Typing of ARMS2 and CFH in Age-Related Macular Degeneration: Title and subTitle BreakCase-Control Study and Assessment of Frequency in the Italian PopulationAssociation of CFH and ARMS2 in AMD FREE

Age-related macular degeneration (AMD) is the most common irreversible cause of severe vision loss throughout the world in people aged 50 years or older.¹ Thirty percent of people older than 75 years show early signs of the disease.^{2 - 3} Laser surgery, photodynamic therapy, and intraocular injections offer some chance of visual improvement, but they require invasive delivery methods and cannot always prevent the progress of the disease. Although several risk factors for AMD such as smoking, being white, and having a family history of AMD have been reported, the risk of developing advanced AMD is assessed on the basis of ocular findings in those who already have the early stages.^{2 ,4 - 6} Accordingly, early identification of individuals at greatest risk for vision loss due to AMD prior to the development of any signs of the disease can be clinically important. In this respect, the identification and validation of genetic predisposing variants to be used as biomarkers would help to identify people at increased risk for more advanced stages of AMD. Hence, heredity is a primary contributor to AMD susceptibility as suggested by family and twin studies.^{7 - 8} First-degree relatives of patients with AMD are at increased risk (odds ratio [OR] = 2.4) for the condition compared with first-degree relatives in families without the disorder. They are also affected at a younger age and have an increased lifetime risk of late AMD (risk ratio = 4.2).^{9 - 11} In 2005, several groups^{12 - 16} independently reported that AMD is strongly associated with the polymorphism Tyr402His in the complement factor H gene (CFH) (GenBank NG_007259), located in chromosome 1q31. CFHis implicated in all stages of AMD from early hallmarks such as drusen to vision-disabling late AMD.^{11 ,17}

The average ORs for the CFHTyr402His variant are 2- to 7-fold depending on the number of risk alleles. Also, it was calculated that individuals homozygous for the risk allele C at the CFHTyr402His polymorphism have a 48% risk of developing late AMD by age 95 years, whereas this risk does not exceed 22% for noncarriers.⁹ A second polymorphism (Ala69Ser) in the age-related maculopathy susceptibility 2 gene (ARMS2,also known as LOC387715) (GenBank NG_011725) located on chromosome 10q26 was reported as another major locus contributing to the pathogenesis of AMD.^{18 - 20} Rivera et al¹⁹ found the strongest association with LOC387715, reporting a 7.6-fold increased risk for individuals homozygous for the risk allele T of rs10490924. Association at chromosome 10q26 is located between 2 nearby genes, ARMS2and HTRA1(high-temperature requirement factor A1; GenBank NG_011554), suggesting 2 equally probable candidates. Recently it was reported that a deletion-insertion polymorphism in ARMS2(del443ins54) was strongly associated with AMD, directly affecting the transcript by removing the polyadenylation signal and inserting a 54–base pair (bp) element known to mediate rapid messenger RNA turnover.²¹ As a consequence, ARMS2shows no detectable expression in homozygous carriers of the deletion-insertion variant. In this article, we present the first confirmation to our knowledge of a deletion-insertion at chromosome 10q26 (del443ins54) in the Italian population, we confirm the association of CFHsingle-nucleotide polymorphism rs1061170 (Tyr402His), we assess the joint effects of both variations on AMD, and we assess the frequencies of the single and complex genotypes in the general Italian population.

One hundred fifty-nine consecutive white patients (73 men and 86 women) harboring exudative AMD in 1 eye were prospectively recruited at the Medical Retina Centre (Table 1). All study subjects underwent a detailed eye examination including best-corrected visual acuity and slitlamp biomicroscopy of the fundus. Color fundus photography and fluorescein angiography were performed. Exudative AMD was diagnosed by the investigators (F.R., F.D., and F.M.) according to the international classification guidelines.²²

Inclusion criteria were being older than 55 years and having unilateral or bilateral exudative AMD. Exclusion criteria were presence of other retinal disease (eg, diabetic retinopathy, high myopia, or retinal dystrophies), having the atrophic form of AMD, association of atrophic and exudative forms of AMD, and having fibrovascular scarring that did not allow the choroidal neovascularization (CNV) angiographic type to be precisely graded.

To classify the initial lesions before any treatment, exudative AMD was graded on the earliest fluorescein angiography examination results available. The grading was performed before the genetic testing and classified as either classic CNV (including prevalently classic) or occult CNV (including minimally classic). The CNV was classified by the type of fluorescein leakage based on guidelines from the Macular Photocoagulation Study and other studies.^{23 - 24}

There were 286 control subjects (126 men and 160 women). They had no family history of AMD, signs of AMD, or any other major eye disease except mild senile cataracts and low refractive defects. In this group, fundus examination excluded the presence of drusen, focal atrophy, and abnormal retinal pigment epithelium change.

A third group of samples was also obtained from 182 blood donors from the same geographical areas as the patients and control subjects. All of the patients and control subjects were sex matched. The study was conducted according to the Declaration of Helsinki, and informed consent was obtained from the participating subjects after the nature of the study had been explained. The study protocol was approved by the ethics committee of the University of Rome “Tor Vergata,” Rome, Italy.

Genotyping of rs1061170 was performed by TaqMan assays (Applied Biosystems, Foster City, California). Reactions were run in an AB7500 (Applied Biosystems) and interpreted using Sequence Detection System 2.1 software (Applied Biosystems). Each plate contained 3 positive control samples (samples previously confirmed by direct sequencing as heterozygous and both homozygous) and 2 negative control samples. No departure from Hardy-Weinberg equilibrium was detected. Results from genotype assessment of rs1061170 and del443ins54 were confirmed by postgenotyping direct resequencing of 10 random samples. Typing of del443ins54 was performed by polymerase chain reaction and agarose gel electrophoresis. Polymerase chain reaction was performed in a 25-μL volume containing 5mM magnesium chloride, 2 μL of 10X buffer (Applied Biosystems), 1 U of Taq Gold polymerase (Applied Biosystems), 1.25mM deoxynucleoside triphosphate (Invitrogen Corp, Carlsbad, California), 80 ng of template DNA, and 10 pmol of each primer (forward: 5′-TCCTAACATCTGGATTCCTC-3′; reverse: 5′- TGAAGTCCAAGCTTCTTACC-3′). Polymerase chain reaction amplification included a 10-minute hot start at 94°C, 30 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes. Amplified polymerase chain reaction products were resolved by electrophoresis on 1.8% agarose (the size of the deleted allele is 239 bp, whereas the size of the nondeleted allele is 628 bp).

Statistical analyses were performed by a standard 2 × 2 table and Fisher exact tests. Allele and genotype frequencies of rs1061170 and del443ins54 were compared with those of the clinically examined control subjects (n = 286) and then separately with those of the clinically unexamined control subjects (n = 186). Allele analysis in unrelated samples was performed using the software UNPHASED (http://www.litbio.org). The ORs were calculated using the online software “Calculator for Confidence Intervals of Odds Ratio in an Unmatched Case Control Study” (http://www.hutchon.net/ConfidOR.htm). Joint analyses of rs1061170 and del443ins54 were performed by comparing the frequencies of combined genotypes in the 3 groups (patients, clinically examined control subjects, and clinically unexamined control subjects).

Allele and genotype frequencies of rs1061170 and del443ins54 were characterized in a cohort of 159 patients with AMD and 286 healthy control subjects.

In our Italian case-control cohort consisting of patients and healthy control subjects who underwent clinical examinations, the deletion-insertion at chromosome 10q26 (del443ins54) showed the strongest association with AMD in terms of both Pvalue and OR (P = 2.7 × 10⁻¹⁵; OR = 3.25) (Table 2). Genotype association revealed OR values of 3.18 and 20.61 if 1 or 2 risk alleles were present, respectively, with 124 patients (78%) having at least 1 risk allele with respect to 130 control subjects (45%). A highly significant association was also confirmed for the risk allele C of rs1061170 at the CFHlocus, generating a Pvalue of 9.9 × 10⁻¹³and an OR of 2.86. Genotype association revealed OR values of 2.88 and 13.06 if 1 or 2 risk alleles were present, respectively, with 122 patients (77%) having at least 1 risk allele with respect to 144 control subjects (50%) (Table 2). We found no differences in allele and genotype association between classic CNV and occult CNV.

Table 3shows the joint OR for the ARMS2and CFHvariations. Joint analyses revealed 5 risk diplotype classes with ORs ranging from 5.46 (in samples homozygous for the T allele of rs1061170 and heterozygous for del443ins54) to 59.50 and 65.17 (in samples homozygous for both risk alleles and in samples homozygous for the C allele of rs1061170 and heterozygous for del443ins54, respectively). Diplotype analyses in control samples failed to observe samples homozygous for del443ins54 and heterozygous for rs1061170, making it unfeasible to assess the OR for this diplotype class. The only protective diplotype classes were observed for samples homozygous for the nondeleted allele of del443ins54 independently from the genotype of rs1061170. According to the genotype of rs1061170, we observed ORs ranging from 0.15 (for individuals homozygous for the nonrisk allele T of rs1061170) to 0.44 (for individuals homozygous for the risk allele C of rs1061170).

After this determination of contributing genetic factors in Italian patients with AMD, we decided to assess the frequency of combined genotypes at the CFHand ARMS2loci in the general population, ie, samples that were not clinically examined. We typed 182 DNA samples from blood donors of the same geographical areas as the patients and control subjects. We observed risk allele frequencies of 36% and 20% for rs1061170 (risk allele C) and del443ins54, respectively (Table 2). The risk allele frequency of rs1061170 differed somewhat from that reported in the HapMap database of white individuals (28%), but the frequency of del443ins54 did not differ from that reported in the HapMap database calculated using a marker of complete linkage disequilibrium (rs3750848) (22%). Table 4shows the frequency of diplotypes at the CFHand ARMS2loci in this general population. Interestingly, 39% of the blood donors are at least 5.4 times more likely than control subjects to develop AMD.

In particular, on the basis of our screening, 18% and 6% of the population have risks 13 and 60 times greater than control subjects, respectively, for developing the disease. On the other hand, 61% of people do not have increased risks for AMD related to the rs1061170 and del443ins54 genotypes, with most of them showing reduced risk with respect to the general population.

To our knowledge, this is the first confirmation of the association of del443ins54 with AMD in the Italian population. We also confirmed the association with rs1061170 in CFHand assessed the frequency of these 2 variations in a control Italian population. With respect to the allele frequency of rs1061170 (C allele) previously reported in the Italian population,²⁵ we observed similar frequencies in the cohort of patients (57% vs 54%, respectively), whereas lower frequencies were observed in the healthy control samples (39% vs 29%, respectively). As a consequence, we observed higher ORs with respect to those previously reported (13.06 vs 3.90, respectively).²⁵ We believe that such discrepancies may be due to the selection of healthy control subjects and/or to the size of the case-control cohort analyzed. Similarly, we observed higher frequencies for the risk allele of del443ins54 in Italian patients with respect to German patients.²¹ These 2 variations appear to independently contribute to the susceptibility to AMD in the Italian patients. Such highly penetrant genetic susceptibility factors in AMD might be used as diagnostic or prognostic molecular markers and/or for drug response. While genetic screening has become a routine practice for many mendelian diseases, population screening for risk assessment for a complex disease is still unexploited. The reason for this is that susceptibility to common diseases results from complex and unpredictable interactions of environment and genes, limiting the possibility of assessing individual risks. However, unlike most common diseases in which many susceptibility genes each have a small effect on the phenotype, AMD shows highly penetrant variations that are quite common in the population. Genetic analysis of the general population suggests that the analysis of the ARMS2and CFHrisk alleles alone may be used to differentiate high- risk individuals (OR > 5.00) from low-risk individuals (OR < 0.45).

Most people with early signs of AMD have little or no decrease in visual acuity or other aspects of vision. Independent studies have demonstrated that persons with early-stage AMD are more likely to have a stroke incident than persons without AMD.^{26 - 28} The association is independent of age, sex, ethnicity, history of diabetes, blood pressure, cigarette smoking, and other stroke risk factors.²⁶ Our findings, demonstrating the possibility of differentiating between high-risk individuals (OR > 5.00) and low-risk individuals (OR < 0.45) in the general population, have potential clinical implications. Recently, it was suggested that specific proteomic biomarkers can be useful for predicting AMD susceptibility in combination with genomic markers.²⁹ In particular, plasma carboxyethylpyrrole marker levels in combination with genomic markers discriminate between patients with AMD and control subjects with up to approximately 80% accuracy. On this basis, a prevention program could be suggested or developed for people at high risk for developing AMD (and/or stroke), ie, based on dietary antioxidants³⁰ or on close monitoring of at-risk individuals. In this perspective, the availability of a true predictive genetic screening for AMD may represent a bold step toward personalized and/or preventive medicine for the disease.

Correspondence: Emiliano Giardina, PhD, Department of Biopathology, University of Rome “Tor Vergata,” Via Montpellier 1, 00133 Rome, Italy (emiliano.giardina@uniroma2.it).

Submitted for Publication: January 30, 2009; final revision received May 29, 2009; accepted June 6, 2009.

Author Contributions: Dr Giardina had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Financial Disclosure: None reported.