Typing of ARMS2 and CFH in Age-Related Macular Degeneration Case-Control Study and Assessment of Frequency in the Italian Population

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD Typing of ARMS2 and CFH in Age-Related Macular Degeneration Case-Control Study and Assessment of Frequency in the Italian Population

Federico Ricci, MD; Stefania Zampatti, MD; Francesca D’Abbruzzi, MD; Filippo Missiroli, MD; Claudia Martone; Tiziana Lepre; Ilenia Pietrangeli; Cecilia Sinibaldi, PhD; Cristina Peconi; Giuseppe Novelli, PhD, MD; Emiliano Giardina, PhD

Objectives: To determine the effects of the polymor- ences in allele and genotype association between classic phisms CFH Tyr402His and ARMS2 del443ins54 on sus- and occult choroidal neovascularization. We also ob- ceptibility to age-related macular degeneration (AMD) served that 39% of the samples in the general Italian popu- and to find the frequencies of these single-nucleotide poly- lation were at least 5.4 times more likely than control sub- morphisms in an Italian population that was not exam- jects to develop AMD. ined clinically. Conclusions: To our knowledge, this is the first confir- Methods: A total of 286 control subjects (126 men and mation of the association of del443ins54 in Italian pa- 160 women) and 159 white patients (73 men and 86 wom- tients with AMD, and we also confirmed the association en) harboring exudative AMD in 1 eye were recruited. of Tyr402His with CFH. Genetic analysis of the general A third group of 182 DNA samples from blood donors population suggested that analysis of the ARMS2 and CFH of the same geographical areas were also typed to assess risk alleles alone may be helpful in differentiating high- the frequency of CFH Tyr402His and ARMS2 del443ins54 risk individuals (odds ratioϾ5.00) from low-risk indi- polymorphisms in the general population. The data were viduals (odds ratioϽ0.45). analyzed statistically by a standard 2ϫ2 table, Fisher exact tests, and odds ratios. Clinical Relevance: Individuals at high risk for developing AMD could be identified and selected for specific Results: The deletion-insertion at chromosome 10q26 prevention programs. In this context, the development (del443ins54) showed the strongest association with AMD of prevention programs based on dietary antioxidants or in terms of both P value and odds ratio (P=2.7ϫ10−15; on close monitoring of at-risk individuals should be con- odds ratio=3.25), and a highly significant association was sidered or suggested. also confirmed for Tyr402His at the CFH locus (P=9.9ϫ10−13; odds ratio=2.86). We found no differ- Arch Ophthalmol. 2009;127(10):1368-1372

GE-RELATED MACULAR DE- the early stages.2,4-6 Accordingly, early iden- generation (AMD) is the tification of individuals at greatest risk for Author Affiliations: Unità most common irreversible vision loss due to AMD prior to the devel- Operative Semplici cause of severe vision loss opment of any signs of the disease can be Dipartimentali Patologia throughout the world in clinicallyimportant.Inthisrespect,theiden- Retinica Fondazione PTV people aged 50 years or older.1 Thirty per- tification and validation of genetic predis- “Policlinico Tor Vergata” A centofpeopleolderthan75yearsshowearly posing variants to be used as biomarkers (Drs Ricci, D’Abbruzzi, and 2,3 Missiroli), and Department of signs of the disease. Laser surgery, pho- would help to identify people at increased Biopathology, Centre of todynamic therapy, and intraocular injec- risk for more advanced stages of AMD. Excellence for Genomic Risk tions offer some chance of visual improve- Hence, heredity is a primary contributor to Assessment in Multifactorial ment, but they require invasive delivery AMD susceptibility as suggested by family and Complex Diseases, School methods and cannot always prevent the and twin studies.7,8 First-degree relatives of Medicine, University of progress of the disease. Although several of patients with AMD are at increased risk Rome “Tor Vergata” and risk factors for AMD such as smoking, being (odds ratio [OR]=2.4) for the condition Fondazione PTV “Policlinico Tor Vergata” (Drs Zampatti, white, and having a family history of AMD compared with first-degree relatives in fami- Martone, Lepre, Pietrangeli, have been reported, the risk of developing lies without the disorder. They are also af- Sinibaldi, Peconi, Novelli, and advanced AMD is assessed on the basis of fectedatayoungerageandhaveanincreased Giardina), Rome, Italy. ocular findings in those who already have lifetime risk of late AMD (risk ratio=4.2).9-11

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 In 2005, several groups12-16 independently reported that AMD is strongly associated with the polymorphism Table 1. Clinical Characteristics of Patients With Tyr402His in the complement factor H gene (CFH) (Gen- Age-Related Macular Degeneration and Control Subjects Bank NG_007259), located in chromosome 1q31. CFH is Healthy implicated in all stages of AMD from early hallmarks such Patients Control 11,17 as drusen to vision-disabling late AMD. With AMD Subjects The average ORs for the CFH Tyr402His variant are Characteristic (n = 159) (n = 286) 2- to 7-fold depending on the number of risk alleles. Also, Age, y it was calculated that individuals homozygous for the risk Range 50-91 50-86 allele C at the CFH Tyr402His polymorphism have a 48% Mean (SD) 75.0 (7.8) 69.2 (11.1) risk of developing late AMD by age 95 years, whereas this Sex, No. (%) risk does not exceed 22% for noncarriers.9 A second poly- Male 73 (46) 126 (44) morphism (Ala69Ser) in the age-related maculopathy sus- Female 86 (54) 160 (56) Family history of AMD, No. (%) 82 (52) NA ceptibility 2 gene (ARMS2, also known as LOC387715) CNV, No. (%) (GenBank NG_011725) located on chromosome 10q26 Classic 59 (37) NA was reported as another major locus contributing to the Occult 100 (63) NA pathogenesis of AMD.18-20 Rivera et al19 found the stron- Bilateral neovascular AMD, No. (%) 15 (9) NA gest association with LOC387715, reporting a 7.6-fold in- Age at diagnosis, mean (SD) 72.8 (7.8) NA creased risk for individuals homozygous for the risk al- Abbreviations: AMD, age-related macular degeneration; CNV, choroidal lele T of rs10490924. Association at chromosome 10q26 neovascularization; NA, not applicable. is located between 2 nearby genes, ARMS2 and HTRA1 (high-temperature requirement factor A1; GenBank NG_011554), suggesting 2 equally probable candi- fore the genetic testing and classified as either classic CNV (including prevalently classic) or occult CNV (including mini- dates. Recently it was reported that a deletion-insertion mally classic). The CNV was classified by the type of fluorescein polymorphism in ARMS2 (del443ins54) was strongly as- leakage based on guidelines from the Macular Photocoagula- sociated with AMD, directly affecting the transcript by tion Study and other studies.23,24 removing the polyadenylation signal and inserting a 54– There were 286 control subjects (126 men and 160 wom- base pair (bp) element known to mediate rapid messen- en). They had no family history of AMD, signs of AMD, or any ger RNA turnover.21 As a consequence, ARMS2 shows no other major eye disease except mild senile cataracts and low detectable expression in homozygous carriers of the de- refractive defects. In this group, fundus examination excluded letion-insertion variant. In this article, we present the first the presence of drusen, focal atrophy, and abnormal retinal pig- confirmation to our knowledge of a deletion-insertion at ment epithelium change. chromosome 10q26 (del443ins54) in the Italian popu- A third group of samples was also obtained from 182 blood donors from the same geographical areas as the patients and lation, we confirm the association of CFH single- control subjects. All of the patients and control subjects were nucleotide polymorphism rs1061170 (Tyr402His), we as- sex matched. The study was conducted according to the Dec- sess the joint effects of both variations on AMD, and we laration of Helsinki, and informed consent was obtained from assess the frequencies of the single and complex geno- the participating subjects after the nature of the study had been types in the general Italian population. explained. The study protocol was approved by the ethics com- mittee of the University of Rome “Tor Vergata,” Rome, Italy. METHODS GENETIC ANALYSIS

STUDY SUBJECTS Genotyping of rs1061170 was performed by TaqMan assays (Applied Biosystems, Foster City, California). Reactions were One hundred fifty-nine consecutive white patients (73 men and run in an AB7500 (Applied Biosystems) and interpreted using 86 women) harboring exudative AMD in 1 eye were prospec- Sequence Detection System 2.1 software (Applied Biosystems). tively recruited at the Medical Retina Centre (Table 1). All Each plate contained 3 positive control samples (samples previ- study subjects underwent a detailed eye examination includ- ously confirmed by direct sequencing as heterozygous and both ing best-corrected visual acuity and slitlamp biomicroscopy of homozygous) and 2 negative control samples. No departure the fundus. Color fundus photography and fluorescein angi- from Hardy-Weinberg equilibrium was detected. Results from ography were performed. Exudative AMD was diagnosed by the genotype assessment of rs1061170 and del443ins54 were con- investigators (F.R., F.D., and F.M.) according to the interna- firmed by postgenotyping direct resequencing of 10 random tional classification guidelines.22 samples. Typing of del443ins54 was performed by polymerase Inclusion criteria were being older than 55 years and hav- chain reaction and agarose gel electrophoresis. Polymerase ing unilateral or bilateral exudative AMD. Exclusion criteria chain reaction was performed in a 25-µL volume containing were presence of other retinal disease (eg, diabetic retinopa- 5mM magnesium chloride, 2 µL of 10X buffer (Applied Biosys- thy, high myopia, or retinal dystrophies), having the atrophic tems),1UofTaqGold polymerase (Applied Biosystems), form of AMD, association of atrophic and exudative forms of 1.25mM deoxynucleoside triphosphate (Invitrogen Corp, Carls- AMD, and having fibrovascular scarring that did not allow the bad, California), 80 ng of template DNA, and 10 pmol of each choroidal neovascularization (CNV) angiographic type to be primer (forward: 5Ј-TCCTAACATCTGGATTCCTC-3Ј; re- precisely graded. verse: 5Ј- TGAAGTCCAAGCTTCTTACC-3Ј). Polymerase chain To classify the initial lesions before any treatment, exuda- reaction amplification included a 10-minute hot start at 94°C, 30 tive AMD was graded on the earliest fluorescein angiography cycles of denaturation at 94°C for 1 minute, annealing at 58°C examination results available. The grading was performed be- for 1 minute, extension at 72°C for 1 minute, and a final exten-

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 Table 2. Allele and Genotype Frequency of del443ins54 and rs1061170 in Italian Patients vs Control Subjects and vs the General Population

Control Control Risk Subjects, OR Subjects, Gene Marker Allele AMD, % % P Value (95% CI) Genotype AMD, % % P Value OR (95% CI) Italian Patients vs Control Subjects ARMS2 del443ins54 Del 51 24 2.7 ϫ 10−15 3.25 (2.36-4.41) Del/del 23 3 2.5 ϫ 10−16 20.61 (8.83-48.11) Het 55 43 7.4 ϫ 10−7 3.18 (2.01-5.03) Wt/wt 22 54 5.9 ϫ 10−11 0.24 (0.15-0.37) CFH rs1061170 C 54 29 9.9 ϫ 10−13 2.86 (2.14-3.84) C/C 27 5 1.1 ϫ 10−13 13.06 (6.27-27.19) C/T 55 48 3.0 ϫ 10−5 2.88 (1.76-4.72) T/T 19 47 1.3 ϫ 10−8 0.26 (0.16-0.41) Italian Patients vs General Population ARMS2 del443ins54 Del 51 20 4.9 ϫ 10−16 3.99 (2.77-5.73) Del/del 23 2 1.3 ϫ 10−13 26.96 (8.96-81.04) Het 55 37 1.1 ϫ 10−8 4.23 (2.54-7.00) Wt/wt 22 61 6.1 ϫ 10−13 0.18 (0.10-0.28) CFH rs1061170 C 54 36 2.0 ϫ 10−6 2.12 (1.55-2.91) C/C 27 15 5.1 ϫ 10−6 4.52 (2.32-8.80) C/T 55 46 1.3 ϫ 10−4 2.78 (1.63-4.73) T/T 19 39 4.6 ϫ 10−6 0.31 (0.19-0.52)

Abbreviations: AMD, age-related macular degeneration; CI, confidence interval; del, deletion; het, heterozygous; OR, odds ratio; wt, wild type.

Table 3. Joint Odds Ratios for the ARMS2 and CFH Variations in the Patient Populationa

CFH Genotype

ARMS2 Genotype C/C C/T T/T Del/del 59.50 (9.20-384.98) NA 25.50 (5.06-128.58) Het 65.17 (16.88-251.53) 13.05 (4.42-38.59) 5.46 (1.75-17.08) Wt/wt 0.44 (0.12-1.59) 0.20 (0.10-0.37) 0.15 (0.05-0.45)

Abbreviations: del, deletion; het, heterozygous; NA, not applicable; wt, wild type. a The values are expressed as odds ratios.

sion at 72°C for 10 minutes. Amplified polymerase chain reac- cal examinations, the deletion-insertion at chromo- tion products were resolved by electrophoresis on 1.8% agarose some 10q26 (del443ins54) showed the strongest (the size of the deleted allele is 239 bp, whereas the size of the association with AMD in terms of both P value and OR nondeleted allele is 628 bp). (P=2.7ϫ10−15; OR=3.25) (Table 2). Genotype association revealed OR values of 3.18 and 20.61 if 1 or 2 risk STATISTICAL ANALYSIS alleles were present, respectively, with 124 patients (78%) Statistical analyses were performed by a standard 2ϫ2 table having at least 1 risk allele with respect to 130 control and Fisher exact tests. Allele and genotype frequencies of subjects (45%). A highly significant association was also rs1061170 and del443ins54 were compared with those of the confirmed for the risk allele C of rs1061170 at the CFH clinically examined control subjects (n=286) and then sepa- locus, generating a P value of 9.9ϫ10−13 and an OR of rately with those of the clinically unexamined control sub- 2.86. Genotype association revealed OR values of 2.88 jects (n=186). Allele analysis in unrelated samples was per- and 13.06 if 1 or 2 risk alleles were present, respec- formed using the software UNPHASED (http://www.litbio tively, with 122 patients (77%) having at least 1 risk al- .org). The ORs were calculated using the online software “Calculator for Confidence Intervals of Odds Ratio in an Un- lele with respect to 144 control subjects (50%) (Table 2). matched Case Control Study” (http://www.hutchon.net We found no differences in allele and genotype associa- /ConfidOR.htm). Joint analyses of rs1061170 and del443ins54 tion between classic CNV and occult CNV. were performed by comparing the frequencies of combined geno- Table 3 shows the joint OR for the ARMS2 and CFH types in the 3 groups (patients, clinically examined control sub- variations. Joint analyses revealed 5 risk diplotype classes jects, and clinically unexamined control subjects). with ORs ranging from 5.46 (in samples homozygous for the T allele of rs1061170 and heterozygous for del443ins54) RESULTS to 59.50 and 65.17 (in samples homozygous for both risk alleles and in samples homozygous for the C allele of Allele and genotype frequencies of rs1061170 and rs1061170 and heterozygous for del443ins54, respec- del443ins54 were characterized in a cohort of 159 pa- tively). Diplotype analyses in control samples failed to ob- tients with AMD and 286 healthy control subjects. serve samples homozygous for del443ins54 and heterozy- In our Italian case-control cohort consisting of pa- gous for rs1061170, making it unfeasible to assess the OR tients and healthy control subjects who underwent clini- for this diplotype class. The only protective diplotype classes

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©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 were observed for samples homozygous for the nonde- Table 4. Frequency of Diplotypes at the CFH and ARMS2 leted allele of del443ins54 independently from the geno- a type of rs1061170. According to the genotype of rs1061170, Loci in the Italian General Population we observed ORs ranging from 0.15 (for individuals homozygous for the nonrisk allele T of rs1061170) to 0.44 CFH Genotype (for individuals homozygous for the risk allele C of ARMS2 Genotype C/C C/T T/T rs1061170). Del/del 1 1 0 After this determination of contributing genetic fac- Het41815 tors in Italian patients with AMD, we decided to assess Wt/wt 10 27 24 the frequency of combined genotypes at the CFH and Abbreviations: del, deletion; het, heterozygous; wt, wild type. ARMS2 loci in the general population, ie, samples that a were not clinically examined. We typed 182 DNA samples The frequency values are expressed as percentages. from blood donors of the same geographical areas as the patients and control subjects. We observed risk allele fre- environment and genes, limiting the possibility of assess- quencies of 36% and 20% for rs1061170 (risk allele C) ing individual risks. However, unlike most common dis- and del443ins54, respectively (Table 2). The risk allele eases in which many susceptibility genes each have a small frequency of rs1061170 differed somewhat from that re- effect on the phenotype, AMD shows highly penetrant varia- ported in the HapMap database of white individuals (28%), tions that are quite common in the population. Genetic but the frequency of del443ins54 did not differ from that analysis of the general population suggests that the analy- reported in the HapMap database calculated using a sis of the ARMS2 and CFH risk alleles alone may be used marker of complete linkage disequilibrium (rs3750848) to differentiate high-risk individuals (ORϾ5.00) from low- (22%). Table 4 shows the frequency of diplotypes at the risk individuals (ORϽ0.45). CFH and ARMS2 loci in this general population. Inter- Most people with early signs of AMD have little or no estingly, 39% of the blood donors are at least 5.4 times decrease in visual acuity or other aspects of vision. Inde- more likely than control subjects to develop AMD. pendent studies have demonstrated that persons with early- In particular, on the basis of our screening, 18% and stage AMD are more likely to have a stroke incident than 6% of the population have risks 13 and 60 times greater persons without AMD.26-28 The association is independent than control subjects, respectively, for developing the dis- of age, sex, ethnicity, history of diabetes, blood pressure, ease. On the other hand, 61% of people do not have in- cigarette smoking, and other stroke risk factors.26 Our find- creased risks for AMD related to the rs1061170 and ings,demonstratingthepossibilityofdifferentiatingbetween del443ins54 genotypes, with most of them showing re- high-risk individuals (ORϾ5.00) and low-risk individu- duced risk with respect to the general population. als (ORϽ0.45) in the general population, have potential clinical implications. Recently, it was suggested that specific proteomic biomarkers can be useful for predicting AMD COMMENT susceptibility in combination with genomic markers.29 In particular,plasmacarboxyethylpyrrolemarkerlevelsincom- To our knowledge, this is the first confirmation of the as- bination with genomic markers discriminate between pa- sociation of del443ins54 with AMD in the Italian popula- tients with AMD and control subjects with up to approxi- tion. We also confirmed the association with rs1061170 mately 80% accuracy. On this basis, a prevention program in CFH and assessed the frequency of these 2 variations in could be suggested or developed for people at high risk for a control Italian population. With respect to the allele fre- developing AMD (and/or stroke), ie, based on dietary an- quency of rs1061170 (C allele) previously reported in the tioxidants30 or on close monitoring of at-risk individuals. Italian population,25 we observed similar frequencies in the In this perspective, the availability of a true predictive ge- cohort of patients (57% vs 54%, respectively), whereas lower netic screening for AMD may represent a bold step toward frequencies were observed in the healthy control samples personalized and/or preventive medicine for the disease. (39% vs 29%, respectively). As a consequence, we observed higher ORs with respect to those previously re- Submitted for Publication: January 30, 2009; final revi- ported (13.06 vs 3.90, respectively).25 We believe that such sion received May 29, 2009; accepted June 6, 2009. discrepancies may be due to the selection of healthy con- Correspondence: Emiliano Giardina, PhD, Department trol subjects and/or to the size of the case-control cohort of Biopathology, University of Rome “Tor Vergata,” Via analyzed. Similarly, we observed higher frequencies for the Montpellier 1, 00133 Rome, Italy (emiliano.giardina risk allele of del443ins54 in Italian patients with respect @uniroma2.it). to German patients.21 These 2 variations appear to inde- Author Contributions: Dr Giardina had full access to all pendently contribute to the susceptibility to AMD in the of the data in the study and takes responsibility for the in- Italian patients. Such highly penetrant genetic susceptibil- tegrity of the data and the accuracy of the data analysis. ity factors in AMD might be used as diagnostic or prog- Financial Disclosure: None reported. nostic molecular markers and/or for drug response. While genetic screening has become a routine practice for many REFERENCES mendelian diseases, population screening for risk assessment for a complex disease is still unexploited. The rea- 1. 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