A Functional Polymorphism at 3UTR of the PAX6 Gene May Confer Risk for Extreme Myopia in the Chinese
- PAX6 polymorphism and myopia
- case–control study
- functional assay
Conflicting data have been reported from recent genetic association studies regarding the association between PAX6 and myopia. To further clarify this relationship, we conducted a large case–control study of more than 2000 subjects and a functional assay, to obtain a more reliable result.
- 1083 cases (≤ −6.0 D) and 1096 controls (≥ −1.5 D) from a Chinese population residing in Taiwan
- Four common tagSNPs——HapMap Project ([www.hapmap.org](http://www.hapmap.org/))Tagger Pairwise method[ r 2 ≥ 0.8 ; MAF(CHB) ≥ 10%]
- rs628224 (intron 7)
- rs644242 (intron 11)——significant
- rs2071754 (intron 11)
- rs3026393 (intron 12)
- an SNP at the 3′UTR：SNP rs662702 at the 3′UTR is tagged by rs644242!(C:\Users\thinkpad\AppData\Roaming\Typora\typora-user-images\1553059078358.png)
- 基因分型：Taqman探针法(TaqMan; Applied Biosystems, Inc. [ABI], Foster City, CA)
- 3utr SNP rs662702 ——双荧光素酶报告基因实验——分析SNP对基因表达的影响
- contain 17 bp surrounding SNP rs662702；
- restriction enzymes SpeI and MluI for cloning sites
- (pMIR-REPORT miRNA Expression Reporter Vector System; ABI)
- T4 DNA ligase (New England BioLabs, Boston, MA)
- One reporter construct carries risk allele C (denoted as rs662702C)；
- the other carries protective allele T (denoted as rs662702T).
The study was approved by the Institutional Review Board at the Kaohsiung Medical University Hospital. The research adhered to the tenets of the Declaration of Helsinki.
SNPs rs644242 and rs662702 had marginal significance (P = 0.063)
SNPs rs644242 and rs662702 were associated with extreme myopia (≤ −11 D)；
The difference in genotype distribution between cases and controls was marginally significant (P = 0.063 from the χ2 analysis) for SNP <u>rs644242</u>.
The results showed that the frequency of the common C allele as well as the CC genotype increased with the severity of myopia；
The most significant result was from the comparison between subjects with spherical refractions ≤ −11 and ≥ −0.5 D；
The CC genotype had an OR of 2.3 (nominal P = 0.009) for extreme myopia when compared to the combination of AA and AC genotypes.
- SNP rs662702 (r 2 = 0.88 with rs644242) at the 3′ UTR [HapMap data]
- the risk allele C was significant only for extreme myopia!(C:\Users\thinkpad\AppData\Roaming\Typora\typora-user-images\1553059492844.png)
- 重新分组：The empiric P value was 0.0448 for the association between extreme cases (≤ −11 D) and controls (≥ −0.5D) for rs662702.
- SNP rs662702(CC) at the 3′UTR：OR值=2.1 (empiric P = 0.007) ；
- Luciferase Assays：
- the rs662702T clone had a significantly higher expression level of firefly luciferase than did the rs662702C clone (P = 0.0001).!(C:\Users\thinkpad\AppData\Roaming\Typora\typora-user-images\1553059683016.png)
- These results suggested that the risk C allele had lower protein expression levels than did the protective T allele.
SNP rs662702：microRNA-328 binding site
- Although 3′UTR SNP rs662702 would not alter the structure of the PAX6 protein, it may influence stability of PAX6 mRNA
- microRNA software:
- microRNA-328 (miR-328)——三个数据库预测结果取交集
- > The position of rs662702 is next to the seed region of miR-328 and the C-to-T substitution leads to a mismatch between miR-328 and PAX6 mRNA paring.Our functional assay indicates that the risk C allele causes a lower PAX6 expression level than the T allele, which can be due to the knockdown effect by miR-328 on the C allele. If so, SNP rs662702 also belongs to a new category of SNPs called mirSNP. A decreased expression of PAX6 can result in an increased expression of MMP2, and an increase of MMP2 expression has been implicated in the pathogenesis of myopia. 34 Accordingly, our initial finding based on the statistical significance can be further supported by these functional studies.
a large-scale study to systematically evaluate the genetic effect of the PAX6 gene
a functional SNP was identified at the 3′UTR that influences the risk for extreme myopia
risk allele can reduce PAX6 protein levels which significantly increases the risk for myopia.
This functional SNP is located at the miR-328 binding site, and the risk allele may be downregulated by miR-328, leading to a lower level of PAX6 protein.
> Tsai et al. investigated a Chinese population residing in Taiwan and reported a significant association between the common C allele of rs667773 at PAX6 and extreme myopia (≤ −10 D), whereas no significant association was found for high myopia, defined as ≤ −6.0 D. Although we did not genotype rs667773, it can be tagged by our initial tSNP rs644242 (r 2 = 0.92) and our functional SNP rs662702 (r 2 = 0.86) according to the Han Chinese data in HapMap. In concert with the finding of Tsai et al., our data suggest that both rs644242 and rs662702 are more related to extreme myopia but less associated with high myopia. We conducted a meta-analysis using data from Tsai et al., and the present studies, and the results showed that the common alleles had an OR of 2.9 (P < 0.0001) for extreme myopia in the Chinese population. Using the family data set, the Han et al. 16 study revealed a weak association (P = 0.0458) between rs667773 and high myopia (≤ −6.0 D). However, they did not specifically examine the association with extreme myopia. On the other hand, they did report the most significant SNP, rs3026393, with a P < 0.001, which was not significant in our study.
> Our study design has strengths and limitations. We used a large number of study subjects to reduce both type I and II error rates. The statistical finding is further supported by the functional experiments, and the significant 3′UTR SNP can be one of the causal variants. Our study population was relatively homogenous in terms of ethnicity, geographic location, and age. As a result, several undetectable confounding factors can be minimized. A previous study investigating the population admixture of Taiwanese also indicated high homogeneity among the Taiwanese subpopulations. 38 In addition, we have assessed population stratification in subjects recruited from the same geographic locations by our team, and the inflation factor was 1. 39 Therefore, population stratification is unlikely to be an explanation for our association results. Using permutation, we also found a significant P value under the consideration of multiple testing. However, the main finding of a significant association was based on only 55 cases of extreme myopia. A meta-analysis is warranted to provide a sufficient power to validate our finding.