171209. However, in the replication stage (rs2171209, p = 0.75; rs290481, p = 0.19) and in the combined discovery and replication analysis the SNP–ABI associations were no longer significant (rs2171209, p = 1.14 × 10−3; rs290481, p = 8.88 × 10−5). In African Americans, none of the SNP associations for ABI or PAD achieved an experiment-wide level of significance.
Genetic determinants of ABI and PAD remain elusive. Follow-up of these preliminary findings may uncover important biology given the known gene-risk factor associations. New and more powerful approaches to PAD gene discovery are warranted.
PMCID: PMC3596171  PMID: 22361517
Ankle brachial index; Peripheral artery disease; Genetics; Candidate gene array; Meta-analysis; Ethnicity
17.  Meta-analysis and imputation refines the association of 15q25 with smoking quantity 
Liu, Jason Z. | Tozzi, Federica | Waterworth, Dawn M. | Pillai, Sreekumar G. | Muglia, Pierandrea | Middleton, Lefkos | Berrettini, Wade | Knouff, Christopher W. | Yuan, Xin | Waeber, Gérard | Vollenweider, Peter | Preisig, Martin | Wareham, Nicholas J | Zhao, Jing Hua | Loos, Ruth J.F. | Barroso, Inês | Khaw, Kay-Tee | Grundy, Scott | Barter, Philip | Mahley, Robert | Kesaniemi, Antero | McPherson, Ruth | Vincent, John B. | Strauss, John | Kennedy, James L. | Farmer, Anne | McGuffin, Peter | Day, Richard | Matthews, Keith | Bakke, Per | Gulsvik, Amund | Lucae, Susanne | Ising, Marcus | Brueckl, Tanja | Horstmann, Sonja | Wichmann, H.-Erich | Rawal, Rajesh | Dahmen, Norbert | Lamina, Claudia | Polasek, Ozren | Zgaga, Lina | Huffman, Jennifer | Campbell, Susan | Kooner, Jaspal | Chambers, John C | Burnett, Mary Susan | Devaney, Joseph M. | Pichard, Augusto D. | Kent, Kenneth M. | Satler, Lowell | Lindsay, Joseph M. | Waksman, Ron | Epstein, Stephen | Wilson, James F. | Wild, Sarah H. | Campbell, Harry | Vitart, Veronique | Reilly, Muredach P. | Li, Mingyao | Qu, Liming | Wilensky, Robert | Matthai, William | Hakonarson, Hakon H. | Rader, Daniel J. | Franke, Andre | Wittig, Michael | Schäfer, Arne | Uda, Manuela | Terracciano, Antonio | Xiao, Xiangjun | Busonero, Fabio | Scheet, Paul | Schlessinger, David | St Clair, David | Rujescu, Dan | Abecasis, Gonçalo R. | Grabe, Hans Jörgen | Teumer, Alexander | Völzke, Henry | Petersmann, Astrid | John, Ulrich | Rudan, Igor | Hayward, Caroline | Wright, Alan F. | Kolcic, Ivana | Wright, Benjamin J | Thompson, John R | Balmforth, Anthony J. | Hall, Alistair S. | Samani, Nilesh J. | Anderson, Carl A. | Ahmad, Tariq | Mathew, Christopher G. | Parkes, Miles | Satsangi, Jack | Caulfield, Mark | Munroe, Patricia B. | Farrall, Martin | Dominiczak, Anna | Worthington, Jane | Thomson, Wendy | Eyre, Steve | Barton, Anne | Mooser, Vincent | Francks, Clyde | Marchini, Jonathan
Nature genetics  2010;42(5):436-440.
Smoking is a leading global cause of disease and mortality1. We performed a genomewide meta-analytic association study of smoking-related behavioral traits in a total sample of 41,150 individuals drawn from 20 disease, population, and control cohorts. Our analysis confirmed an effect on smoking quantity (SQ) at a locus on 15q25 (P=9.45e-19) that includes three genes encoding neuronal nicotinic acetylcholine receptor subunits (CHRNA5, CHRNA3, CHRNB4). We used data from the 1000 Genomes project to investigate the region using imputation, which allowed analysis of virtually all common variants in the region and offered a five-fold increase in coverage over the HapMap. This increased the spectrum of potentially causal single nucleotide polymorphisms (SNPs), which included a novel SNP that showed the highest significance, rs55853698, located within the promoter region of CHRNA5. Conditional analysis also identified a secondary locus (rs6495308) in CHRNA3.
PMCID: PMC3612983  PMID: 20418889
18.  Translational studies of lipoprotein-associated phospholipase A2 in inflammation and atherosclerosis 
To examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in human inflammation and coronary atherosclerosis.
Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease (CHD). Data supporting Lp-PLA2 are indirect and confounded by species differences; whether Lp-PLA2 is causal in CHD remains in question.
We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in human, probed the source of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of variation in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification (CAC).
In contrast to circulating TNFα and CRP, blood and monocyte Lp-PLA2 mRNA decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia. In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation and further in inflammatory macrophages and foam like-cells. Despite only a marginal association of SNPs in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 SNPs were associated with CAC. In contrast, several SNPs in CRP were significantly associated with plasma CRP levels but had no relation with CAC.
Circulating Lp-PLA2 did not increase during acute phase response in human, while inflammatory macrophages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common genetic variation in PLA2G7 is associated with sub-clinical coronary atherosclerosis. These data link Lp-PLA2 to atherosclerosis in human while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2 actions in the vasculature.
PMCID: PMC3285416  PMID: 22340269
19.  Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study 
Voight, Benjamin F | Peloso, Gina M | Orho-Melander, Marju | Frikke-Schmidt, Ruth | Barbalic, Maja | Jensen, Majken K | Hindy, George | Hólm, Hilma | Ding, Eric L | Johnson, Toby | Schunkert, Heribert | Samani, Nilesh J | Clarke, Robert | Hopewell, Jemma C | Thompson, John F | Li, Mingyao | Thorleifsson, Gudmar | Newton-Cheh, Christopher | Musunuru, Kiran | Pirruccello, James P | Saleheen, Danish | Chen, Li | Stewart, Alexandre FR | Schillert, Arne | Thorsteinsdottir, Unnur | Thorgeirsson, Gudmundur | Anand, Sonia | Engert, James C | Morgan, Thomas | Spertus, John | Stoll, Monika | Berger, Klaus | Martinelli, Nicola | Girelli, Domenico | McKeown, Pascal P | Patterson, Christopher C | Epstein, Stephen E | Devaney, Joseph | Burnett, Mary-Susan | Mooser, Vincent | Ripatti, Samuli | Surakka, Ida | Nieminen, Markku S | Sinisalo, Juha | Lokki, Marja-Liisa | Perola, Markus | Havulinna, Aki | de Faire, Ulf | Gigante, Bruna | Ingelsson, Erik | Zeller, Tanja | Wild, Philipp | de Bakker, Paul I W | Klungel, Olaf H | Maitland-van der Zee, Anke-Hilse | Peters, Bas J M | de Boer, Anthonius | Grobbee, Diederick E | Kamphuisen, Pieter W | Deneer, Vera H M | Elbers, Clara C | Onland-Moret, N Charlotte | Hofker, Marten H | Wijmenga, Cisca | Verschuren, WM Monique | Boer, Jolanda MA | van der Schouw, Yvonne T | Rasheed, Asif | Frossard, Philippe | Demissie, Serkalem | Willer, Cristen | Do, Ron | Ordovas, Jose M | Abecasis, Gonçalo R | Boehnke, Michael | Mohlke, Karen L | Daly, Mark J | Guiducci, Candace | Burtt, Noël P | Surti, Aarti | Gonzalez, Elena | Purcell, Shaun | Gabriel, Stacey | Marrugat, Jaume | Peden, John | Erdmann, Jeanette | Diemert, Patrick | Willenborg, Christina | König, Inke R | Fischer, Marcus | Hengstenberg, Christian | Ziegler, Andreas | Buysschaert, Ian | Lambrechts, Diether | Van de Werf, Frans | Fox, Keith A | El Mokhtari, Nour Eddine | Rubin, Diana | Schrezenmeir, Jürgen | Schreiber, Stefan | Schäfer, Arne | Danesh, John | Blankenberg, Stefan | Roberts, Robert | McPherson, Ruth | Watkins, Hugh | Hall, Alistair S | Overvad, Kim | Rimm, Eric | Boerwinkle, Eric | Tybjaerg-Hansen, Anne | Cupples, L Adrienne | Reilly, Muredach P | Melander, Olle | Mannucci, Pier M | Ardissino, Diego | Siscovick, David | Elosua, Roberto | Stefansson, Kari | O'Donnell, Christopher J | Salomaa, Veikko | Rader, Daniel J | Peltonen, Leena | Schwartz, Stephen M | Altshuler, David | Kathiresan, Sekar
Lancet  2012;380(9841):572-580.
High plasma HDL cholesterol is associated with reduced risk of myocardial infarction, but whether this association is causal is unclear. Exploiting the fact that genotypes are randomly assigned at meiosis, are independent of non-genetic confounding, and are unmodified by disease processes, mendelian randomisation can be used to test the hypothesis that the association of a plasma biomarker with disease is causal.
We performed two mendelian randomisation analyses. First, we used as an instrument a single nucleotide polymorphism (SNP) in the endothelial lipase gene (LIPG Asn396Ser) and tested this SNP in 20 studies (20 913 myocardial infarction cases, 95 407 controls). Second, we used as an instrument a genetic score consisting of 14 common SNPs that exclusively associate with HDL cholesterol and tested this score in up to 12 482 cases of myocardial infarction and 41 331 controls. As a positive control, we also tested a genetic score of 13 common SNPs exclusively associated with LDL cholesterol.
Carriers of the LIPG 396Ser allele (2·6% frequency) had higher HDL cholesterol (0·14 mmol/L higher, p=8×10−13) but similar levels of other lipid and non-lipid risk factors for myocardial infarction compared with non-carriers. This difference in HDL cholesterol is expected to decrease risk of myocardial infarction by 13% (odds ratio [OR] 0·87, 95% CI 0·84–0·91). However, we noted that the 396Ser allele was not associated with risk of myocardial infarction (OR 0·99, 95% CI 0·88–1·11, p=0·85). From observational epidemiology, an increase of 1 SD in HDL cholesterol was associated with reduced risk of myocardial infarction (OR 0·62, 95% CI 0·58–0·66). However, a 1 SD increase in HDL cholesterol due to genetic score was not associated with risk of myocardial infarction (OR 0·93, 95% CI 0·68–1·26, p=0·63). For LDL cholesterol, the estimate from observational epidemiology (a 1 SD increase in LDL cholesterol associated with OR 1·54, 95% CI 1·45–1·63) was concordant with that from genetic score (OR 2·13, 95% CI 1·69–2·69, p=2×10−10).
Some genetic mechanisms that raise plasma HDL cholesterol do not seem to lower risk of myocardial infarction. These data challenge the concept that raising of plasma HDL cholesterol will uniformly translate into reductions in risk of myocardial infarction.
US National Institutes of Health, The Wellcome Trust, European Union, British Heart Foundation, and the German Federal Ministry of Education and Research.
PMCID: PMC3419820  PMID: 22607825
20.  A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility 
Human Molecular Genetics  2011;20(15):3109-3117.
Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10−5] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10−7) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.
PMCID: PMC3131044  PMID: 21551455
21.  A Genome-wide Association Study Identifies LIPA as a Susceptibility Gene for Coronary Artery Disease 
Wild, Philipp S | Zeller, Tanja | Schillert, Arne | Szymczak, Silke | Sinning, Christoph R | Deiseroth, Arne | Schnabel, Renate B | Lubos, Edith | Keller, Till | Eleftheriadis, Medea S | Bickel, Christoph | Rupprecht, Hans J | Wilde, Sandra | Rossmann, Heidi | Diemert, Patrick | Cupples, L Adrienne | Perret, Claire | Erdmann, Jeanette | Stark, Klaus | Kleber, Marcus E | Epstein, Stephen E | Voight, Benjamin F | Kuulasmaa, Kari | Li, Mingyao | Schäfer, Arne S | Klopp, Norman | Braund, Peter S | Sager, Hendrik B | Demissie, Serkalem | Proust, Carole | König, Inke R | Wichmann, Heinz-Erich | Reinhard, Wibke | Hoffmann, Michael M | Virtamo, Jarmo | Burnett, Mary Susan | Siscovick, David | Wiklund, Per Gunnar | Qu, Liming | El Mokthari, Nour Eddine | Thompson, John R | Peters, Annette | Smith, Albert V | Yon, Emmanuelle | Baumert, Jens | Hengstenberg, Christian | März, Winfried | Amouyel, Philippe | Devaney, Joseph | Schwartz, Stephen M | Saarela, Olli | Mehta, Nehal N | Rubin, Diana | Silander, Kaisa | Hall, Alistair S | Ferrieres, Jean | Harris, Tamara B | Melander, Olle | Kee, Frank | Hakonarson, Hakon | Schrezenmeir, Juergen | Gudnason, Vilmundur | Elosua, Roberto | Arveiler, Dominique | Evans, Alun | Rader, Daniel J | Illig, Thomas | Schreiber, Stefan | Bis, Joshua C | Altshuler, David | Kavousi, Maryam | Witteman, Jaqueline CM | Uitterlinden, Andre G | Hofman, Albert | Folsom, Aaron R | Barbalic, Maja | Boerwinkle, Eric | Kathiresan, Sekar | Reilly, Muredach P | O'Donnell, Christopher J | Samani, Nilesh J | Schunkert, Heribert | Cambien, Francois | Lackner, Karl J | Tiret, Laurence | Salomaa, Veikko | Munzel, Thomas | Ziegler, Andreas | Blankenberg, Stefan
eQTL analyses are important to improve the understanding of genetic association results. Here, we performed a genome-wide association and global gene expression study to identify functionally relevant variants affecting the risk of coronary artery disease (CAD).
Methods and Results
In a genome-wide association analysis of 2,078 CAD cases and 2,953 controls, we identified 950 single nucleotide polymorphisms (SNPs) that were associated with CAD at P<10-3. Subsequent in silico and wet-lab replication stages and a final meta-analysis of 21,428 CAD cases and 38,361 controls revealed a novel association signal at chromosome 10q23.31 within the LIPA (Lysosomal Acid Lipase A) gene (P=3.7×10-8; OR 1.1; 95% CI: 1.07-1.14). The association of this locus with global gene expression was assessed by genome-wide expression analyses in the monocyte transcriptome of 1,494 individuals. The results showed a strong association of this locus with expression of the LIPA transcript (P=1.3×10-96). An assessment of LIPA SNPs and transcript with cardiovascular phenotypes revealed an association of LIPA transcript levels with impaired endothelial function (P=4.4×10-3).
The use of data on genetic variants and the addition of data on global monocytic gene expression led to the identification of the novel functional CAD susceptibility locus LIPA, located on chromosome 10q23.31. The respective eSNPs associated with CAD strongly affect LIPA gene expression level, which itself was related to endothelial dysfunction, a precursor of CAD.
PMCID: PMC3157552  PMID: 21606135
coronary artery disease; genome-wide association studies; gene expression; genetic variation; genomics; eQTL; eSNP; LIPA
22.  A genome-wide association meta-analysis identifies new childhood obesity loci 
Bradfield, Jonathan P. | Taal, H. Rob | Timpson, Nicholas J. | Scherag, André | Lecoeur, Cecile | Warrington, Nicole M. | Hypponen, Elina | Holst, Claus | Valcarcel, Beatriz | Thiering, Elisabeth | Salem, Rany M. | Schumacher, Fredrick R. | Cousminer, Diana L. | Sleiman, Patrick M.A. | Zhao, Jianhua | Berkowitz, Robert I. | Vimaleswaran, Karani S. | Jarick, Ivonne | Pennell, Craig E. | Evans, David M. | St. Pourcain, Beate | Berry, Diane J. | Mook-Kanamori, Dennis O | Hofman, Albert | Rivadeinera, Fernando | Uitterlinden, André G. | van Duijn, Cornelia M. | van der Valk, Ralf J.P. | de Jongste, Johan C. | Postma, Dirkje S. | Boomsma, Dorret I. | Gauderman, William J. | Hassanein, Mohamed T. | Lindgren, Cecilia M. | Mägi, Reedik | Boreham, Colin A.G. | Neville, Charlotte E. | Moreno, Luis A. | Elliott, Paul | Pouta, Anneli | Hartikainen, Anna-Liisa | Li, Mingyao | Raitakari, Olli | Lehtimäki, Terho | Eriksson, Johan G. | Palotie, Aarno | Dallongeville, Jean | Das, Shikta | Deloukas, Panos | McMahon, George | Ring, Susan M. | Kemp, John P. | Buxton, Jessica L. | Blakemore, Alexandra I.F. | Bustamante, Mariona | Guxens, Mònica | Hirschhorn, Joel N. | Gillman, Matthew W. | Kreiner-Møller, Eskil | Bisgaard, Hans | Gilliland, Frank D. | Heinrich, Joachim | Wheeler, Eleanor | Barroso, Inês | O'Rahilly, Stephen | Meirhaeghe, Aline | Sørensen, Thorkild I.A. | Power, Chris | Palmer, Lyle J. | Hinney, Anke | Widen, Elisabeth | Farooqi, I. Sadaf | McCarthy, Mark I. | Froguel, Philippe | Meyre, David | Hebebrand, Johannes | Jarvelin, Marjo-Riitta | Jaddoe, Vincent W.V. | Smith, George Davey | Hakonarson, Hakon | Grant, Struan F.A.
Nature Genetics  2012;44(5):526-531.
Multiple genetic variants have been associated with adult obesity and a few with severe obesity in childhood; however, less progress has been made to establish genetic influences on common early-onset obesity. We performed a North American-Australian-European collaborative meta-analysis of fourteen studies consisting of 5,530 cases (≥95th percentile of body mass index (BMI)) and 8,318 controls (<50th percentile of BMI) of European ancestry. Taking forward the eight novel signals yielding association with P < 5×10−6 in to nine independent datasets (n = 2,818 cases and 4,083 controls) we observed two loci that yielded a genome wide significant combined P-value, namely near OLFM4 on 13q14 (rs9568856; P=1.82×10−9; OR=1.22) and within HOXB5 on 17q21 (rs9299; P=3.54×10−9; OR=1.14). Both loci continued to show association when including two extreme childhood obesity cohorts (n = 2,214 cases and 2,674 controls). Finally, these two loci yielded directionally consistent associations in the GIANT meta-analysis of adult BMI1.
PMCID: PMC3370100  PMID: 22484627
23.  ANGPT2 Genetic Variant Is Associated with Trauma-associated Acute Lung Injury and Altered Plasma Angiopoietin-2 Isoform Ratio 
Rationale: Acute lung injury (ALI) acts as a complex genetic trait, yet its genetic risk factors remain incompletely understood. Large-scale genotyping has not previously been reported for ALI.
Objectives: To identify ALI risk variants after major trauma using a large-scale candidate gene approach.
Methods: We performed a two-stage genetic association study. We derived findings in an African American cohort (n = 222) using a cardiopulmonary disease–centric 50K single nucleotide polymorphism (SNP) array. Genotype and haplotype distributions were compared between subjects with ALI and without ALI, with adjustment for clinical factors. Top performing SNPs (P < 10−4) were tested in a multicenter European American trauma-associated ALI case-control population (n = 600 ALI; n = 2,266 population-based control subjects) for replication. The ALI-associated genomic region was sequenced, analyzed for in silico prediction of function, and plasma was assayed by ELISA and immunoblot.
Measurements and Main Results: Five SNPs demonstrated a significant association with ALI after adjustment for covariates in Stage I. Two SNPs in ANGPT2 (rs1868554 and rs2442598) replicated their significant association with ALI in Stage II. rs1868554 was robust to multiple comparison correction: odds ratio 1.22 (1.06–1.40), P = 0.0047. Resequencing identified predicted novel splice sites in linkage disequilibrium with rs1868554, and immunoblots showed higher proportion of variant angiopoietin-2 (ANG2) isoform associated with rs1868554T (0.81 vs. 0.48; P = 0.038).
Conclusions: An ANGPT2 region is associated with both ALI and variation in plasma angiopoietin-2 isoforms. Characterization of the variant isoform and its genetic regulation may yield important insights about ALI pathogenesis and susceptibility.
PMCID: PMC3114062  PMID: 21257790
acute lung injury; acute respiratory distress syndrome; functional genetic polymorphism; genetic association study
24.  Fractalkine Is a Novel Human Adipochemokine Associated With Type 2 Diabetes 
Diabetes  2011;60(5):1512-1518.
Leukocyte infiltration of adipose is a critical determinant of obesity-related metabolic diseases. Fractalkine (CX3CL1) and its receptor (CX3CR1) comprise a chemokine system involved in leukocyte recruitment and adhesion in atherosclerosis, but its role in adipose inflammation and type 2 diabetes is unknown.
CX3CL1 mRNA and protein were quantified in subcutaneous adipose and blood during experimental human endotoxemia and in lean and obese human adipose. CX3CL1 cellular source was probed in human adipocytes, monocytes, and macrophages, and CX3CL1-blocking antibodies were used to assess its role in monocyte-adipocyte adhesion. The association of genetic variation in CX3CR1 with metabolic traits was determined in a community-based sample. Finally, plasma CX3CL1 levels were measured in a case-control study of type 2 diabetes.
Endotoxemia induced adipose CX3CL1 mRNA (32.7-fold, P < 1 × 10−5) and protein (43-fold, P = 0.006). Obese subjects had higher CX3CL1 levels in subcutaneous adipose compared with lean (0.420 ± 0.387 vs. 0.228 ± 0.187 ng/mL, P = 0.04). CX3CL1 was expressed and secreted by human adipocytes and stromal vascular cells. Inflammatory cytokine induction of CX3CL1 in human adipocytes (27.5-fold mRNA and threefold protein) was completely attenuated by pretreatment with a peroxisome proliferator–activated receptor-γ agonist. A putative functional nonsynonymous single nucleotide polymorphism (rs3732378) in CX3CR1 was associated with adipose and metabolic traits, and plasma CX3CL1 levels were increased in patients with type 2 diabetes vs. nondiabetics (0.506 ± 0.262 vs. 0.422 ± 0.210 ng/mL, P < 0.0001).
CX3CL1-CX3CR1 is a novel inflammatory adipose chemokine system that modulates monocyte adhesion to adipocytes and is associated with obesity, insulin resistance, and type 2 diabetes. These data provide support for CX3CL1 as a diagnostic and therapeutic target in cardiometabolic disease.
PMCID: PMC3292325  PMID: 21525510
25.  The novel atherosclerosis locus at 10q11 regulates plasma CXCL12 levels 
European Heart Journal  2011;32(8):963-971.
Two single-nucleotide polymorphisms (SNPs), rs1746048 and rs501120, from genome wide association studies of coronary artery disease (CAD) map to chromosome 10q11 ∼80 kb downstream of chemokine CXCL12. Therefore, we examined the relationship between these two SNPs and plasma CXCL12 levels.
Methods and Results
We tested the association of two SNPs with plasma CXCL12 levels in a two-stage study (n= 2939): first in PennCath (n= 1182), a Caucasian, angiographic CAD case–control study, and second in PennCAC (n= 1757), a community-based study of CAD risk factors. Plasma CXCL12 levels increased with age and did not vary by gender. There was no linkage disequilibrium between these two SNPs and SNPs within CXCL12 gene. However, CAD risk alleles at rs1746048 (C allele, P= 0.034; CC 2.33 ± 0.49, CT 2.27 ± 0.46, and TT 2.21 ± 0.52 ng/mL) and rs501120 (T allele, P= 0.041; TT 2.34 ± 0.49, CT 2.28 ± 0.46, and CC 2.23 ± 0.53 ng/mL) were associated with higher plasma levels of CXCL12 in age and gender adjusted models. In Stage 2, we confirmed this association (rs501120, T allele, P= 0.007), and meta-analysis strengthened this finding (n= 2939, P= 6.0 × 10−4). Finally, in exploratory analysis, the rs1746048 risk allele tended to have higher transcript levels of CXCL12 in human natural killer cells and the liver.
Coronary artery disease risk alleles downstream of CXCL12 are associated with plasma protein levels of CXCL12 and appear to be related to CXCL12 transcript levels in two human cell lines. This implicates CXCL12 as potentially causal and supports CXCL12 as a potential therapeutic target for CAD.
PMCID: PMC3076669  PMID: 21415067
Myocardial infarction; Cardiovascular genomics; Chemokines; CXCL12; Inflammation

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