PITX2 gene and minigene structure. A. Schematic of PITX2 gene organization (drawn to scale). Open boxes represent exons, lines represent introns. The sizes of exons and introns are shown. The PITX2 gene has two major promoters (arrows) and six exons. Alternative splicing and transcription start sites give three major isoforms (not shown). PITX2c uses the downstream promoter and produces an mRNA containing exon 1b, 4, and 5. B. Schematic of PITX2c minigene. The minigene contains a portion of exon 1b, exon 4, part of exon 5, and uses the CMV promoter and bGH polyadenylation site from the pcDNA3.1/myc-His(-)A vector (transcribed vector sequences are represented by shaded boxes). The lengths of intron regions associated with each exon are shown with vertical lines indicating the fusion of two fragments. The positions of the T7 primer to vector sequences, and primers to PITX2 exons 1b and 5 that were used for RT-PCR are shown below the diagram. C. Expansion of (B) showing the names and locations of three different intronic point mutations identified in ARS patients. The sequence surrounding the 5' and 3' splice sites of exon 4, and the 3' ss of exon 5 is shown. Mutations in patients 0175 (IVS4-1G>T), 689 or 2 (IVS4+5G>C), and 4 (IVS5-11A>G) are indicated by arrows and the nucleotide changes are shown.