High-temperature requirement factor A1 rs11200638 polymorphism and age-related macular degenerative from a comprehensive analysis about 15316 subjects

Background: The high-temperature requirement factor A1 (HTRA1) gene at the 10q26 locus has been associated with age-related macular degenerative (AMD) risk, with the significantly associated polymorphism being (rs11200638, -625G/A), however, above association is not consistent. We investigated an updated meta-analysis to evaluate the association between rs11200638 polymorphism and AMD risk thoroughly addressing this issue. Methods: An identification was covered with the Pubmed and Chinese Wanfang databases through 27th Jan, 2020. Odds ratios (OR) and 95% confidence intervals (CI) were used to assess the strength of associations. After a thorough and meticulous search, 35 different articles (33 case-control studies with HWE, 22 case-control studies about wet/dry AMD) were retrieved. Results: Individuals carrying A-allele or AA genotype may have an increased risk to be AMD disease. For example, there has a significantly increased relationship between rs11200638 polymorphism and AMD both for Asians (OR: 2.51, 95%CI: 2.22-2.83 for A-allele vs. G-allele) and Caucasians (OR: 2.63, 95%CI: 2.29-3.02 for A-allele vs. G-allele). Moreover, a similar trend in the source of control subgroup was detected. To classify the type of AMD, increased association was also observed in both wet (OR: 3.40, 95%CI: 2.90-3.99 for dominant model) and dry (OR: 2.08, 95%CI: 1.24-3.48 for dominant model) AMD. Finally, based on the different genotyping methods, increased relationships were identified by sequencing, TaqMan, PCR-RFLP and RT-PCR. Conclusions: Our present meta-analysis suggests that the HTRA1 rs11200638 polymorphism are potentially associated with the risk of AMD development, especially about individuals carrying A-allele or AA genotype, who may be as identified targets to detect and intervene in advance. Further studies using larger sample sizes and including information about gene-environment interactions should be conducted to elucidate. and systematic meta-analysis exploring the associations between HTRA1 gene rs11200638 polymorphism and AMD risk; it involved about 8101 AMD individuals and 7215 controls. Increased associations were found in the whole group, in Asian and Caucasian subgroups, source of control subgroup, and dry/wet sub-types of AMD, different genotyping methods (Sequencing, TaqMan, PCR-RFLP, RT-PCR and MassARRAY MALDI-TOF), which


Introduction
Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly people in developed countries [1,2]. The number of individuals affected by AMD may be estimated to reach 17.8 million by 2050 [3]. The visual loss of AMD attributes to dead or non-functional photoreceptors 3 and the underlying retinal pigment epithelium (RPE) [4]. Clinically, AMD is divided into two forms: The early stages, dry (atrophic, non-exudative) that can progress to geographic atrophy (GA) and the late stages, wet (exudative) AMD characterized by choroidal neovascularization (CNV) [5,6].
Age, ethnicity, family history, smoking and sun exposure are common risk factors [7,8], another important factor for AMD etiology is the genetic predisposition [9]. A genome-wide association study (GWAS) showed a clearer view about significant links between AMD risk and genetic variations in 2005, suggesting AMD is a polygenic disease [10], which triggered numerous studies involving the genetic associations of AMD in the following 15 years [11][12][13]. So far, AMRS2 rs10490924 polymorphism, SNPs from complement factor H, C2/CFB, complement component C3, APOE haplotypes have been confirmed as being associated with susceptibility of AMD [14][15][16][17][18].
It is well known that vascular endothelial growth factor (VEGF) is involved in wet AMD development because that the formation of angiogenesis and vascular permeability can lead to fluid leakage across the blood vessels, and visual loss in the final [19]. Anti-VEGF agents such as ranibizumab and bevacizumab have been widely applied in the clinic [20,21], in addition, have been proved to effectively slow the progress of CNV, however, heterogeneity was observed among patients in terms of the invalid samples and who have shorter duration of treatment [22]. It was hypothesized that genetic factors may participate in this period of this heterogeneous response, such as the variants of CHF, VEGFA, ARMS2 and high-temperature requirement factor A1 (HTRA1) genes [23][24][25][26].
HTRA1 encodes a member of the trypsin family of serine proteases and regulates the availability of insulin-like growth factors (IGFs) by cleaving IGF-binding proteins and transforming growth factor-β (TGF-β), which have been suggested to be a regulator of cell growth, angiogenesis and extracellular matrix deposition. In addition, the inhibition of TGF-β may lead to the overexpression of HTRA1 gene in wet AMD [27] (https://www.ncbi.nlm.nih.gov/gene/5654).
One of common polymorphisms in HTRA1 gene is rs11200638 (wide allele G to mutation allele A) In view of the foregoing, we realized that the important role of HTRA1 gene and its common rs11200638 polymorphism, we used the meta-analysis method to perform comprehensive conclusions including 28 different publications (33 case-control studies) [26, 30-57].

Search Strategy
We searched relative studies from PubMed and Wanfang databases before 27th Jan, 2020. The keywords were "age-related macular degeneration," "AMD," "polymorphism or variant," and "HTRA1 or high-temperature requirement factor A1." With these terms, a total of 35 different articles were included from above databases based on our inclusion criteria. Stages of AMD were assigned based on the classification of the Age-Related Eye Disease Study (AREDS) [58].

Inclusion and Exclusion Criteria
Included studies were according with (i) the correlation between AMD risk and HTRA1 gene rs11200638 polymorphism; (ii) case-control studies, and (iii) sufficient numbers of each genotypes (AA, AG, and GG) in case and control groups. Studies were excluded if they (i) included no control information; (ii) didn't contain genotype frequency data, and (iii) were duplicated studies with some other papers.

Data Extraction
Two authors (Ying Liu and Dong Wei) independently screened all papers that according with the selection criteria. These data included the first author's last name, publication year, country of origin, ethnicity, Hardy-Weinberg equilibrium (HWE) of control group, genotyping method and AMD disease types (dry and wet AMD). Ethnicity was categorized as Caucasian or Asian. The control subgroups were classified to population-based (PB) and hospital-based (HB).

Statistical Analysis
Based on the genotype frequencies for cases and controls, odds ratios (OR) with 95% confidence intervals (CI) were used to measure the strengths of associations. The statistical significance of the OR was determined with the Z test [59]. The heterogeneity assumption among studies was evaluated using a χ 2 -square-based Q test. If P-value > 0.10 for the Q test was indicated, a lack of heterogeneity among studies, other words, Mantel-Haenszel (fixed-effects model) was chosen, otherwise, the DerSimonian-Laird (random-effects model) was applied [60,61]. We investigated the correlation between rs11200638 polymorphism and AMD risk by testing whole five genetic models: A versus G, AG versus GG, AA + AG versus GG, AA versus GG and AA versus AG+GG. A sensitivity analysis was performed by omitting studies, one after another, to assess the stability of results. The departure of frequencies of the rs11200638 polymorphism from expectation under HWE was assessed by the Pearson's χ 2 test, P < 0.05 was considered significant [62]. The funnel plot was evaluated by Begg's test, and the publication bias was evaluated by Egger's test, whose P-value < 0.05 was considered significant [63]. All statistical tests for this meta-analysis were performed using version 10.

Network of gene-interaction of HTRA1 gene
To more complete understanding of the role of HTRA1 in AMD, the network of gene-gene interactions for HTRA1 gene was utilized through String online server (http://string-db.org/) [64].

Study searching and their basic information
Using various combinations of key terms, a total of 262 article titles were garnered by a document search using the PubMed (222 titles) and Wanfang (40 titles) databases. As shown in Figure 1, 178 articles were excluded after screening the Abstract sections of the manuscripts. The full texts were then evaluated, and 49 additional articles were excluded due to duplication (7), meta-analysis or systematic analysis (26), clinical trial (10), randomized controlled trial (6). Finally, 35 different articles [26, 30-57, [65][66][67][68][69] were included in our meta-analysis, including 38 case-control studies about HTRA1 gene rs11200638 polymorphism and AMD risk ( Table 1) and 27 case-control studies about HTRA1 gene rs11200638 polymorphism and wet or dry AMD risk ( Table 2). Five case-control studies [65][66][67][68][69] were not consistent with HWE in control groups. To make our analysis to more strict, we deleted above five studies, so there were about 33 case-control studies (

Total analysis
Results of the overall meta-analysis were suggestive of increasing associations between this polymorphism and AMD susceptibility in all five genetic models (for example: AA vs. GG: OR = 5.45, 95CI% = 4.26-6.98, P < 0.001) ( Table 3). In order to make this study more convincing and reliable, we detected five studies, which were not according with HWE, finally, we tested the 33 case-control studies. Also significantly increasing correlations were observed in whole genetic models (for example: A-allele vs. G-allele: OR = 2.56, 95%CI = 2.34-2.80, P < 0.001; AA+AG vs. GG: OR = 2.80, 95%CI = 2.49-3.15, P < 0.001) (Figure 4) (Table 3).

8
The publication bias was evaluated by both Begg's funnel plot and Egger's test. At beginning, the shape of the funnel plots seemed asymmetrical in allele comparison for rs11200638 by Begg's test, suggesting no publication bias was existed. Then, Egger's test was applied to provide statistical evidence of funnel plot symmetry. As a result, no obvious evidence of publication bias was observed (A-allele vs. G-allele, t = 0.89, P = 0.38 for Egger's test; z = 0.85, P = 0.396 for Begg's test, Figure 1A,B) (Table 4).

Supplementary
To delete studies which may influence the power and stability of whole study, we applied the sensitive analysis, finally, no sensitive case-control studies were found (Supplementary Figure 2).

Gene-gene network diagram and interaction of online website
String online server indicated that HTRA1 gene interacts with numerous genes. The network of genegene interaction has been illustrated in Figure 8.

Discussion
Because of the critical consequences about the visual loss caused by AMD, especially advanced AMD (atrophic/dry or neovascular/wet), it is necessary to study its etiology and mechanism, then to development early diagnostic methods and effective treatment. Nowadays, VEGF inhibitors are widely recognized as effective drugs in clinical application for CNV (wet AMD) [3, 70,71], instead of dry AMD. Therefore, to identify some novel detection markers and target drugs for different types of AMD is the current and future research focus on the direction. In the introduction section, we have enunciated the genetic factors may help us to search potential high-risk group about AMD, which can be prevented and treated in advance.
In our analysis, we chose the HTRA1 gene, which can regulate some kinds of growth factors.
Rs11200638 polymorphism in HTRA1 is the most common SNP, which is getting noticed. However, Kanda et al. [35] demonstrated that there was no HTRA1 gene involved in related SNPs for AMD, and its rs11200638 polymorphism seemed to have no effect on the transcript. On the contrary, they found in the minus strand there had a putative mitochondrial protein LOC105378525 probably expressing in the retina, which could be a candidate gene. In fact, they showed that rs11200638 is in strong linkage disequilibrium with rs10490924, a nonsynonymous A69S alteration in the predicted protein named LOC105378525(LOC387715)/ARMS2. From their study, rs10490924 was a strong candidate SNP related to AMD risk instead of rs11200638. In addition, Bonyadi et al. [72] performed a meta-analysis about rs10490924, and revealed combined cigarette smoking and rs10490924 polymorphism may have significant association with AMD risk. We believed rs10490924 was a valuable SNP for AMD, nevertheless, conclusions based on a single study can not negate the potential functions for HTRA1 and its SNPs, which need more evidences and support from published and future researches.
Mori et al. first investigated the association between HTRA1 gene rs11200638 polymorphism and risk of AMD [47]. Other more following researchers duplicated their work in different populations and different types of AMD. However, results were confounding, even within same populations, though two published meta-analysis. It is well known that meta-analysis provides a means for effectively increasing the size of the sample by pooling data from individual correlation studies, thus enhancing the statistical power of the analysis to estimate genetic effects [73], which used this method to demonstrate statistically significant genetic associations.
Two previous meta-analysis [74,75] about rs11200638 polymorphism and AMD have been reported, however, each study has its limitations. For example, Tang et al. just included fourteen case-control studies, two studies [65,67] were not consistent with HWE, and Tuo et al. actually reported foursource case-control studies, which shouldn't be combined together [75]. Chen et al. also performed a meta-analysis in the same year including 14 case-control studies, similar limitations were existed [74]. After year of 2008, newly added studies have been published, and to perfect the above deficiencies, we performed an updated meta-analysis to come to a more convincing conclusion about HTRA1 gene rs11200638 polymorphism and AMD susceptibility.
To the best of our knowledge, this is the comprehensive and systematic meta-analysis exploring the associations between HTRA1 gene rs11200638 polymorphism and AMD risk; it involved about 8101 AMD individuals and 7215 controls. Increased associations were found in the whole group, in Asian and Caucasian subgroups, source of control subgroup, and dry/wet sub-types of AMD, different genotyping methods (Sequencing, TaqMan, PCR-RFLP, RT-PCR and MassARRAY MALDI-TOF), which means that A-allele or AA genotype is the risk factor for AMD, in other words, if individuals carry on this SNP from peripheral blood test, which may indicate that it is possible to increase the occurrence of AMD for them in present time or at some point in the future. Therefore, this polymorphism may be helpful in screening vulnerable populations for AMD in advance. In addition, the power of present study was 1.00, which suggested our conclusions were stable and convincing.
In addition, the online analysis system-String was applied to predict potential and functional partners related to HTRA1, which can help us to better understand the value for detection and concern. Finally, ten genes were predicted. Among them, the highest score of association was ACAN (0.943), however, so far, no research has been reported between this gene and AMD and interaction between this gene and HTRA1. Future research should be payed attention to above information, which may be in favor of AMD early detection/prevention and intervention. In other partners, ARMS2 and CFH have been shown to associate with AMD. The ARMS2 and HTRA1 genes are located nearby on the 10q26 chromosome in a strong linkage disequilibrium. Significant association was observed that ARMS2 rs10490924 was related response to ranibizumab treatment among wet AMD subjects [68]. CFH gene T1277C polymorphism is strong associated with both wet and dry AMD and may be contribute to the inflammation in the pathogenesis of AMD [76]. As for the rest interaction genes (CLPP, CTRC, YME1L1, HSPD1, RPL34, CLPX and PLEKHG4) both had moderate score and no literature to support. It seems that above ten genes associated with HTRA1 came from text mining scores, which were derived from the co-occurrence of gene/protein names in related abstracts. In addition, it was important considered the occurrence of the LOC105378525 (LOC387715) and its polymorphism (A69S, rs10490924) as the main factor for AMD reported by Kanda et al (2007) [35], which should be added in the network of HTRA1 related genes. In a word, we should deep explore these partners of HTRA1 gene, and genegene interactions in the development of AMD in the next step.
There are some inherent limitations of our study should be declared. First, further studies should focus on Mixed and African populations, which was vacant in present analysis. Second, gene-gene and gene-environment interactions were not well analyzed. It is possible that specific environmental and lifestyle factors alter the associations between HTRA1 rs11200638 polymorphism and AMD, including 11 age, diabetes, smoking, familial history, and hypertension. Third, vision is the most concerned-clinical indicator of AMD, future studies should include the value of the vision and analyze the relationships between rs11200638 polymorphism and the degree of visual impairment, which may help us to better detect disease progression.

Conclusions
In conclusion, our present meta-analysis suggests that HTRA1 rs11200638 polymorphism may be a risk factor for the susceptibility of AMD, larger and more comprehensive studies should be performed in the future

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Higgins JP, Thompson SG: Quantifying heterogeneity in a meta-analysis.  Figure 1 Flowchart illustrating the search strategy used to identify association studies for HTRA1 gene rs11200638 polymorphism and AMD risk.
28 Figure 2 The