Association of complement factor H gene polymorphisms with age-related macular egeneration susceptibility

Abstract

Objective: This study was aimed to confirm whether I62V and Y402H polymorphisms of complement factor H (CFH) were risk factors for age-related macular degeneration (AMD). Method: 109 AMD patients and 165 AMD-free controls were enrolled in the study. The I62V and Y402H polymorphisms were analyzed by polymerase chain reaction-restriction fragment length of polymorphism (PCR-RFLP). Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by the X² test to assess the relationship of I62V and Y402H polymorphisms with AMD risk. Analysis of haplotype and stratification by age and smoking status was conducted as well. Results: AA genotype and A allele of I62V polymorphism was significantly associated with increased risk for AMD (OR = 3.75, 95% CI = 1.70-8.30; OR = 1.64, 95% CI = 1.14-2.36). For Y402H polymorphism, CT genotype showed strong effects on the occurrence of AMD (OR = 2.10, 95% CI = 1.04-4.27). Moreover, C allele was also a risk factor for AMD (OR = 1.95, 95% CI = 1.02-3.72). The haplotypes analysis suggested that the risk for AT haplotype carriers was high, compared with GT haplotype (OR = 3.91, 95% CI = 2.58-5.94). In addition, we found that smoking status could affect the genotype distribution of Y402H polymorphism (P < 0.05). Conclusions: Our results revealed that CFH polymorphisms I62V and Y402H might be associated with the susceptibility to AMD in Chinese population.

Introduction

Age-related macular degeneration (AMD) is a major factor resulting in irreversible vision loss, and more than 50 million people have been disturbed all over the world [1,2]. It is characterized by lateonset, chronicity and progressive degeneration of vision in the central region of the retina [3]. There were two stages in AMD progression: the early stage and the advanced stage. Signs like soft drusen, increasing areas of pigment or hyperpigmentation (depigmentation or hypopigmentation) on the retinal pigment epithelium are indicated for the early stage, which would develop into advanced stage in the forms of atrophic AMD or neovascular AMD [4].

The pathogenesis of AMD is complex and multifactorial. Epidemiologic and laboratory studies have demonstrated genetic and environmental factors are primary causes for AMD [5]. Familial aggregation studies identified about a quarter of AMD cases were closely relevant to heredity [6]. Thus exploring the genetic effects of AMD pathogenesis is very necessary.

Complement factor H (CFH) is a multi-domains and functional protein. The association of complement factor H (CFH) polymorphism with AMD susceptibility has been extensively studied in past years. It has been reported that CFH Y402H polymorphism shows negative effects on AMD in Caucasians [7-13], while it shows weak relationship with AMD risk in Asian populations [14-16]. Meanwhile, some studies found that CFH I62V polymorphism was also associated with AMD susceptibility [17,18].

Based on the previous studies, we concluded that Y402H and I62V might play different roles in AMD susceptibility in different ethnicities. Hence, we conducted an updated study to further confirm the effects of the two polymorphisms on the susceptibility of AMD in Chinese and Caucasian population.

Methods and materials

Study population

A case-control study was conducted to assess the relevance of CFH polymorphism with AMD risk. A total of 109 AMD patients were enrolled from Eye Hospital of China Academy of Chinese Medical Sciences (Beijing, China) as case group. They all underwent fluorescein and/or indocyanine green fundus angiography. 165 AMD-free volunteers who usually take a physical examination in the same hospital were enrolled as controls. They were required no sign of AMD or any other eye diseases. The unrelated controls and cases were matched on gender and age. Written informed consent was obtained before blood samples extraction and information collection. A person who smoked at least 5 cigarettes a day for more than one year was defined as a smoker [19]. The study protocol was approved by the Ethics Committee of Eye Hospital of China Academy of Chinese Medical Sciences. All the procedures rigorously were conducted following the terms of the Declaration of Helsinki.

Genotyping

Genome DNA was extracted from the peripheral blood of all subjects using DNA extraction kit (Wizard Genomic DNA Purification Kit; Promega, Madison, WI), according to the manufacturer’s instructions. Then the DNA samples were stored at -20°C.

Polymerase chain reaction-restriction fragment length of polymorphism (PCR-RFLP) technology was used as genotyping method. PCR primers were designed by primer premier 5.0 software. 20 μl PCR reaction solution included 1 μl genome DNA (10 ng/μl) template, 10 μl 2 × Tap PCR Master Mix, 0.5 μl forward and backward primers (6 mm/L) respectively and redistilled water. PCR reaction procedure: first initial denaturation at 95°C for 5 min, 33 cycles of denaturation at 95°C for 45 s, annealing at 62°C for 30 s, extending 72°C for 45 s and final extension at 72°C for 10 min. Then, PCR products were digested by restriction enzyme and digested products were detected by 2.0% agarose gel electrophoresis.

Statistical analysis

X² test was used to assess whether genotypes distribution of the controls was in agreement with Hardy-Weinberg equilibrium (HWE). Comparison on genotype and allele frequencies between cases and controls were conducted by χ² analysis. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by the chi-squared test to evaluate the association of CFH polymarphisms with the risk of AMD. Pairwise linkage disequilibrium (LD, D’) between CFH polymorphisms were analyzed by Haploview (Version 3.31). Comparson on the haplotypes between cases and controls were assessed by χ² test. Statistical analysis was performed with PASW Statistics version 18.0. P < 0.05 was considered statistically significant.

Results

A total of 109 patients with AMD and 165 AMD-free controls were included in this study. Demographic and clinical characteristics of the subjects were listed in Table 1. Of the subjects, 47.7% of the patients (aged 62.1 ± 7.2 with 58.7% males) were less than 60 years old, which was a little lower than the controls (47.9%) (aged 61.4 ± 5.3 with 60.0% males; P > 0.05). There was no significant difference for smoking status in cases and controls (P > 0.05). In addition, we also found no differences on hypertension and heart disease between the two groups (P > 0.05 for both).

Table 1.

Demographic and clinical characteristics of the subjects

Variables	AMD patients (109)	Controls (165)	X2	P
	n (%)	n (%)
Age (mean age) (year)	62.1 ± 7.2	61.4 ± 5.3
			0.001	1.000
< 60	52 (47.7)	79 (47.9)
≥ 60	57 (52.3)	86 (52.1)
Gender
Male	64 (58.7)	99 (60.0)	0.045	0.900
Female	45 (41.3)	66 (40.0)
Smoking status			1.306	0.521
Nonsmokers	42 (38.5)	75 (45.4)
Light smokers1	32 (29.4)	44 (26.7)
Heavy smokers2	35 (32.1)	46 (27.9)
Hypertension
Yes	51 (46.8)	79 (47.9)	0.031	0.902
No	58 (53.2)	86 (52.1)	-	-
Heart disease
Yes	26 (23.9)	34 (20.6)	0.405	0.553
No	83 (76.1)	131 (79.4)	-	-

¹Smoking less than 20 packs per year;

²Smoking more than 20 packs per year.

The association between CFH (Y402H and I62V) polymorphisms with AMD risk was analyzed in Table 2. Generally, CFH polymorphisms were observed to be risk factors for AMD. The AA genotype of I62V polymorphism was significantly associated with increased risk for AMD (OR = 3.75, 95% CI = 1.70-8.30). Meanwhile, an allele was also a risk factor for AMD (OR = 1.64, 95% CI = 1.14-2.36). For Y402H polymorphism, CT genotype was implicated in the pathogenesis of AMD (OR = 2.10, 95% CI = 1.04-4.27. Moreover, C allele appeared to be genetic-susceptibility factor for AMD (OR = 1.95, 95% CI = 1.02-3.72).

Table 2.

Association between CFH polymorphisms and AMD

SNPs	Designation	Genotypes		OR (95% CI)	P	X2	Alleles		OR (95% CI)	P	X2
		Cases	Controls				Cases	Controls
		n (%)	n (%)				n (%)	n (%)
rs800292	Exon 2 (I62V)	50 (45.9)	86 (52.1)	1	-	-	135 (61.9)	240 (72.7)	1	-	-
		35 (32.1)	68 (41.2)	0.89 (0.52-1.51)	0.198	0.656	83 (38.1)	90 (27.3)	1.64 (1.14-2.36)	0.008	7.089
		24 (22.0)	11 (6.7)	3.75 (1.70-8.30)	0.001	11.472
rs1061170	Exon 9 (Y420H)	88 (80.7)	148 (89.7)	1	-	-	196 (89.9)	312 (94.5)	1	-	-
		20 (18.3)	16 (9.7)	2.10 (1.04-4.27)	4.354	0.037	22 (20.1)	18 (5.5)	1.95 (1.02-3.72)	0.041	4.172
		1 (0.9)	1 (0.6)	1.68 (0.10-27.23)	0.137	0.711

The analysis of pairwise LD between I62V and Y402H polymorphisms was conducted (D’ = 1.0, r² = 0.062) in the study. The results indicated that there were four haplotypes for I62V and Y402H. While, we selected two haplotypes (GT and AT) among them with more than 10% frequency. High risk for AMD was observed in the AT haplotype carriers (OR = 3.91, 95% CI = 2.58-5.94) (Table 3).

Table 3.

Association analysis of CFH haplotypes in AMD patients and controls

	I62V	Y420H	Frequency		OR (95% CI)	P	X 2
			Cases	Controls
			2n (%)	2n (%)
Haplotypes
K1	G	T	109 (50.0)	257 (77.9)	1	-	-
K2	A	T	83 (38.1)	50 (15.2)	3.91 (2.58-5.94)	0.000	43.862
K3	G	C	22 (10.0)	18 (5.4)	-	-	-
K4	G	T	4 (1.9)	5 (1.5)	-	-	-

In the study, we also analyzed the effects of age and smoking status on the genotype distribution. The results were shown in Table 4. In the study, we found that smoking status showed strong effects on the genotype distribution of Y402H polymorphism (χ² = 25.521, P < 0.05).

Table 4.

The effects of age and smoking status on the genotypes distribution of CFH polymorphisms

Genotypes	Age (years)		X 2	P	Smoking status
	< 60/n	≥ 60/n			Non-smokers/n	Light smokers/n	Heavy smokers/n	X 2	P
I62V			0.711	0.682				7.230	0.124
GG	13	37			22	11	17
AG	12	23			11	16	8
AA	7	17			9	5	10
Y402H			0.761	0.684				25.521	0.000
TT	25	63			24	29	35
CT	7	13			17	3	0
CC	0	1			1	0	0

Discussion

AMD is a major factor resulting in visual impairment. Many studies have reported age and ethnicity were two major risk factors for AMD susceptibility. Less than 1% of AMD patients are younger than 65 years old; approximately 30% AMD patients are over 75 years [20-22]. Besides, a large number of studies have revealed CFH gene polymorphisms (I62V and Y402H) play crucial roles in pathogenesis of AMD. While, the effects varies with the change of ethnicity. Y402H, a common missense variant, confers significant effects on the elevated risk of AMD in Caucasians [23], while it has no relationship with AMD in Japanese [14,15]. Another common missense variant I62V has also been reported to be significantly associated with the increased risk of AMD in Caucasians, Chinese, Korean as well as Japanese [14,23-25]. The information above suggested the genetic effects of I62V and Y402H on AMD risk among diverse ethnic groups should be identified in future studies.

The present study based on 109 patients with AMD and 165 AMD-free controls was conducted to further identify the effects of I62V and Y402H polymorphisms on AMD risk in Chinese population. In addition, the effects of age and smoking status were evaluated as well. The analysis indicated that CFH I62V and Y402H polymorphisms were both related with increased risk for AMD, which were supported by previous publications [23-25]. On the basis of the strong correlation of I62V and Y402H and the AMD susceptibility, association of CFH haplotypes between risks of AMD was analyzed. Of the four haplotypes, K2 (AT) was related with AMD susceptibility. For age and smoking status, both of them were suggestive of a negative interaction with the risk of AMD. The results indicated that smoking status could influence the genotype distribution of Y402H polymorphism. However, age and smoking status showed no effects on I62V polymorphism.

In summary, the analyses of this study appeared that I62V and Y402H might be associated with AMD risk in Chinese population This finding was consistent with some of the previous publications, while differed from certain published findings as well, which might attribute to the small sample size of our study. Thus, larger genetic association studies with different ethnic groups are needed to be further identifying the findings.

Disclosure of conflict of interest

None.