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Recent progress in human disease genetics is leading to rapid advances in understanding pathobiological mechanisms. However, the sheer number of risk-conveying genetic variants being identified demands in vivo model systems that are amenable to functional analyses at scale. Here we provide a practical guide for using the diploid frog species Xenopus tropicalis to study many genes and variants to uncover conserved mechanisms of pathobiology relevant to human disease. We discuss key considerations in modelling human genetic disorders: genetic architecture, conservation, phenotyping strategy and rigour, as well as more complex topics, such as penetrance, expressivity, sex differences and current challenges in the field. As the patient-driven gene discovery field expands significantly, the cost-effective, rapid and higher throughput nature of Xenopus make it an essential member of the model organism armamentarium for understanding gene function in development and in relation to disease.
Fig. 1. Relationship between rare and common variants with respect to allele frequency and effect size. Rare variants are typically detected in specific, clinically indicated patient trio (i.e. involving testing of the affected individual and both biological parents) studies of exome sequencing, and enriched for de novo variants with individually large effects. When perturbed, the genes that carry these variants often produce phenotypes in model organisms. By contrast, common variants are usually identified in much broader genome-wide association studies (GWAS) of large unrelated cohorts. Individually, these common variants carry very small risk and are unlikely to produce a measurable phenotype when recapitulated in model organisms.
Fig. 2. Approaches and tools that facilitate human genetic disease modelling in Xenopus. Once a gene of interest is selected, the locus conservation between X. tropicalis and humans can be explored in Xenbase, NCBI and gnomAD databases. The expression profile of the gene can be queried by using RNA in situ hybridization and on Xenbase. Then the protein can be located in cells and embryos through expression of a tagged human cDNA clone or validated antibody staining. Finally, the function of this gene can be inferred through loss-of-function (LoF) analysis, rescue experiments, overexpression and generating mutant lines with patient-derived variants. This kind of work can quickly point to mechanisms of disease, and tissues and structures for successful studies.
Fig. 3. Phenotyping opportunities in Xenopus. Studying how genetic perturbation disrupts organ function is essential to successful human disease modelling. Various methods are available in Xenopus and, due to the functional similarities, can be applied to characterising disorders within organs as diverse as lung, heart, brain, kidney and gut.
