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1.  Comparing gene set analysis methods on single-nucleotide polymorphism data from Genetic Analysis Workshop 16 
BMC Proceedings  2009;3(Suppl 7):S96.
Recently, gene set analysis (GSA) has been extended from use on gene expression data to use on single-nucleotide polymorphism (SNP) data in genome-wide association studies. When GSA has been demonstrated on SNP data, two popular statistics from gene expression data analysis (gene set enrichment analysis [GSEA] and Fisher's exact test [FET]) have been used. However, GSEA and FET have shown a lack of power and robustness in the analysis of gene expression data. The purpose of this work is to investigate whether the same issues are also true for the analysis of SNP data. Ultimately, we conclude that GSEA and FET are not optimal for the analysis of SNP data when compared with the SUMSTAT method. In analysis of real SNP data from the Framingham Heart Study, we find that SUMSTAT finds many more gene sets to be significant when compared with other methods. In an analysis of simulated data, SUMSTAT demonstrates high power and better control of the type I error rate. GSA is a promising approach to the analysis of SNP data in GWAS and use of the SUMSTAT statistic instead of GSEA or FET may increase power and robustness.
PMCID: PMC2796000  PMID: 20018093
2.  RS-SNP: a random-set method for genome-wide association studies 
BMC Genomics  2011;12:166.
Background
The typical objective of Genome-wide association (GWA) studies is to identify single-nucleotide polymorphisms (SNPs) and corresponding genes with the strongest evidence of association (the 'most-significant SNPs/genes' approach). Borrowing ideas from micro-array data analysis, we propose a new method, named RS-SNP, for detecting sets of genes enriched in SNPs moderately associated to the phenotype. RS-SNP assesses whether the number of significant SNPs, with p-value P ≤ α, belonging to a given SNP set is statistically significant. The rationale of proposed method is that two kinds of null hypotheses are taken into account simultaneously. In the first null model the genotype and the phenotype are assumed to be independent random variables and the null distribution is the probability of the number of significant SNPs in greater than observed by chance. The second null model assumes the number of significant SNPs in depends on the size of and not on the identity of the SNPs in . Statistical significance is assessed using non-parametric permutation tests.
Results
We applied RS-SNP to the Crohn's disease (CD) data set collected by the Wellcome Trust Case Control Consortium (WTCCC) and compared the results with GENGEN, an approach recently proposed in literature. The enrichment analysis using RS-SNP and the set of pathways contained in the MSigDB C2 CP pathway collection highlighted 86 pathways rich in SNPs weakly associated to CD. Of these, 47 were also indicated to be significant by GENGEN. Similar results were obtained using the MSigDB C5 pathway collection. Many of the pathways found to be enriched by RS-SNP have a well-known connection to CD and often with inflammatory diseases.
Conclusions
The proposed method is a valuable alternative to other techniques for enrichment analysis of SNP sets. It is well founded from a theoretical and statistical perspective. Moreover, the experimental comparison with GENGEN highlights that it is more robust with respect to false positive findings.
doi:10.1186/1471-2164-12-166
PMCID: PMC3079664  PMID: 21450072
3.  SNP-based pathway enrichment analysis for genome-wide association studies 
BMC Bioinformatics  2011;12:99.
Background
Recently we have witnessed a surge of interest in using genome-wide association studies (GWAS) to discover the genetic basis of complex diseases. Many genetic variations, mostly in the form of single nucleotide polymorphisms (SNPs), have been identified in a wide spectrum of diseases, including diabetes, cancer, and psychiatric diseases. A common theme arising from these studies is that the genetic variations discovered by GWAS can only explain a small fraction of the genetic risks associated with the complex diseases. New strategies and statistical approaches are needed to address this lack of explanation. One such approach is the pathway analysis, which considers the genetic variations underlying a biological pathway, rather than separately as in the traditional GWAS studies. A critical challenge in the pathway analysis is how to combine evidences of association over multiple SNPs within a gene and multiple genes within a pathway. Most current methods choose the most significant SNP from each gene as a representative, ignoring the joint action of multiple SNPs within a gene. This approach leads to preferential identification of genes with a greater number of SNPs.
Results
We describe a SNP-based pathway enrichment method for GWAS studies. The method consists of the following two main steps: 1) for a given pathway, using an adaptive truncated product statistic to identify all representative (potentially more than one) SNPs of each gene, calculating the average number of representative SNPs for the genes, then re-selecting the representative SNPs of genes in the pathway based on this number; and 2) ranking all selected SNPs by the significance of their statistical association with a trait of interest, and testing if the set of SNPs from a particular pathway is significantly enriched with high ranks using a weighted Kolmogorov-Smirnov test. We applied our method to two large genetically distinct GWAS data sets of schizophrenia, one from European-American (EA) and the other from African-American (AA). In the EA data set, we found 22 pathways with nominal P-value less than or equal to 0.001 and corresponding false discovery rate (FDR) less than 5%. In the AA data set, we found 11 pathways by controlling the same nominal P-value and FDR threshold. Interestingly, 8 of these pathways overlap with those found in the EA sample. We have implemented our method in a JAVA software package, called SNP Set Enrichment Analysis (SSEA), which contains a user-friendly interface and is freely available at http://cbcl.ics.uci.edu/SSEA.
Conclusions
The SNP-based pathway enrichment method described here offers a new alternative approach for analysing GWAS data. By applying it to schizophrenia GWAS studies, we show that our method is able to identify statistically significant pathways, and importantly, pathways that can be replicated in large genetically distinct samples.
doi:10.1186/1471-2105-12-99
PMCID: PMC3102637  PMID: 21496265
4.  i-GSEA4GWAS: a web server for identification of pathways/gene sets associated with traits by applying an improved gene set enrichment analysis to genome-wide association study 
Nucleic Acids Research  2010;38(Web Server issue):W90-W95.
Genome-wide association study (GWAS) is nowadays widely used to identify genes involved in human complex disease. The standard GWAS analysis examines SNPs/genes independently and identifies only a number of the most significant SNPs. It ignores the combined effect of weaker SNPs/genes, which leads to difficulties to explore biological function and mechanism from a systems point of view. Although gene set enrichment analysis (GSEA) has been introduced to GWAS to overcome these limitations by identifying the correlation between pathways/gene sets and traits, the heavy dependence on genotype data, which is not easily available for most published GWAS investigations, has led to limited application of it. In order to perform GSEA on a simple list of GWAS SNP P-values, we implemented GSEA by using SNP label permutation. We further improved GSEA (i-GSEA) by focusing on pathways/gene sets with high proportion of significant genes. To provide researchers an open platform to analyze GWAS data, we developed the i-GSEA4GWAS (improved GSEA for GWAS) web server. i-GSEA4GWAS implements the i-GSEA approach and aims to provide new insights in complex disease studies. i-GSEA4GWAS is freely available at http://gsea4gwas.psych.ac.cn/.
doi:10.1093/nar/gkq324
PMCID: PMC2896119  PMID: 20435672
5.  Variable set enrichment analysis in genome-wide association studies 
Complex diseases such as hypertension are inherently multifactorial and involve many factors of mild-to-minute effect sizes. A genome-wide association study (GWAS) typically tests hundreds of thousands of single-nucleotide polymorphisms (SNPs), and offers opportunity to evaluate aggregated effects of many genetic variants with effects that are too small to detect individually. The gene-set-enrichment analysis (GSEA) is a pathway-based approach that tests for such aggregated effects of genes that are linked by biological functions. A key step in GSEA is the summary statistic (gene score) used to measure the overall relevance of a gene based on all SNPs tested in the gene. Existing GSEA methods use maximum statistics sensitive to gene size and linkage equilibrium. We propose the approach of variable set enrichment analysis (VSEA) and study new gene score methods that are less dependent on gene size. The new method treats groups of variables (SNPs or other variants) as base units for summarizing gene scores and relies less on gene definition itself. The power of VSEA is analyzed by simulation studies modeling various scenarios of complex multiloci interactions. Results show that the new gene scores generally performed better, some substantially so, than existing GSEA extension to GWAS. The new methods are implemented in an R package and when applied to a real GWAS data set demonstrated its practical utility in a GWAS setting.
doi:10.1038/ejhg.2011.46
PMCID: PMC3172928  PMID: 21427759
gene set enrichment; pathway-based analysis; SNP; genome-wide association
6.  Use of Wrapper Algorithms Coupled with a Random Forests Classifier for Variable Selection in Large-Scale Genomic Association Studies 
Modern large-scale genetic association studies generate increasingly high-dimensional datasets. Therefore, some variable selection procedure should be performed before the application of traditional data analysis methods, for reasons of both computational efficiency and problems related to overfitting. We describe here a “wrapper” strategy (SIZEFIT) for variable selection that uses a Random Forests classifier, coupled with various local search/optimization algorithms. We apply it to a large dataset consisting of 2,425 African-American and non-Hispanic white individuals genotyped for 4,869 single-nucleotide polymorphisms (SNPs) in a coronary heart disease (CHD) case–cohort association study (Atherosclerosis Risk in Communities), using incident CHD and plasma low-density lipoprotein (LDL) cholesterol levels as the dependent variables. We show that most SNPs can be safely removed from the dataset without compromising the predictive (classification) accuracy, with only a small number of SNPs (sometimes less than 100) containing any predictive signal. A statistical (SUMSTAT) approach is also applied to the dataset for comparison purposes. We describe a novel method for refining the subset of signal-containing SNPs (FIXFIT), based on an Extremal Optimization algorithm. Finally, we compare the top SNP rankings obtained by different methods and devise practical guidelines for researchers trying to generate a compact subset of predictive SNPs from genome-wide association datasets. Interestingly, there is a significant amount of overlap between seemingly very heterogeneous rankings. We conclude by constructing compact optimal predictive SNP subsets for CHD (less than 150 SNPs) and LDL (less than 300 SNPs) phenotypes, and by comparing various rankings for two well-known positive control SNPs for LDL in the apolipoprotein E gene.
doi:10.1089/cmb.2008.0037
PMCID: PMC2980837  PMID: 20047492
coronary heart disease; genome-wide association studies; Random Forests classifier; SNPs; variable selection
7.  Use of Wrapper Algorithms Coupled with a Random Forests Classifier for Variable Selection in Large-Scale Genomic Association Studies 
Journal of Computational Biology  2009;16(12):1705-1718.
Abstract
Modern large-scale genetic association studies generate increasingly high-dimensional datasets. Therefore, some variable selection procedure should be performed before the application of traditional data analysis methods, for reasons of both computational efficiency and problems related to overfitting. We describe here a “wrapper” strategy (SIZEFIT) for variable selection that uses a Random Forests classifier, coupled with various local search/optimization algorithms. We apply it to a large dataset consisting of 2,425 African-American and non-Hispanic white individuals genotyped for 4,869 single-nucleotide polymorphisms (SNPs) in a coronary heart disease (CHD) case-cohort association study (Atherosclerosis Risk in Communities), using incident CHD and plasma low-density lipoprotein (LDL) cholesterol levels as the dependent variables. We show that most SNPs can be safely removed from the dataset without compromising the predictive (classification) accuracy, with only a small number of SNPs (sometimes less than 100) containing any predictive signal. A statistical (SUMSTAT) approach is also applied to the dataset for comparison purposes. We describe a novel method for refining the subset of signal-containing SNPs (FIXFIT), based on an Extremal Optimization algorithm. Finally, we compare the top SNP rankings obtained by different methods and devise practical guidelines for researchers trying to generate a compact subset of predictive SNPs from genome-wide association datasets. Interestingly, there is a significant amount of overlap between seemingly very heterogeneous rankings. We conclude by constructing compact optimal predictive SNP subsets for CHD (less than 150 SNPs) and LDL (less than 300 SNPs) phenotypes, and by comparing various rankings for two well-known positive control SNPs for LDL in the apolipoprotein E gene.
doi:10.1089/cmb.2008.0037
PMCID: PMC2980837  PMID: 20047492
coronary heart disease; genome-wide association studies; Random Forests classifier; SNPs; variable selection
8.  A comparative study on gene-set analysis methods for assessing differential expression associated with the survival phenotype 
BMC Bioinformatics  2011;12:377.
Background
Many gene-set analysis methods have been previously proposed and compared through simulation studies and analysis of real datasets for binary phenotypes. We focused on the survival phenotype and compared the performances of Gene Set Enrichment Analysis (GSEA), Global Test (GT), Wald-type Test (WT) and Global Boost Test (GBST) methods in a simulation study and on two ovarian cancer data sets. We considered two versions of GSEA by allowing different weights: GSEA1 uses equal weights, yielding results similar to the Kolmogorov-Smirnov test; while GSEA2's weights are based on the correlation between genes and the phenotype.
Results
We compared GSEA1, GSEA2, GT, WT and GBST in a simulation study with various settings for the correlation structure of the genes and the association parameter between the survival outcome and the genes. Simulation results indicated that GT, WT and GBST consistently have higher power than GSEA1 and GSEA2 across all scenarios. However, the power of the five tests depends on the combination of correlation structure and association parameter. For the ovarian cancer data set, using the FDR threshold of q < 0.1, the GT, WT and GBST detected 12, 6 and 8 significant pathways, respectively, whereas neither GSEA1 nor GSEA2 detected any significant pathways. In addition, among the pathways found significant by GT, WT, and GBST, three pathways - Purine metabolism, Leukocyte transendothelial migration and Jak-STAT signaling pathway - overlapped with those reported in previous ovarian cancer microarray studies.
Conclusion
Simulation studies and a real data example indicate that GT, WT and GBST tend to have high power, whereas GSEA1 and GSEA2 have lower power. We also found that the power of the five tests is much higher when genes are correlated than when genes are independent, when survival is positively associated with genes. It seems that there is a synergistic effect in detecting significant gene sets when significant genes have within-class correlation and the association between survival and genes is positive or negative (i.e., one-direction correlation).
doi:10.1186/1471-2105-12-377
PMCID: PMC3196970  PMID: 21943316
9.  Genetic Association Analysis of Complex Diseases Incorporating Intermediate Phenotype Information 
PLoS ONE  2012;7(10):e46612.
Genetic researchers often collect disease related quantitative traits in addition to disease status because they are interested in understanding the pathophysiology of disease processes. In genome-wide association (GWA) studies, these quantitative phenotypes may be relevant to disease development and serve as intermediate phenotypes or they could be behavioral or other risk factors that predict disease risk. Statistical tests combining both disease status and quantitative risk factors should be more powerful than case-control studies, as the former incorporates more information about the disease. In this paper, we proposed a modified inverse-variance weighted meta-analysis method to combine disease status and quantitative intermediate phenotype information. The simulation results showed that when an intermediate phenotype was available, the inverse-variance weighted method had more power than did a case-control study of complex diseases, especially in identifying susceptibility loci having minor effects. We further applied this modified meta-analysis to a study of imputed lung cancer genotypes with smoking data in 1154 cases and 1137 matched controls. The most significant SNPs came from the CHRNA3-CHRNA5-CHRNB4 region on chromosome 15q24–25.1, which has been replicated in many other studies. Our results confirm that this CHRNA region is associated with both lung cancer development and smoking behavior. We also detected three significant SNPs—rs1800469, rs1982072, and rs2241714—in the promoter region of the TGFB1 gene on chromosome 19 (p = 1.46×10−5, 1.18×10−5, and 6.57×10−6, respectively). The SNP rs1800469 is reported to be associated with chronic obstructive pulmonary disease and lung cancer in cigarette smokers. The present study is the first GWA study to replicate this result. Signals in the 3q26 region were also identified in the meta-analysis. We demonstrate the intermediate phenotype can potentially enhance the power of complex disease association analysis and the modified meta-analysis method is robust to incorporate intermediate phenotype or other quantitative risk factor in the analysis.
doi:10.1371/journal.pone.0046612
PMCID: PMC3477105  PMID: 23094028
10.  Finding type 2 diabetes causal single nucleotide polymorphism combinations and functional modules from genome-wide association data 
Background
Due to the low statistical power of individual markers from a genome-wide association study (GWAS), detecting causal single nucleotide polymorphisms (SNPs) for complex diseases is a challenge. SNP combinations are suggested to compensate for the low statistical power of individual markers, but SNP combinations from GWAS generate high computational complexity.
Methods
We aim to detect type 2 diabetes (T2D) causal SNP combinations from a GWAS dataset with optimal filtration and to discover the biological meaning of the detected SNP combinations. Optimal filtration can enhance the statistical power of SNP combinations by comparing the error rates of SNP combinations from various Bonferroni thresholds and p-value range-based thresholds combined with linkage disequilibrium (LD) pruning. T2D causal SNP combinations are selected using random forests with variable selection from an optimal SNP dataset. T2D causal SNP combinations and genome-wide SNPs are mapped into functional modules using expanded gene set enrichment analysis (GSEA) considering pathway, transcription factor (TF)-target, miRNA-target, gene ontology, and protein complex functional modules. The prediction error rates are measured for SNP sets from functional module-based filtration that selects SNPs within functional modules from genome-wide SNPs based expanded GSEA.
Results
A T2D causal SNP combination containing 101 SNPs from the Wellcome Trust Case Control Consortium (WTCCC) GWAS dataset are selected using optimal filtration criteria, with an error rate of 10.25%. Matching 101 SNPs with known T2D genes and functional modules reveals the relationships between T2D and SNP combinations. The prediction error rates of SNP sets from functional module-based filtration record no significance compared to the prediction error rates of randomly selected SNP sets and T2D causal SNP combinations from optimal filtration.
Conclusions
We propose a detection method for complex disease causal SNP combinations from an optimal SNP dataset by using random forests with variable selection. Mapping the biological meanings of detected SNP combinations can help uncover complex disease mechanisms.
doi:10.1186/1472-6947-13-S1-S3
PMCID: PMC3618247  PMID: 23566118
11.  Pathway Analysis for Genome-Wide Association Study of Basal Cell Carcinoma of the Skin 
PLoS ONE  2011;6(7):e22760.
Background
Recently, a pathway-based approach has been developed to evaluate the cumulative contribution of the functionally related genes for genome-wide association studies (GWASs), which may help utilize GWAS data to a greater extent.
Methods
In this study, we applied this approach for the GWAS of basal cell carcinoma (BCC) of the skin. We first conducted the BCC GWAS among 1,797 BCC cases and 5,197 controls in Caucasians with 740,760 genotyped SNPs. 115,688 SNPs were grouped into gene transcripts within 20 kb in distance and then into 174 Kyoto Encyclopedia of Genes and Genomes pathways, 205 BioCarta pathways, as well as two positive control gene sets (pigmentation gene set and BCC risk gene set). The association of each pathway with BCC risk was evaluated using the weighted Kolmogorov-Smirnov test. One thousand permutations were conducted to assess the significance.
Results
Both of the positive control gene sets reached pathway p-values<0.05. Four other pathways were also significantly associated with BCC risk: the heparan sulfate biosynthesis pathway (p  =  0.007, false discovery rate, FDR  =  0.35), the mCalpain pathway (p  =  0.002, FDR  =  0.12), the Rho cell motility signaling pathway (p  =  0.011, FDR  =  0.30), and the nitric oxide pathway (p  =  0.022, FDR  =  0.42).
Conclusion
We identified four pathways associated with BCC risk, which may offer new insights into the etiology of BCC upon further validation, and this approach may help identify potential biological pathways that might be missed by the standard GWAS approach.
doi:10.1371/journal.pone.0022760
PMCID: PMC3145747  PMID: 21829505
12.  Comparative study of gene set enrichment methods 
BMC Bioinformatics  2009;10:275.
Background
The analysis of high-throughput gene expression data with respect to sets of genes rather than individual genes has many advantages. A variety of methods have been developed for assessing the enrichment of sets of genes with respect to differential expression. In this paper we provide a comparative study of four of these methods: Fisher's exact test, Gene Set Enrichment Analysis (GSEA), Random-Sets (RS), and Gene List Analysis with Prediction Accuracy (GLAPA). The first three methods use associative statistics, while the fourth uses predictive statistics. We first compare all four methods on simulated data sets to verify that Fisher's exact test is markedly worse than the other three approaches. We then validate the other three methods on seven real data sets with known genetic perturbations and then compare the methods on two cancer data sets where our a priori knowledge is limited.
Results
The simulation study highlights that none of the three method outperforms all others consistently. GSEA and RS are able to detect weak signals of deregulation and they perform differently when genes in a gene set are both differentially up and down regulated. GLAPA is more conservative and large differences between the two phenotypes are required to allow the method to detect differential deregulation in gene sets. This is due to the fact that the enrichment statistic in GLAPA is prediction error which is a stronger criteria than classical two sample statistic as used in RS and GSEA. This was reflected in the analysis on real data sets as GSEA and RS were seen to be significant for particular gene sets while GLAPA was not, suggesting a small effect size. We find that the rank of gene set enrichment induced by GLAPA is more similar to RS than GSEA. More importantly, the rankings of the three methods share significant overlap.
Conclusion
The three methods considered in our study recover relevant gene sets known to be deregulated in the experimental conditions and pathologies analyzed. There are differences between the three methods and GSEA seems to be more consistent in finding enriched gene sets, although no method uniformly dominates over all data sets. Our analysis highlights the deep difference existing between associative and predictive methods for detecting enrichment and the use of both to better interpret results of pathway analysis. We close with suggestions for users of gene set methods.
doi:10.1186/1471-2105-10-275
PMCID: PMC2746222  PMID: 19725948
13.  Large-Scale Pathway-Based Analysis of Bladder Cancer Genome-Wide Association Data from Five Studies of European Background 
PLoS ONE  2012;7(1):e29396.
Pathway analysis of genome-wide association studies (GWAS) offer a unique opportunity to collectively evaluate genetic variants with effects that are too small to be detected individually. We applied a pathway analysis to a bladder cancer GWAS containing data from 3,532 cases and 5,120 controls of European background (n = 5 studies). Thirteen hundred and ninety-nine pathways were drawn from five publicly available resources (Biocarta, Kegg, NCI-PID, HumanCyc, and Reactome), and we constructed 22 additional candidate pathways previously hypothesized to be related to bladder cancer. In total, 1421 pathways, 5647 genes and ∼90,000 SNPs were included in our study. Logistic regression model adjusting for age, sex, study, DNA source, and smoking status was used to assess the marginal trend effect of SNPs on bladder cancer risk. Two complementary pathway-based methods (gene-set enrichment analysis [GSEA], and adapted rank-truncated product [ARTP]) were used to assess the enrichment of association signals within each pathway. Eighteen pathways were detected by either GSEA or ARTP at P≤0.01. To minimize false positives, we used the I2 statistic to identify SNPs displaying heterogeneous effects across the five studies. After removing these SNPs, seven pathways (‘Aromatic amine metabolism’ [PGSEA = 0.0100, PARTP = 0.0020], ‘NAD biosynthesis’ [PGSEA = 0.0018, PARTP = 0.0086], ‘NAD salvage’ [PARTP = 0.0068], ‘Clathrin derived vesicle budding’ [PARTP = 0.0018], ‘Lysosome vesicle biogenesis’ [PGSEA = 0.0023, PARTP<0.00012], ’Retrograde neurotrophin signaling’ [PGSEA = 0.00840], and ‘Mitotic metaphase/anaphase transition’ [PGSEA = 0.0040]) remained. These pathways seem to belong to three fundamental cellular processes (metabolic detoxification, mitosis, and clathrin-mediated vesicles). Identification of the aromatic amine metabolism pathway provides support for the ability of this approach to identify pathways with established relevance to bladder carcinogenesis.
doi:10.1371/journal.pone.0029396
PMCID: PMC3251580  PMID: 22238607
14.  Gene set enrichment analysis for non-monotone association and multiple experimental categories 
BMC Bioinformatics  2008;9:481.
Background
Recently, microarray data analyses using functional pathway information, e.g., gene set enrichment analysis (GSEA) and significance analysis of function and expression (SAFE), have gained recognition as a way to identify biological pathways/processes associated with a phenotypic endpoint. In these analyses, a local statistic is used to assess the association between the expression level of a gene and the value of a phenotypic endpoint. Then these gene-specific local statistics are combined to evaluate association for pre-selected sets of genes. Commonly used local statistics include t-statistics for binary phenotypes and correlation coefficients that assume a linear or monotone relationship between a continuous phenotype and gene expression level. Methods applicable to continuous non-monotone relationships are needed. Furthermore, for multiple experimental categories, methods that combine multiple GSEA/SAFE analyses are needed.
Results
For continuous or ordinal phenotypic outcome, we propose to use as the local statistic the coefficient of multiple determination (i.e., the square of multiple correlation coefficient) R2 from fitting natural cubic spline models to the phenotype-expression relationship. Next, we incorporate this association measure into the GSEA/SAFE framework to identify significant gene sets. Unsigned local statistics, signed global statistics and one-sided p-values are used to reflect our inferential interest. Furthermore, we describe a procedure for inference across multiple GSEA/SAFE analyses. We illustrate our approach using gene expression and liver injury data from liver and blood samples from rats treated with eight hepatotoxicants under multiple time and dose combinations. We set out to identify biological pathways/processes associated with liver injury as manifested by increased blood levels of alanine transaminase in common for most of the eight compounds. Potential statistical dependency resulting from the experimental design is addressed in permutation based hypothesis testing.
Conclusion
The proposed framework captures both linear and non-linear association between gene expression level and a phenotypic endpoint and thus can be viewed as extending the current GSEA/SAFE methodology. The framework for combining results from multiple GSEA/SAFE analyses is flexible to address practical inference interests. Our methods can be applied to microarray data with continuous phenotypes with multi-level design or the meta-analysis of multiple microarray data sets.
doi:10.1186/1471-2105-9-481
PMCID: PMC2636811  PMID: 19014579
15.  Improved Detection of Common Variants Associated with Schizophrenia and Bipolar Disorder Using Pleiotropy-Informed Conditional False Discovery Rate 
PLoS Genetics  2013;9(4):e1003455.
Several lines of evidence suggest that genome-wide association studies (GWAS) have the potential to explain more of the “missing heritability” of common complex phenotypes. However, reliable methods to identify a larger proportion of single nucleotide polymorphisms (SNPs) that impact disease risk are currently lacking. Here, we use a genetic pleiotropy-informed conditional false discovery rate (FDR) method on GWAS summary statistics data to identify new loci associated with schizophrenia (SCZ) and bipolar disorders (BD), two highly heritable disorders with significant missing heritability. Epidemiological and clinical evidence suggest similar disease characteristics and overlapping genes between SCZ and BD. Here, we computed conditional Q–Q curves of data from the Psychiatric Genome Consortium (SCZ; n = 9,379 cases and n = 7,736 controls; BD: n = 6,990 cases and n = 4,820 controls) to show enrichment of SNPs associated with SCZ as a function of association with BD and vice versa with a corresponding reduction in FDR. Applying the conditional FDR method, we identified 58 loci associated with SCZ and 35 loci associated with BD below the conditional FDR level of 0.05. Of these, 14 loci were associated with both SCZ and BD (conjunction FDR). Together, these findings show the feasibility of genetic pleiotropy-informed methods to improve gene discovery in SCZ and BD and indicate overlapping genetic mechanisms between these two disorders.
Author Summary
Genome-wide association studies (GWAS) have thus far identified only a small fraction of the heritability of common complex disorders, such as severe mental disorders. We used a conditional false discovery rate approach for analysis of GWAS data, exploiting “genetic pleiotropy” to increase discovery of common gene variants associated with schizophrenia and bipolar disorders. Leveraging the increased power from combining GWAS of two associated phenotypes, we found a striking overlap in polygenic signals, allowing for the discovery of several new common gene variants associated with bipolar disorder and schizophrenia that were not identified in the original analysis using traditional GWAS methods. Some of the gene variants have been identified in other studies with large targeted replication samples, validating the present findings. Our pleiotropy-informed method may be of significant importance for detecting effects that are below the traditional genome-wide significance level in GWAS, particularly in highly polygenic, complex phenotypes, such as schizophrenia and bipolar disorder, where most of the genetic signal is missing (i.e., “missing heritability”). The findings also offer insights into mechanistic relationships between bipolar disorder and schizophrenia pathogenesis.
doi:10.1371/journal.pgen.1003455
PMCID: PMC3636100  PMID: 23637625
16.  Association and interaction analysis of variants in CHRNA5/CHRNA3/CHRNB4 gene cluster with nicotine dependence in African and European Americans 
Several previous genome-wide and targeted association studies revealed that variants in the CHRNA5-CHRNA3-CHRNB4 (CHRNA5/A3/B4) gene cluster on chromosome 15 that encode the α5, α3 and β4 subunits of the nicotinic acetylcholine receptor (nAChRs) are associated with nicotine dependence (ND) in European Americans (EAs) or others of European origin. Considering the distinct linkage disequilibrium patterns in European and other ethnic populations such as African Americans (AAs), it would be interesting to determine whether such associations exist in other ethnic populations. We performed a comprehensive association and interaction analysis of the CHRNA5/A3/B4 cluster in two ethnic samples to investigate the role of variants in the risk for ND, which was assessed by Smoking Quantity, Heaviness Smoking Index, and Fagerström test for ND. Using a family-based association test, we found a nominal association of single nucleotide polymorphisms (SNPs) rs1317286 and rs8040868 in CHRNA3 with ND in the AA and combined AA and EA samples. Furthermore, we found that several haplotypes in CHRNA5 and CHRNA3 are nominally associated with ND in AA, EA, and pooled samples. However, none of these associations remained significant after correction for multiple testing. In addition, we performed interaction analysis of SNPs within the CHRNA5/A3/B4 cluster using the pedigree-based generalized multifactor dimensionality reduction method and found significant interactions within CHRNA3 and among the three subunit genes in the AA and pooled samples. Together, these results indicate that variants within CHRNA3 and among CHRNA5, CHRNA3, and CHRNB4 contribute significantly to the etiology of ND through gene-gene interactions, although the association of each subunit gene with ND is weak in both the AA and EA samples.
doi:10.1002/ajmg.b.31043
PMCID: PMC2924635  PMID: 19859904
Association analysis; CHRNA5; CHRNA3; CHRNB4; Interaction analysis; Nicotine dependence; Smoking
17.  Pathways of Distinction Analysis: A New Technique for Multi–SNP Analysis of GWAS Data 
PLoS Genetics  2011;7(6):e1002101.
Genome-wide association studies (GWAS) have become increasingly common due to advances in technology and have permitted the identification of differences in single nucleotide polymorphism (SNP) alleles that are associated with diseases. However, while typical GWAS analysis techniques treat markers individually, complex diseases (cancers, diabetes, and Alzheimers, amongst others) are unlikely to have a single causative gene. Thus, there is a pressing need for multi–SNP analysis methods that can reveal system-level differences in cases and controls. Here, we present a novel multi–SNP GWAS analysis method called Pathways of Distinction Analysis (PoDA). The method uses GWAS data and known pathway–gene and gene–SNP associations to identify pathways that permit, ideally, the distinction of cases from controls. The technique is based upon the hypothesis that, if a pathway is related to disease risk, cases will appear more similar to other cases than to controls (or vice versa) for the SNPs associated with that pathway. By systematically applying the method to all pathways of potential interest, we can identify those for which the hypothesis holds true, i.e., pathways containing SNPs for which the samples exhibit greater within-class similarity than across classes. Importantly, PoDA improves on existing single–SNP and SNP–set enrichment analyses, in that it does not require the SNPs in a pathway to exhibit independent main effects. This permits PoDA to reveal pathways in which epistatic interactions drive risk. In this paper, we detail the PoDA method and apply it to two GWAS: one of breast cancer and the other of liver cancer. The results obtained strongly suggest that there exist pathway-wide genomic differences that contribute to disease susceptibility. PoDA thus provides an analytical tool that is complementary to existing techniques and has the power to enrich our understanding of disease genomics at the systems-level.
Author Summary
We present a novel method for multi–SNP analysis of genome-wide association studies. The method is motivated by the intuition that, if a set of SNPs is associated with disease, cases and controls will exhibit more within-group similarity than across-group similarity for the SNPs in the set of interest. Our method, Pathways of Distinction Analysis (PoDA), uses GWAS data and known pathway–gene and gene–SNP associations to identify pathways that permit the distinction of cases from controls. By systematically applying the method to all pathways of potential interest, we can identify pathways containing SNPs for which the cases and controls are distinguished and infer those pathways' role in disease. We detail the PoDA method and describe its results in breast and liver cancer GWAS data, demonstrating its utility as a method for systems-level analysis of GWAS data.
doi:10.1371/journal.pgen.1002101
PMCID: PMC3111473  PMID: 21695280
18.  Pathways-Driven Sparse Regression Identifies Pathways and Genes Associated with High-Density Lipoprotein Cholesterol in Two Asian Cohorts 
PLoS Genetics  2013;9(11):e1003939.
Standard approaches to data analysis in genome-wide association studies (GWAS) ignore any potential functional relationships between gene variants. In contrast gene pathways analysis uses prior information on functional structure within the genome to identify pathways associated with a trait of interest. In a second step, important single nucleotide polymorphisms (SNPs) or genes may be identified within associated pathways. The pathways approach is motivated by the fact that genes do not act alone, but instead have effects that are likely to be mediated through their interaction in gene pathways. Where this is the case, pathways approaches may reveal aspects of a trait's genetic architecture that would otherwise be missed when considering SNPs in isolation. Most pathways methods begin by testing SNPs one at a time, and so fail to capitalise on the potential advantages inherent in a multi-SNP, joint modelling approach. Here, we describe a dual-level, sparse regression model for the simultaneous identification of pathways and genes associated with a quantitative trait. Our method takes account of various factors specific to the joint modelling of pathways with genome-wide data, including widespread correlation between genetic predictors, and the fact that variants may overlap multiple pathways. We use a resampling strategy that exploits finite sample variability to provide robust rankings for pathways and genes. We test our method through simulation, and use it to perform pathways-driven gene selection in a search for pathways and genes associated with variation in serum high-density lipoprotein cholesterol levels in two separate GWAS cohorts of Asian adults. By comparing results from both cohorts we identify a number of candidate pathways including those associated with cardiomyopathy, and T cell receptor and PPAR signalling. Highlighted genes include those associated with the L-type calcium channel, adenylate cyclase, integrin, laminin, MAPK signalling and immune function.
Author Summary
Genes do not act in isolation, but interact in complex networks or pathways. By accounting for such interactions, pathways analysis methods hope to identify aspects of a disease or trait's genetic architecture that might be missed using more conventional approaches. Most existing pathways methods take a univariate approach, in which each variant within a pathway is separately tested for association with the phenotype of interest. These statistics are then combined to assess pathway significance. As a second step, further analysis can reveal important genetic variants within significant pathways. We have previously shown that a joint-modelling approach using a sparse regression model can increase the power to detect pathways influencing a quantitative trait. Here we extend this approach, and describe a method that is able to simultaneously identify pathways and genes that may be driving pathway selection. We test our method using simulations, and apply it to a study searching for pathways and genes associated with high-density lipoprotein cholesterol in two separate East Asian cohorts.
doi:10.1371/journal.pgen.1003939
PMCID: PMC3836716  PMID: 24278029
19.  Bayesian Test for Colocalisation between Pairs of Genetic Association Studies Using Summary Statistics 
PLoS Genetics  2014;10(5):e1004383.
Genetic association studies, in particular the genome-wide association study (GWAS) design, have provided a wealth of novel insights into the aetiology of a wide range of human diseases and traits, in particular cardiovascular diseases and lipid biomarkers. The next challenge consists of understanding the molecular basis of these associations. The integration of multiple association datasets, including gene expression datasets, can contribute to this goal. We have developed a novel statistical methodology to assess whether two association signals are consistent with a shared causal variant. An application is the integration of disease scans with expression quantitative trait locus (eQTL) studies, but any pair of GWAS datasets can be integrated in this framework. We demonstrate the value of the approach by re-analysing a gene expression dataset in 966 liver samples with a published meta-analysis of lipid traits including >100,000 individuals of European ancestry. Combining all lipid biomarkers, our re-analysis supported 26 out of 38 reported colocalisation results with eQTLs and identified 14 new colocalisation results, hence highlighting the value of a formal statistical test. In three cases of reported eQTL-lipid pairs (SYPL2, IFT172, TBKBP1) for which our analysis suggests that the eQTL pattern is not consistent with the lipid association, we identify alternative colocalisation results with SORT1, GCKR, and KPNB1, indicating that these genes are more likely to be causal in these genomic intervals. A key feature of the method is the ability to derive the output statistics from single SNP summary statistics, hence making it possible to perform systematic meta-analysis type comparisons across multiple GWAS datasets (implemented online at http://coloc.cs.ucl.ac.uk/coloc/). Our methodology provides information about candidate causal genes in associated intervals and has direct implications for the understanding of complex diseases as well as the design of drugs to target disease pathways.
Author Summary
Genome-wide association studies (GWAS) have found a large number of genetic regions (“loci”) affecting clinical end-points and phenotypes, many outside coding intervals. One approach to understanding the biological basis of these associations has been to explore whether GWAS signals from intermediate cellular phenotypes, in particular gene expression, are located in the same loci (“colocalise”) and are potentially mediating the disease signals. However, it is not clear how to assess whether the same variants are responsible for the two GWAS signals or whether it is distinct causal variants close to each other. In this paper, we describe a statistical method that can use simply single variant summary statistics to test for colocalisation of GWAS signals. We describe one application of our method to a meta-analysis of blood lipids and liver expression, although any two datasets resulting from association studies can be used. Our method is able to detect the subset of GWAS signals explained by regulatory effects and identify candidate genes affected by the same GWAS variants. As summary GWAS data are increasingly available, applications of colocalisation methods to integrate the findings will be essential for functional follow-up, and will also be particularly useful to identify tissue specific signals in eQTL datasets.
doi:10.1371/journal.pgen.1004383
PMCID: PMC4022491  PMID: 24830394
20.  Gene Set of Nuclear-Encoded Mitochondrial Regulators Is Enriched for Common Inherited Variation in Obesity 
PLoS ONE  2013;8(2):e55884.
There are hints of an altered mitochondrial function in obesity. Nuclear-encoded genes are relevant for mitochondrial function (3 gene sets of known relevant pathways: (1) 16 nuclear regulators of mitochondrial genes, (2) 91 genes for oxidative phosphorylation and (3) 966 nuclear-encoded mitochondrial genes). Gene set enrichment analysis (GSEA) showed no association with type 2 diabetes mellitus in these gene sets. Here we performed a GSEA for the same gene sets for obesity. Genome wide association study (GWAS) data from a case-control approach on 453 extremely obese children and adolescents and 435 lean adult controls were used for GSEA. For independent confirmation, we analyzed 705 obesity GWAS trios (extremely obese child and both biological parents) and a population-based GWAS sample (KORA F4, n = 1,743). A meta-analysis was performed on all three samples. In each sample, the distribution of significance levels between the respective gene set and those of all genes was compared using the leading-edge-fraction-comparison test (cut-offs between the 50th and 95th percentile of the set of all gene-wise corrected p-values) as implemented in the MAGENTA software. In the case-control sample, significant enrichment of associations with obesity was observed above the 50th percentile for the set of the 16 nuclear regulators of mitochondrial genes (pGSEA,50 = 0.0103). This finding was not confirmed in the trios (pGSEA,50 = 0.5991), but in KORA (pGSEA,50 = 0.0398). The meta-analysis again indicated a trend for enrichment (pMAGENTA,50 = 0.1052, pMAGENTA,75 = 0.0251). The GSEA revealed that weak association signals for obesity might be enriched in the gene set of 16 nuclear regulators of mitochondrial genes.
doi:10.1371/journal.pone.0055884
PMCID: PMC3568071  PMID: 23409076
21.  Pathway-based Approaches for Sequencing-based Genome-wide Association Studies 
Genetic epidemiology  2013;37(5):10.1002/gepi.21728.
For analyzing complex trait association with sequencing data, most current studies test aggregated effects of variants in a gene or genomic region. While gene-based tests have insufficient power even for moderately sized samples, pathway-based analyses combine information across multiple genes in biological pathways and may offer additional insight. However, most existing pathway association methods are originally designed for genome-wide association studies (GWAS), and are not comprehensively evaluated for sequencing data. Moreover, region-based rare variant association methods, although potentially applicable to pathway-based analysis by extending their region definition to gene sets, have never been rigorously tested.
In the context of exome-based studies, we use simulated and real data sets to evaluate pathway-based association tests. Our simulation strategy adopts a genome-wide genetic model that distributes total genetic effects hierarchically into pathways, genes, and individual variants, allowing the evaluation of pathway-based methods with realistic quantifiable assumptions on the underlying genetic architectures.
The results show that, while no single pathway-based association method offers superior performance in all simulated scenarios, a modification of GSEA approach using statistics from single-marker tests without gene-level collapsing (WKS-Variant method) is consistently powerful. Interestingly, directly applying rare variant association tests (e.g., SKAT) to pathway analysis offers a similar power, but its results are sensitive to assumptions of genetic architecture. We applied pathway association analysis to an exome sequencing data of the chronic obstructive pulmonary disease (COPD), and found that the WKS-Variant method confirms associated genes previously published.
doi:10.1002/gepi.21728
PMCID: PMC3856324  PMID: 23650134
Pathway analysis; sequencing data; genome-wide association studies; simulation framework; chronic obstructive pulmonary disease
22.  Appearance frequency modulated gene set enrichment testing 
BMC Bioinformatics  2011;12:81.
Background
Gene set enrichment testing has helped bridge the gap from an individual gene to a systems biology interpretation of microarray data. Although gene sets are defined a priori based on biological knowledge, current methods for gene set enrichment testing treat all genes equal. It is well-known that some genes, such as those responsible for housekeeping functions, appear in many pathways, whereas other genes are more specialized and play a unique role in a single pathway. Drawing inspiration from the field of information retrieval, we have developed and present here an approach to incorporate gene appearance frequency (in KEGG pathways) into two current methods, Gene Set Enrichment Analysis (GSEA) and logistic regression-based LRpath framework, to generate more reproducible and biologically meaningful results.
Results
Two breast cancer microarray datasets were analyzed to identify gene sets differentially expressed between histological grade 1 and 3 breast cancer. The correlation of Normalized Enrichment Scores (NES) between gene sets, generated by the original GSEA and GSEA with the appearance frequency of genes incorporated (GSEA-AF), was compared. GSEA-AF resulted in higher correlation between experiments and more overlapping top gene sets. Several cancer related gene sets achieved higher NES in GSEA-AF as well. The same datasets were also analyzed by LRpath and LRpath with the appearance frequency of genes incorporated (LRpath-AF). Two well-studied lung cancer datasets were also analyzed in the same manner to demonstrate the validity of the method, and similar results were obtained.
Conclusions
We introduce an alternative way to integrate KEGG PATHWAY information into gene set enrichment testing. The performance of GSEA and LRpath can be enhanced with the integration of appearance frequency of genes. We conclude that, generally, gene set analysis methods with the integration of information from KEGG PATHWAY performs better both statistically and biologically.
doi:10.1186/1471-2105-12-81
PMCID: PMC3213687  PMID: 21418606
23.  A towards-multidimensional screening approach to predict candidate genes of rheumatoid arthritis based on SNP, structural and functional annotations 
BMC Medical Genomics  2010;3:38.
Background
According to the Genetic Analysis Workshops (GAW), hundreds of thousands of SNPs have been tested for association with rheumatoid arthritis. Traditional genome-wide association studies (GWAS) have been developed to identify susceptibility genes using a "most significant SNPs/genes" model. However, many minor- or modest-risk genes are likely to be missed after adjustment of multiple testing. This screening process uses a strict selection of statistical thresholds that aim to identify susceptibility genes based only on statistical model, without considering multi-dimensional biological similarities in sequence arrangement, crystal structure, or functional categories/biological pathways between candidate and known disease genes.
Methods
Multidimensional screening approaches combined with traditional statistical genetics methods can consider multiple biological backgrounds of genetic mutation, structural, and functional annotations. Here we introduce a newly developed multidimensional screening approach for rheumatoid arthritis candidate genes that considers all SNPs with nominal evidence of Bayesian association (BFLn > 0), and structural and functional similarities of corresponding genes or proteins.
Results
Our multidimensional screening approach extracted all risk genes (BFLn > 0) by odd ratios of hypothesis H1 to H0, and determined whether a particular group of genes shared underlying biological similarities with known disease genes. Using this method, we found 6614 risk SNPs in our Bayesian screen result set. Finally, we identified 146 likely causal genes for rheumatoid arthritis, including CD4, FGFR1, and KDR, which have been reported as high risk factors by recent studies. We must denote that 790 (96.1%) of genes identified by GWAS could not easily be classified into related functional categories or biological processes associated with the disease, while our candidate genes shared underlying biological similarities (e.g. were in the same pathway or GO term) and contributed to disease etiology, but where common variations in each of these genes make modest contributions to disease risk. We also found 6141 risk SNPs that were too minor to be detected by conventional approaches, and associations between 58 candidate genes and rheumatoid arthritis were verified by literature retrieved from the NCBI PubMed module.
Conclusions
Our proposed approach to the analysis of GAW16 data for rheumatoid arthritis was based on an underlying biological similarities-based method applied to candidate and known disease genes. Application of our method could identify likely causal candidate disease genes of rheumatoid arthritis, and could yield biological insights that not detected when focusing only on genes that give the strongest evidence by multiple testing. We hope that our proposed method complements the "most significant SNPs/genes" model, and provides additional insights into the pathogenesis of rheumatoid arthritis and other diseases, when searching datasets for hundreds of genetic variances.
doi:10.1186/1755-8794-3-38
PMCID: PMC2939610  PMID: 20727150
24.  Genetic Association of CHRNB3 and CHRNA6 Gene Polymorphisms with Nicotine Dependence Syndrome Scale in Korean Population 
Psychiatry Investigation  2014;11(3):307-312.
Objective
Cholinergic nicotinic receptor (CHRN) gene family has been known to mediate the highly additive effects of nicotine in the body, and implicated nicotine dependence (ND) and related phenotypes. Previous studies have found that CHRNA6-CHRNB3 cluster polymorphisms were significantly associated with the risk of ND and various tobacco behaviors. The aim of study was to evaluate the genetic association of CHRNB3 and CHRNA6 polymorphisms with the risk of ND based on the Fagerstrom Test for Nicotine Dependence (FTND) score and five subscales of nicotine dependence syndrome scale (NDSS) in Korean population.
Methods
Six SNPs in CHRNA6-CHRNB3 cluster were analyzed in 576 Korean subjects. Association analysis using logistic models and regression analysis with NDSS were performed.
Results
There was no association in the case-control analysis, whereas all six SNPs were significantly associated with drive factor among NDSS in subgroup based on the FTND score. CHRNB3 rs4954 and CHRNA6 rs16891604 showed significant associations with NDSSF1 (drive) in dominant models among moderate to severe ND among smokers after correction (pcorr=0.02 and 0.001, respectively), whereas other four SNPs showed significant associations among mild ND after correction (pcorr=0.03-0.02 in dominant model).
Conclusion
This study showed that the genetic influence of CHRNB3-CHRNA6 cluster polymorphisms are found in a ND endophenotype (drive) using NDSS subscales, rather than the risk of ND in Korean population. Our findings might be the first report for the association of CHRNB3-CHRNA6 cluster with ND-related phenotypes in Korean and might offer an approach to elucidating the molecular mechanisms of ND and ND-related phenotypes.
doi:10.4306/pi.2014.11.3.307
PMCID: PMC4124190  PMID: 25110504
CHRNB3; CHRNA6; SNP; Nicotine dependence; NDSS
25.  Pathway-Based Analysis Using Genome-wide Association Data from a Korean Non-Small Cell Lung Cancer Study 
PLoS ONE  2013;8(6):e65396.
Pathway-based analysis, used in conjunction with genome-wide association study (GWAS) techniques, is a powerful tool to detect subtle but systematic patterns in genome that can help elucidate complex diseases, like cancers. Here, we stepped back from genetic polymorphisms at a single locus and examined how multiple association signals can be orchestrated to find pathways related to lung cancer susceptibility. We used single-nucleotide polymorphism (SNP) array data from 869 non-small cell lung cancer (NSCLC) cases from a previous GWAS at the National Cancer Center and 1,533 controls from the Korean Association Resource project for the pathway-based analysis. After mapping single-nucleotide polymorphisms to genes, considering their coding region and regulatory elements (±20 kbp), multivariate logistic regression of additive and dominant genetic models were fitted against disease status, with adjustments for age, gender, and smoking status. Pathway statistics were evaluated using Gene Set Enrichment Analysis (GSEA) and Adaptive Rank Truncated Product (ARTP) methods. Among 880 pathways, 11 showed relatively significant statistics compared to our positive controls (PGSEA≤0.025, false discovery rate≤0.25). Candidate pathways were validated using the ARTP method and similarities between pathways were computed against each other. The top-ranked pathways were ABC Transporters (PGSEA<0.001, PARTP = 0.001), VEGF Signaling Pathway (PGSEA<0.001, PARTP = 0.008), G1/S Check Point (PGSEA = 0.004, PARTP = 0.013), and NRAGE Signals Death through JNK (PGSEA = 0.006, PARTP = 0.001). Our results demonstrate that pathway analysis can shed light on post-GWAS research and help identify potential targets for cancer susceptibility.
doi:10.1371/journal.pone.0065396
PMCID: PMC3675130  PMID: 23762359

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