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The majority of gene expression studies focus on the search for genes whose mean expression is different between two or more populations of samples in the so-called “differential expression analysis” approach. However, a difference in variance in gene expression may also be biologically and physiologically relevant. In the classical statistical model used to analyze RNA-sequencing (RNA-seq) data, the dispersion, which defines the variance, is only considered as a parameter to be estimated prior to identifying a difference in mean expression between conditions of interest. Here, we propose to evaluate four recently published methods, which detect differences in both the mean and dispersion in RNA-seq data. We thoroughly investigated the performance of these methods on simulated datasets and characterized parameter settings to reliably detect genes with a differential expression dispersion. We applied these methods to The Cancer Genome Atlas datasets. Interestingly, among the genes with an increased expression dispersion in tumors and without a change in mean expression, we identified some key cellular functions, most of which were related to catabolism and were overrepresented in most of the analyzed cancers. In particular, our results highlight autophagy, whose role in cancerogenesis is context-dependent, illustrating the potential of the differential dispersion approach to gain new insights into biological processes and to discover new biomarkers. Author summary Gene expression is the process by which genetic information is translated into functional molecules. Transcription is the first step of this process, consisting of synthesizing messenger RNAs. During recent decades, genome-wide transcriptional profiling technologies have made it possible to assess the expression levels of thousands of genes in parallel in a variety of biological contexts. In statistical analyses, the expression of a gene is estimated by counting sequencing reads over a set of samples and is defined by two dimensions: mean and variance. The overwhelming majority of gene expression studies focus on identifying genes whose mean expression significantly changes when comparing samples of different conditions of interest to gain knowledge of biological processes. In this classical approach, the variance is usually considered only as a noise parameter to be estimated before assessing the mean expression. However, finely estimating the variance of expression may be biologically relevant since a modification of this parameter may reflect a change in gene expression regulation. Here, we propose to evaluate the performance of statistical methods that identify such differentially variant genes. We highlighted the potential of this approach by analyzing cancer datasets, thus identifying key cellular functions in tumor progression.

Genome-wide association studies (GWAS) explore the genetic causes of complex diseases. However, classical approaches ignore the biological context of the genetic variants and genes under study. To address this shortcoming, one can use biological networks, which model functional relationships, to search for functionally related susceptibility loci. Many such network methods exist, each arising from different mathematical frameworks, pre-processing steps, and assumptions about the network properties of the susceptibility mechanism. Unsurprisingly, this results in disparate solutions. To explore how to exploit these heterogeneous approaches, we selected six network methods and applied them to GENESIS, a nationwide French study on familial breast cancer. First, we verified that network methods recovered more interpretable results than a standard GWAS. We addressed the heterogeneity of their solutions by studying their overlap, computing what we called the consensus. The key gene in this consensus solution was COPS5, a gene related to multiple cancer hallmarks. Another issue we observed was that network methods were unstable, selecting very different genes on different subsamples of GENESIS. Therefore, we proposed a stable consensus solution formed by the 68 genes most consistently selected across multiple subsamples. This solution was also enriched in genes known to be associated with breast cancer susceptibility (BLM, CASP8, CASP10, DNAJC1, FGFR2, MRPS30, and SLC4A7, P-value = 3 × 10-4). The most connected gene was CUL3, a regulator of several genes linked to cancer progression. Lastly, we evaluated the biases of each method and the impact of their parameters on the outcome. In general, network methods preferred highly connected genes, even after random rewirings that stripped the connections of any biological meaning. In conclusion, we present the advantages of network-guided GWAS, characterize their shortcomings, and provide strategies to address them. To compute the consensus networks, implementations of all six methods are available at https://github.com/hclimente/gwas-tools.