AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia

Abstract

Purpose: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls.

Methods: High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities.

Results: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths.

Conclusions: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.

Figure 1

Transcriptional activity of dinucleotide repeats in the PAX6 P1 promoter. A 1,851 bp genomic fragment (from –1278 to +573) containing the PAX6 P1 promoter with different dinucleotide repeats was cloned into an empty pGL3-Basic vector (pGL3) and transfected into ARPE-19 cells. The activity of each allelic construct is expressed relative to the construct (AC)₂₀(AG)₆. Data are represented as mean±SD for five independent experiments. A and B: Immunoblotting results and a bar chart show relative luciferase activity for grouped (AC)_m repeats with a stable (AG)₆. C and D: Immunoblotting results and a bar chart show relative luciferase activity for (AG)_n repeats with (AC)₂₁. E and F: Immunoblotting results and a bar chart show relative luciferase activity for combined (AC)_m(AG)_n repeats.

Figure 2

Transcription factor binding site prediction for dinucleotide repeats in the PAX6 P1 promoter. The cloned PAX6 P1 promoter DNA sequence was used to predict transcription factor binding sites. Predicted transcription factor binding sites around the region of the dinucleotide repeats are shown, and different lengths of AC and AG repeats are assessed. As in the immunoblotting analysis, [(AC)₂₀(AG)₆] was set as a reference. A: Predicted transcription factor binding sites for (AC)₁₅(AG)₄ are shown. B: Predicted transcription factor binding sites for (AC)₂₀(AG)₆ are shown. C: Predicted transcription factor binding sites for (AC)₂₆(AG)₈ are shown.