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

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.

Characterization of an eye field-like state during optic vesicle organoid development.

Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors. This crucial event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate. Using optic vesicle organoids to model the onset of the EF, we generate time-course transcriptomic data allowing us to identify dynamic gene expression programmes that characterize this cellular-state transition. Integrating this with chromatin accessibility data suggests a direct role of canonical EF transcription factors in regulating these gene expression changes, and highlights candidate cis-regulatory elements through which these transcription factors act. Finally, we begin to test a subset of these candidate enhancer elements, within the organoid system, by perturbing the underlying DNA sequence and measuring transcriptomic changes during EF activation.

Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use.

Recurrent heterozygous PAX6 missense variants cause severe bilateral microphthalmia via predictable effects on DNA-protein interaction.

PURPOSE: Most classical aniridia is caused by PAX6 haploinsufficiency. PAX6 missense variants can be hypomorphic or mimic haploinsufficiency. We hypothesized that missense variants also cause previously undescribed disease by altering the affinity and/or specificity of PAX6 genomic interactions. METHODS: We screened PAX6 in 372 individuals with bilateral microphthalmia, anophthalmia, or coloboma (MAC) from the Medical Research Council Human Genetics Unit eye malformation cohort (HGUeye) and reviewed data from the Deciphering Developmental Disorders study. We performed cluster analysis on PAX6-associated ocular phenotypes by variant type and molecular modeling of the structural impact of 86 different PAX6 causative missense variants. RESULTS: Eight different PAX6 missense variants were identified in 17 individuals (15 families) with MAC, accounting for 4% (15/372) of our cohort. Seven altered the paired domain (p.[Arg26Gln]x1, p.[Gly36Val]x1, p.[Arg38Trp]x2, p.[Arg38Gln]x1, p.[Gly51Arg]x2, p.[Ser54Arg]x2, p.[Asn124Lys]x5) and one the homeodomain (p.[Asn260Tyr]x1). p.Ser54Arg and p.Asn124Lys were exclusively associated with severe bilateral microphthalmia. MAC-associated variants were predicted to alter but not ablate DNA interaction, consistent with the electrophoretic mobility shifts observed using mutant paired domains with well-characterized PAX6-binding sites. We found no strong evidence for novel PAX6-associated extraocular disease. CONCLUSION: Altering the affinity and specificity of PAX6-binding genome-wide provides a plausible mechanism for the worse-than-null effects of MAC-associated missense variants.