Abstract
Many antibacterial drugs have multiple mechanisms of resistance, which are often represented simultaneously by a mixture of resistance mutations (some more frequent than others) in a clinical population. This presents a challenge for Genome-Wide Association Studies (GWAS) methods, making it difficult to detect less prevalent resistance mechanisms purely through (weak) statistical associations. Homoplasy, or the occurrence of multiple independent mutations at the same site, is often observed with drug resistance mutations and can be a strong indicator of positive selection. However, traditional GWAS methods, such as those based on allele counting or linear regression, are not designed to take homoplasy into account. In this article, we present a new method, called ECAT (for Evolutionary Cluster-based Association Test), that extends traditional regression-based GWAS methods with the ability to take advantage of homoplasy. This is achieved through a preprocessing step which identifies hypervariable regions in the genome exhibiting statistically significant clusters of distinct evolutionary changes, to which association testing by a linear mixed model (LMM) is applied using GEMMA (a well-established LMM-based GWAS tool). Thus, the approach can be viewed as extending GEMMA from the usual site- or gene-level analysis to focusing on clustered regions of mutations. This approach was evaluated on a large collection of more than 600 clinical isolates of multidrug-resistant (MDR) Mycobacterium tuberculosis from Lima, Peru. We show that ECAT does a better job of detecting known resistance mutations for several antitubercular drugs (including less prevalent mutations with weaker associations), compared with (site- or gene-based) GEMMA, as representative of existing GWAS methods. The power of the multiphase approach in ECAT comes from focusing association testing on the hypervariable regions of the genome, which reduces complexity in the model and increases statistical power.