|Molecular Vision 2007;
Received 24 January 2007 | Accepted 5 November 2007 | Published 26 November 2007
PLEKHA1-LOC387715-HTRA1 polymorphisms and exudative age-related macular degeneration in the French population
Eric H. Souied,1,3 Florence
Richard,4 Véronique Barbu,5 Alain Zourdani,1 Gilles
Morineau,2 Jennyfer Zerbib,1 Gabriel Coscas,1 Gisèle
Soubrane,1,3 Pascale Benlian2
1Creteil University Eye Clinic, Faculte de Medecine Henri Mondor, Creteil; 2Universite Pierre et Marie Curie, Paris 6, Faculte de Medecine Pierre et Marie Curie, Paris; Department of Molecular Biology and Biochemistry, Hopital Saint-Antoine, Paris, France; 3Unite Fonctionnelle de Recherche Clinique, Creteil, France; 4INSERM UMR744, Institut Pasteur de Lille; Université Lille 2, Lille, France; 5Universite Pierre et Marie Curie, Hopital Saint-Antoine, LCBGM, Paris, Paris, France
Correspondence to: Eric H. Souied, Creteil University Eye Clinic, 40 Avenue de Verdun, 94000 Creteil, France; Phone: 33 1 45 17 59 08; FAX: 33 1 45 17 52 27; email: firstname.lastname@example.org
Purpose: Identification of genetic factors for age-related macular degeneration (AMD) is of crucial importance in this common cause of blindness. Exudative AMD is rapidly progressive and usually associated with severe prognosis. Our purpose was to investigate this association on locus 10q26 in a case-control study including French patients specifically affected with exudative AMD.
Methods: Polymorphisms rs4146894:G>A of Pleckstrin Homology Domain-containing Protein Family A member 1 (PLEKHA1) gene, rs10490924:G>T at LOC387715, and rs11200638:G>A of HTRA1 (HTRA serine peptidase 1) gene were analysed in AMD cases (n=118, age=72.3±3.8 years old) and healthy controls (n=116, age=72.0±3.8 years old).
Results: PLEKHA1 polymorphism was associated with AMD. The A allele frequency was 0.67 in cases versus 0.41 in controls, (p=0.0001). After age and sex adjustment, the odds ratio for risk of AMD was 9.1 (4.0-20.9, 95% CI, p=0.0001) for the AA genotype and 2.6 (1.3-5.5, 95% CI, p=0.04) for the AG genotype, conditional on HTRA1. Association was even stronger and independent with HTRA1. The A allele frequency was 0.51 in cases versus 0.22 in controls, (p=0.0001). The odds ratio was 15.5 (5.5-43.9, 95% CI, p=0.0001) for the AA genotype and 3.4 (1.9-6.1, 95% CI, p=0.0001) for the AG genotype. No further information was obtained from LOC387715 due to virtually complete linkage disequilibrium with HTRA1 polymorphism in cases (D'=1.0) and controls (D'=0.98). Although a role for PLEKHA1 could not be totally excluded, there was a four times higher AMD risk was associated with haplotype "A-T-A" involving "PLEKHA1-LOC387715-HTRA1" risk alleles.
Conclusions: Compared to PLEKHA1, HTRA1/LOC387715 genetic variations were independently and strongly associated with exudative AMD in the French population. Chromosome-10 genetic variants appear as potentially useful risk markers for early detection of AMD.
With increasing global prevalence, age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly population . There are two major subgroups of advanced AMD: atrophic and exudative . Exudative AMD is the most frequent and rapidly progressive form of AMD in European populations . Identification of risk factors for exudative AMD is of major importance for understanding disease pathogenesis and for establishing strategies to prevent blindness. While the etiology of AMD remains unclear, it is considered multifactorial, with environmental and polygenic components. Environmental (e.g. smoking, diet rich in saturated fat or poor in antioxidants), or biological risk factors (e.g. high plasma cholesterol), were identified [4-7]. In addition, a genetic predisposition was suggested, based on familial aggregation and twin-studies [8,9]. Because of the extreme phenotypic heterogeneity of AMD, a monogenic model is unlikely in this disease, thus research for predisposing genetic polymorphisms is of major importance. Genetic studies have demonstrated or suggested a role for apolipoprotein E (APOE), ATP-Binding Cassette subfamily A member 4 (ABCA4), hemicentin-1, and fibulin-5 genes in AMD [10-16]. Although investigated in sporadic AMD, level of risk was found stronger for genetic markers than for other classical risk factors in populations of different ancestry or ethnic background [3,17].
Linkage studies identified several genomic regions of interest in susceptibility for AMD [18-22]. Significant associations with the Y402H single nucleotide polymorphism (SNP) of complement factor H (CFH) gene consistently support this genetic variation as a risk factor for AMD in different populations of Caucasian ancestry [23-30]. However, the CFH Y402H polymorphism was not found as a genetic risk factor of AMD in Japanese patients [31,32].
Subsequently, a whole-genome association study in familial cases with AMD, identified a linkage peak for AMD at 10q26, most highly significant between (a pleckstrin-homology-domain-containing protein involved in phospho-inositide metabolism, loci PLEKHA1) and LOC387715 [33,34]. Susceptibility for AMD at putative genomic locus LOC387715 was further confirmed [35-38], for the rs10490924 SNP located 6096 bp upstream of the gene encoding secreted heat-shock serine protease involved in matrix remodeling (HTRA1). Recently, a promoter variant located 512 bp upstream of the transcription start site of the HTRA1 gene was demonstrated as functional in AMD pathogenesis  and a strong predictor for advanced-stage AMD in patients of Asian or Caucasian ancestry [39-41]. Because PLEKHA1, LOC387715, and HTRA1 may play independent and possibly synergistic roles, we analyzed these genetic variants at chromosome-10q26 loci, in a case-control study designed to explore a homogeneous group of individuals from a French population with exudative AMD.
200 consecutive exudative AMD patients were recruited at the Eye University Clinic of Creteil. Criteria for inclusion were: unilateral or bilateral choroidal well-defined, occult, minimally classic or predominantly classic neovascularization, or vascularized pigment epithelium detachment; presence of any type of drusen (small or large); women or men aged 65 or older; and no association with other retinal disease. Exudative AMD was diagnosed by investigators, according to the guidelines of the international classification . Patients with exudative AMD underwent complete ophthalmological examination including visual acuity measurement, fundus examination, and a a fluorescein angiography (Topcon 50IA camera, Tokyo, Japan) in our retinal department.
116 healthy men and women, aged 65 or older, were recruited as controls at the local department for clinical research (UFRC) at Creteil. All controls underwent a bilateral fundus examination in order to ascertain they had no signs of any type of drusen, geographic atrophy, or exudative AMD. Fundus photographs were performed in all controls, using a Topcon camera.
Cases and controls were recruited in the same center, and all participants resided in the same area of France. European ancestry was assigned by country of birth. Each study participant gave informed consent according to the French and European bioethical legislation, in agreement with the Declaration of Helsinki for Research involving human subjects. Protocols for DNA extraction and collection for clinical research were in agreement with the French and European legislation.
From a 10 ml peripheral venous blood sample, genomic DNA was extracted from leukocytes after lysis of red blood cells by a non phenolic solvant method using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Genotyping of each SNP was performed by real-time polymerase chain reaction (RT-PCR) and allelic discrimination using reagents and conditions from Custom Taqman SNP Genotyping Assays (Applera Corp., France). Primers and probes were synthesized by Applera SA France for the following SNPs: rs4146894:G>A PLEKHA1, rs10490924:G>T LOC387715, and rs11200638:G>A HTRA1. Primers and probes solution (0.625 ml) and 12.5 ml of 2X qPCR Master Mix No ROX (Eurogentec, Seraing, Belgium) were brought to 25 ml with 50 ng genomic DNA. PCR reaction (40 cycles) and allelic discrimination was processed on 96-well microtiter plates with an Mx3000P QPCR System (Stratagene Europe, Amsterdam Zuidoost, The Netherlands). For each SNP, three pairs of DNA samples exhibiting representative genotypes ascertained by DNA sequencing, were used as references for quantitative PCR genotyping. Results were obtained from duplicate samples of test DNA.
For quality-control purposes, reference genotypes for each SNP were obtained by direct sequencing of 20 randomly selected PCR amplified DNA samples. Target sequences surrounding each SNP were amplified by PCR using the following primer pairs: 5'-TAC CAT CAG GTT CGA CTG GA-3' (forward) and 5'-ACT TTG GGG CTT CCT GTG TT-3' (reverse) for SNP rs4146894 of PLEKHA1; 5'-GTG GAG AAG GAG CCA GTG AC-3' (forward) and 5'-CAG TGT CAG GTG GTG CTG AG-3' (reverse) for SNP rs10490924 of LOC387715; 5'-TCG AAT TAC TTC TGC TCT CTG C-3' (forward) and 5'-GGG GAA AGT TCC TGC AAA TC-3' (reverse) for SNP rs11200638 of HTRA1. Amplification cycles (n=35) consisted of a denaturation step at 94 °C for 30 s, annealing at 60°C for 30 s, and extension at 72 °C for 30 s. DNA sequencing was performed using the Big-Dye terminator cycle sequencing method on a 16-capillary DNA sequencer 3130 (Applied Biosystems, Courtaboeuf, France). Sequence tracks were analyzed by software Seqscape v3.2 (Applied Biosystems). In addition, RT-PCR genotyping of independent groups of 42 randomly selected individuals were replicated at least twice for each SNP, providing 100% reproducibility of genotype calling for all SNPs.
Hardy-Weinberg assumption was assessed by a Pearson goodness-of-fit statistic with the χ test with 1 ° of freedom.
Allelic and genotype distributions were compared between cases and controls using the χ2 test. Logistic regression models were used to estimate odds ratio (OR) with 95% confidence interval (CI) for AMD risk associated with a given SNP. Adjustment variables were age and sex. Bonferroni correction was applied for allelic and genotypic analysis. We presented corrected p for these results (p multiplied by 3). To compare the dominant, recessive, and codominant models and to select the best-fitting genetic model, we used Akaike Information Criterion because these models are not nested. The model with the lowest AIC reflected the best balance of goodness-of-fit and parsimony . Statistical interactions between the two genotypes and sex or age were systematically explored. Significance levels were set at p<0.05. Analyses were performed with the SAS software release 8.02 (SAS Institute INC, Cary, NC).
Haplotypes analyses were based on the maximum likelihood model previously described  and linked to the SEM algorithm  implemented in the THESIAS program (THESIAS release 2, Paris, France). By definition, the reference haplotype corresponded to the more frequent haplotype in control group. Haplotypes with a frequency of less than 5% were excluded from analysis as recommended.
Results & Discussion
The present study was designed to analyze the potential association between three newly found genetic markers of chromosome 10, as well as exudative AMD in a French population. A homogeneous group of 118 well defined AMD cases, with choroidal neovascularization was compared with a group of 116 healthy ontrols. There was no statistical age difference (p=0.59) between cases (72.3±3.8 years) and controls (72.0±3.8 years), and there was no statistical difference (p=0.82) in male to female ratio between cases (45/73; 38.1%) and controls (46/70; 39.7%). Genotype distribution for SNPs at gene loci PLEKHA1, LOC387715, and HTRA1 were in Hardy-Weinberg equilibrium in either case or control groups (χ2 less than or equal to 0.31, p greater than or equal to 0.58). Consistent with previous reports [33,34], SNPs at PLEKHA1 and LOC387715 were in linkage disequilibrium (LD) in cases (D'=0.636, r2=0.292) and controls (D'=0.328, r2=0.269). In agreement with previous studies done in Caucasians or Asians [37,39-41], LOC387715 and HTRA1 polymorphisms were almost in complete LD in cases (D'=1.000, r2=0.873) and controls (D'=0.980, r2=0.975).
Strong associations were found for all polymorphisms with an allelic dosage effect, for risk of AMD (see Table 1). OR, estimated by logistic regression analysis, were similar for all SNPs, before or after adjustment for age and sex. In keeping, there were no significant interactions with age (p>0.57) or sex (p>0.23), for any of the three markers. Consistent with previous reports [33,34], risk of AMD risk was higher with rare alleles T of LOC387715 or A of HTRA1 [37,39-41]. High levels of significance persisted after adjustment for multiple comparisons (Bonferroni correction), whereas OR for PLEKHA1 genotypes were no longer significant after adjustment for HTRA1 or LOC387715 genotypes. The AA genotype was 3.3 (1.3-8.9) with a 95% CI and a p=0.05; and the AG genotype was 1.5 (0.7-3.4) with a 95% CI and p=nonsignificant. A p=0.09 was used for global comparison.
Haplotype estimation revealed a strong association between risk alleles, at PLEKHA1 and HTRA1/LOC387715 loci (Table 2). Due to high LD between the three markers, and singularly between LOC387715 and HTRA1, three major haplotypes, accounting for 93% of genotypes in cases and for 97% in controls, could be analyzed for their potential risk for AMD. Haplotype "A-T-A" of PLEKHA1-LOC387715-HTRA1 was the most prevalent in cases (0.475) and was present in less than 1/5 controls (0.190; p<0.0001). Adjusted OR revealed a four times higher AMD risk for "A-T-A" haplotype carriers as, compared with "G-G-G" haplotype carriers. An increment of disease risk was observed with every added "A" allele of PLEKHA1 (OR=2.1 (1.1-4.1), 95% CI p=0.04), in carriers of the G-G haplotype of LOC387715-HTRA1. However, the "A-G-G" haplotype was not associated with AMD, suggesting that HTRA1/LOC387715 gene variants had stronger effects than PLEKHA1 on risk of exudative AMD in the present population. Therefore, in agreement with several reports in study populations selected for different stages of disease [33,36-39] or ethnic background [40,41], genetic variations at the HTRA1/LOC387715 locus seemed to play a predominant role on the risk of exudative AMD, over variations at the PLEKHA1 locus.
We further analyzed for consistency of allelic frequencies for the three SNPs of locus 10q26, in the present study-population with those previously reported in AMD case-control studies (see Table 3). In the present sample of French healthy elderly controls, allelic frequencies and genotypic distributions for all markers were similar with those previously reported in Caucasian populations of comparable age and sex ratio [33-39] and about half that observed for LOC387715 and HTRA1 SNPs in Asians [40,41]. Prevalence of risk allele "A" of PLEKHA1 was similar in French patients with exudative AMD, with those reported in other Caucasian populations with any form of AMD. Consistent with previous reports [40,41], risk alleles of LOC387715 appeared more prevalent in AMD population than controls. It is notable that the LOC387715 A69S polymorphism was recently found to be related to progression from early to advanced stage of AMD . A prospective analysis of the Age-Related-Eye Disease Study (AREDS) cohort showed that the effect of LOC387715 was stronger for progression to exudative AMD (OR: 6.1) compared with geographic atrophy (OR: 3.0). Our purpose was to confirm the association between exudative AMD and HTRA1 in French population. Deliberately, we did not analyzed early stages of AMD, geographic atrophy AMD, or disease progression. A comparison between these subgroups of AMD was recently performed in large series of patients by Cameron et al .
The present study focused on three SNPs located on neighboring loci within chromosomal region 10q26. A meta-analysis  and a review  observed this genomic region has demonstrated strong evidence for linkage to AMD at genome-wide significance level in families affected with AMD [33,49]. Subsequent studies pointed LOC387715 as a second putative genetic risk locus for AMD in conjunction with SNP Y402H of the CFH gene [33-37,50]. Indeed, significant association was found across a 60 kb region of high LD harboring loci PLEKHA1 and LOC387715. Protein PLEKHA1 is a ubiquitous cytoplasmic adaptator containing a pleckstrin homology-domain, recruited to the plasma membrane in response to the activation of phosphoinositol 3-kinase [51,52], a key component of multiple signaling pathways, including regulation of cell survival, cellular growth and cellular motility . However, rs4146894 is an intronic polymorphism with no impact on primary PLEKHA1 protein structure, and no proven role so far on PLEKHA1 protein expression. Our findings of a minor independent effect of a genetic variant at this locus on the risk of AMD are in agreement with those of other researchers. We feel these results warrant further studies on the biologic role of PLEKHA1 expression in normal or diseased retina.
Similarly, SNP rs10490924 of LOC387715 is located within a genomic region of unknown function, which remains to be definitely proven as an expressed gene in human. However, in keeping with previous reports in populations of different ancestries, we found SNP at LOC387715 in almost complete LD with SNP rs11200638 of HTRA1 gene. This tightly linked physical association between both genomic variations, suggest that concerted regulations might operate on both risk alleles, possibly through DNA interactions yet to be discovered with trans-acting factors. Apart from a putative and proper role in AMD, LOC387715 SNP is known to reside 6 kb upstream of HTRA1. This gene encodes a secreted heat shock serine protease involved in the modulation of various malignancies, chronic diseases, such as Duchenne muscular dystrophy, and osteoarthritis [54,55]. HTRA1 is expressed in a variety of human tissues and cells, including retinal pigment epithelium. Located within a CpG island of the proximal promoter, 512 bp upstream of HTRA1 transcription start site, the risk allele "A" of HTRA1 SNP was shown to enhance HTRA1 transcription in vitro in retinal pigment epithelium cell-lines . In the same study, homozygous "AA" individuals expressed higher levels of HTRA1 in their circulating lymphocytes in vivo, compared with "GG" individuals. HTRA1 was suggested to play dual functions in extracellular protein degradation and in cellular growth or survival. In chronic inflammatory conditions, extracellular protease activities of HTRA1 may potentially favor neovascularization by enhancing extracellular matrix components degradation, through increased expression of matrix metalloproteases , or binding to transforming growth factor-α, an important key regulator of angiogenesis . In conditions of cellular stress, intracellular proapototic properties of HTRA1 may accelerate cell-death and tissue degeneration, with potential implications in the development of retinal atrophy . Further studies are underway to clarify the role of this important stress-inducible protein in the pathogenesis of AMD.
In conclusion, we found an association of three neighboring genomic variants of chromosome 10 with exudative AMD in a French population. However, the association was stronger and more specific with HTRA1/LOC387715, than with PLEKHA1 polymorphism.
We thank Josseline Kaplan for scientific support and Chantal Bernard, Marjorie Jodar, Patrick Ledudal, Christine Morel, and Laure Muller for expert technical assistance. This study was supported by the Darty Foundation, The Fondation Retina France, the Association DMLA, INSERM, and PHRC program of Assistance Publique Hôpitaux de Paris.
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