Related Experiment Video
Updated: Aug 11, 2026
In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila
Published on: August 20, 2019
Molecular analysis of a human PAX6 homeobox mutant
Angela Valentina D'Elia1, Cinzia Puppin, Lucia Pellizzari
1Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Udine, Italy.
Insights
The PAX6 R242T mutation, found in a child with aniridia, increases PAX6 protein levels. This suggests that elevated PAX6 dosage may cause congenital eye abnormalities like aniridia.
Area of Science:
- Developmental Biology
- Genetics
- Ophthalmology
Background:
- Pax6 is crucial for eye, pancreas, and brain development.
- PAX6 mutations in humans cause aniridia and other congenital eye disorders.
- Most PAX6 missense mutations occur in the paired domain (PD), fewer in the homeodomain (HD).
Purpose of the Study:
- To molecularly analyze the human PAX6 R242T missense mutation in the HD.
- To investigate the functional impact of the R242T mutation on PAX6 protein.
Main Methods:
- Gel-retardation assays to assess DNA-binding.
- Cell transfection assays to evaluate protein levels and promoter activation.
- In vitro limited proteolysis assays to determine protein stability.
Main Results:
- The R242T mutant HD binds DNA similarly to wild-type HD.
- The mutation does not affect the DNA-binding properties of the PD.
- Mutant PAX6 protein exhibits higher steady-state levels and increased promoter activation compared to wild-type.
- The R242T mutation reduces protein sensitivity to trypsin digestion, indicating increased stability.
Conclusions:
- The R242T mutation leads to increased PAX6 protein levels and activity.
- This elevated PAX6 dosage is a potential cause for the observed partial aniridia and pseudo-coloboma.
- Findings align with the known sensitivity of eye development to PAX6 dosage.
Abstract:
Pax6 controls eye, pancreas and brain morphogenesis. In humans, heterozygous PAX6 mutations cause aniridia and various other congenital eye abnormalities. Most frequent PAX6 missense mutations are located in the paired domain (PD), while very few missense mutations have been identified in the homeodomain (HD). In the present report, we describe a molecular analysis of the human PAX6 R242T missense mutation, which is located in the second helix of the HD. It was identified in a male child with partial aniridia in the left eye, presenting as a pseudo-coloboma. Gel-retardation assays revealed that the mutant HD binds DNA as well as the wild-type HD. In addition, the mutation does not modify the DNA-binding properties of the PD. Cell transfection assays indicated that the steady-state levels of the full length mutant protein are higher than those of the wild-type one. In cotransfection assays a PAX6 responsive promoter is activated to a higher extent by the mutant protein than by the wild-type protein. In vitro limited proteolysis assays indicated that the presence of the mutation reduces the sensitivity to trypsin digestion. Thus, we suggest that the R242T human phenotype could be due to abnormal increase of PAX6 protein, in keeping with the reported sensitivity of the eye phenotype to increased PAX6 dosage.
