Lack of Association between Interleukin-1 Gene Cluster Polymorphisms and Glaucoma in Chinese Subjects

Abstract

purpose. A recent study identified single nucleotide polymorphisms (SNPs) within the IL-1 gene cluster at chromosomal locus 2q13 that were associated with reduced risk for primary open-angle glaucoma (POAG) in whites. The purpose of this study was to investigate the association between IL-1 SNPs and glaucoma in Chinese patients with either POAG or primary-angle closure glaucoma (PACG).

methods. Patients with POAG with a mean IOP without treatment that was consistently <21 mm Hg on diurnal testing were classified as having normal-tension glaucoma (NTG) and those with higher IOP were classified as having high-tension glaucoma (HTG). Subjects with PACG had at least 180° of angle closure on gonioscopy. Genotypes were determined by polymerase chain reaction and restriction digest enzymes at the following loci: IL1A (−889C/T), IL1B (+3953C/T), and IL1B (−511C/T). The association of individual SNPs with glaucoma was evaluated by using χ² testing. Haplotype analysis was performed with the PHASE program, with haplotype frequency estimated for combined cases and controls, assuming Hardy-Weinberg equilibrium (HWE) of haplotypes.

results. Of the Chinese subjects studies, 194 had POAG (94 NTG and 100 HTG), 125 had PACG, and 79 were normal control subjects. There was no significant difference in IL-1 SNP or allele frequencies for in subjects with POAG or PACG compared with control subjects, or between NTG and HTG. None of the common haplotypes showed any significant difference between the HTG, NTG, PACG, and normal control subjects.

conclusions. This study did not find an association between IL-1 gene cluster polymorphisms and glaucoma in this sample of Chinese subjects.

Glaucoma affects approximately 70 million people and represents a leading cause of irreversible blindness worldwide. ^{1 2} In an aging population, an increase in glaucoma morbidity can be expected. This probability has important public health implications, as once visual loss from glaucoma occurs, it cannot be reversed. Primary open-angle glaucoma (POAG) is the main form of glaucoma in Western countries in contrast to Asian countries, where primary angle-closure glaucoma (PACG) is the major type of the disease. ³  

Glaucoma has a substantial heritable basis, as illustrated by the large proportion of patients with a positive family history, and the many loci and glaucoma-causing genes identified to date. ⁴ Genetic mechanisms found to induce open-angle and developmental glaucomas include coding mutations, particularly in transcription factors ⁵ ; altered gene dosage ⁶ ; and dominant negative effects. ⁷ An increase in intraocular pressure (IOP) is the major risk factor for development of glaucoma, and the risk of progression of optic nerve damage increases exponentially with increasing pressure. ^{8 9}  

The role of interleukin (IL)-1, an important mediator of inflammation, in glaucoma is a subject of recent interest. It has been shown that IL-1 mediates a stress response activated endogenously by trabecular meshwork cells and confers a protective response against glaucoma. ¹⁰ There are two proinflammatory IL-1 cytokines, IL-1α and IL-1β, and the genes encoding IL-1 are located within a 430-kb region on chromosome 2 at q14.2 (Wang CY et al. IOVS 2005;46:ARVO E-Abstract 2369). ^{11 12}  

A recent study identified single nucleotide polymorphisms (SNPs) within the IL-1 gene cluster at chromosomal locus 2q13 that were associated with reduced risk for POAG in whites (Wang CY et al. IOVS 2005;46:ARVO E-Abstract 2369). Three SNPs were investigated (IL1A −889C/T, IL1B+3953C/T, and IL1B −511C/T), and a single SNP (IL1B +3953) as well as a haplotype within the IL-1 gene cluster were found to be more common in normal control subjects than in those with POAG. 

We wanted to investigate the role of these IL-1 SNPs, to establish a link between inflammation and genetics in relation to the pathogenesis of glaucoma in Chinese patients. We also evaluated whether there were differences in the distribution of these SNPs in relation to the two main forms of glaucoma, POAG and PACG, and in normal tension glaucoma (NTG), a subtype of POAG associated with normal IOP. 

Methods

Patient Recruitment and Assessment

Study subjects were recruited from clinics at the Singapore National Eye Centre, a tertiary ophthalmology referral center. Written, informed consent was obtained, and the study had the approval of the Ethics Committee of the Singapore Eye Research Institute/Singapore National Eye Centre and was conducted in compliance with the tenets of the Declaration of Helsinki. 

Subjects had to be 60 years of age or older and underwent a complete ophthalmic examination that included best corrected Snellen visual acuity, gonioscopy, IOP measurement by Goldmann applanation tonometer, slit lamp examination of the anterior segment, and lens and dilated slit lamp biomicroscopy of the vitreous and retina. Patients were also subjected to Humphrey SITA standard 24-2 perimetry (Carl Zeiss Meditec AG, Jena, Germany) and Heidelberg Retinal Tomography (HRT; Heidelberg Engineering, Dossenheim, Germany) if not previously performed. 

Standardized inclusion criteria for glaucoma were used, including the presence of glaucomatous optic neuropathy (defined as loss of neuroretinal rim with a vertical cup-to-disc ratio of ≥0.7) with compatible visual field loss. PACG was defined as the presence of at least 180° of angle in which the trabecular meshwork was not visible on gonioscopy, along with the presence of glaucoma. Patients with POAG had open angles on gonioscopy, and those with a mean IOP without treatment that was consistently <21 mm Hg on diurnal testing were classified as having NTG, whereas those with a mean IOP without treatment that was consistently >21 mm Hg on diurnal testing were classified as having high-tension glaucoma (HTG). All normal control subjects had IOP of <21 mm Hg, normal optic nerve heads with cup-to-disc ratios of 0.5 or less, normal HRT, no other ocular disease or previous surgeries, and no family history of glaucoma. 

DNA Preparation and Genotyping

Ten milliliters of blood was obtained from each patient by venipuncture. Genomic DNA was extracted from leukocytes of the peripheral blood (Nucleon DNA Extraction kits; Tepnel Life Sciences, Manchester, UK). We performed genotyping for the IL-1 gene cluster in three polymorphic loci to determine the presence of single nucleotide. The IL-1α (−889C/T), IL-1β (−511C/T), and IL-1β (+3953C/T) variants were detected by specific polymerase chain reaction (PCR; using primers in Table 1 ) with a DNA thermocycler (model 9700; Applied Biosystems, Foster City, CA). 

PCR reactions were performed in 50-μL reaction volumes containing 10 mM Tris HCl (pH 8.9), 50 mM KCl, 1.5 mM MgCl₂, 25 picomoles of each primer, 200 μM each dNTP, 50 to 100 ng of patient genomic DNA and 0.7 units of Taq thermostable DNA polymerase (Promega, Madison, WI). Cycling parameters were 3 minutes at 95°C, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at the annealing temperature (T_m) of the primers (58–60°C), and 30 seconds at 72°C with a final 5-minute extension at 72°C. The PCR products for IL-1β (−511C/T) and IL-1β (+3953C/T) were then subject to restriction enzyme digestion, to determine the presence of polymorphisms. 

The PCR products were incubated at the optimal temperature for enzyme digestion for 8 hours and then separated on 2% agarose gels. The separation of the DNA products were visualized by staining with ethidium bromide, and the size of the expected DNA products was compared against a DNA ladder (Hyperladder IV; Bioline, London, UK) which was run in an adjacent gel lane. Confirmation of polymorphisms at these two loci was performed by direct sequencing. 

The IL-1α (−889) PCR products were not subject to restriction enzyme digest but were sequenced. The PCR products were purified on PCR columns (GFX purification column; GE Healthcare, Amersham, UK). Sequence variations were identified by automated bidirectional sequencing (BigDye terminator ver. 3.1; Applied Biosystems) on an automated DNA sequencer (Prism 3100; Applied Biosystems). 

Statistical Analysis

Genotype and allele frequencies between glaucoma groups and control subjects were compared by using χ² and Fisher exact tests. Statistical significance was defined as a P < 0.05. Statistical analyses were performed with commercial software (SPSS 11.5; SPSS Inc., Chicago, IL). 

Haplotype analysis was performed by the PHASE program ¹³ (developed by Matthew Stephens, Department of Statistics, University of Washington, Seattle, WA, and provided by the in the public domain at www.stat.washington.edu/stephens/software.html/), with haplotype frequency estimated for combined cases and controls assuming Hardy-Weinberg equilibrium (HWE) of haplotypes. Haplotype dosages were then included as independent variables in the logistic regression models to test for association between common haplotypes and different phenotypes. These analyses were performed with commercial software (Stata SE8 Statistical Software; Stata Corp., College Station, TX). 

Results

For this study, 194 patients with POAG (100 with HTG and 94 with NTG), 125 patients with PACG, and 79 normal control subjects were recruited. All the patients were of Chinese descent. There was no difference between the normal control group and the patients with glaucoma in gender and age, as shown in Table 2 . 

The distribution of the allele frequency and genotype frequency of IL-1 SNPs in patients with HTG, NTG, or PACG compared with control subjects are shown in Tables 3 and 4 , respectively. Although the frequency of the IL-1α (−889T) allele was higher in the control group (10.8%) than the in POAG (7.0%) or PACG (6.8%) groups, the difference was not statistically significant. The distributions of the other two polymorphisms, IL-1β (−511) and IL-1β (+3953), were not significantly different between the HTG, NTG, PACG, and normal control groups, or between HTG and NTG. 

Analysis showed that all groups were in HWE. Haplotype analysis was also performed on IL-1α (−889), IL1β (+3953), and IL1β (−511) together, and the results are shown in Table 5 . None of the common haplotypes showed any significant difference between the HTG, NTG, PACG, and normal control groups. 

Discussion

Molecular mechanisms giving rise to glaucomatous disease are complex and have yet to be understood. SNPs of the IL-1 gene cluster have been shown to alter protein expression and have been linked with other diseases such as pre-eclampsia, end-stage renal disease, systemic sclerosis, and rheumatoid arthritis. ^{14 15 16} There are two proinflammatory cytokines, IL-1α and IL-1β, that are produced by monocytes, macrophages, and epithelial cells as host responses to tissue injury. Wang et al. (IOVS 2005;46:ARVO E-Abstract 2369) found that IL-1 produced endogenously by glaucomatous cells inhibits the apoptotic response to oxidative stress. IL-1 has also been reported to increase outflow facility by stimulating the expression of matrix metalloproteinase enzymes, which in turn reduces extracellular resistance. ^{17 18 19 20} The effect of IL-1 on the synthesis of nitric oxide causing a relaxation in ciliary muscle tone may also contribute to increasing aqueous outflow. ²¹ It has been shown that IL-1β SNPs are associated with increased secretion of IL-1β, and it has therefore been suggested that IL-1 SNP confers a protective effect against glaucoma, either at the outflow facility level or at the optic nerve head. ²²  

In the recent study by Wang et al. (IOVS 2005;46:ARVO E-Abstract 2369) the three polymorphic loci were selected based on the fact that the IL-1α (−889C/T) and IL-1β (−511T/C) sites are located within the transcriptional promoter regions of the IL-1α and IL-1β genes, respectively, and the IL-1β (+3953C/T) site is within the coding region of the IL-1β gene. Their study found that IL-1 SNPs reduce the risk of POAG in whites (Wang CY et al. IOVS 2005;46:ARVO E-Abstract 2369). The TT haplotype of ILB (+3953) and IL1B (−511) together and the TTT haplotype of IL1A (−889), IL1B (+3953) and IL1B (−511) was significantly more common in normal control subjects than in those with POAG. 

In this study, we sought to investigate the role of these IL-1 SNPs, to establish a link between inflammation and genetics in relation to glaucoma pathogenesis in Chinese patients. We also hoped to confirm that IL-1 SNPs confer a protective effect against glaucoma in Chinese subjects, but we were unable to duplicate these findings. Our study’s strengths were that we analyzed the two major glaucoma types, POAG and PACG, and we also stratified the POAG population into those with normal IOP and those with high IOP. Our results demonstrated that polymorphisms in the IL-1β (−511T/C) gene loci were not protective against glaucoma in patients in our study population. 

Of interest, the IL-1β (+3953T) allele found to be protective in the white population was found to occur at a much lower frequency in this Singaporean Chinese population. In addition, the frequency of the T allele in the loci IL-1α (−889C/T) and IL-1β (+3953C/T) in the white population studied by Wang et al. (IOVS 2005;46:ARVO E-Abstract 2369) was much higher. Thus, although there was no significant difference between the control group and the glaucoma groups for the two loci IL-1α (−889C/T) and IL-1β (+3953C/T), we were unable to conclude that there is no association, as the frequency of the T allele in these two regions was low in our sample of Chinese subjects. To confirm whether there is a true absence of association, a much larger sample of the population would have to be investigated. Lin et al. ²³ also found an opposing effect of IL-1β SNP on Chinese patients with glaucoma in Taiwan. The Taiwanese study, however, involved only a small Chinese cohort of 58 patients with POAG and 105 healthy volunteers. Taking this into account together with our findings, it seems likely that there are other factors in the role of IL-1 in glaucoma that may include environmental, racial, and ethnic influences. Population diversity may also explain the differences in the results of these studies. 

To date, there have been limited studies identifying the genetic risk variants for glaucoma. Whereas association studies are appropriate for identifying interactions between environmental and genetic variables, there are frequently differing results between different groups of patients. ²⁴ Hence, it is not unusual that our study did not find the same association as the ones by Wang et al. (IOVS 2005;46:ARVO E-Abstract 2369) and Lin et al. ²³ There is a need for association studies to be repeatedly replicated and further confirmed by meta-analyses. 

In summary, our study could not replicate the finding of the protective effect of the IL-1β gene polymorphisms for glaucoma in our Chinese patients. There were also no differences in the distribution of polymorphisms of the IL-1 gene cluster in patients with NTG versus HTG. Our results may reflect the genetic variability and the varying effects of single gene polymorphisms on different ethnic groups with the same disease. Further research should be performed to investigate the role of the immune system in IOP response and glaucoma pathogenesis. 

 Supported by a grant from the Singapore Eye Research Institute (SERI). TA is supported by the National Medical Research Council, Singapore.

 Submitted for publication October 10, 2006; revised December 28, 2006, and January 12, 2007; accepted March 8, 2007

 Disclosure: A.C.S. How, None; T. Aung, None; X. Chew, None; V.H.K. Yong, None; M.C.C. Lim, None; K.Y.C. Lee, None; J.-Y. Toh, None; Y. Li, None; J. Liu, None; E.N. Vithana, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Primer Sequences, PCR Conditions, and Enzymes for the Polymorphic Loci

Polymorphism	Primer Sequence	Annealing Temp. (°C)	Enzyme and Temp. (°C)	Expected DNA Products (bp)
IL-1α (−889 C/T)	Forward
	5′-GCATGCCATCACACCTAGTT-3′	58	NA	NA
	Reverse			NA
	5′-TTACATATGAGCCTTCCATG-3′
IL-1β (−511 T/C)	Forward
	5′-TGGCATTGATCTGGTTCATC-3′	60	Bsu361,37	C:304
	Reverse			T:190,114
	5′-GTTTAGGAATCTTCCCACTT-3′
IL-1β (+3953 C/T)	Forward
	5′-GTTGTCATCAGACTTTGACC-3′	60	TagI, 60	C:135,114
	Reverse			T:249
	5′-TTCAGTTCATATGGACCAGA-3′

Table 2.

 

Demographic Features of Patients with Glaucoma and Control Subjects and the Vertical Cup-to-Disc Ratio of Patients with Glaucoma in the Study

	Control	HTG	NTG	PACG
Mean age (y)	67.7	68.2	72.9	64.1
SD	4.7	12.3	8.5	10.9
Minimum age	61	43	52	35
Maximum age	86	98	89	88
Male (%)	40.5	74.7	67.9	45.5
Mean vertical cup- disc ratio (± SD)	NA	0.84 ± 0.18	0.82 ± 0.12	0.89 ± 0.14

Table 3.

 

Allele Frequency of IL-1 SNPs in Patients with HTG, NTG, or PACG, Compared with Control Subjects

	Patient	Control	P
HTG
IL1A (−889)
C	186 (93.0%)	141 (89.2%)	0.22
T	14 (7.0%)	17 (10.8%)
IL1B (+3953)
C	194 (97.0%)	156 (98.7%)	0.47*
T	6 (3.0%)	2 (1.3%)
IL1B (−511)
C	116 (58.0%)	90 (57.0%)	0.84
T	84 (42.0%)	68 (43.0%)
NTG
IL1A (−889)
C	159 (89.3%)	141 (89.2%)	0.98
T	19 (10.7%)	17 (10.8%)
IL1B (+3953)
C	187 (99.5%)	156 (98.7%)	0.59*
T	1 (0.5%)	2 (1.3%)
IL1B (−511)
C	92 (48.9%)	90 (57.0%)	0.14
T	96 (51.1%)	68 (43.0%)
PACG
IL1A (−889)
C	233 (93.2%)	141 (89.2%)	0.16
T	17 (6.8%)	17 (10.8%)
IL1B (+3953)
C	245 (98.0%)	156 (98.7%)	0.71*
T	5 (2.0%)	2 (1.3%)
IL1B (−511)
C	144 (57.6%)	90 (57.0%)	0.90
T	106 (42.4%)	68 (43.0%)

*  Fisher exact test.

Table 4.

 

Genotype Frequency of IL-1 SNPs

	Patients, n (%)	Control	P
HTG
IL1A (−889)
CC	87 (87.0%)	64 (81.0%)
CT	12 (12.0%)	13 (16.5%)	0.37
TT	1 (1.0%)	2 (2.5%)	0.58*
IL1B (+3953)
CC	94 (94.0%)	77 (97.5%)
CT	6 (6.0%)	2 (2.5%)	0.47*
TT	0 (0%)	0 (0%)	—
IL1B (−511)
CC	35 (35.0%)	24 (30.4%)
CT	46 (46.0%)	42 (53.1%)	0.40
TT	19 (19.0%)	13 (16.5%)	1.00
NTG
IL1A (−889)
CC	71 (79.8%)	64 (81.0%)
CT	17 (19.1%)	13 (16.5%)	0.69
TT	1 (1.1%)	2 (2.5%)	0.61*
IL1B (+3953)
CC	93 (98.9%)	77 (97.5%)
CT	1 (1.1%)	2 (2.5%)	0.59*
TT	0 (0%)	0 (0%)	—
IL1B (−511)
CC	21 (22.3%)	24 (30.4%)
CT	50 (53.2%)	42 (53.1%)	0.40
TT	23 (24.5%)	13 (16.5%)	0.12
PSCG
IL1A (−889)
CC	110 (88.0%)	64 (81.0%)
CT	13 (10.4%)	13 (16.5%)	0.20
TT	2 (1.6%)	2 (2.5%)	0.63*
IL1B (+3953)
CC	120 (96.0%)	77 (97.5%)
CT	5 (4.0%)	2 (2.5%)	0.71*
TT	0 (0%)	0 (0%)	—
IL1B (−511)
CC	42 (33.6%)	24 (30.4%)
CT	60 (48.0%)	42 (53.1%)	0.53
TT	23 (18.4%)	13 (16.5%)	0.98

*  Fisher’s exact test.

Table 5.

 

Haplotype Analyses

IL1A −889	IL1B +3953	IL1B −511	Haplotype Proportions			P
							Patient	Control (n = 79)
HTG (n = 100)
C	C	C	51.50%	49.37%	—
C	C	T	39.50%	39.24%	0.88
T	C	C	5.50%	6.96%	0.55
Rare*			3.50%	4.43%	0.64
NTG (n = 94)
C	C	C	46.81%	52.53%	—
C	C	T	43.09%	36.08%	0.21
T	C	T	7.98%	6.96%	0.46
T	C	C	1.60%	3.16%	0.50
Rare, †			0.53%	1.27%	0.51
PACG (n = 129)
C	C	C	52.00%	48.73%	—
C	C	T	40.80%	39.87%	0.89
T	C	C	3.60%	6.96%	0.12
Rare, ‡			3.60%	4.43%	0.72

*  Four rare haplotypes (TCT, CTC, CTT, and TTC) combined.

†  Four rare haplotypes (CTC, TTC, CTT and TTT) combined.

‡  Five rare haplotypes (TCT, TTC, CTC, CTT and TTT) combined. Each haplotype has a frequency below 4% among the combined cases and controls.