NONPARAMETRIC ESTIMATION OF GENEWISE VARIANCE FOR MICROARRAY DATA

Estimation of genewise variance arises from two important applications in microarray data analysis: selecting significantly differentially expressed genes and validation tests for normalization of microarray data. We approach the problem by introducing a two-way nonparametric model, which is an extension of the famous Neyman-Scott model and is applicable beyond microarray data. The problem itself poses interesting challenges because the number of nuisance parameters is proportional to the sample size and it is not obvious how the variance function can be estimated when measurements are correlated. In such a high-dimensional nonparametric problem, we proposed two novel nonparametric estimators for genewise variance function and semiparametric estimators for measurement correlation, via solving a system of nonlinear equations. Their asymptotic normality is established. The finite sample property is demonstrated by simulation studies. The estimators also improve the power of the tests for detecting statistically differentially expressed genes. The methodology is illustrated by the data from MicroArray Quality Control (MAQC) project.     

Omnibus testing and gene filtration in microarray data analysis

When thousands of tests are performed simultaneously to detect differentially expressed genes in microarray analysis, the number of Type I errors can be immense if a multiplicity adjustment is not made. However, due to the large scale, traditional adjustment methods require very stringen significance levels for individual tests, which yield low power for detecting alterations. In this work, we describe how two omnibus tests can be used in conjunction with a gene filtration process to circumvent difficulties due to the large scale of testing. These two omnibus tests, the D-test and the modified likelihood ratio test (MLRT), can be used to investigate whether a collection of P-values has arisen from the Uniform(0,1) distribution or whether the Uniform(0,1) distribution contaminated by another Beta distribution is more appropriate. In the former case, attention can be directed to a smaller part of the genome; in the latter event, parameter estimates for the contamination model provide a frame of reference for multiple comparisons. Unlike the likelihood ratio test (LRT), both the D-test and MLRT enjoy simple limiting distributions under the null hypothesis of no contamination, so critical values can be obtained from standard tables. Simulation studies demonstrate that the D-test and MLRT are superior to the AIC, BIC, and Kolmogorov-Smirnov test. A case study illustrates omnibus testing and filtration.     

Hadamard matrix methods in identifying differentially expressed genes from microarray experiments

Identifying differentially expressed genes is a basic objective in microarray experiments. Many statistical methods for detecting differentially expressed genes in multiple-slide experiments have been proposed. However, sometimes with limited experimental resources, only a single cDNA array or two Oligonuleotide arrays could be made or only insufficient replicated arrays could be conducted. Many current statistical models cannot be used because of the non-availability of replicated data. Simply using fold changes is also unreliable and inefficient [Chen et al. 1997. Ratio-based decisions and the quantitative analysis of cDNA microarray images. J. Biomed. Optics 2, 364–374; Newton et al. 2001. On differential variability of expression ratios: improving statistical inference about gene expression changes from microarray data. J. Comput. Biol. 8, 37–52; Pan et al. 2002. How many replicates of arrays are required to detect gene expression changes in microarray experiments? a mixture model approach. Genome Biol. 3, research0022.1-0022.10]. We propose a new method. If the log-transformed ratios for the expressed genes as well as unexpressed genes have equal variance, we use a Hadamard matrix to construct a t-test from a single array data. Basically, we test whether each doubtful gene has significantly differential expression compared to the unexpressed genes. We form some new random variables corresponding to the rows of a Hadamard matrix using the algebraic sum of gene expressions. A one-sample t-test is constructed and the p-value is calculated for each doubtful gene based on these random variables. By using any method for multiple testing, adjusted p-values could be obtained from original p-values and significance of doubtful genes can be determined. When the variance of expressed genes differs from the variance of unexpressed genes, we construct a z-statistic based on the result from application of Hadamard matrix and find the confidence interval to retain the null hypothesis. Using the interval, we determine differentially expressed genes. This method is also useful for multiple microarrays, especially when sufficient replicated data are not available for a traditional t-test. We apply our methodology to ApoAI data. The results appear to be promising. They not only confirm the early known differentially expressed genes, but also indicate more genes to be differentially expressed.     

A modified two-sample t-test based on permutation method for large-scale data

In large-scale data, for example, analyzing microarray data, which includes hypothesis testing for equality of means in order to discover differentially expressed genes, often deals with a large number of features versus a few number of replicates. Furthermore, some genes are differentially expressed and some others not. Thus, a usual permutation method, which is applied facing these situations, estimates the p-value poorly. This is because two types of genes are mixed. To overcome this obstacle, the null permutation samples are suggested in the literatures. We propose a modified uniformly most powerful unbiased test for testing the null hypothesis.     

Comparisons of estimators of the number of true null hypotheses and adaptive FDR procedures in multiplicity testing

Many exploratory studies such as microarray experiments require the simultaneous comparison of hundreds or thousands of genes. It is common to see that most genes in many microarray experiments are not expected to be differentially expressed. Under such a setting, a procedure that is designed to control the false discovery rate (FDR) is aimed at identifying as many potential differentially expressed genes as possible. The usual FDR controlling procedure is constructed based on the number of hypotheses. However, it can become very conservative when some of the alternative hypotheses are expected to be true. The power of a controlling procedure can be improved if the number of true null hypotheses (m ₀) instead of the number of hypotheses is incorporated in the procedure [Y. Benjamini and Y. Hochberg, On the adaptive control of the false discovery rate in multiple testing with independent statistics, J. Edu. Behav. Statist. 25(2000), pp. 60–83]. Nevertheless, m ₀ is unknown, and has to be estimated. The objective of this article is to evaluate some existing estimators of m ₀ and discuss the feasibility of these estimators in incorporating into FDR controlling procedures under various experimental settings. The results of simulations can help the investigator to choose an appropriate procedure to meet the requirement of the study.     

Searching for differential expression: a non-parametric approach

Microarray experiments are being widely used in medical and biological research. The main features of these studies are the large number of variables (genes) involved and the low number of replicates (arrays). It seems clear that the most appropriate models, when looking for detecting differences in gene expression are those that exploit the most useful information to compensate for the lack of replicates. On the other hand, the control of the error in the decision process plays an important role for the high number of simultaneous statistical tests (one for each gene), so that concepts such as the false discovery rate (FDR) take a special importance. One of the alternatives for the analysis of the data in these experiments is based on the calculation of statistics derived from modifications of the classical methods used in this type of problems (moderated-t, B-statistic). Nonparametric techniques have been also proposed [B. Efron, R. Tibshirani, J.D. Storey, and V. Tusher, Empirical Bayes analysis of a microarray experiment, J. Amer. Stat. Assoc. 96 (2001), pp. 1151–1160; W. Pan, J. Lin, and C.T. Le, A mixture model approach to detecting differentially expressed genes with microarray data, Funct. Integr. Genomics 3 (2003), pp. 117–124], allowing the analysis without assuming any prior condition about the distribution of the data, which make them especially suitable in such situations. This paper presents a new method to detect differentially expressed genes based on non-parametric density estimation by a class of functions that allow us to define a distance between individuals in the sample (characterized by the coordinates of the individual (gene) in the dual space tangent to the manifold of parameters) [A. Miñarro and J.M. Oller, Some remarks on the individuals-score distance and its applications to statistical inference, Qüestiió, 16 (1992), pp. 43–57]. From these distances, we designed the test to determine the rejection region based on the control of FDR.     

A new class of mixture models for differential gene expression in DNA microarray data

One of the fundamental issues in analyzing microarray data is to determine which genes are expressed and which ones are not for a given group of subjects. In datasets where many genes are expressed and many are not expressed (i.e., underexpressed), a bimodal distribution for the gene expression levels often results, where one mode of the distribution represents the expressed genes and the other mode represents the underexpressed genes. To model this bimodality, we propose a new class of mixture models that utilize a random threshold value for accommodating bimodality in the gene expression distribution. Theoretical properties of the proposed model are carefully examined. We use this new model to examine the problem of differential gene expression between two groups of subjects, develop prior distributions, and derive a new criterion for determining which genes are differentially expressed between the two groups. Prior elicitation is carried out using empirical Bayes methodology in order to estimate the threshold value as well as elicit the hyperparameters for the two component mixture model. The new gene selection criterion is demonstrated via several simulations to have excellent false positive rate and false negative rate properties. A gastric cancer dataset is used to motivate and illustrate the proposed methodology.     

Randomization tests for small samples: an application for genetic expression data

Summary. An advantage of randomization tests for small samples is that an exact P -value can be computed under an additive model. A disadvantage with very small sample sizes is that the resulting discrete distribution for P -values can make it mathematically impossible for a P -value to attain a particular degree of significance. We investigate a distribution of P -values that arises when several thousand randomization tests are conducted simultaneously using small samples, a situation that arises with microarray gene expression data. We show that the distribution yields valuable information regarding groups of genes that are differentially expressed between two groups: a treatment group and a control group. This distribution helps to categorize genes with varying degrees of overlap of genetic expression values between the two groups, and it helps to quantify the degree of overlap by using the P -value from a randomization test. Moreover, a statistical test is available that compares the actual distribution of P -values with an expected distribution if there are no genes that are differentially expressed. We demonstrate the method and illustrate the results by using a microarray data set involving a cell line for rheumatoid arthritis. A small simulation study evaluates the effect that correlated gene expression levels could have on results from the analysis.     

Mixture model on the variance for the differential analysis of gene expression data

Summary.  In microarray experiments, accurate estimation of the gene variance is a key step in the identification of differentially expressed genes. Variance models go from the too stringent homoscedastic assumption to the overparameterized model assuming a specific variance for each gene. Between these two extremes there is some room for intermediate models. We propose a method that identifies clusters of genes with equal variance. We use a mixture model on the gene variance distribution. A test statistic for ranking and detecting differentially expressed genes is proposed. The method is illustrated with publicly available complementary deoxyribonucleic acid microarray experiments, an unpublished data set and further simulation studies.     

Identification of genomic markers correlated with sensitivity in solid tumors to Dasatinib using sparse principal components

Differential analysis techniques are commonly used to offer scientists a dimension reduction procedure and an interpretable gateway to variable selection, especially when confronting high-dimensional genomic data. Huang et al. used a gene expression profile of breast cancer cell lines to identify genomic markers which are highly correlated with in vitro sensitivity of a drug Dasatinib. They considered three statistical methods to identify differentially expressed genes and finally used the results from the intersection. But the statistical methods that are used in the paper are not sufficient to select the genomic markers. In this paper we used three alternative statistical methods to select a combined list of genomic markers and compared the genes that were proposed by Huang et al. We then proposed to use sparse principal component analysis (Sparse PCA) to identify a final list of genomic markers. The Sparse PCA incorporates correlation into account among the genes and helps to draw a successful genomic markers discovery. We present a new and a small set of genomic markers to separate out the groups of patients effectively who are sensitive to the drug Dasatinib. The analysis procedure will also encourage scientists in identifying genomic markers that can help to separate out two groups.     

Spatial model fitting for large datasets with applications to climate and microarray problems

Many problems in the environmental and biological sciences involve the analysis of large quantities of data. Further, the data in these problems are often subject to various types of structure and, in particular, spatial dependence. Traditional model fitting often fails due to the size of the datasets since it is difficult to not only specify but also to compute with the full covariance matrix describing the spatial dependence. We propose a very general type of mixed model that has a random spatial component. Recognizing that spatial covariance matrices often exhibit a large number of zero or near-zero entries, covariance tapering is used to force near-zero entries to zero. Then, taking advantage of the sparse nature of such tapered covariance matrices, backfitting is used to estimate the fixed and random model parameters. The novelty of the paper is the combination of the two techniques, tapering and backfitting, to model and analyze spatial datasets several orders of magnitude larger than those datasets typically analyzed with conventional approaches. Results will be demonstrated with two datasets. The first consists of regional climate model output that is based on an experiment with two regional and two driver models arranged in a two-by-two layout. The second is microarray data used to build a profile of differentially expressed genes relating to cerebral vascular malformations, an important cause of hemorrhagic stroke and seizures.     

A Bayesian False Discovery Rate for Multiple Testing

Case-control studies of genetic polymorphisms and gene-environment interactions are reporting large numbers of statistically significant associations, many of which are likely to be spurious. This problem reflects the low prior probability that any one null hypothesis is false, and the large number of test results reported for a given study. In a Bayesian approach to the low prior probabilities, Wacholder et al. (2004) suggest supplementing the p-value for a hypothesis with its posterior probability given the study data. In a frequentist approach to the test multiplicity problem, Benjamini & Hochberg (1995) propose a hypothesis-rejection rule that provides greater statistical power by controlling the false discovery rate rather than the family-wise error rate controlled by the Bonferroni correction. This paper defines a Bayes false discovery rate and proposes a Bayes-based rejection rule for controlling it. The method, which combines the Bayesian approach of Wacholder et al. with the frequentist approach of Benjamini & Hochberg, is used to evaluate the associations reported in a case-control study of breast cancer risk and genetic polymorphisms of genes involved in the repair of double-strand DNA breaks.     

Evaluation of false discovery rate and power via sample size in microarray studies

Microarray studies are now common for human, agricultural plant and animal studies. False discovery rate (FDR) is widely used in the analysis of large-scale microarray data to account for problems associated with multiple testing. A well-designed microarray study should have adequate statistical power to detect the differentially expressed (DE) genes, while keeping the FDR acceptably low. In this paper, we used a mixture model of expression responses involving DE genes and non-DE genes to analyse theoretical FDR and power for simple scenarios where it is assumed that each gene has equal error variance and the gene effects are independent. A simulation study was used to evaluate the empirical FDR and power for more complex scenarios with unequal error variance and gene dependence. Based on this approach, we present a general guide for sample size requirement at the experimental design stage for prospective microarray studies. This paper presented an approach to explicitly connect the sample size with FDR and power. While the methods have been developed in the context of one-sample microarray studies, they are readily applicable to two-sample, and could be adapted to multiple-sample studies.     

Bayesian curve fitting and clustering with Dirichlet process mixture models for microarray data

In the field of molecular biology, it is often of interest to analyze microarray data for clustering genes based on similar profiles of gene expression to identify genes that are differentially expressed under multiple biological conditions. One of the notable characteristics of a gene expression profile is that it shows a cyclic curve over a course of time. To group sequences of similar molecular functions, we propose a Bayesian Dirichlet process mixture of linear regression models with a Fourier series for the regression coefficients, for each of which a spike and slab prior is assumed. A full Gibbs-sampling algorithm is developed for an efficient Markov chain Monte Carlo (MCMC) posterior computation. Due to the so-called “label-switching” problem and different numbers of clusters during the MCMC computation, a post-process approach of Fritsch and Ickstadt (2009) is additionally applied to MCMC samples for an optimal single clustering estimate by maximizing the posterior expected adjusted Rand index with the posterior probabilities of two observations being clustered together. The proposed method is illustrated with two simulated data and one real data of the physiological response of fibroblasts to serum of Iyer et al. (1999).     

A Bayesian mixture model for differential gene expression

Summary.  We propose model-based inference for differential gene expression, using a nonparametric Bayesian probability model for the distribution of gene intensities under various conditions. The probability model is a mixture of normal distributions. The resulting inference is similar to a popular empirical Bayes approach that is used for the same inference problem. The use of fully model-based inference mitigates some of the necessary limitations of the empirical Bayes method. We argue that inference is no more difficult than posterior simulation in traditional nonparametric mixture-of-normal models. The approach proposed is motivated by a microarray experiment that was carried out to identify genes that are differentially expressed between normal tissue and colon cancer tissue samples. Additionally, we carried out a small simulation study to verify the methods proposed. In the motivating case-studies we show how the nonparametric Bayes approach facilitates the evaluation of posterior expected false discovery rates. We also show how inference can proceed even in the absence of a null sample of known non-differentially expressed scores. This highlights the difference from alternative empirical Bayes approaches that are based on plug-in estimates.     

Simultaneous rank tests for detecting differentially expressed genes

Rank tests are known to be robust to outliers and violation of distributional assumptions. Two major issues besetting microarray data are violation of the normality assumption and contamination by outliers. In this article, we formulate the normal theory simultaneous tests and their aligned rank transformation (ART) analog for detecting differentially expressed genes. These tests are based on the least-squares estimates of the effects when data follow a linear model. Application of the two methods are then demonstrated on a real data set. To evaluate the performance of the aligned rank transform method with the corresponding normal theory method, data were simulated according to the characteristics of a real gene expression data. These simulated data are then used to compare the two methods with respect to their sensitivity to the distributional assumption and to outliers for controlling the family-wise Type I error rate, power, and false discovery rate. It is demonstrated that the ART generally possesses the robustness of validity property even for microarray data with small number of replications. Although these methods can be applied to more general designs, in this article the simulation study is carried out for a dye-swap design since this design is broadly used in cDNA microarray experiments.     

A guide to the design and analysis of small clinical studies

Clinical studies, which have a small number of patients, are conducted by pharmaceutical companies and research institutions. Examples of constraints that lead to a small clinical study include a single investigative site with a highly specialized expertise or equipment, rare diseases, and limited time and budget. We consider the following topics, which we believe will be helpful for the investigator and statistician working together on the design and analysis of small clinical studies: definitions of various types of small studies (exploratory, pilot, proof of concept); bias and ways to mitigate the bias; commonly used study designs for randomized and nonrandomized studies, and some less commonly used designs; potential ethical issues associated with small underpowered clinical studies; sample size for small studies; statistical analysis methods for different types of variables and multiplicity issues. We conclude the paper with recommendations made by an Institute of Medicine committee, which was asked to assess the current methodologies and appropriate situations for conducting small clinical studies.     

LM Test of Neglected Correlated Random Effects and Its Application

This article aims at achieving two distinct goals. The first is to extend the existing LM test of overdispersion to the situation where the alternative hypothesis is characterized by the correlated random effects model. We obtain a result that the test against the random effects model has a certain max-min type optimality property. We will call such a test the LM test of overdispersion. The second goal of the article is to draw a connection between panel data analysis and the analysis of multiplicity of equilibrium in games. Because such multiplicity can be viewed as a particular form of neglected heterogeneity, we propose an intuitive specification test for a class of two-step game estimators.     

A selection procedure for estimating the number of signal components

This paper proposes a selection procedure to estimate the multiplicity of the smallest eigenvalue of the covariance matrix. The unknown number of signals present in a radar data can be formulated as the difference between the total number of components in the observed multivariate data vector and the multiplicity of the smallest eigenvalue. In the observed multivariate data, the smallest eigenvalues of the sample covariance matrix may in fact be grouped about some nominal value, as opposed to being identically equal. We propose a selection procedure to estimate the multiplicity of the common smallest eigenvalue, which is significantly smaller than the other eigenvalues. We derive the probability of a correct selection, P(CS), and the least favorable configuration (LFC) for our procedures. Under the LFC, the P(CS) attains its minimum over the preference zone of all eigenvalues. Therefore, a minimum sample size can be determined from the P(CS) under the LFC, P(CS|LFC), in order to implement our new procedure with a guaranteed probability requirement. Numerical examples are presented in order to illustrate our proposed procedure.     

The Use of Multiplicity in a Household Survey of Disabilities

The multiplicity technique has been proposed (Sirken, 1970) as a means of improving estimates of the number of rare “events” in a population. This paper describes one use of this technique, namely estimation of the number of persons with certain types of disability living in private dwellings in Canberra. The results are taken from three household surveys conducted in Canberra during 1978–79 in which a multiplicity rule linking parents, siblings and children of residents of sample households who were also living in private dwellings in Canberra was used. While no direct evidence on the level of non-sampling error is available here, it appears that net response errors at least are not substantial using this multiplicity rule and that a reasonable gain in sampling efficiency results from the use of multiplicity.