Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants.
Mol Syst Biol
; 20(7): 825-844, 2024 Jul.
Article
in En
| MEDLINE
| ID: mdl-38849565
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 PAX6DNA 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 PAX6DNA association, and establishes Y1H as a generalisable assay for the interpretation of variant effects in transcription factors.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA
/
PAX6 Transcription Factor
Limits:
Humans
Language:
En
Journal:
Mol Syst Biol
/
Molecular systems biology
Year:
2024
Document type:
Article