A Population Proportion approach for ranking differentially expressed genes
© Gadgil; licensee BioMed Central Ltd. 2008
Received: 12 June 2008
Accepted: 18 September 2008
Published: 18 September 2008
DNA microarrays are used to investigate differences in gene expression between two or more classes of samples. Most currently used approaches compare mean expression levels between classes and are not geared to find genes whose expression is significantly different in only a subset of samples in a class. However, biological variability can lead to situations where key genes are differentially expressed in only a subset of samples. To facilitate the identification of such genes, a new method is reported.
The key difference between the Population Proportion Ranking Method (PPRM) presented here and almost all other methods currently used is in the quantification of variability. PPRM quantifies variability in terms of inter-sample ratios and can be used to calculate the relative merit of differentially expressed genes with a specified difference in expression level between at least some samples in the two classes, which at the same time have lower than a specified variability within each class.
PPRM is tested on simulated data and on three publicly available cancer data sets. It is compared to the t test, PPST, COPA, OS, ORT and MOST using the simulated data. Under the conditions tested, it performs as well or better than the other methods tested under low intra-class variability and better than t test, PPST, COPA and OS when a gene is differentially expressed in only a subset of samples. It performs better than ORT and MOST in recognizing non differentially expressed genes with high variability in expression levels across all samples. For biological data, the success of predictor genes identified in appropriately classifying an independent sample is reported.
DNA microarrays are used to monitor the expression level of thousands of genes simultaneously, and are extensively used in various areas of biological research [1–4]. The reader is referred to Schena  and Bowtell and Sambrook  for a detailed introduction to microarray technology. A biological problem which is being increasingly addressed through the use of microarray assays is the identification of differences in gene expression between two or more classes of samples e.g. between disease and normal tissue [7–18]. The methods for identifying differentially expressed genes vary greatly [19–27], but all have a goal of identifying genes with a significant difference in expression level between samples in the two classes. A simple method to analyze such data is to compare the sample means of the expression level of each gene in the two classes to obtain a 'fold-change'  in the expression level of the gene between the two classes. However, fold change calculations fail to account for variability in expression levels between samples within a class. As aptly pointed out by Simon et al , "some twofold average effects represent statistically significant differences and some do not". Statistical methods like t-test [30, 31] and ANOVA [32–34] are used to assess the significance of differential expression by incorporating data on variability between samples. Many alternative approaches of incorporating data on variability have also been developed [19–21, 26, 27, 35].
Unlike the case of replicate in vitro data which are expected to have extremely low intra-class variability under ideal conditions, the expression level of a gene can vary significantly within samples obtained from different individuals in one class due to biological variation . Also, clinically similar phenotypes can be caused by different molecular mechanisms . Genes which are differentially expressed in only a subset of samples in a class can be important in such cases [38–40]. Most analysis methods compare the means of intra-class expression levels and are not likely to find genes whose expression is significantly different in only a subset of samples in a class, or have high intra-class variability.
A few approaches have been previously proposed to identify such genes [38, 39, 41–43]. One approach to identify such genes proposed by Lyons-Weiler et al , is the Permutation Percentile Separability Test (PPST). This test identifies genes for which a statistically significant number of samples in group A exhibit expression intensities beyond a particular percentile of the observed expression intensities of that gene in group B. Another approach is proposed by Bijlani et al  who compare the expression level of a gene in every sample in one class to the mean of the expression level in the other class. The proposed application of this method is to select genes which can be used for class distinction. Tomlins et al , Tibshirani et al , Wu et al  and Lian et al  use variations of transformation of gene expression values using the sample median and median absolute deviation in the Cancer Outlier Profile Analysis (COPA), Outlier sums (OS), Outlier Robust t-statistics (ORT) and Maximum Ordered Subset t-statistics (MOST) methods respectively. The performance of COPA and OS has been shown to deteriorate as the number of outliers increase .
All the methods listed above except PPST use some normalized form of the algebraic difference between expression levels as a measure of heterogeneity to identify 'outliers'. These methods might not be suitable for cases where a subset of samples in a class are responsible for significantly increasing the variability in the class, and are spread over a large range. Consider the following hypothetical example; a group of 10 samples have expression levels of a gene as [50, 50, 75, 80, 100, 120, 120, 300, 500, and 700]. Defining an outlier as a value more than the interquartile range above the third quartile, as used by some researchers , only one sample (700) is identified as an outlier. However a closer look at the data indicates that the last three samples are responsible for the increased variability in the class. This motivated the need to explore alternative ways to quantify variability.
Intra-class variability for a class is similarly quantified by calculating the ratios of expression level of a sample in the class to its expression level in every other sample in the same class (referred to henceforth as intra-class ratios). Analogous to the inter-class ratio cutoff, an intra-class ratio cutoff is chosen based on acceptable level of variability within a class. The fraction of intra-class ratios calculated above which are greater than the cutoff is calculated (fTT & fNN). Genes in which these fractions are significantly smaller than fTN are ranked based on an established statistical method of comparing population proportions .
Simulated data sets where the truly differentially expressed genes are known are used to test the ability of PPRM to identify differentially expressed genes. The performance of PPRM is compared to the t test, PPST, COPA , OS , ORT  and MOST  for the simulated data, and is found to be comparable or better under the conditions tested. Thus, PPRM could be a valuable addition to the repertoire of existing methods for detecting genes differentially expressed in a subset of samples in a class. However, simulated data sets do not necessarily mimic the variability in real biological data sets. Hence, this method is also applied to three publicly available cancer data sets to identify differentially expressed genes.
Since there is no gold standard of true differentially expressed genes in an experimental study, an approach of using differentially expressed genes identified by the method as predictors to test their ability to successfully classify independent sample(s) is used for validation of the method in real-world data. This approach was also used by Jeffery et al for evaluation of lists of differentially expressed genes identified . The method proposed in this paper is tested on 3 publicly available cancer data sets: leukemia , colon cancer  and prostate cancer . In case of the leukemia data set, an independent sample set is available to test whether the top differentially expressed genes identified can correctly classify independent samples. For the other two data sets, leave-one-out cross-validation (LOOCV) is implemented to test the accuracy of classification.
The particular method of choice for identifying differentially expressed genes depends on the biological question, and PPRM provides an additional tool to rank genes complying with a given set of constraints.
In this section, the Population Proportion Ranking Method (PPRM) is described, followed by a discussion on the assumptions used in PPRM and results of testing of this method on simulated and experimental data.
Population Proportion Ranking Method
Let the number of samples in class T (for 'Treated') be mT and the number of samples in class N (for Normal) be mN. Ti, for i = 1 to mT, are the expression levels of a gene in the mT samples of class T and Nj, for j = 1 to mN, are the expression levels of the gene in the mN samples of class N.
A ratio-cutoff is chosen based on biological knowledge of the magnitude of difference in expression level required between groups (CTN) and amount of variability that is acceptable within groups (CTT and CNN). For example, an inter-class ratio cutoff of 3 implies that there should be at least a 3 fold difference in expression between a sample in class T and another sample in class N for the gene to be identified as differentially expressed for that pair of samples and an intra-class ratio cutoff of 1.5 means that the maximum acceptable difference in expression between any two samples in a class is 1.5 fold. Increasing CTN will lead to identification of genes which have a larger magnitude of difference between the two classes, while changing intra-class ratios (CTT and CNN) allows the user to change the magnitude of variability acceptable within a given class. Naturally, since increasing CTN or decreasing CTT or CNN leads to a decrease in the number of genes identified as differentially expressed, these parameters can be used to identify a tractable number of differentially expressed genes of a certain nature, for further analysis.
To identify differentially expressed genes, the fraction of the inter-class ratios which are either greater than the ratio-cutoff CTN or smaller than 1/CTN is calculated as fTN. Similarly the fraction of intra-class ratios and which are either greater than the ratio-cutoff CTT and CNN respectively or smaller than 1/CTT and 1/CNN respectively are calculated as fTT and fNN.
mT is the number of samples in class T
mN is the number of samples in class N
NTN is the number of ratios which are greater than the ratio-cutoff CTN or smaller than 1/CTN
NTT is the number of ratios which are greater than the ratio-cutoff CTT or smaller than 1/CTT
NNN is the number of ratios which are greater than the ratio-cutoff CNN or smaller than 1/CNN
Parameters used in the Population Proportion Ranking Method
Ratio cutoff for inter-class ratios
Chosen based on the required magnitude of difference in expression between the two classes
Ratio cutoff for intra-class ratios
Chosen based on allowable heterogeneity in expression within a class
Significance value cutoff for significance of difference between the proportions of inter-class and intra-class populations greater than respective ratio cutoffs
Chosen based on required stringency in difference between the proportions of inter-class and intra-class populations greater than respective ratio cutoffs
The test makes an assumption of 1) Random and independent selection of inter-class and intra-class ratios and 2) Large sample size of the inter-class ratios and inter-class ratios, so the sampling distributions of differences of proportions are very closely normally distributed. Though the samples within each class are reasonably expected to be selected randomly and independently, all inter- and intra- group ratios are not independent. Specifically, there are only (mT + mN -1) independent inter-class ratios and (mT -1) or (mN -1) independent intra-class ratios. Hence the effective sample size is smaller leading to smaller reported significance values. However, in order to capture the true variability between all samples in a group or between groups, it is essential to use all inter-class and intra-class ratios. Hence the reported significance values are not exact and should only be used to calculate the relative merit of genes, and not the actual distance between them.
PPRM is tested on 5 sets of simulated data representing various intra and inter-class variability situations and compared to the t test, PPST, COPA, OS, ORT and MOST. PPST is implemented through the online implementation provided by Lyons-Weiler et al.  available at http://bioinformatics.upmc.edu/GE2/GEDA.html. COPA, OS, ORT and MOST were implemented using the R code by Lian  available at http://www.ntu.edu.sg/home/henglian/most.htm. PPRM is also tested on three publicly available cancer datas and used to identify predictor genes that can be used for classification. The classification accuracy using predictors identified by the PPRM is comparable to other reported classification accuracies.
Parameters used to generate simulated data for the 5 cases tested
Number of Samples
Number of Samples
True positives = Number of truly differentially expressed genes identified
False positives = Number of genes identified which are not differentially expressed
False negatives = Number of truly differentially expressed genes not identified
True negatives = Number of genes which are not differentially expressed, which are correctly not identified
FPR = False positive rate
Inter-class and intra-class ratio cutoffs used in the analysis of simulated data using PPRM
Case 1 is an example of a case of differential expression, with low variability within samples. As seen in Figure 2B, all intra-class ratios have small values while the inter-class ratios are higher. PPRM, t test, ORT and MOST identify most differentially expressed genes, with PPRM having the lowest FPR.
Case 2 is an example of genes which do not have a significant difference in expression level in the two classes and have larger variability as compared to case 1. Here again, PPRM has the lowest FPR among all methods tested.
Case 3 is an example of genes which have a low variability in one class, but very high variability in the other due to a subset of samples. Here, the intra-class ratios for class T are small, while those for class N are high (Figure 2B). In this case, COPA, OS, ORT and MOST have a 100% Recall. PPRM does not identify any differentially expressed gene when heterogeneity in both classes is controlled (i.e. both conditions pTT < pcutoff and pNN < pcutoff used; data not shown). However, if heterogeneity in class T is allowed by only using the condition pNN < pcutoff, PPRM has a 96% Recall and 1% FPR, which is similar to the other methods. This is an example of the application of PPRM allowing the control of heterogeneity in any one class only.
Case 4 is an example of genes which have moderate variability in one class and high variability in the other. This is different from case 3 in having the magnitude of expression level between the two classes lower (average 2-fold) than that in case 3 (average 3-fold). Again, the t test, PPST and OS have a poor Recall. ORT and MOST have a Recall of 99% and 94% with a FPR of 1%. PPRM does not identify any differentially expressed gene when heterogeneity in both classes is controlled (data not shown), but when heterogeneity in class T is allowed (pNN < pcutoff is the only condition used), a 98% Recall is obtained, but at the cost of 6% FPR. There is thus a trade-off between identifying all truly differentially expressed genes and obtaining false positives. Increasing the stringency of the parameters (e.g. increase in CTN, decrease in pcutoff) can reduce FPR at the expense of Recall (data not shown).
Case 5 is an example of a gene with high variability in both classes, which should ideally not be identified as differentially expressed. Here, there does not appear to be a significant difference in the distribution of inter-class and intra-class ratios, as seen in Figure 2B. PPRM has FPR of 0.02% which is the lowest, followed by the t test and PPST at 1%. COPA, OS, ORT and MOST have a FPR of 11%. (Note: Not accounting for variability in class N by PPRM has a FPR of 7%. This FPR decreases as the values of CTN and CTT are increased)
In summary, in cases where the heterogeneity in the sample population is low as exemplified by Case 1, all tests except COPA and OS perform reasonably well in identifying true positives. The t test, PPST, COPA and OS fail to identify differentially expressed genes in most cases, whereas PPRM, ORT and MOST can identify most differentially expressed genes in all cases. However, though ORT and MOST give lower FPR for case 4, they give higher FPRs than PPRM in Case 2 and 5 representing non differentially expressed genes.
In the case of simulated data, the inter-class and intra-class ratios were chosen based on knowledge of expression levels of truly differentially expressed genes, which will clearly not be the case in real world data. However, for real-world data, these parameters will be chosen based on the requirement of specific types of genes. More than one set of parameters can be used for an analysis to obtain different groups of differentially expressed genes. For example, using low intra-class cutoffs allows the identification of differentially expressed genes with low intra-class variability whereas using a higher value of one intra-class cutoff (CTT or CNN) also identifies genes with higher heterogeneity in that group (T or N, respectively).
Variability in simulated data cannot mimic the heterogeneity in real biological data, and hence PPRM is also tested on the following three publicly available experimental data sets. Since there is no gold standard of a list of differentially expressed genes in real world data, simply identifying differentially expressed genes in a data set is not adequate to test the method. Though the distinguishing feature of PPRM lies in its ability to identify differentially expressed genes with greater variability between samples in a class, the method is also able to identify differentially expressed with low variability within groups based on the choice of parameters used for the test. Hence, in analyzing real biological data, an approach of identifying a relatively small number of 'predictor' genes is adopted and their accuracy in being able to predict the class of an unknown sample is tested. This approach of validation of new methods of identification of differentially expressed genes has also been used by other researchers . The classification accuracy is expected to be similar to other reported values, but not necessarily better since the primary goal of this report is not to identify genes for classification.
In order to identify biomarkers, stringent conditions are used (i.e. higher values of inter-class ratio cut-off, lower value of intra-class ratio cutoff and lower values of cutoff of the p-value) to select a small number of genes with low heterogeneity in expression within a class. For the biological data sets used below, misclassification rates reported using some other methods are included for the sake of general comparison. For the leukemia data set, the independent data set available is used to test the prediction power of selected genes. For all other data sets, a LOOCV technique is used. To avoid bias in gene selection from the sample which is left out, the list of differentially expressed genes is calculated separately every time with the same parameters, and this list is used to predict the class of the sample that is left out. Classification is performed using Discriminant Analysis in Matlab (The Mathworks, Inc., Natick, MA, USA).
Gene expression profiles of two types of leukemia samples were derived from 47 patients with acute lymphoblastic leukemia (ALL) and 25 patients with acute myeloblastic leukemia by Golub et al . Data is obtained from the Broad Institute website at http://www.broad.mit.edu/cgi-bin/cancer/datasets.cgi.
Parameters used for the analysis of the three cancer data sets
Prostate cancer data
The prostate cancer data set generated by Singh et al  consists of 92 samples, 45 of which were non-tumor prostate samples and 47 of which were prostate tumor. The data set is publicly available and is obtained from the Broad Institute website http://www.broad.mit.edu/cgi-bin/cancer/datasets.cgi. Genes for which less than 20 samples had a "Present" call were not used in the analysis. A LOOCV technique is used for this data set. In the original paper, a 10% error rate in sample classification using LOOCV is obtained, while Dettling et al  reported misclassification rates between 5%–14% using supervised clustering. In this study, using the parameters listed in Table 4, an 8% error rate in sample classification using LOOCV is obtained. The number of biomarker genes identified in all LOOCV runs is between 9 and 18.
Colon cancer data
The colon cancer data set generated by Alon et al. consists of 62 samples, 40 tumor samples and 22 normal controls. The gene expression data were downloaded from http://microarray.princeton.edu/oncology/affydata/index.html. LOOCV is also used for this data set. Other researchers have obtained misclassification rates (including unclassified samples) between 8% to 34% [50–54] using various methods like nearest neighbor classifiers, SVM, boosting, 'Minimum Redundancy- Maximum Relevancy', Bayes error filter for gene selection and supervised clustering.
In this study, using the parameters listed in Table 4, a 16% error rate in sample classification using the LOOCV is obtained. The number of biomarker genes identified in all LOOCV validation runs is between 7 and 13, with one exception where 23 genes were identified.
DNA microarray analysis is being increasingly used to identify differences between two or more classes like diseased and healthy tissue. Most methods used for the identification of differentially expressed genes between two classes identify genes where the variability between samples in a class is low. However there can be significant variability among samples in a class due to differences between individual subjects and their environment . PPRM uses inter-sample ratios to quantify variability in expression. This method allows for the identification of genes where the user can define the allowable heterogeneity within one or both classes and required difference in expression between samples in the two classes. Since all inter-class and intra-class ratios used in this method are not independent, the significance values calculated by PPRM are not exact and should be used only for ranking and prioritizing genes. The mean and standard deviation of the test statistic are reported for the simulated data sets to facilitate the assessment of the impact of violation of the assumptions for the sample size of 10 samples in each class (i.e. 100 inter-class ratios and 45 intra-class ratios for each class, out of which 19 and 9 respectively are independent).
PPRM works as well or better than all other methods tested in data sets where the heterogeneity in samples is low. In simulated cases tested where variability is high, ORT, MOST and PPRM successfully identify most differentially expressed genes. In addition to a high Recall, it is necessary for any method to minimize the number of false positives identified. Genes with high variability in expression levels among samples in both classes should not be identified as differentially expressed simply because the expression level in some samples in one class is different than the expression level of some samples in the other class. This is tested in case 2 and 5 in the simulated data, where reassuringly a very low FPR of 0.2 and 0.02% is obtained using PPRM. However, for these cases ORT and MOST consistently resulted in higher values of the test statistic for the 1000 non differentially expressed genes resulting in high FPRs. This is likely due to the lack of an additional constraint of relative difference in these methods as available in PPRM.
PPRM is also able to identify differentially expressed with low variability within groups, based on the choice of parameters used for the test. Hence, it is possible to test it on publicly available cancer data sets by assessing the success of the genes identified in correctly classifying samples in the two groups. The classification accuracies obtained for the three publicly available cancer data sets used for testing are similar to those reported using other methods.
The Population Proportion Ranking Method (PPRM) presented here quantifies variability in terms of inter-sample ratios and allows for the identification of genes where the user can define the allowable heterogeneity within one or both classes and required difference in expression between samples in the two classes for ranking differentially expressed genes.
The motivation for this problem was obtained while MG was a post doctoral research associate in Wei-Shou Hu's laboratory in the Chemical Engineering and Materials Science Department at the University of Minnesota. This research was supported by a start up grant MLP011026 from the National Chemical Laboratory.
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