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

高通量DNA测序的能力已经完全改变了我们诊断遗传疾病的能力。通过比较受影响儿童的DNA序列与其父母和兄弟姐妹的DNA序列，可以确定受影响儿童的某些基因序列与其家庭的基因序列不同;这些基因被称为变体，其中一个或多个可能是疾病的原因。一旦这些变异之一被确定为致病原因，这一知识将指导对儿童的临床干预，并就家庭其他现有或未来子女的健康问题向家庭提供咨询意见，还将促进级联筛查和产前诊断。然而，在大约一半的情况下，不可能从患者体内发现的一组变体中识别出致病基因，并且无法有足够的确定性来做出临床决策。细胞或“类器官”培养物中的实验（复制器官中存在的一些细胞-细胞相互作用的细胞的复杂混合物）可以告知临床医生哪些变体是致病的，但在其他情况下，仍然需要在动物中测试基因变异的效果。这只能在老鼠身上进行，而这些实验在伦理和经济上都很昂贵。包括我们在内的几个实验室最近表明，蝌蚪实验也可以为变异人类基因的功能提供非常有力的证据。该项目由青蛙遗传学家、医学基因组研究科学家和临床遗传学家联合开展，旨在发现从含有患者基因变异的蝌蚪中获得的信息对照顾它们的临床医生有多大的用处。青蛙作为人类生物学模型的有用性常常令人惊讶，但我们对胚胎发育的了解以及我们对许多疾病的理解所依赖的生物学往往首先在青蛙中发现。在基因相似性方面，几乎80%已知在人类疾病中被改变的基因在青蛙中被发现，并且大多数基因在青蛙和人类的染色体上以相同的顺序被发现。到目前为止，我们已经分析了80%的基因变异，在青蛙和人类中受疾病影响的区域非常相似，很可能是因为基因产物的这些部分对生物体的健康至关重要，因此进化使它们保持不变。在蝌蚪中重建某些类型的基因变异已经非常简单;通常在不到3天的时间内发生变化。这些随后对蝌蚪发育的改变可以在几天后进行测定。通过对蝌蚪进行自动计算机断层扫描（CT），以及在大量的“转基因”动物（其中特定细胞类型发出荧光）中制造基因变体的能力，这些测定变得更容易;这使得很容易看到变化。在患者身上发现的许多基因变异比迄今为止测试的基因变异更微妙，需要使用更新的技术来制造它们。这些方法改变DNA中的一个字母或一组非常特定的字母，存在于每个细胞中的基因的一个或两个拷贝中。目前在青蛙中快速高效地完成这项工作的技术效果最好。利用这些实验方法，我们将在蝌蚪中重现罕见遗传病患者的变异体，并测试每种变异体对它们发育的影响。如果蝌蚪显示出人类疾病的特征，例如我们的试验显示蝌蚪中的白内障具有导致儿童白内障的基因变异，这支持基因是因果关系，因此允许临床团队照顾患者及其家人。如果临床研究人员发现这些信息足够有用，那么我们将继续共同努力，扩大青蛙基因功能分析的规模，以便它可以用于指导大量患者的有效干预。

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.