The Association between Copy Number Variations in Glutathione S-transferase M1 and T1 and Age-Related Cataract in a Han Chinese Population

Abstract

Purpose.: To determine the contribution of copy number variation (CNV) in glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1) to the susceptibility to age-related cataract (ARC) and its subtypes in a Han Chinese population.

Methods.: ARC cases (n = 279) and controls (n = 145) were included from a Han Chinese population–based prospective study. Quantitative RT-PCR and the ΔCt method were used to determine the presence of no, one, or multiple alleles of GSTM1 and GSTT1. The RNaseP gene was used as the internal control.

Results.: The GSTT1 null genotype was associated with ARC with an odds ratio (OR) of 1.56 (P < 0.05). Deletion of at least one GSTT1 allele also influenced the onset of ARC (OR = 2.16, P < 0.01). Consistent associations were observed between GSTT1 CNV and cortical cataract. The deletion of at least one GSTT1 allele was associated with an OR of 4.81 for developing cortical ARC (P < 0.001), although individuals who had more than two copies of GSTT1 had a reduced risk of cortical ARC (OR = 0.19, P < 0.05). The frequency of the GSTT1 null genotype in the Han Chinese population differed dramatically from that in Caucasians. No association was detected between GSTM1 CNV and ARC.

Conclusions.: An association was observed between GSTT1 CNV and ARC in a Han Chinese population. The GSTT1 CNV is most closely associated with cortical cataract risk. The loss of at least one GSTT1 allele increases the risk of cortical cataract, whereas gain in GSTT1 copy number reduces the risk.

Age-related cataract (ARC) is one of the leading causes of visual impairment and blindness among the elderly worldwide. ¹ The global burden of blindness due to ARC is increasing as a result of a growing elderly population. ² Clinically, ARC can be classified as nuclear, cortical, or posterior subcapsular cataract according to the degenerative region of lens. 

ARC is a complex disease with a broad spectrum of risk factors including age, sex, smoking, exposure to sunlight, estrogen sufficiency or deficiency, cardiovascular factors, and genetic background. ³ Various studies have indicated a significant genetic component to ARC risk. ^4,5 A study on the familial aggregation of ARC reported that the probability of the development of nuclear cataract was significantly increased (OR = 2.07) among individuals with a sibling with nuclear cataract. ⁶ A family-based linkage study reported multiple ARC loci at 1q31, 2p24, 2q11, 4q28, 15q13, and 6p12-q12. ⁷ The potential role of gene structure variation in ARC development has been considered. For example, several studies have demonstrated the association between ARC and gene polymorphisms of αA-crystallin and EPHA2. ^8,9  

Involvement of oxidative stress appears to be one of the critical events in the pathogenesis of ARC. Oxidative damage can result in several molecular changes, such as degradation, cross-linking, and aggregation of lens proteins that contribute to the development of cataracts. ^5,8–11 Crystallins and other proteins in lens fiber cells do not turn over and must serve the lens for a lifetime. Thus, the lens must have efficient reducing and detoxification systems. Enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase (GST) are thought to be essential in protecting the eye from oxidative damage. ^8,9,12,13  

GSTs are a superfamily of phase II drug-metabolizing enzymes that catalyze the conjugation of reduced glutathione (GSH) with a variety of electrophilic compounds, thereby protecting the cell against xenobiotics and oxidative stress. ^8,9,14,15 The members of the GST family include various isoforms, including α (GSTA), μ (GSTM), π (GSTP), κ (GSTK), θ (GSTT), and ζ (GSTZ), based on sequence identity. ¹⁶ Copy number variations (CNV) in GSTM1 and GSTT1 have been reported. ^17–19 The GSTM1*0/0 null allele may result from homologous unequal crossing over between two highly identical 4.2-kb repeated sequences flanking the GSTM1 gene, causing a 15-kb deletion including the entire GSTM1 gene. ^18,19 A similar mechanism causes the generation of the GSTT1*0/0 null allele. ¹⁷  

Gene dosage effects between CNV and enzyme activity have been reported for both GSTM1 and GSTT1. ^20,21 The null genotypes GSTM1*0/0 and GSTT1*0/0 are associated with complete loss of catalytic activity ^17,22 and have been suggested to be associated with diseases that have oxidative stress in their pathogenesis. ^23–26 As mediators of protection against oxidative stress, GSTM1 and GSTT1 serve as excellent candidates for studies of ARC. Accordingly, several recent studies have investigated the influence of the presence or absence of GST gene deletions on human cataract with controversial results. ^27–30 By measuring protein level of GSTM1, GSTM1-present phenotype was reported to be associated with the development of cortical cataract in Estonians. ²⁸ However, the GSTM1-null genotype or a combination of the GSTM1-null and GSTT1-present genotypes were found to be associated with increased risk of cataract development in a female Turkish population. ³¹ Results in another study in an Italian population showed no association between GSTM1 CNV and cataract. ²⁸ A significant increase in GSTM1-null genotype frequency was observed in ARC cases in comparison with healthy controls in a Chinese population. ²⁹ Conversely, in another study in a Chinese population, it was reported that the GSTM1-null genotype was not related to ARC. ³⁰ These discrepancies may be partially explained by the fact that most studies were designed as hospital-based case–control studies that might have population stratification. Moreover, all studies were conducted by using traditional CNV assays that were unable to discriminate between carriers of one, two, or more than two gene copies. Another reason of the inconsistent conclusions may be the small sample sizes in some studies, which failed to afford sufficient power for statistically significant results. 

Quantitative real-time PCR assays have been recently devised to determine CNV. The method can discriminate between carriers of one or multiple GSTM1 and GSTT1 alleles with RNaseP as the endogenous calibrator beside the homogenous deletion of 2 alleles. ^25,32,33 In this study, we recruited subjects with ARC and without ARC from a population-based prospective cohort in the Nantong area of Eastern China that was of sufficient size. We tested the hypothesis that CNV in GSTM1 and GSTT1 may contribute to ARC susceptibility in the Han Chinese population. 

Methods

Study Subjects

The research adhered to the tenets of the Declaration of Helsinki. All participants signed the respective informed consent forms. The study was approved by the Ethics Committee of Nantong University. 

The study was a part of the Nantong Eye Study, a population-based cohort study that focuses on common eye diseases and health-related parameters among 57,843 urban residents with 6,421 people older than 60 years according to the census data from 2000. The covered area of the Nantong Eye Study has a stable and ethnically homogenous population. The participants have been followed up for 10 years. The design and initial results of the study have been published. ^34,35 The study participants were unrelated, self-identified Han Chinese (at least all four grandparents were ethnically Han Chinese). 

ARC was defined as the appearance of the clinical sign of cataract in one or both eyes in a person older than 50 years. ³⁶ The clinical sign was characterized as opacity of the lens resulting in visual acuity of 20/40 or worse measured with Early Treatment of Diabetic Retinopathy Study (ETDRS) chart. The patients with systemic diseases, a history of ocular trauma, secondary cataract due to glaucoma, age-related macular degeneration, uveitis, or diabetes and other known causes were excluded. We paid particular attention to the glycometabolic status of all participants and excluded those with abnormal blood glucose levels (fasting blood glucose >6.05 mmol/L) because a disorder of the galactose metabolic pathway was reported to be associated with cataract formation in an East Asian population. ³⁷ The average fasting blood glucose levels are presented in Table 1. All patients with ARC and control subjects underwent a full ophthalmic examination, including visual acuity, lens examination in transient and side illumination with a slit lamp biomicroscope after mydriasis and ophthalmoscopic examination. Lens photographs from all participants were recorded by the same ophthalmologist at the same dark room. The opacity of the lens was classified using the lens opacities classification system II (LOCS II). ³⁸ The grading was conducted by comparing slit lamp observations to standard LOCS II photos. The grading was later cross-checked by the group leader by comparing lens photographs with the same standard photos. We included only the cases with grades ≥NII of nuclear cataract, ≥CII of cortical cataract, or ≥PI of posterior subcapsular cataract. Individuals with a mixture of subtypes in one both eyes were excluded. The case group consisted of 279 ARC patients, among whom there were 131 with cortical cataract, 70 with nuclear cataract, and 78 with posterior subcapsular cataract. 

Table 1.

 

Demographics of Study Participants

Group	n	Sex		Age (y)		Smoking n (%)	Drinking n (%)	Hypertension n (%)	Blood Glucose (mmol/L)
		Male n (%)	Female n (%)	Mean ± SD	Range
Control	145	72 (49.7)	73 (50.3)	64.5 ± 4.2	59–82	32 (22.1)	40 (27.6)	38 (26.2)	4.9 ± 3.1
ARC	279	103 (36.9)*	176 (63.1)*	69.0 ± 8.4	50–92	89 (31.9)*	51 (18.3)*	109 (39.1)*	5.1 ± 4.0

*   P > 0.01 and <0.05 in the comparisons between the case and control groups.

The control group included 145 unrelated individuals with transparent lenses and visual acuity better than 20/25 in both eyes. The opacities in the lenses of both eyes corresponded to a grade of ≤N0 of the nuclear area, ≤Ctr of the cortical area, and ≤P0 of the posterior subcapsular area, according to LOCS II. The individuals with other major eye diseases such as dislocated lenses, glaucoma, age-related macular degeneration, diabetic retinopathy, uveitis, or systemic diseases such as diabetes, kidney diseases, and cancers were excluded from the control group. The cases and controls were matched as closely as possible for age and other factors. The demographic information for the study participants is summarized in Table 1. 

Blood glucose level, blood pressure, and liver and renal function were obtained and chest x-rays were performed in all subjects. Whole-blood samples were collected from every participant for genomic DNA extraction. 

Purification and Measurement of DNA

Genomic DNA was isolated from 200 μL of peripheral blood (QIAamp DNA Blood Mini Kit; Qiagen, Hilden, Germany) according to the manufacturer's protocol, eluted in a total volume of 200 μL, and stored at −20°C. ³⁹ DNA concentrations were measured by UV absorbance (Gene Quant 100; Applied Biosystems [ABI], Foster City, CA). The samples from the cases and controls were collected during the same time period, and they were processed and stored using the same methods. 

Copy Number Determination by Relative Quantitative Real-Time PCR

All assay reagents were purchased from ABI. The primers and 6-FAM-labeled probes that were used to amplify GSTM1 and GSTT1 are listed in Table 2. The RNaseP control kit containing VIC-labeled probes and gene-specific primers was used as an endogenous control. The amplification of RNaseP and the GST genes in the same sample normalized the differences in DNA concentration between samples and ensured that no false-negative GST*0/0 genotypes due to PCR or pipetting failure or insufficient DNA concentrations in the original sample were generated. The initial step of the PCR reaction was set at 50°C for 2 minutes, and the denaturation step was performed at 95°C for 10 minutes. The amplification was performed for 40 cycles at 95°C for 15 seconds, and at 60°C for 1 minute. PCR was performed (model 7500 PCR system; ABI) in duplicate in 96-well plates with a final sample volume of 10 μL, including 1× universal PCR master mix (TaqMan; ABI), 25 ng DNA, and proper dilutions of probes/primers according to the manufacturer's instruction. Data were then collected (Absolute Quantification; SDS software, ver. 1.3.1, SDS, Cary, NC). 

Table 2.

 

Primers and Probes for the Quantization of the Target Gene Copy Numbers by Real-Time PCR

Gene	PCR Primers	Hybridization Probes	Amplicon Size (bp)
GSTM1	5′-CACCTGCATTCGTTCATGTGAC-3′	6FAM-TTCAGTCCTGCCATGAGCAGGCACA-TAMRA	81
	5′-CACCTGCATTCGTTCATGTGAC-3′
GSTT1	5′-AAGTCCCAGAGCACCTCACCTCC-3′	6FAM-CACCATCCCCACCCTGTCTTCCA-TAMRA	82
	5′-CAGTGTGCATCATTCTCATTGTGGC-3′

Copy number estimation was conducted by the ΔCt method (CopyCaller software, ver. 1.0; ABI). The detection of RNaseP, known to exist only in two copies in a diploid genome, was used as the calibrator to estimate the copy number of GSTM1 and GSTT1. ^32,33,40 The calculation was performed by a maximum-likelihood algorithm built into the software. 

Statistical Analysis

A χ² test was performed (Stata 8.0; Stata Corp., College Station, TX) to compare the genotype distributions of CNV between the cases and controls and estimate the odds ratio (OR) and 95% confidence interval (CI) of the comparison. P < 0.05 was considered to be statistically significant. 

Results

The assay afforded 100% call rates and 100% call accuracy (consistency of duplicate wells) for both genes. The readings of the genotypes were randomly distributed across the plates, thus excluding the possibility of batch effects. All samples with null genotypes showed a Ct value >39 for the target genes, whereas the Ct of RNaseP was ∼27. The mean ΔCt values for GSTT1*1/0, GSTT1*1/1, and GSTT1*>1/1 were 0.125169, −0.78088, and > −1.44014, respectively. The mean ΔCt for GSTM1*1/0, GSTM1*1/1, and GSTM1*>1/1 was −0.13458, −0.98676, and > −1.69163, respectively. 

The genotype distributions and ORs of GSTM1 and GSTT1 in patients with cataracts and control individuals are summarized in Table 3. The GSTM1 copy numbers were evenly distributed in the cases and controls. However, a significantly higher percentage with double deletion for GSTT1 (GSTT1*0/0 null) was found in the cataract patients in comparison with the control (P < 0.05). The GSTT1 null genotype conferred a significantly higher risk of developing ARC (OR = 1.56). Further analysis of the individuals with at least one GSTT1 allele deleted also revealed a significant impact on ARC risk (OR = 2.16, P < 0.01; Table 3). 

Table 3.

 

Association between GST Genotypes and Cataract Development

Gene/Copy Number	Control n (%) 145 (100)	Case n (%) 279 (100)	P	OR (95% CI)
GSTM1
0/0	95 (65.5)	171 (61.3)	0.39*	0.83 (0.54–1.29)
1/0	28 (19.3)	67 (24.0)	0.47†	0.78 (0.39–1.54)
1/1	14 (9.7)	28 (10.0)
>1/1	8 (5.5)	13 (4.7)	0.70*	0.84 (0.31–2.39)
GSTT1
0/0	60 (41.4)	146 (52.3)	0.03*	1.56 (1.02–2.38)
1/0	54 (37.2)	100 (35.9)	0.07†	0.57 (0.32–1.04)
1/1	20 (13.8)	23 (8.2)
>1/1	11 (7.6)	10 (3.6)	0.07*	0.45 (0.17–1.21)
0/0 + 1/0	114 (78.6)	246 (88.2)	0.004†	2.16 (1.27–3.67)

*  Compared with the remaining three genotypes combined.

†  Compared with 1/0 + >1/1 genotypes combined.

After the stratification of the cases to the cortical, nuclear, and posterior subcapsular subtypes, no association was detected between the GSTT1 genotype and the nuclear type of ARC (Table 4). Consistent associations were observed between the GSTT1 genotype and the cortical type of ARC. The individuals with at least one deleted GSTT1 allele had a 4.81-fold increased risk of cortical ARC (P < 0.001), whereas individuals with GSTT1 >1/1 had a lower risk of the disease (OR = 0.19, P < 0.05; Table 4). There was no consistent association between GSTT1 CNV and posterior subcapsular cataract (Table 4). 

Table 4.

 

Association between GSTT1 Genotypes and the Subtypes of ARC

Copy Number	Control n (%) 145	Cortical			Nuclear			Posterior Subcapsular
		n (%) 131 (100)	OR (95% CI)	P	n (%) 70 (100)	OR (95% CI)	P	n (%) 78 (100)	OR (95% CI)	P
0/0	60 (41.4)	67 (51.2)	1.48 (0.92–2.39)	0.10*	33 (47.2)	1.26 (0.68–2.33)	0.42*	46 (59.0)	2.04 (1.16–3.56)	0.01*
1/0	54 (37.2)	57 (43.5)	4.98 (1.80–13.53)	0.0002**	21 (30.0)	0.80 (0.37–1.75)	0.31**	22 (28.2)	1.34 (0.57–3.19)	0.5**
1/1	20 (13.8)	5 (3.8)			12 (17.1)			6 (7.7)
>1/1	11 (7.6)	2 (1.5)	0.19 (0.04–0.87)	0.018*	4 (5.7)	0.74 (0.23–2.41)	0.61*	4 (5.1)	0.66 (0.20–2.14)	0.48*
0/0 + 1/0	114 (78.6)	124 (94.7)	4.81 (2.04–11.37)	0.0001**	54 (77.2)	0.92 (0.46–1.82)	0.81**	68 (87.2)	1.85 (0.85–4.01)	0.12**

*  Compared with the remaining three genotypes combined.

†  Compared with 1/0 + >1/1 genotypes combined.

Discussion

In this study, we found an association between CNV in GSTT1, but not in GSTM1, and ARC susceptibility. This result was not in agreement with previous studies which reported a more significant impact for CNV in GSTM1. Moreover, the GSTT1 association with ARC was more closely linked to the cortical type of the disease. The novel genotyping method adopted in this study discriminated between carriers of one or multiple copies of the genes, which provided more insight into the impact of CNVs in GSTT1/GSTM1 on ARC susceptibility. Our data show that GSTT1 >1/1 was protective against the development of the cortical type of ARC. 

Even though we tried to balance the possible confounding factors between the cases and controls, significant differences remained between the sexes, smoking, drinking, and hypertension. However, there was no report on the independent association of GST CNV with the individual's sex, drinking, or hypertension. There was also no report on the predisposition to smoking addiction in the presence of GST CNV, even though smoking often plays a role as a modifying factor in the association of GST CNV and various tumors. ^41,42 Therefore, we believe that it is impossible for these imbalances to seriously distort the major conclusions of the study. 

Information on CNVs in different ethnic groups and subpopulation groups is seldom available. We anticipate that significant differences in the distribution of CNVs exist from population to population, mirroring the differences in the distribution of single nucleotide polymorphisms (SNPs), another common form of DNA sequence variation. The differences in CNV between ethnic groups could be one explanation for the discrepancy in the reported results on GST–ARC association. The minimum age of the control subjects in this study was 50 years, with an average age of 64.5 years. One characteristic of the residents older than 50 years in this geographic area is that they are less migratory and that they are the offspring of people who have lived here for generations due to unique geographic features, including the long distance from major transportation routes, geographic isolation due to rivers, and a conservative ideology. Because the study provided detailed CNV information for two genes in an ethnically homogenous population, it can serve as a reference for similar studies. Moreover, the data from a population-based study can minimize the selection bias. 

In Caucasians, enzymatic activity of GSTM1 and GSTT1 is absent in approximately 53.1% and 19.7% of the population, respectively. ⁴³ The frequencies of homozygous deletions for GSTM1 and GSTT1 in a Korean population are 58.3% and 54.3%, respectively, ⁴⁴ whereas the frequencies in a Japanese population are 51.3% and 54.0%, respectively. ⁴⁵ We observed that 62.7% and 48.6% of the Han Chinese were homozygous for the GSTM1 and GSTT1 deletions, respectively. The frequency of the GSTT1 null genotype in the Han Chinese in our study was closer to that of the Japanese and Korean populations, but different from the Caucasian population. 

CNV in the human genome and its potential role in contributing to diseases has been gaining attention. ^21,46–49 In general, a gain in copy number results in increased gene expression. GSTs are important enzymes in the induction of endogenous and exogenous compounds. ^40,50,51 They catalyze the nucleophilic addition of the thiol of GSH to many possibly harmful compounds, making them water-soluble so that they may be discharged more easily. ⁵² Oxidative damage of the lens is the major risk factor for the development of ARC. ^5,10,11 The high GSH in the lens is believed to preserve the proper biological functions of thiols in structural proteins and enzymes, to detoxify xenobiotics, and thus to protect the lens from oxidative damage. ⁵³ A significant decrease was recorded in GST activity in cataract lenses compared with age-matched normal lenses. ⁵⁴  

In this study, we found that CNV with GSTT1 had a consistent association with cortical cataract but not other types of cataract. This finding may relate to the distinct distribution of GST activity in lens. The highest level of GST activity was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. ⁵⁴  

In conclusion, a clear association was established between the GSTT1 genotype and cataract susceptibility in a Han Chinese population. The association between GSTT1 CNV and ARC is more closely linked to cortical cataract. Individuals having at least one GSTT1 allele deleted will have a 4.81-fold increased risk of this subtype of the disease, whereas individuals with more than two copies of the GSTT1 allele will have a 5.26-fold (OR = 0.19) increased protection against the disease. 

Footnotes

 Supported by the Research Program of Nantong University and by the Key Medical Laboratory Program of Jiangsu Province, China.

Footnotes

 Disclosure: J. Zhou, None; J. Hu, None; H. Guan, None