Epigenetic regulation of αA-crystallin in high myopia-induced dark nuclear cataract

Abstract

Purpose: To assess the etiology of early-onset dark nucleus in high-myopic patients and its relationship with the epigenetic regulation of αA-crystallin (CRYAA).

Methods: We reviewed clinical data from patients who underwent cataract surgery at our center in 2012. Lens epithelial samples were collected during capsulorhexis, whereas young lens epithelium was donated. Cataract type and severity were graded according to the Lens Opacity Classification System III (LOCS III). DNA methylation was analyzed by pyrosequencing the CpG islands of the CRYAA promoter in the following groups: Age-Related Cataract (ARC) Nuclear Color (NC) 2-3; High-Myopic Cataract (HMC) NC2-3; ARC NC5-6; HMC NC5-6; and in young lenses graded NC1. We analyzed CRYAA expression by real-time polymerase chain reaction (PCR), reverse transcription PCR, and immunohistochemistry.

Results: The odds ratio of dark nucleus in high-myopic patients was 5.16 (95% confidence interval: 3.98-6.69; p<0.001). CpG islands in lens epithelial CRYAA promoter in the HMC NC5-6 Group exhibited the highest methylation of all the groups, but no statistically significant differences were evident between the HMC NC2-3 and ARC NC2-3 Groups. Likewise, CRYAA mRNA and protein levels in the HMC NC5-6 Group were significantly lower than the ARC NC5-6 Group and high-myopic controls.

Conclusions: High myopia is a risk factor for dark nucleus. Downregulation of CRYAA via the hypermethylation of CpG islands in its promoter could underlie the earlier onset of dark nucleus in high-myopic patients.

Figure 1. Prevalence of Grade Nuclear Color 5–6 cataracts in different age groups among age-related cataract and high-myopic cataract.

Statistically significant differences were evident in the 40–55, 56–65, and 66–75 years age groups (Pearson’s chi-squared test; *all p<0.05).

Figure 2. CpG island methylation in the αA-crystallin promoter was similar in age-related cataracts and high-myopic cataracts classified as Lens Opacity Classification System III Grade Nuclear Color 2–3.

(A) Representative Pyrosequencing results of the Control, Age-Related Cataract (ARC) Nuclear Color (NC) 2–3 and High-Myopic Cataract (HMC) NC2–3 Groups. (B) Anterior segment photos of patients in each group. (C) The six selected CpG islands in the ARC NC2–3 (44±8.3%) and HMC NC2–3 (43.7±3.8%) Groups displayed higher methylation compared with the Control Group (34±6.1%). (D) Percentage of methylation at each individual CpG site in each group. (*Statistically significant differences were found between the Control and ARC NC2–3 Groups; ^&statistically significant differences were found between the Control and HMC NC2–3 Groups; *^,&all p<0.05. However, no statistically significant differences were found between the two cataract groups.)

Figure 3. Expression of αA-crystallin was similar in age-related cataracts and high-myopic cataracts classified as Lens Opacity Classification System III Grade Nuclear Color 2–3.

Alpha-crystallin expression in the lens epithelium of each group was detected by (A) real-time polymerase chain reaction (PCR; also showing levels of αA-crystallin relative to β-actin) and (B) reverse transcription PCR (C, D, E) Representative immunohistochemistry images of the Control, Age-Related Cataract Nuclear Color (NC) 2–3, and High-Myopic Cataract NC2–3 Groups. (F) Mean staining density in each group (*all p<0.05).

Figure 4. CpG island methylation in the αA-crystallin promoter was significantly greater in high-myopic cataracts than in age-related cataracts classified as Lens Opacity Classification System III Grade Nuclear Color 5–6.

(A) Representative pyrosequencing results of the Age-Related Cataract (ARC) Nuclear Color (NC) 5–6 and High-Myopic Cataract (HMC) NC5–6 Groups. (B) Anterior segment photos of patients in each group. (C) The six selected CpG islands in the HMC NC5–6 Group (57.6±2.6%) displayed hypermethylation compared with the ARC NC5–6 Group (48.1±3.4%). (D) Percentage of methylation at each individual CpG site in each group. (*Statistically significant differences were found between the ARC NC5–6 and HMC NC5–6 Groups; *all p<0.05).

Figure 5. αΑ-crystallin expression in the lens epithelium was significantly lower in the High-Myopic Cataract Nuclear Color 5–6 Group than in the Age-Related Cataract Nuclear Color 5–6 Group.

αA-crystallin expression in the lens epithelium of each group was detected by (A) real-time polymerase chain reaction (PCR; also showing levels of αA-crystallin relative to β-actin) and (B) reverse transcription PCR. (C, D) Representative immunohistochemistry images of the Age-Related Cataract Nuclear Color (NC) 5–6 and High-Myopic Cataract NC5–6 Groups. (E) Mean staining density in each group (*both p<0.05).

Figure 6. Illustration for the possible mechanism of high-myopia-induced dark nuclear cataract.