IGF-1 rs6218 polymorphisms modulate the susceptibility to age-related cataract

Study participants

The study was granted approval from the Ethics Committee of Changzhou Third People’s Hospital (ethical approval number: 2021012) and followed the Declaration of Helsinki. The participants were aware of the study’s aim and subsequently provided their informed consent by signing the appropriate documentation.

All participants underwent a comprehensive ophthalmic assessment, which included examinations of visual acuity, lens using a slit lamp biomicroscope under transient and side illumination after mydriasis, and ophthalmoscopic. The classification of ARC based on the opacity region of the lens includes four subtypes: cortical cataract (CC), nuclear cataract (NC), posterior sub-capsular cataract (PSC), and mixed cataract (MC) (Klein, Klein & Linton, 1992). The diagnosis and grading of lens opacities were conducted following the Lens Opacities Classification System III (LOCS III) (Feng et al., 2022).

This study adopts a case-control design, involving cases and controls from a population based epidemiologic cohort of the Jiangsu Eye Study located in Jiangsu Eye Study in Qidong country. In addition, individuals who served as controls and were matched with the experimental terms of age and sex and had transparent lenses were selected from the same communities. The geographical region under investigation exhibits a relatively stable and ethnically homogenous population. The participants were individuals sharing no familial relationships and self-identified as belonging to the Han Chinese ethnic group. Consequently, 716 ARC patients (CC = 377, NC = 223, PSC = 48, MC = 68) and 685 controls were included (Table 1). The inclusion/exclusion details of the case-control design followed the previous study (Kang et al., 2019).

In addition, we included additional 20 ARNC patients and 20 age-, sex- and ethnically matched controls from inpatients in our hospital from Jan 2019 to Dec 2021 (Table 2) to collect not only venous blood but also matched lens anterior capsule samples. Venous blood is used to detect its genotype, while lens anterior capsule samples were used to measure the mRNA levels of LECs and detect their apoptosis. The lens anterior capsule samples from ARC patients were obtained through anterior continuous circular capsulorhexis in phacoemulsification surgery. The lens anterior capsule samples from transparent lenses were acquired from patients who received lens extraction as part of vitrectomy procedures. The study excluded participants with lens trauma, diabetes, uveitis, glaucoma, and high myopia (>6D).

Selection of SNPs

Haplotype-tagging SNPs of genes were chosen by conducting a search in NCBI dbSNP (https://www.ncbi.nlm.nih.gov/snp) using Han Chinese data. The study included SNPs that had a MAF of >10% and were predicted to be potentially functional using the SNP function prediction program (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html) while excluding SNPs that exhibited strong linkage disequilibrium (LD) with adjacent variants, as determined by r² threshold ≤0.80 (Table 3).

DNA preparation and genotyping

The study extracted genomic DNA from the venous blood of all participants through the Qiagen Blood DNA Mini Kit (Qiagen, Valencia, CA, USA), following the protocols.

The SNP genotyping analysis was conducted using the TaqMan genotyping assay (Thermos Fisher, Foster City, CA, USA) per the guidelines as previously described in our published works (Kang et al., 2019; Zou et al., 2018, 2020).

RNA extracting and quantification of IGF1 mRNA expression

Total RNA was isolated by Trizol reagent from LECs (Invitrogen, Carlsbad, CA, USA). Then cDNAs were performed by PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The total RNA extracted in this experiment was detected using a UV spectrophotometer, and the UV absorption values (260/280) of the RNA were all between 1.9 and 2.1, indicating a high purity of the extracted RNA.

The study employed TaqMan gene expression assay probes (Thermos Fisher, Waltham, MA, USA) to quantify IGF1 mRNA (assay ID: Hs01547656_m1). The housekeeping gene control utilized in this study was Human GAPDH (assay ID: Hs02786624_g1). The study also conducted real-time PCR analysis using the ABI StepOne plus real-time PCR system (Applied Biosystems, Foster City, CA, USA) as well as utilized the 2^(−ΔΔCt) algorithm for calculating the fold change of gene mRNA level.

Annexin V/PI apoptosis detection

Apoptosis was measured by the Annexin V-FITC/PI apoptosis detection kitt (BD, Franklin Lakes, NJ, USA). LECs were seprarated from the lens anterior capsule by using trypsin. The LECs were stained cells through the per protocols. After staining 15 min, flow cytometry was employed for cell detection on a CytoFLEX system (Beckman Coulter, Brea, CA, USA).

Statistical analysis

The study utilized a commercial statistical software program (Stata 8.0) for performing the statistical analyses, reporting the data as means ± SD. The t-test was utilized to conduct statistical comparisons between the average values of the two groups. The χ2 test was employed for evaluating the relationship between the allele frequencies of ARC patients and normal controls, different ARC subtypes, odds ratios (OR), and 95% confidence intervals (CI), and also for testing Hardy-Weinberg Equilibriums (HWE) of genotype distributions. Upon detecting any positive correlation during the initial allele analysis, a Bonferroni correction was applied. The results only presented the most significant model. The qRT-PCR assays were conducted in a minimum of three independent replicates. p < 0.05 is indicative of a significant difference.

The participant features for the association study

This study enrolled participants from the hospital and epidemiologic, and the general demographic details of the participants were listed in Tables 1 and 2, respectively. A total of 716 patients with ARCs were included in the study, among which the numbers of CC, NC, PSC, and MC were 377, 223, 48, and 68, respectively. There were 685 age-, sex- and ethnically matched healthy control subjects. The general demographic details of the study participants are summarized in Table 1. Among the hospitalized population, there were 20 ARNC patients (AA genotype = 10; AG genotype = 4; GG genotype = 6) and 20 controls (AA genotype = 10; AG genotype = 5; GG genotype = 5) (Table 2). The average age is 62.6 ± 8.24 years in controls and 64.3 ± 6.42 years in ARNC patients. The ratio of sex is 0.55 in ARNC patients and 0.5 in controls. There is no difference between age and sex.

Bioinformatics selection of candidate SNPs

The SNPs of IGF1 were chosen for genotyping, listing their basic features in Table 3. Among them, rs6218, rs5742714, rs12579108 were located in the 3′-UTR region; rs2288377, rs35767, rs5742612, rs12579077 were located in the intron region of the IGF1 gene.

SNPs and ARC risk correlation

Between the 7 SNPs, the IGF1-rs6218 allele frequency of ARCs differed significantly from controls (p = 0.003). This significance remained even after applying multiple comparison corrections (Bonferroni correction) (p = 0.021). The findings indicated that the minor allele G frequency in the IGF1-rs6218 was higher in the ARCs than in the controls (Table 4). Subsequently, stratification analysis was further conducted to investigate the SNP contribution in ARC subtypes, revealing that the minor allele frequency of IGF1-rs6218 was only higher in the nuclear type of ARCs (ARNCs) than in the controls (p = 0.021) (Table 5).

rs6218 impacts on the mRNA levels of IGF1 in biopsy samples

IGF1 mRNA expression was higher in LECs of the ARNCs than in the controls (Fig. 1A). Additionally, IGF1 mRNA expression of GG-genotype individuals was higher than AA-genotype individuals in both ARNCs and controls. In the ARNCs, IGF1 mRNA levels were significantly higher in the GG or AG group than in the AA group. In the controls, IGF1 mRNA levels were higher in the GG than in the AA group (*: p < 0.05; **: p < 0.01) (Fig. 1B).

Apoptosis of LECs in biopsy samples

Apoptosis was a normal physiological process that orderly controls cell death to maintain stable homeostasis. The experimental results showed that cell apoptosis was present in both the ARNCs and the control group of LECs. However, The apoptosis of LECs in the anterior capsules of ARNCs was significantly higher than in the controls (Fig. 2). *: p < 0.05.