Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld-Rieger syndrome

The specific role of PITX2 in the pathogenesis: of anterior segment dysgene sis has yet to tie clearly defined. We provide here new insight into PITX2 pathogenesis through mutational and functional analyses. Three PITX2 mutati ons were found in a screen of 38 unrelated individuals affected with anteri or segment anomalies (8%). All three mutations were found among the 21 indi viduals affected with Axenfeld-Rieger syndrome (ARS). We have identified tw o novel mutations, a valine --> leucine (V45L) missense mutation at positio n 45 within the PITX2 homeodomain, and a seven amino acid duplication (7aaD up) of residues 6-12 of the homeodomain. DNA-binding studies of the two mut ant PITX2 proteins demonstrated a < 10-fold reduction in the DNA-binding ac tivity of the V45L mutant, and a > 100-fold reduction in activity of the 7a aDup mutant. Luciferase reporter assays showed a > 200% increase in PITX2 t ransactivation activity of the V45L mutant, while the 7aaDup mutant was una ble to transactivate at detectable levels. Our analyses of the V45L PITX2 m utant reveal that the DNA-binding domain of PITX2 can influence transactiva tion activity independently, of DNA binding. Furthermore, our findings expa nd the hypothesis that the amount of residual PITX2 activity underlies the variable severity of ocular phenotypes that result from PITX2 mutation. For the first time, we present evidence that increased PITX2 activity may unde rlie the severe ARS ocular phenotype. We conclude that increased activity o f one PITX2 allele may be as physiologically disruptive as a mutation that nullifies a PITX2 allele, with either condition resulting in ARS.