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

Alexander F. McDonnell, Marcin Plech, Benjamin J. Livesey, Lukas Gerasimavicius, Liusaidh J. Owen, Hildegard Nikki Hall, David R. FitzPatrick, Joseph A. Marsh, Grzegorz Kudla*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)825-844
Number of pages20
JournalMolecular Systems Biology
Volume20
Issue number7
Early online date7 Jun 2024
DOIs
Publication statusPublished - 2 Jul 2024

Keywords / Materials (for Non-textual outputs)

  • Deep Mutational Scanning
  • Eye Development
  • Transcription Factor

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