Studies of factors involved in PITX2 regulation and their role in anterior segment dysgenesis phenotypes

Axenfeld-Rieger syndrome (ARS) is characterized by malformations of the anterior segment of the eye, craniofacial and dental defects, and redundant periumbilical skin. Mutations in PITX2 are associated with ARS; however, mutations in PITX2 or other known genes explain only ∼40% of ARS cases. Identification of cis-regulatory elements of PITX2 can aid in elucidating the mechanisms of disease. Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Conserved non-coding elements surrounding PITX2/ pitx2 were identified and examined through transgenic analysis in zebrafish. Thirteen conserved non-coding sequences positioned within a gene desert as far as 1.1-Mb upstream of the human PITX2 gene were identified. One region, CE4, located approximately 111-kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, consistent with the sites affected in ARS. Screening of ARS patients identified two patients with genomic deletions located 50-kb and 106-108-kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4-CE13. These data support the significance of distant upstream regulatory elements in normal gene function and offer a possible mechanism for ARS in patients with balanced translocations and deletions involving the upstream PITX2 region. The CE4 sequence was examined for putative interacting proteins, and multiple forkhead protein consensus binding sites were identified within or near CE4. FOXD3 encodes a forkhead-domain transcription factor that is expressed in precursor and early migratory neural crest cells which contribute to the developing anterior segment structures of the eye. A role for FOXD3 in human eye disease has not been reported. In zebrafish, foxd3 is expressed in the neural-crest derived periocular mesenchyme, as well as in the hindbrain and pharyngeal arches. A novel foxd3-deficient zebrafish line harboring a 5.7-kb viral insertion incorporated into the forkhead DNA-binding domain and predicted to encode a mutant protein containing 33% of normal foxd3 sequence (including only 30% of the forkhead domain) and 12 erroneous amino acids encoded by the viral sequence was characterized. Homozygous mutant fish display craniofacial and cardiac defects with embryonic lethality by 7-10 days post-fertilization (dpf), while heterozygous embryos appear normal. While no major structural changes in the eye were observed in up to 5-dpf mutant embryos, a reduction in expression of several genes known to be important for anterior segment development (including pitx2 ) was observed. FOXD3 was next evaluated for its contribution to human ocular disease. 310 probands with developmental ocular conditions were screened for variation in FOXD3. Six nonsynonymous FOXD3 variants were identified. Four of these changes, c.47C>T (p.Thr16Met), c.359C>T (p.Pro120Leu), c.517A>C (p.Asn173His), and c.818_829dup (p.Arg273_Gly276dup), affected conserved regions and were observed primarily in probands with aniridia or Peters anomaly; out of these four variants, p.Arg273_Gly276dup was not detected in control populations and p.Pro120Leu and p.Asn173His were statistically enriched in cases with aniridia or Peters anomaly. The p.Asn173His missense mutation affects a 100% conserved residue among vertebrate orthologs located within the forkhead domain and is predicted to be involved in protein-protein interactions rather than direct contact with DNA. In summary, these data suggest that a complex distant regulatory matrix located upstream of PITX2 plays an essential role in gene activity and its deletion provides a novel mechanism for ARS. FOXD3/foxd3 may play a role in normal eye development and contribute to human disease. Identification of co-factors that may modulate FOXD3 interactions and further analysis of foxd3-deficient zebrafish lines are necessary to better understand the contribution of FOXD3/foxd3 to ocular phenotypes, as well as determine relationships between FOXD3 and PITX2..