Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants
Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants
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深度突变扫描可量化 DNA 结合并预测 PAX6 变体的临床结果
DOI：
10.1101/2023.07.25.550478
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
McDonnell A
中科院分区：
文献类型：
--
作者：
McDonnell A
转录因子 PAX6 的无义和错义突变会导致多种眼睛发育缺陷，包括无虹膜、小眼球和缺损。为了了解 PAX6:DNA 结合的变化如何导致这些表型，我们将 PAX6 配对结构域的饱和诱变与酵母单杂交 (Y1H) 测定相结合，其中 PAX6-GAL4 融合基因的表达驱动抗生素耐药性。我们量化了 2700 多个单一氨基酸变体与两个 DNA 序列元件的结合。 N 末端子结构域和接头区域的面向 DNA 的残基的突变是最有害的，脯氨酸和带负电残基的突变也是如此。许多变体引起序列特异性分子功能获得效应，包括 71 位的变体增加了与 LE9 增强子的结合，但减少了与 SELEX 衍生的结合位点的结合。在没有抗生素选择的情况下，保留 DNA 结合的变体减缓了酵母的生长，可能是因为这些变体扰乱了酵母转录组。通过对已知患者变异进行基准测试并将 ACMG/AMP 指南应用于变异分类，我们获得了支持性至中等程度的证据，表明 977 种变异可能是致病性的，1306 种变异可能是良性的。我们的分析表明，PAX6 配对结构域中的大多数致病性突变可以简单地通过这些突变对 PAX6:DNA 关联的影响来解释，并建立 Y1H 作为解释转录因子变异效应的通用分析方法。
Nonsense and missense mutations in the transcription factor PAX6 cause a wide range of eye development defects, including aniridia, microphthalmia and coloboma. To understand how changes of PAX6:DNA binding cause these phenotypes, we combined saturation mutagenesis of the paired domain of PAX6 with a yeast one-hybrid (Y1H) assay in which expression of a PAX6-GAL4 fusion gene drives antibiotic resistance. We quantified binding of more than 2700 single amino-acid variants to two DNA sequence elements. Mutations in DNA-facing residues of the N-terminal subdomain and linker region were most detrimental, as were mutations to prolines and to negatively charged residues. Many variants caused sequence-specific molecular gain-of-function effects, including variants in position 71 that increased binding to the LE9 enhancer but decreased binding to a SELEX-derived binding site. In the absence of antibiotic selection, variants that retained DNA binding slowed yeast growth, likely because such variants perturbed the yeast transcriptome. Benchmarking against known patient variants and applying ACMG/AMP guidelines to variant classification, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1306 are likely benign. Our analysis shows that most pathogenic mutations in the paired domain of PAX6 can be explained simply by the effects of these mutations on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.
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作者：
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影响因子：
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