Can precise re-creations of disease gene variants be made in Xenopus that are useful to inform clinical interventions?

The ability to sequence DNA at high throughput has completely transformed our ability to diagnose genetic diseases. By comparing the DNA sequence of an affected child with those of its parents and siblings it is possible to identify some genes in the affected child whose sequences vary from those of their family; these are called variants and one or more of them may be the cause of the disease. Once one of these variants has been identified as causing the disease, this knowledge guides the clinical interventions for the child and advice to the family on the health of the family's other existing or future children, also facilitating cascade screening and prenatal diagnosis. In around half of cases however it is not possible to identify the causative gene from the set of variants found in a patient with sufficient certainty to make clinical decisions.

Experiments in cell or "organoid" cultures (complex mixtures of cells that replicate some of the cell-cell interactions present in an organ) can inform clinicians about which of the variants is causative, but in other cases it is still necessary to test the effect of the gene variants in animals. This was only possible in mice, experiments that are both ethically and financially expensive. Several labs, including ours, have recently shown that experiments in tadpoles can also provide very strong evidence about the function of variant human genes. This programme, which is joint between frog geneticists, medical genomic research scientists and clinical geneticists, is to discover how useful the information gained from tadpoles containing a patient's gene variant can be to the clinicians caring for them.

Quite how useful frogs have been as models for human biology often surprises, but much of what we know about how embryos develop and the biology that underpins our understanding of many diseases was often first discovered in the frog. In terms of gene similarity, almost 80% of the genes known to be altered in human disease are found in the frog and most genes are found in the same order on the chromosomes of frogs and humans. For 80% of the gene variants we have analysed so far, the regions affected in disease are extremely similar in frogs and humans, most likely because these parts of the gene product are critically important for the health of the organism, and evolution has therefore kept them the same.

Already it is very straightforward to re-create some types of gene variants in tadpoles; normally changes are made in less than 3 days. The alterations that these subsequently make to the tadpoles' development can be assayed a few days later. These assays are made easier by automated computerised tomography (CT) scans of the tadpoles and the ability to make the gene variants in a large bank of "transgenic" animals in which specific cell types fluoresce; this makes it easy to see alterations. Many of the gene variants found in patients are more subtle than those tested so far, and more recent techniques need to be used to make them. These methods change a single letter in the DNA or a very specific group of letters, in either one or both copies of a gene that exist in each cell. The technique to do this quickly and with high efficiency currently works best in frogs.

Using these experimental approaches, we will re-create rare genetic disease patients' variants in tadpoles and test the effects that each has on their development. If the tadpole shows the human disease characteristics, for example our trials revealed cataracts in tadpoles with a gene variant causing childhood cataracts, this supports the gene being causal, so allowing the clinical team to care for the patient and their family. If the clinical researchers find that this information is sufficiently useful, then we will continue to work together to scale up the pipeline of gene function analysis in the frog so it can be used to direct effective interventions for a significant number of patients.

Next generation DNA sequencing (NGS) has revolutionised our understanding and mitigation of human genetic diseases by enabling clinical research to identify causal variants within a gene. NGS has produced many successes in the unambiguous identification of disease-causing gene variants that have directed effective interventions for patients and their families. A challenge in making use of this sequence data is however interpretation of variants of uncertain significance (VUS). For around half of patients, the link between the gene(s) identified by sequence comparisons and their disease is insufficiently robust for clinical decision making.

Assessing VUS function needs rapid ways to test variant pathogenicity. While non-animal studies can be informative, they do not reflect the complex interactions in organisms. The main approach uses mouse models but, due to the financial and ethical costs involved, alternatives are sought. Highly efficient gene editing is routine in Xenopus frogs and analysis of resulting phenotypic changes is very efficient; tadpoles are transparent, fluorescently-labelled transgenic lines are available and there is robust microCT analysis. Collaborating between clinical and research staff, we will re-create prioritsed VUS from patients. As proof of principle we made and analysed 10 disease-related gene changes in X. tropicalis, producing phenotypes very similar to the patients in 9 of them. To produce data robust enough for clinical use it is necessary to make highly specific mutations in the appropriate zygosity. Homology-directed repair works very efficiently in frog oocytes with up to 75% correct insertion reported and we have overcome the bottleneck in this technique. By using this together with lssDNA injection in embryos, we will precisely re-create a variety of patient VUS in frogs and work collaboratively to understand how effective these are at informing both clinical judgements and mechanistic processes of rare diseases.