| Purpose:To deep analyse the association between high myopia and PAX6 gene with its regulatory regions in Han Chinese, in order to setup the pathogenesis basis of myopia for further research.Methods:Case-control association study was used in this research. We recruitd 300 high myopia (SE≤-8.00D) and 300 normal control subjects (SE within±1.00D) for the initial association study (Group 1); and recruited another 300 high myopia (SE≤-8.00D) and 300 normal control subjects (SE in±1.00D) (Group 2) for follow-up haplptype analysis with expanding sample size, the same entry criteria were used for recruiting subjects of Group1 and Group2. Then we collected blood samples and extracted DNA.Tag SNPs were selected by HapMap project, and added 5 SNPs more into analysis, we analyzed total 16 SNPs in the intial study. We used PCR-RFLP to genotype rs3026354, rs3026393, rs1506, rs12421026, rs3026401, rs7125966. rs2863231, rs964112, rs7947424, rs509628; used unlabeled probe melting analysis to genotype the other 6 SNPs:rs667773, rs3026390, rs2071754, rs662702, rs11031423, rs11031419. Ocular data were analysed using the SPSS package; PLINK was used for the analysis of genetic data:Hardy-Weinberg equilibrium on unrelated subjects and association analysis under 5 different genetic models. Haploview was used for constructing linkage disequilibrium block and haplotype analysis; Haplotype analysis with sliding windows was also performed with PLINK and multiple comparisons was corrected by generating empirical P (Pemp) values based on 50,000 permutations. A follow-up analysis with expanding sample size did for the significant results obtained in the initial association study.Results:The initial association study (Group 1) results showed 16 single SNPs were not associated with high myopia or extra high myopia. All subjects in Group 1 constructed three linkage disequilibrium blocks, and two haplotypes were significantly different between cases and controls in the block 1:one high-risk haplotype CGAGTGG (P=0.0000) and one protective haplotype CGAGTAG (P=0.0003). Exhaustive haplotype analyses showed at least one window showing significant results among sliding windows of a given size, the crucial importance of rs12420126 was obvious; the 4-SNPs window of rs2071754, rs3026393, rs1506 and rs12421026 and the 3-SNPs window of rs3026393, rs1506 and rs12421026 gave the most impressive Pemp value for the omnibus test. These reulsts were consistent with those obtained for LD-block-based haplotype analysis by Haploview.A follow-up haplotype analysis with expanding sample size also included Group 2 which tested 5 SNPs (rs3026390, rs2071754, rs3026393, rs1506, and rs12421026), there were no significant association between single SNP and high myopia in Group2. With sliding-window haplotype analysis, Group1, Group2 or the combined group (Group1 and Group2) obtained two high-risk 4-SNPs haplotypes constructed by rs2071754. rs3026393, rs1506 and rs12421026:GTAA and AGTG; and one protective haplotype AGTA (P≤0.0118). We also obtained 3-SNPs haplotypes constructed by rs3026393, rs1506 and rs12421026:TAA和GTG;and one protective haplotype GTA (P≤0.0045). The two high-risk haplotypes were each found at～1% in the combined control group, but at～9% in the combined case group. On the contrary, the protective haplotypes were found at～42% in the combined control group, but at 30% in the combined case group. Conclusion:We used case-control study to test the association between candidate gene PAX6 and high myopia. Results showed several haplotypes in the 3’end of the PAX6 locus were associated with high myopia both in the initial study and the follow-up study with expanding sample size. The study support PAX6 as a susceptibility gene for high myopia and provides the foundation for further investigation to identify the genuine causal variants. |
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