Interleukin Gene Polymorphisms in Age-Related Macular Degeneration

Abstract

purpose. To investigate polymorphisms in a candidate gene of interleukin (IL) in unrelated Taiwan Chinese patients with late age-related macular degeneration (AMD) and control subjects without AMD.

methods. In this retrospective, case-control study, 312 unrelated Taiwan Chinese patients with late AMD and 180 age- and sex-matched control subjects were enrolled. Late AMD was classified as either atrophic (dry) or neovascular (wet) according to the International ARM Epidemiologic Study. Genomic DNA was prepared from peripheral blood obtained from all patients with AMD and control subjects. Polymerase chain reactions were used to analyze 14 single-nucleotide polymorphisms (SNPs) in candidate genes of 5 ILs: IL-1β(2q14): −511 T/C; IL-6 (7p21): −572 C/G and −596 G/A; IL-8 (4q13-q21): −251 A/T, +781 C/T, +1633 T/C, and +2767 A/T; IL-10 (1q31-q32): −592 A/C, −819 C/T, and −1082 G/A; and IL-18 (11q22.2-q22.3): +105 A/C, −137 C/G, −607 A/C, and −656 T/G.

results. In the 312 patients with late AMD, dry AMD was diagnosed in 136 and wet AMD in 176. Among the 14 SNPs in the 5 IL genes studied, only the IL-8 +781 C/T SNP was significantly associated with wet AMD (T allele: 46% in wet AMD versus 28% in the control subjects, P = 1.03 × 10⁻⁶, OR = 2.16, 95% CI = 1.58–2.94). The association analysis based on genotypes at both IL-8 +781 C/T and the CFH Y402H demonstrated that the IL-8 +781 C/T to AMD was not significant when analyzed conditional on the presence of the CFH Y402H C risk allele and vice versa. The IL-8 +781 C/T was in low linkage disequilibrium with CFH Y402H (D′ = 0.02).

conclusions. The data suggest that Taiwan Chinese carriers of the IL-8 +781 T allele, independent of the CFH Y402H polymorphism, are at increased risk of developing wet AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among older individuals throughout the developed world. ^{1 2} More than 10 million people in the United States have AMD. ³ It occurs in less than 1% of people younger than 65, but increases dramatically with age, afflicting 9% of the population over 65 years and 30% over 75 years. ^{4 5 6} The condition is assuming greater importance among researchers because of the increasing aging population. It is estimated that by the year 2020, the number of individuals with AMD will have increased by 50%. ³  

AMD is a complex disease, and its etiology is largely unknown. According to clinical and pathologic features, ^{7 8} there are two subtypes of late AMD, the atrophic (dry) and neovascular (wet) forms, both of which can lead to significant visual loss. 

Previous studies assessing systemic C-reactive protein (CRP), an inflammation marker, have shown that inflammation plays a role in the pathogenesis and progression of AMD. ^{9 10} However, the relationship has been controversial. ^{11 12} Furthermore, consistent with the role of inflammation in AMD, several studies have identified a strong association between a common variant (Y402H) of the gene for complement factor H (CFH) and the risk of AMD. ^{13 14 15 16 17} Besides, several interleukins (IL-6, -8, and -10) have also been found to be associated with AMD. ^{9 12 18 19 20} In fact, interleukins help mediate many of the effector phases of immune and inflammatory responses. Genetic polymorphisms in several cytokine genes have been described and demonstrated to influence gene transcription, leading to interindividual variations in cytokine production. ^{21 22 23 24} Therefore, it is reasonable to speculate that genetic polymorphisms may be important determinants of pathogenesis of AMD. 

Complementing a genomic screen (e.g., location) approach is the direct testing of candidate genes proposed because their putative functions are related to the known AMD pathogenesis. Therefore, we analyzed 14 candidate SNPs in five IL genes that functionally influence transcription levels in previous studies: IL-1β(2q14): −511 T/C (promoter); IL-6 (7p21): −572 C/G (promoter) and −596 G/A (promoter); IL-8 (4q13-q21): −251 A/T (promoter), +781 C/T (intron 1), +1633 T/C (intron 3), and +2767 A/T (3′-untranslated); IL-10 (1q31-q32): −592 A/C (promoter), −819 C/T (promoter), and −1082 G/A (promoter); and IL-18 (11q22.2-q22.3): +105 A/C (exon), −137 C/G (promoter), −607 A/C (promoter), and −656 T/G (promoter). 

Materials and Methods

We recruited 312 unrelated Taiwan Chinese patients with late AMD and 180 age- and sex-matched control subjects from the outpatient clinic, Department of Ophthalmology, China Medical University Hospital. All the patients and control subjects were fully informed of the purpose and procedures of the study, and informed consents were obtained from all patients before they were enrolled, including the collection of blood samples. The Institutional Review Board of China Medical University Hospital approved this study, and the protocol adhered to the Declaration of Helsinki. 

All individuals received a standard examination protocol including comprehensive medical and ophthalmic history review, visual acuity, intraocular pressure measurement, slit-lamp biomicroscopy, dilated fundus photographs, and fluorescein angiography. All individuals were older than 60 years. The diagnosis of AMD was established on the basis of clinical examination, fundus photography, and fluorescein angiography. Fundus findings in each eye were classified based on a standardized set of diagnostic criteria established by the International ARM Epidemiologic Study. ⁷ Late AMD was classified as either dry (atrophic) or wet (neovascular). Individuals who presented with both neovascular and atrophic forms of AMD were classified as having neovascular AMD. Patients with severe macular diseases other than AMD, secondary choroidal neovascular (CNV) disease, such as ocular trauma, angioid streaks, degenerative high myopia, retinal detachment, chorioretinal infective or inflammatory processes, presumed ocular histoplasmosis, and cases of large cicatricial lesions were excluded from our study. Control subjects, who were age-matched volunteers without visual impairment, were recruited from the outpatient department during routine ophthalmic examinations. They did not have a family history of AMD and did not have any type of drusen, geographic atrophy, CNV, or other retinal disorder in either eye. Severe cataracts leading to impaired visualization of the macula excluded patients from recruitment. 

Genomic DNA was prepared from peripheral blood by a DNA extraction kit (WB Wako, Japan). Polymerase chain reactions (PCRs) for 14 polymorphisms in five IL genes (Table 1)were performed to a total volume of 50 μL, containing 50 ng genomic DNA; 2 to 6 picomoles of each primer; 1× Taq polymerase buffer (1.5 mM MgCI₂); and 0.25 U of DNA polymerase (AmpliTaq; Applied Biosystem, Inc. Foster City, CA). The primers, annealing temperature of PCR, and restriction enzymes used to reveal the genotypes are listed in Table 1 . PCR amplification was performed in a programable thermal cycler (GeneAmp PCR System 2400; Perkin Elmer, Waltham, MA). The cycling conditions were set as follows: one cycle at 94°C for 8 minutes, 30 cycles at 94°C for 30 seconds, appropriate annealing temperature for 30 seconds, and 72°C for 60 seconds, and one final cycle of extension at 72°C for 10 minutes. The PCR products were mixed with 2 U of restriction enzymes and the reaction buffer according to the manufacturer’s instructions. The cuttable and uncuttable genotypes and reaction temperatures are shown in Table 1 . The reaction was incubated for 2 hours at 37°C. Then, 10 μL of the products was loaded into a 3% agarose gel containing ethidium bromide for electrophoresis. 

The unpaired Student’s t-test and χ² test were used to assess the differences in the clinical baseline characteristics between groups. The χ² test was used to analyze the genotypes and allele frequencies between patients and control subjects and to test for Hardy-Weinberg equilibrium (HWE). When the assumption of the χ² test was violated (i.e., when one cell had an expected count of <1, or >20% of the cells had an expected count of <5), the Fisher exact test was used. Odds ratios with 95% confidence intervals were determined for disease susceptibility of specific genotypes and alleles in the polymorphism of the IL gene. Haplotypes and linkage disequilibrium (LD) were determined based on the expectation-maximization algorithm using the SNPStats program (available at http://bioinfo.iconcologia.net/SNPstats/ provided in the public domain by the Biostatistics and Bioinformatics Unit, Catalan Institute of Oncology, Barcelona, Spain). Because of the multiple comparisons conducted, the Bonferroni correction was applied and the probability was evaluated at the significance level of 0.025 for two comparisons (see Tables 2 and 3 ) and 0.017 for 3 comparisons (see 4 5 Table 6 ). 

Results

Clinical baseline characteristics of the patients with late AMD and the control subjects are shown in Table 2 . There was no significant difference in age, age distribution, and gender between groups (all P > 0.025). The distribution of other factors, such as smoking status, body mass index, blood pressure, hypertension, diabetes mellitus, cardiovascular disease, and hyperlipidemia, also did not differ significantly between groups (all P > 0.025). Of the 312 participants with late AMD, dry AMD was diagnosed in 136 patients and wet AMD in 176. 

For each IL gene polymorphism, we confirmed that the genotypes and allelic frequencies in the patients with AMD and the control subjects fit the HWE. 

Among the 14 SNPs in 5 IL genes (IL-1β: −511 T/C; IL-6: −572 C/G, and −596 G/A; IL-8: −251 A/T, +781 C/T, +1633 T/C, and +2767 A/T; IL-10: −592 A/C, −819 C/T, and −1082 G/A; and IL-18: +105 A/C, −137 C/G, −607 A/C, and −656 T/G), only IL-8 +781 C/T was significantly associated with wet AMD. Table 3showed the distribution of the IL-8 +781 C/T polymorphism between the patients with AMD and the control subjects. The genotypes and allele frequencies in those with dry AMD and the control subjects were not significantly different (P = 0.06 and 0.58, respectively). In those with wet AMD, both genotypes and alleles frequencies were significantly different when compared with those in the control subjects (P = 1.15 × 10⁻⁷ and 1.03 × 10⁻⁶, respectively). In those with wet AMD, the IL-8 +781T allele was significantly increased compared with control subjects (46% versus 28%, P = 1.03 × 10⁻⁶, OR = 2.16, 95% CI = 1.58–2.94). 

We next considered association analysis based on genotypes at both IL-8 +781 C/T and the CFH rs1061170 variant at chromosome 1, area q31 (Y402H, previously genotyped, Table 4 ). In a global two-locus analysis enumerating all nine two-locus genotype combinations, the association with AMD was significant (χ² = 63.35, 6 df, P < 0.0001). Table 5shows the risk estimates for each two-locus genotype combination compared with the baseline of no-risk genotypes (TT at CFH Y402H and CC at IL-8 +781 C/T). The association of IL-8 +781 C/T with AMD was not significant when analyzed conditional on the presence of the CFH C risk allele and vice versa. The SNPStats program predicted three different haplotypes from these two SNPs (Table 6) . The haplotype TT was significantly associated with wet AMD (P = 4.0 × 10⁻⁴, OR = 2.13, 95% CI = 1.38–3.29), whereas the haplotype CT was significantly associated with the control subjects (P = 5.07 × 10⁻⁶, OR = 2.70, 95% CI = 1.75–4.15). The normal control data set was used for analysis of LD. The IL-8 +781 C/T was in low LD with CFH Y402H (D′ = 0.02). 

Discussion

In this retrospective, case-control study, we examined 14 candidate SNPs of five IL genes to determine whether these polymorphisms are associated with an increased risk for either dry or wet AMD. We found that the presence of the IL-8 +781 T allele was significantly associated with wet AMD. Our data indicated that carriers of the IL-8 +781 T allele may have increased risk of wet AMD in the Taiwan Chinese population. 

Currently, it is not clear what role IL-8 plays in the pathogenesis of wet AMD. The IL-8 gene is located on chromosome 4, area q13-q21, and consists of four exons and three introns. ²⁵ Figure 1shows a representation of the IL-8 gene (74825139–74828297 bp; size, 3158 bases) and detailed positions of four SNPs in our study. IL-8 is a multifunctional cytokine and a member of the family of chemokines. It is involved in acute and chronic inflammatory processes. ²⁶ It also has been shown to have potent proangiogenic properties in vitro and in vivo, ^{27 28} and has been implicated in the formation of CNV in AMD. ^{18 19} Kalayoglu et al. ¹⁹ reported that both IL-8 and VEGF were detected in CNV from patients with AMD. Higgins et al. ¹⁸ found that ingestion by RPE of oxidized bovine photoreceptor outer segments stimulates the expression of IL-8. In reality, the process of CNV is a complex interplay between numerous stimulators and inhibitors. Therefore, we believe that many proangiogenic and angiogenic factors and/or the inflammatory stimuli that induce these factors should be involved. Further studies are needed to determine the exact role of IL-8 in the pathogenesis of CNV. 

In our study, we found that the IL-8 +781 T allele was significantly associated with wet AMD. Previous reports have demonstrated that the −251 A allele within the promoter was significantly associated with higher IL-8 levels and is itself a functional variant. ^{29 30 31} However, the relationship has been controversial. ^{32 33} Hull et al. ³⁰ found that the functional allele of IL-8 may lie on chromosomes of the −251A/+781T haplotype and −251A may not be functional. Currently, it is still unclear which one is the functional variant that directly affects IL-8 production and whether these SNPs are in linkage disequilibrium with a functional variant elsewhere in IL-8 or in a neighboring gene. Therefore, to investigate the functional significance of IL-8 +781 C/T polymorphism, we used enzyme-linked immunoassays (ELISAs) to measure the vitreous IL-8 concentration of six patients with wet AMD carrying the risk allele TT and five normal control subjects carrying the normal allele CC. Significantly higher vitreous levels of IL-8 were found in patients with wet AMD with the TT genotype than in normal control subjects with the CC genotype (57.12 and 7.82 pg/mL, respectively; P = 0.001; Fig. 2 ). Unfortunately, our functional study was limited because we were able to obtain only six AMD donor eyes with TT genotype and vitreous of five normal control subjects. Therefore, these data merely show a trend toward higher expression in the vitreous with the risk T allele. Further rigorous functional studies to investigate this question are warranted. 

A strong association between CFH and wet AMD has been shown in several studies including Taiwan Chinese. ^{14 15 16 17} Therefore, we reanalyzed our original CFH Y402H genotype data to evaluate its association with IL-8 +781 C/T. We found that both IL-8 +781 and CFH Y402H are independent SNPs associated with wet AMD. First, in two-locus analyses enumerating all nine two-locus genotype combinations, no interaction was detected (Table 5) . Second, the IL-8 +781 C/T was in low LD with CFH Y402H (D′ = 0.02). Besides, haplotype analysis of the two SNPs demonstrated that the haplotype TT was associated with wet AMD and the haplotype CT was associated with the control subjects. Closer inspection of two haplotypes at IL-8 +781 confirmed our findings: T allele is risk and C allele is protective for wet AMD. However, the risk-allele (C allele) of CFH Y402H was not shown in these two haplotypes. Besides, we found that the C allele of CFH Y402H was much lower in Asian populations. Chen et al. ³⁴ found the C allele was 5.8% in wet AMD and 3.9% in control subjects in a Chinese population in Hong Kong. In another Taiwan Chinese population study by Lau et al. ¹⁶ found that the C allele frequency was 11.3% in neovascular AMD and 2.8% in the control. The similar frequency was also noted in Japanese. ^{35 36} In our study, the C allele frequency was 11% in wet AMD and 4% in control (white population: 34%–61% in late AMD and 9%–39% in control subjects). ^{37 38 39 40 41} In fact, the low frequency may not allow the conclusion of genetic susceptibility in our Chinese population, because most AMD patients do not carry the risk allele. The genetic influence of Y402H SNP on AMD seems to be different between Chinese and white populations. 

Seddon et al. ⁹ found that a higher level of the systemic inflammatory marker IL-6 was independently associated with the progression of AMD. However, in our study, no specific SNP of the IL-6 gene was related to either dry or wet AMD. Because we did not measure serum IL-6 levels in the individuals in our study, we have no comment on their findings. 

In a recent animal study, Apte et al. ²⁰ found that IL-10 can suppress or inhibit CNV in AMD by regulating macrophage activity. They suggested that IL-10 is an attractive therapeutic target for wet AMD. In fact, IL-10 is an anti-inflammatory cytokine which is involved in downregulating cell-mediated and cytotoxic inflammatory responses. ⁴² However, in our study, no specific SNP of the IL-10 gene was associated with either dry or wet AMD. This finding may have resulted from our examining only three promoter polymorphisms (−592, −819, and −1082). Other functional variants or genetic factors may influence the expression of IL-10 in AMD. 

In summary, our data demonstrated a significant association between the IL-8 +781 C/T polymorphism and wet AMD in a Taiwan Chinese population. Further biological and/or functional evidence is needed to confirm the role IL-8 polymorphisms play in the pathogenesis of AMD. 

 Submitted for publication February 1, 2007; revised August 16, 2007; accepted December 17, 2007.

 Disclosure: Y.-Y. Tsai, None; J.-M. Lin, None; L. Wan, None; H.-J. Lin, None; Y. Tsai, None; C.-C. Lee, None; C.-H. Tsai, None; F.-J. Tsai, None; S.-H. Tseng, 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.

 

Primers and Optimized PCR Conditions

Gene	rs Number	PCR Primers	Annealing Tm (°C)	PCR Product	Restriction Enzyme	Incubation (°C)	Uncut	Cut
IL-1B−511 T/C	16944	Forward: 5′-TGGCATTGATCTGGTTCATC-3′	58	304	Aval	37	T: 304	C: 190+114
		Reverse: 5′-GTTTAGGAATCTTCCCACTT-3′
IL-6−572 C/G	1800796	Forward: 5′-GCAAAGTCCTCACTGGGAGGA-3′	60	296	BsrBI	37	C: 296	G201+95
		Reverse: 5′-TCTGACTCCATCGCAGCCC-3′
IL-6−596 A/G	1800797	Forward: 5′-GGAGTCACACACTCCACCTG-3	57	418	FokI	37	C: 418	A: 352+66
		Reverse: 5′-AAGCAGAACCACTCTTCCTTTACTT-3
IL-8−251 A/T	4073	Forward: 5′-TCATCCATGATCTTGTTCTAA-3′	58	542	MfeI	37	T: 524	A: 449+92
		Reverse: 5′-GGAAAACGCTGTAGGTCAGA-3′
IL-8 781 C/T	2227306	Forward: 5′-CTCTAACTCTTTATATAGGAATT-3	52	203	EcoRI	37	T: 203	C: 184+19
		Reverse: 5′GATTGATTTTATCAACAGGCA-3′
IL-8 1633 C/T	2227543	Forward: 5′-CTG ATG GAA GAG AGC TCT GT-3′	56	397	NlaIII	37	T: 397	C: 234+163
		Reverse: 5′-TGTTAGAAATGCTCTATATTCTC-3
IL-8 2767 C/T	1126647	Forward: 5-CCAGTTAAATTTTCATTT CAGGTA-3	53	222	BstZ171	37	A: 222	T: 198+24
		Reverse: 5-CAACCAGCAAGAAATTACTAA-3
IL-10−592 C/A	1800872	Forward: 5′-GGTGAGCACTACCTGACTAGC-3′	60	412	RsaI	37	C: 412	A: 236+176
		Reverse: 5′-CCTAGGTCACAGTGACGTGG-3′
IL-10−819 C/T	1800871	Forward: 5′-TCATTCTATGTGCTGGAGATGG-3′	55	209	MaeIII	55	T: 209	C: 125+84
		Reverse: 5′-TGGGGGAAGTGGGTAAGAGT-3′
IL-10−1082 G/A	1800896	Forward: 5′-CTCGCTGCAACCCAACTGGC-3′	65–55	139	Mn/I	37	A: 139	G: 106+33
		Reverse: 5′-TCTTACCTATCCCTACTTCC-3′
IL-18−105 A/C	549908	Forward: 5′-TGTTTATTGTAGAAAACCTGGAATT-3′	50	148	TaqI	65	A: 148	C: 123+25
		Reverse: 5′-CCTCTACAGTCAGAATCAGT-3′
IL-18−656 T/G	1946518	Forward: 5′-TAAGATTTACTTTTCAGTGGA-3′	50	200	MseI	37	G: 200	T: 101+99
		Reverse: 5′-AACACTGGAAACTGCAAGTAA-3′
IL-18−607 A/C	1946519	Forward: 5′-TGTTGCAGAAAGTGTAAAAATTTTAA-3′	50
		Reverse: 5′-CGGATACCATCATTAGAATAATAT-3′
IL-18−137 C/G	187238	Forward: 5′-ATGCTTCTAATGGACTAAGGA-3′	50	131	EcoRI	37	C: 131	G: 107+24
		Reverse: 5′-GTAATATCACTATTTTCATGAATT-3′

Table 2.

 

Baseline Characteristics of Study Participants

	Late AMD Cases (n = 312)							Controls (n = 180)
		Dry (n = 136)			Wet (n = 176)
		Values	P	Values	P
Age (y)	71.1 ± 7.5	0.97	71.4 ± 6.4	0.96	71.5 ± 6.9
Age distribution (n)
60–69, 70–79, ≧80	63, 55, 18	0.99	74, 78, 24	0.76	80, 76, 24
Gender (n)
Female, Male	60, 76	0.98	74, 102	0.83	82, 98
Smoking status (n)
Never, past, current	68, 58, 10	0.59	86, 57, 33	0.65	86, 70, 24
Body mass index (kg/m2)	24.1 ± 0.9	0.92	23.8 ± 0.5	0.85	24.5 ± 0.7
Blood pressure (mm Hg)
Systolic	136.2 ± 8.3	0.95	138.9 ± 10.3	0.89	136.8 ± 9.4
Diastolic	88.6 ± 6.7	0.94	87.7 ± 7.6	0.93	86.9 ± 6.9
Hypertension (%)	46.5	0.82	49.3	0.86	48.3
Diabetes (%)	7.7	0.87	9.4	0.85	9.8
Cardiovascular disease (%)	14.6	0.93	13.7	0.86	15.6
Hyperlipidemia (%)	21.8	0.91	20.9	0.97	19.5

 Data are expressed as the mean ± SD.

 Comparisons of age, body mass index, and blood pressure, by unpaired Student’s t-test.

 Comparisons of distributions of age, gender, smoking status, hypertension, diabetes, cardiovascular disease, and hyperlipidemia, by χ² test.

Table 3.

 

Distribution of the IL-8 +781 C/T Gene Polymorphisms in Patients with AMD and Control Subjects

IL-8 +781 C/T	Late AMD (n = 312)			Controls (n = 180)
		Dry (n = 136)	Wet (n = 176)
Genotypes
CC	56 (41)	40 (23)	93 (52)
CT	72 (53)	110 (63)	72 (40)
TT	8 (6)	26 (14)	15 (8)
P	0.06	1.15 × 10−7
Alleles
C	184 (68)	190 (54)	258 (72)
T	88 (32)	162 (46)	102 (28)
P	0.58	1.03 × 10−6

 Data are number of subjects (% of total group).

Table 4.

 

Distribution of the CFH Y402H Gene Polymorphisms in Patients with Wet AMD and Control Subjects

CFH Y402H	Wet (n = 176) Number (%)	Controls (n = 180) Number (%)
Genotypes
TT	144 (82)	164 (91)
CT	24 (14)	16 (9)
CC	8 (5)	0 (0)
P	0.004
Alleles
T	312 (89)	344 (96)
C	40 (11)	16 (4)
P	0.001

 Data are number of subjects (% of total group).

Table 5.

 

Two-Locus Odds Ratios for IL-8 +781 and CFH rs1061170

SNP		IL-8+781 C/T
				CC	CT	TT
CFH rs1061170	TT	1	5.09	6.23
(Y402H)	CT	2.91	29.09	0.59
	CC	38.35	3.56	3.56

 Odds ratios in parentheses were calculated to compare each genotypic combination with the baseline of homozygosity for the common allele at both loci (TT at CFH Y402H and CC at IL-8 +781 C/T).

Table 6.

 

Three Haplotypes Defined by IL-8 +781C/T and CFH Y402H (C/T) Polymorphisms in Patients with Wet AMD and Control Subjects

IL-8 +781C/T/CFH Y402H (C/T) Haplotypes	Wet AMD (n = 176) Number (%)	Controls (n = 180) Number (%)	P
TT	83 (47%)	51 (28%)	4.0 × 10−4
CT	76 (43%)	121 (67%)	5.07 × 10−6
CC	17 (10%)	8 (5%)	0.086

Figure 1.

 

Representation of the IL-8 gene, showing the position of four SNPs analyzed in our study.

Figure 2.

 

ELISA analysis of IL-8 concentration in vitreous from six patients with wet AMD with the TT genotype and five normal control subjects with the CC genotype.