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Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants

ABSTRACT: Mutations in the transcription factor PAX6 cause eye development defects, including aniridia, microphthalmia and coloboma. To understand the molecular mechanisms, 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 2,700 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. Some variants, including Ile71Tyr, increased binding to one of the DNA elements. 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, we obtained supporting-to-moderate evidence that 977 variants are likely pathogenic and 1,306 are likely benign. Our analysis shows that most pathogenic mutations in PAX6 can be explained by effects on PAX6:DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.

SUBMITTER: Alexander, F McDonnell

PROVIDER: S-SCDT-10_1038-S44320-024-00043-8 | biostudies-other |

REPOSITORIES: biostudies-other

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Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants

Project description: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 2,700 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 Ile71 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 to suggest that 1,306 variants are likely benign, and 977, likely pathogenic. 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.

2024-03-21 | GSE253580 | GEO

Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.

Project description: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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Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants

Project description:Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants

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