Purpose

DNA sequencing technology has unmasked a vast number of uncharacterized single-nucleotide variants in disease-associated genes, and efficient methods are needed to determine pathogenicity and enable clinical care.

Methods

We report an E. coli–based solubility assay for assessing the effects of variants on protein domain stability for three disease-associated proteins.

Results

First, we examined variants in the Kv11.1 channel PAS domain (PASD) associated with inherited long QT syndrome type 2 and found that protein solubility correlated well with reported in vitro protein stabilities. A comprehensive solubility analysis of 56 Kv11.1 PASD variants revealed that disruption of membrane trafficking, the dominant loss-of-function disease mechanism, is largely determined by domain stability. We further validated this assay by using it to identify second-site suppressor PASD variants that improve domain stability and Kv11.1 protein trafficking. Finally, we applied this assay to several cancer-linked P53 tumor suppressor DNA-binding domain and myopathy-linked Lamin A/C Ig-like domain variants, which also correlated well with reported protein stabilities and functional analyses.

Conclusion

This simple solubility assay can aid in determining the likelihood of pathogenicity for sequence variants due to protein misfolding in structured domains of disease-associated genes as well as provide insights into the structural basis of disease.

中文翻译：

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蛋白质结构域错误折叠的快速溶解度测定，用于罕见 DNA 序列变异的致病性评估。

目的

DNA 测序技术揭示了疾病相关基因中大量未鉴定的单核苷酸变异，因此需要有效的方法来确定致病性并实现临床护理。

方法

我们报告了一种基于大肠杆菌的溶解度测定，用于评估变体对三种疾病相关蛋白质的蛋白质结构域稳定性的影响。

结果

首先，我们检查了与遗传性长 QT 综合征 2 型相关的 Kv11.1 通道 PAS 结构域 (PASD) 的变体，发现蛋白质溶解度与报告的体外蛋白质稳定性密切相关。对 56 个 Kv11.1 PASD 变体的综合溶解度分析表明，膜运输的中断（主要的功能丧失疾病机制）很大程度上取决于结构域稳定性。我们通过使用它来识别提高域稳定性和 Kv11.1 蛋白质运输的第二位点抑制 PASD 变体进一步验证了该测定。最后，我们将该测定应用于几种与癌症相关的 P53 肿瘤抑制基因 DNA 结合结构域和与肌病相关的 Lamin A/C Ig 样结构域变体，它们也与报告的蛋白质稳定性和功能分析密切相关。

结论

这种简单的溶解度测定可以帮助确定由于疾病相关基因的结构域中的蛋白质错误折叠导致的序列变异的致病性可能性，并提供对疾病结构基础的见解。