Abstract
Objective: To investigate whether haplotypes of rhodopsin (RHO) polymorphisms including rs7984, rs2855552, rs2855557 and rs2410 were associated with age-related macular degeneration (AMD) risk in Chinese Han population. Methods: Genotypes of rs7984, rs2855552, rs2855557 and rs2410 were detected with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 186 cases and 196 healthy controls. Then, the haplotypes were established with Haploview 4.2 software. And the effects of clinical charactersitics on the frequency of GTTG haplotype were also analyzed. Odds ratios (ORs) with 95% confidence interval (95% CI) were utilized to assess the relationship of haplotypes and genotypes of RHO polymorphisms with susceptibility to AMD. Results: Genotype distribution of all polymorphisms in control group were all in agreement with Hardy-Weinberg equilibrium (HWE) (P>0.05). In the analysis, we found that mutant alleles of rs7984 and rs2855557 were both associated with increased risk of AMD. For genotype analysis, rs7984 AA and rs2855557AA, rs2410GG genotypes all could increase the risk for AMD (OR=1.905, 95% CI=1.143-3.174; OR=2.226, 95% CI=1.261-3.932; OR=2.073, 95% CI=1.105-3.888). However, rs2855552 showed no effects on the onset of AMD. Compared with GTTA, the haplotypes of GGTG, ATAA and GTTG were all related with AMD susceptibility. Further analysis suggested that age, hypertension and hyperlipidemia history play important roles in the frequency alteration of GTTG haplotype. Conclusion: RHO polymorphisms (rs7984, rs2855557 and rs2410) and haplotypes may confer remarkable susceptibility to AMD. Further investigation showed that gene and environmental factors may work together in the pathogenesis of AMD.
Keywords: Rhodopsin, age-related macular degeneration, haplotype, polymorphism
Introduction
Age-related macular degeneration (AMD) is the most important eye diseases which can lead to blind in western developed countries currently, and its morbidity increases year by year with the aggravation of aging of population [1-4]. In America, there are nearly 10 million Americans suffer from AMD and the morbidity of AMD ranges from 7% to 10% among the elderly aged over 75 [5,6]. AMD, characterized by the clinical feature of drusen sedimentation in the macular region and nearby retinas between retinal pigmented epithelium and choroidea, contributes to the progressive damage of macular area and further causes the impairment of the central visual field [7]. It has been demonstrated that pathogenesis of AMD involves many genes, of which RHOdopsin (RHO) gene draw more attention [8].
The 6952 bp-length humans RHO gene locates on chromosome 3q21-24, consists of 4 introns and 5 exons and encodes RHO protein, which is one of the receptors of G binding protein related to human diseases [9]. RHO gene was first described in the literature as a causative gene for adRP by Dryja et al [10,11]. Mutations in RHO gene often lead to abnormal pathophysiology of human vision. Molecular biology studies have identified a variety of polymorphisms sites of RHO gene, changes of which can regulate or alter the expression of RHO gene [6,10]. Moreover, it has been reported that mutations in RHO gene account for about 25% of adRP cases in Caucasians [12]. Besides, conformational changes of RHO gene may lead to the hyperpolarization of the rod cells, which play a vital role in vision [13]. However, the function mechanism of ROH mutation in the pathogenesis of AMD is still unclear.
In current study, we selected 4 polymorphisms sites (rs2410, rs7984, rs2855552 and rs2855557) in RHO gene and analyzed the relationship of each SNP and haplotypes with AMD susceptibility in Chinese Han population.
Materials and methods
Subjects
We conducted a multicentric case-control study through respectively selecting a certain number of AMD cases and controls from hospitals during January, 2008 to May, 2010. The inclusion and exclusion criteria for cases and controls were established. The inclusion criteria for cases were as follows: ① aged over 50; ② diagnosed as AMD by fluorescein fundus angiography (FFA) or optical coherence tomography (OCT); ③ with no other retinal diseases; ④ signed informed consent. The cases would be excluded if they meet the following items: ① high myopia (≥6.00D); ② polypoidal choroidal vasculopathy (PCV); ③ macular dystrophia; ④ lesions of central serous retinal choroid; ⑤ vein occlusion; ⑥ diabetic retinopathy; ⑦ uveitis; ⑧ other diseases affecting functions of retinal photoreceptor cells.
The inclusion criteria for controls included: ① aged over 50 (age gap between two groups was no more than five years); ② not suffering from AMD with fundus examination. And the individuals with severe physical defects or chronic diseases were excluded. Meanwhile, the participants who did not comply with the survey and blood test were also precluded from the study.
DNA extraction and primer design
DNA was extracted by the method of phenol/chloroform extraction. Then the DNA was purified with glass milk precipitation and stored at -20°C. Primers were design by Premer 5.0 software and synthesized by GenScript (Nanjing) Co., Ltd. The sequence of primers was showed in Table 1.
Table 1.
Primer sequence of RHO polymorphisms
| Polymorphism | MAF | Site | Primer | Length | Tm (°C) |
|---|---|---|---|---|---|
| rs2410 | 0.489 | Exon 5 | Forward: 5’-AGCCATCCCACCAGGAGCAG-3’ | 293 bp | 65 |
| Reverse: 5’-CTCCTGGTGGGATGGCTGTG-3’ | |||||
| rs7984 | 0.411 | Exon 1 | Forward: 5’-GCTTAGGAGGGGGAGGTCAC-3’ | 238 bp | 60 |
| Reverse: 5’-GTGACCTCCCCCTCCTAAGC-3’ | |||||
| rs2855552 | 0.256 | Intron 1 | Forward: 5’-TGCAAAGCTGGGTGACGGGG-3’ | 200 bp | 68 |
| Reverse: 5’-AAGGCTGGCAGAGAGACGCT-3’ | |||||
| rs2855557 | 0.422 | Intron 4 | Forward: 5’-GCATTTCCAGCAAGCCCTCC-3’ | 217 bp | 64 |
| Reverse: 5’-TGGAGGGCTTGCTGGAAATG-3’ |
PCR reaction
PCR reaction mixture included ddH2O 40.5 μl, 10×PCR buffer (with 1.5 mmol/l Mg2+) 5 μl, 10 mmol/L dNTP 1 μl, Taq polymerase 5 μl (Promega Company, USA), forward and reverse primer respectively 1 μl (20 μmol/l), template DNA 1 μl (500 mg). PCR amplification was performed under the following condition: initial degeneration for 2 min at 94°C, 35 cycles of degeneration for 40 s at 94°C, annealing for 40 s (annealing temperature: 60°C for rs2410; 55°C for rs7984; 63°C for rs2855552; 59°C for rs2855557) and extension for 40 s at 72°C, finally extension for 10 min at 72°C. PCR products were purified by Multiscreen-PCR purification plate (Millipore Company, USA).
Genotype determination
Restricted fragment length polymorphisms (RFLP) was used to determine genotypes of rs2410, rs7984, rs2855552 and rs2855557. The purified PCR products were respectively mixed with, restriction enzymes of Apa I, BamH I, BsaHI and BsmFI (New England Biolabs Beverly, Ma, USA). 20 μl digestion mixture in-cluded DNA 1 μg, incision enzyme 5 U, corresponding 10×Buffer 2 μl and supplemental ddH2O. Then the mixture was treated with water bath at 37°C and 65°C for 12 h sequentially. The digestion products were detected by 2% agarose el electrophoresis (Agarose-1000; Gibco BRL, Rockville, MD, USA).
Haplotype construction
In the analysis, we found that rs2410, rs7984, rs2855552 and rs2855557 show up a block within RHO gene. Then we analyzed their haplotype species with Haploview 4.2 software.
Statistical analysis
Chi-square test was utilized to check the distributional differences of age, smoking, drinking and allele between case and control groups and then verify whether genotype frequencies were consistent with Hardy-Weinberg equilibrium (HWE). MannWhitney-U test was used to compare the difference of age distribution between groups. Association of polymorphisms, haplotypes and the risk of AMD were estimated by odds ratio (OR) with 95% confidence interval (95% CI) which was obtained after the adjustment of age, gender, smoking and drinking by unconditional logistic regression. All the statistical tests were bilateral probability test and conducted with SPSS 11.5 software. It was considered to be significant difference when P value is smaller than 0.05.
Results
Correlation of genotypes and AMD susceptibility
The four polymorphisms sites (rs7984, rs2855552, rs2855557 and rs2410) existed in all samples. In the analysis of genotype distribution, we found that genotype distribution of each polymorphism sites were consistent with HWE (P=0.08, 0.22, 0.14 and 0.10). Compared with control group, mutant allele frequencies of rs7984 and rs2855557 were much higher than corresponding wild allele, which suggested that the mutant alleles were related with the occurrence of AMD (OR=1.469, 95% CI=1.104-1.954; OR=1.469, 95% CI=1.104-1.954). For genotype analysis, rs7984 AA and rs2855557AA, rs2410GG genotypes all showed strong effects on the onset of AMD (OR=1.905, 95% CI=1.143-3.174; OR=2.226, 95% CI=1.261-3.932; OR=2.073, 95% CI=1.105-3.888). There was no significant difference neither in allele frequency of rs2855552 polymorphisms (P=0.917) nor in genotype frequency of it (P=0.986) between two groups (Table 2).
Table 2.
General data and correlation between RHO polymorphisms and AMD risk
| Item | Case | Control | P value | OR (95% CI) |
|---|---|---|---|---|
| Age (x±s) | 63.53±9.7 | 62.95±8.6 | 0.716 | |
| Gender | ||||
| Men | 97 | 110 | 0.473 | |
| Women | 89 | 86 | ||
| rs7984 | ||||
| GG | 63 | 70 | - | 1.00 |
| AG | 51 | 84 | 0.112 | 0.675 (0.415-1.098) |
| AA | 72 | 42 | 0.013 | 1.905 (1.143-3.174) |
| G | 177 | 224 | - | 1.00 |
| A | 195 | 168 | 0.008 | 1.469 (1.104-1.954) |
| rs2855552 | ||||
| TT | 98 (52.7) | 104 | - | 1.00 |
| GT | 79 (42.5) | 82 | 0.917 | 1.022 (0.676-1.547) |
| GG | 9 (4.8) | 10 | 0.924 | 0.955 (0.372-2.450) |
| T | 275 (73.9) | 290 | - | 1.00 |
| G | 97 (26.1) | 102 | 0.986 | 1.003 (0.726-1.386) |
| rs2855557 | ||||
| TT | 53 | 59 | - | 1.00 |
| AT | 71 | 106 | 0.228 | 0.746 (0.463-1.202) |
| AA | 62 | 31 | 0.005 | 2.226 (1.261-3.932) |
| T | 177 | 224 | - | 1.00 |
| A | 195 | 168 | 0.008 | 1.469 (1.104-1.954) |
| rs2410 | ||||
| AA | 82 | 93 | - | 1.00 |
| AG | 71 | 84 | 0.849 | 0.959 (0.621-1.479) |
| GG | 33 | 19 | 0.035 | 1.970 (1.041-3.727) |
| A | 235 | 270 | - | 1.00 |
| G | 137 | 122 | 0.096 | 1.290 (0.956-1.742) |
Association of haplotypes and AMD risk
As shown in Table 3, there are four haplotypes combination: GTTA, GGTG, ATAA and GTTG. And the results indicated that the haplotypes were all significantly associated with increased risk for AMD (OR=2.425, 95% CI=1.532-3.839; OR=2.960, 95% CI=1.945-4.504; OR=5.100, 95% CI=2.664-9.764).
Table 3.
Haplotypes associated with AMD risk
| Haplotype 1-2-3-4 | Case (2n=372) | Control (2n=392) | P value | OR (95% CI) |
|---|---|---|---|---|
| GTTA | 40 | 102 | - | 1.00 |
| GGTG | 97 | 102 | 0.000 | 2.425 (1.532-3.839) |
| ATAA | 195 | 168 | 0.000 | 2.960 (1.945-4.504) |
| GTTG | 40 | 20 | 0.000 | 5.100 (2.664-9.764) |
1: rs7984; 2: rs2855552; 3: rs2855557; 4: rs2410.
Correlation analysis of GTTG haplotype and clinical characteristics
Then we investigated the relationship of clinical characteristics with GTTG hapotype (Table 4). For the individuals older than 71, the frequency of GTTG hapotype was much higher in case group (P=0.008). In addition, hypertension (P=0.005) and hyperlipidemia history also showed effects on the frequency of GTTG hapotype (P=0.002, 0.015). Above results suggested that gene and environmental factor may work together in the pathogenesis of AMD.
Table 4.
GTTG haplotype associated with AMD risk
| Factor | GTTG haplotype | P value | OR (95% CI) | |
|---|---|---|---|---|
|
| ||||
| Case (2n=372) | Control (2n=392) | |||
| Total | 40/372 | 20/392 | 0.000 | 5.100 (2.664-9.764) |
| Gender | ||||
| Men | 24/194 | 10/220 | - | 1.00 |
| Women | 16/178 | 10/172 | 0.461 | 0.667 (0.226-1.965) |
| Age | ||||
| ≤60 | 10/178 | 10/184 | - | 1.00 |
| 61~70 | 10/132 | 6/152 | 0.453 | 1.667 (0.437-6.358) |
| ≥71 | 20/62 | 3/56 | 0.008 | 6.667 (1.492-29.787) |
| Smoking | ||||
| No | 21/212 | 11/228 | - | 1.00 |
| Yes | 19/160 | 9/164 | 0.855 | 1.106 (0.376-3.248) |
| Drinking | ||||
| No | 17/198 | 12/208 | - | 1.00 |
| Yes | 13/174 | 8/184 | 0.815 | 1.147 (0.363-3.621) |
| Hypertension | ||||
| No | 11/188 | 14/200 | - | 1.00 |
| Yes | 29/184 | 6/192 | 0.002 | 1.152 (1.887-20.053) |
| Hyperlipidemia | ||||
| No | 11/202 | 12/210 | - | 1.00 |
| Yes | 29/170 | 8/182 | 0.015 | 3.955 (1.275-12.269) |
Discussion
In western countries, macular degeneration is the main cause of blindness among populations aged over 50. The number of blind cases with macular degeneration is lager than the total number of those with glaucoma, cataract and diabetes retinopathy [14,15]. In China, the morbidity of macular degeneration is 6.04%-11.19% in people aged 60 to 69, and the incidence trends to increase with the rapid aging of population [16,17]. Macular degeneration has been identified as one of the most serious eye diseases in international ophthalmology. The herapeutic treatment for macular degeneration at present are urgently needed to improve.
RHO gene encodes RHO protein, a transmembrane protein, contains three functional domains, that is, extracellular domain, transmembrane domain and cytoplasmic domain [18]. RHO protein expresses specifically in rod photoreceptor cells and plays a crucial role in the transformation process of optical signal to nervous impulse. Once activated by the photon, the protein can result in cascade reactions in the transduction process of vision signal and thus promote the production of vision signal [11,19,20]. Katagiri et al., have investigated the genetic and clinical features of patients with RHOdopsin (RHO) mutations and found RHO mutations can precise predicte disease severity [21]. Until now, the relationship of haplotypes of RHO and AMD susceptibilityhave not been studied.
Our study demonstrated that rs7984, rs2855552 rs2855557 and rs2410 in RHO promoter region were prevalent in Chinese Han population. And the analysis showed that rs7984 AA and rs2855557 AA, rs2410 GG genotypes were all related with AMD susceptibility. However, only mutant alleles of rs7984 and rs2855557 were associated with risk of AMD.
As respect to genetic factors for diseases, not only genetic polymorphisms individually result in the occurrence and development of diseases, but also certain interactions among them show effects on the disease [22]. Haplotypes exactly reflect genetic inter-relation and study on haplotypes is helpful for discovering multiple genetic polymorphisms involved in diseases [23,24]. Our study established haplotypes for RHO polymorphisms sites and analyzed the correlations between haplotypes of RHO polymorphisms and susceptibility to AMD. Based on the results, the four haplotypes composed of rs7984, rs2855552, rs2855557 and rs2410 were all associated with AMD susceptibility. Then we analyzed the effects of clinical characteristics on the frequency of GTTG haplotype. As a result, age, hypertension and hyperlipidemia all could alter the frequency of GTTG haplotype.
In conclusion, haplotypes formed by RHO rs7984, rs2855552, rs2855557 and rs2410 polymorphisms sites alone or combination with environmental factors may be likely to influence the susceptibility of AMD. Nonetheless, how RHO polymorphisms and their haplotypes regulate serum levels and expression of RHO and how they participate in the occurrence and development of AMD is needed to be further investigated in subsequent studies.
Disclosure of conflict of interest
None.
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