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Association of Complement Factor H and LOC387715 Genotypes with Response of Exudative Age-Related Macular Degeneration to Intravitreal Bevacizumab
Association of Complement Factor H and LOC387715 Genotypes with Response of Exudative Age-Related Macular Degeneration to Intravitreal Bevacizumab Milam A. Brantley, Jr, MD, PhD,1,2 Amy M. Fang, MD,1 Jennifer M. King, BS,1 Asheesh Tewari, MD,1,2 Steven M. Kymes, PhD, MHA,1 Alan Shiels, PhD1 Purpose: To investigate whether there is an association between complement factor H (CFH) or LOC387715 genotypes with response to treatment with intravitreal bevacizumab for exudative age-related macular degeneration (AMD). Design: Retrospective cohort study. Participants: The study cohort consisted of 86 patients being treated for neovascular AMD with bevacizumab alone. Methods: Genotype determination for the CFH Y402H and LOC387715 A69S polymorphisms was performed by allele-specific digestion of polymerase chain reaction products. All patients were treated with 1.25 mg intravitreal bevacizumab at 6-week intervals until choroidal neovascularization was no longer active. Main Outcome Measures: CFH Y402H and LOC387715 A69S polymorphisms. Choroidal neovascular lesion characteristics were ascertained by fluorescein angiography. Snellen visual acuity (VA) was measured before and after treatment. Results: For the CFH Y402H polymorphism, patients with the CFH TT genotype had the largest choroidal neovascular lesions (P ⫽ 0.02). With treatment, VA improved from 20/248 to 20/166 for the CFH TT genotype and from 20/206 to 20/170 for the TC genotype, but fell from 20/206 to 20/341 for the CFH CC genotype (P ⫽ 0.016). Only 10.5% of patients with the CFH CC genotype demonstrated improved VA with treatment, compared with 53.7% of CFH TT and TC genotypes (P ⫽ 0.004). For the LOC387715 A69S variant, patients with the TT genotype had the largest choroidal neovascular lesions (P ⫽ 0.012). There was no significant difference in response to bevacizumab treatment according to LOC387715 genotype. Conclusions: The AMD-associated CFH Y402H and LOC387715 A69S variants were associated with differences in choroidal neovascular lesion size in this study. Patients with the CFH CC genotype fared significantly worse with intravitreal bevacizumab than did those with the CFH TC and TT genotypes, suggesting a potential pharmacogenetic relationship. Prospective studies to confirm or refute this observation should be considered. Ophthalmology 2007;114:2168 –2173 © 2007 by the American Academy of Ophthalmology.
Age-related macular degeneration (AMD) is the most common irreversible cause of severe vision loss throughout the world in individuals ⬎50 years old. Advanced AMD includes geographic atrophy, characterized by extensive loss of the choriocapillaris and overlying retinal pigment epithelium, and exudative (neovascular) AMD, characterized by Originally received: June 8, 2007. Final revision: September 12, 2007. Accepted: September 12, 2007. Manuscript no. 2007-771. 1 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri. 2 Barnes Retina Institute, St. Louis, Missouri. Presented at: Association of Research in Vision and Ophthalmology meeting, May 2007, Fort Lauderdale, Florida. Supported by the National Eye Institute, Bethesda, Maryland (grant no. EY012284, NEI Core Grant 5 P30 EY02687), and a grant from Research to Prevent Blindness, New York, New York, to the Department
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© 2007 by the American Academy of Ophthalmology Published by Elsevier Inc.
invasion of blood vessels into subretinal spaces. Along with dietary and environmental risk factors for AMD, heredity is a primary contributor to AMD susceptibility.1 Recently, a coding variation in the complement factor H (CFH) gene on chromosome 1q32 was found to be strongly associated with AMD.2–5 The predicted tyrosine-to-histidine substitution at amino acid position 402 (Y402H) in the CFH protein is the result of a T-to-C transition at nucleotide position 1277 in exon-9 of the gene. Additional reports have confirmed this association in numerous populations of Ophthalmology and Visual Sciences, Washington University School of Medicine. No conflicting relationship exists for any author. Correspondence to Milam A. Brantley, Jr, MD, PhD, Department of Ophthalmology and Visual Sciences/Campus Box 8096, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: [email protected]. ISSN 0161-6420/07/$–see front matter doi:10.1016/j.ophtha.2007.09.008
Brantley et al 䡠 CFH and LOC387715 Genotypes and Response to Intravitreal Bevacizumab throughout the world,6 –23 although this relationship seems to be absent in the Japanese population.24 –26 The CFH Y402H polymorphism has been associated with both exudative8,15 and advanced atrophic10 AMD, as well as AMD progression,20 but limited data are available evaluating AMD phenotypes with regard to CFH genotype.21–23 Linkage studies have identified a region within chromosome 10q26 as a second major locus contributing to AMD pathogenesis. In initial reports,27,28 a single nucleotide polymorphism (rs10490924) within the hypothetical gene LOC387715 was found to confer an increased risk for development of AMD. Further studies have supported the association of this alanine-to-serine substitution (A69S) with AMD,17,18,20,29 –33 and have suggested that cigarette smoking may influence susceptibility to AMD conferred by LOC387715.29 This two-exon gene encodes a distinct 107-amino acid protein with no matches in public protein or protein motif databases.27 LOC387715 appears to be a phylogenetically recent gene with conservation restricted to the primate lineage.27 Expression of the mRNA is abundant in placental tissue and has been found in human retina. This locus has been renamed ARMS2 (age-related maculopathy susceptibility gene 2), but biochemical data still are necessary to confirm that this putative protein plays a role in the pathogenesis of AMD. A recent genome-wide association study in patients from the United States and Hong Kong identified another single nucleotide polymorphism (rs11200638), located approximately 5.5 kilobases downstream of the LOC387715 rs10490924 single nucleotide polymorphism in the putative GC-rich promoter region of the HTRA1 gene, which codes for a serine protease.34,35 Additional studies have linked HTRA1 with AMD,36,37 but it remains unclear if either of the polymorphisms in the 10q26 region (rs10490924 and rs11200638) is causally associated with AMD. The addition of antivascular endothelial growth factor (anti-VEGF) therapy to the treatment armamentarium for choroidal neovascularization has revolutionized treatment of exudative AMD. Bevacizumab (Avastin, Genentech, South San Francisco, CA) is a recombinant, humanized monoclonal anti-VEGF antibody that binds all VEGF isoforms and exerts its neutralizing effect by inhibiting the VEGF–receptor interaction, thus blocking both increased vascular permeability and angiogenesis. The drug is approved by the United States Food and Drug Administration for intravenous use for metastatic colorectal cancer. Bevacizumab has been administered off label for the treatment of neovascular AMD38 – 42 and other retinal vascular conditions43– 45 with encouraging results. Genotype–phenotype correlation studies have suggested that the AMD-associated CFH genetic variant may play a role in the type or size of neovascular complex that forms in exudative AMD.21–23 The purpose of this study was investigate whether there is an association between response to treatment for neovascular AMD with bevacizumab and the CFH Y402H variant. For comparison, we also evaluated one of the chromosome 10q26 polymorphic loci, LOC387715 A69S, for potential relationships to treatment response.
Materials and Methods Patients, Clinical Examination, and Treatment This retrospective cohort study was approved by the Washington University Human Research Protection Office and the Barnes Retina Institute Study Center. Research adhered to the tenets of the Declaration of Helsinki and was conducted in accordance with Health Insurance Portability and Accountability Act regulations. All participants were enrolled from the clinical offices of the Barnes Retina Institute and signed written informed consent before participation. Mouthwash samples for genotyping were collected from 86 Caucasian patients with a diagnosis of exudative AMD who were undergoing treatment for an active neovascular lesion with intravitreal bevacizumab. Only eyes that had received no previous therapeutic intervention for AMD were enrolled in the study. For each patient, an intravitreal injection of 1.25 mg bevacizumab was performed at the initial presentation of an active choroidal neovascular complex, and subsequent injections were performed at 6-week intervals until there was no longer evidence of active neovascularization. Each patient was followed for a minimum of 6 months for inclusion in the study. The AMD phenotypes were characterized by clinical examination, including dilated fundus examination, fundus photography, and fluorescein angiography. Fluorescein angiograms obtained at initial presentation of active choroidal neovascular lesions were classified as either predominantly classic (⬎50% classic, a definable vascular complex appearing early in the angiogram followed by late leakage), minimally classic (⬍50% classic), or occult (leakage appearing only late in the angiogram). Angiograms were read independently by two retina specialists (MAB and AT) masked to patients’ genotype (intergrader ⫽ 0.92). Any discrepancies in lesion classification were openly adjudicated. Fluorescein angiograms were obtained digitally with a Zeiss fundus camera and imaging software (OIS, Sacramento, CA). Snellen visual acuity (VA) was recorded in a standardized manner for all patients at initial presentation and follow-up visits. For all calculations and comparisons, Snellen acuities were converted to log minimal angle of resolution values. These values were converted back to Snellen acuities for reporting the results.
DNA Preparation and Genotyping Participants provided buccal tissue samples by expectorating into 50-ml Falcon conical tubes (BD Biosciences, Franklin Lakes, NJ) after vigorously rinsing for 30 seconds with ⬃20 ml Scope mouthwash (Procter & Gamble, Cincinnati, OH). Genomic DNA was prepared from buccal cells using the Puregene mouthwash kit (Gentra Systems, Minneapolis, MN) and quantified by absorbance at 260 nm (GeneQuant pro, GE Healthcare, Waukesha, WI). Exon-9 of CFH was amplified by polymerase chain reaction using AmpliTaq Gold Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and gene-specific primers located in intron-8 (5=-ctttgttagtaactttagttcgtcttcag) and intron-9 (5=-acaaggtgacataaacattttgcc). Similarly, exon-1 of LOC387715 was amplified using primers located in the 5=-untranslated region (5=-tgagtgagatggcagctgg) and intron-1 (5=-tccagctattcaaccagagg). For restriction fragment length analysis, CFH amplicons (443 base pairs) and LOC387715 amplicons (547 base pairs) were digested with Hsp92 II (Promega, Madison, WI) and Pvu II (New England Biolabs, Ipswich, MA), respectively, according to the manufacturers’ instructions, then visualized on 2.5% agarose-gels stained with SYBR Gold (Molecular Probes, Eugene, OR).
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Ophthalmology Volume 114, Number 12, December 2007 Table 1. Demographics and Age-related Macular Degeneration (AMD) Phenotypes of Study Participants Variable
Value
Number of patients Mean age (yr) Female Exudative AMD in contralateral eye (%) Predominantly classic lesions (% of gradable lesions) Mean GLD Mean disc area Mean number of treatments Mean follow-up (mos) Treatments/month
86 79.8 64.0% 23 (26.7) 31/77 (40.3) 3104 3.90 2.85 9.33 0.305
GLD ⫽ greatest linear dimension.
Data Analysis Descriptive statistics for all demographic and clinical variables were calculated and comparisons made using the t test for means with continuous data (e.g., age, VA) and the chi-square test for categorical data (e.g., gender). Comparisons between genotypes were adjusted for multiple comparisons using the Bonferroni approach. The association between genotype and VA was assessed using generalized linear modeling techniques, with VA expressed in log minimal angle of resolution units. The posttreatment VA was modeled after adjustment for pretreatment acuity. Univariate analysis was conducted and any risk factor for change in VA with an association at the Pⱕ0.10 level was included in the final multivariate model. Genotype was included in the model with a dummy variable for the homozygous candidate allele (CC for CFH, TT for LOC387715) with the P-value associated with the coefficient indicating if posttreatment VA was influenced by the candidate allele (Pⱕ0.05 indicating a significant association).
Results Eighty-six patients with exudative macular degeneration undergoing treatment with intravitreal bevacizumab in one eye were enrolled in the study. Demographic information and characterization of AMD phenotypes are presented in Table 1. Neovascularization
in the study eye was the first evidence of exudative AMD in 73.3% of patients. Subjects received a mean of 2.8 injections over a mean follow-up period of 9.3 months. Table 2 displays the distribution of demographic and clinical phenotype data for the AMD patients according to CFH Y402H genotype. The high-risk CFH CC genotype was seen in 22.1% of AMD patients, and the overall frequency of the C allele in this population was 55.2%. Mean age and gender distributions were similar among the 3 CFH genotypes. The percentage of predominantly classic lesions did not differ among the CFH genotypes. Choroidal neovascular lesions in patients with he CFH TT genotype had the largest mean greatest linear dimension (GLD; P ⫽ 0.02) and disc area (P ⫽ 0.03) on fluorescein angiograms obtained at the time of initial presentation (n ⫽ 77). There was no difference in the mean follow-up period (P ⫽ 0.17) or mean number of treatments per months of follow-up (P ⫽ 0.82) among the CFH genotypes. We examined the pretreatment and posttreatment VA for all eyes in the study by CFH genotype (Fig 1). Mean pretreatment VA for the CFH CC genotype (n ⫽ 19) and for the CFH TC genotype (n ⫽ 57) was 20/206 (Snellen acuity converted from log minimal angle of resolution). The mean pretreatment VA (20/248) for the CFH TT genotype (n ⫽ 10) was somewhat worse than this, but this difference was not significant (P ⫽ 0.86). After treatment, mean VA for the CFH TC genotype (20/170) and the CFH TT genotype (20/166) improved substantially, whereas the mean posttreatment VA for the CFH CC group fell to 20/341. Adjusting for age, pretreatment VA, and lesion size, we found that the mean posttreatment VA for the CFH CC genotype was significantly worse than mean posttreatment VA of the TC and TT genotypes (P ⫽ 0.016), indicating that bevacizumab was much less effective as measured by VA in patients with the CFH CC genotype than in patients with the CFH TC or TT genotypes. In fact, although a majority (53.7%) of patients in the CFH TC and TT groups showed improvement in VA with bevacizumab treatment, only 10.5% of CFH CC patients demonstrated VA improvement (P ⫽ 0.004). Comparing patients with known exudative AMD in the contralateral eye at the time of study enrollment to those with a new diagnosis of exudative AMD, we found no effect of contralateral eye status on posttreatment VA. Table 3 displays the distribution of demographic and clinical phenotype data for the AMD patients according to LOC387715 A69S genotype. The high-risk LOC387715 TT genotype was seen in 17.4% of AMD patients and the overall frequency of the T allele in this population was 39.5%. Mean age and gender distributions
Table 2. Clinical Phenotypes of Age-related Macular Degeneration Patients by Complement Factor H Genotype Characteristic
CC
TC
TT
P
Total number of patients (%) Mean age Female (%) Eyes with predominantly classic lesions (% of gradable lesions) GLD of lesions (m) Disc area Follow-up period (mos) Mean treatments/month Pretreatment VA Posttreatment VA Eyes with improved VA (%)
19 (22.1) 78.2 52.6 6/16 (37.5)
57 (66.3) 79.9 64.9 22/52 (42.3)
10 (11.6) 82.0 80.0 3/9 (33.3)
0.35 0.27 0.94
3528 5.00 8.68 0.27 20/206 20/341 2 (10.5)
2718 2.88 9.79 0.32 20/206 20/170 31 (54.4)
4414 7.56 7.90 0.28 20/248 20/166 5 (50.0)
0.02 0.03 0.17 0.82 0.86 0.016 0.004
GLD ⫽ greatest linear dimension; VA ⫽ visual acuity.
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Brantley et al 䡠 CFH and LOC387715 Genotypes and Response to Intravitreal Bevacizumab (P ⫽ 0.021) on angiograms obtained at the time of initial presentation (n ⫽ 77). There was no difference in the mean follow-up period (P ⫽ 0.20) or mean number of treatments per month of follow-up (P ⫽ 0.76) among the LOC387715 genotypes. Mean pretreatment VA for the LOC387715 GG genotype (n ⫽ 33) was 20/197 and for the GT genotype (n ⫽ 38), 20/196. The mean pretreatment VA (20/288) for the LOC387715 TT genotype (n ⫽ 15) was worse than this, but this difference was not significant (P ⫽ 0.51). Mean posttreatment VA for the LOC387715 GG group (20/191) and the GT group (20/166) improved, whereas the mean posttreatment VA for the TT group fell to 20/344. However, when adjusting for age, pretreatment VA, and lesion size, we found that the mean posttreatment VA for the LOC387715 TT genotype was not significantly different than for those without the TT genotype (P ⫽ 0.18). A similar percentage of patients in each LOC387715 genotype group demonstrated improvement in VA with treatment (P ⫽ 0.94). As with the CFH genotypes, we found no effect of contralateral eye status on posttreatment VA for LOC387715. We examined the combination of CFH and LOC387715 genotypes to determine if the total number of risk alleles (CFH “C” and LOC387715 “T”) was associated with a difference in posttreatment VA. Evaluating 3 groups (0 –1 risk allele, 2 risk alleles, or 3– 4 risk alleles), we found no relationship between the number of risk alleles and posttreatment VA.
Discussion
Figure 1. Graphs demonstrating visual acuity (VA) before (e) and after () treatment with intravitreal bevacizumab. A, Pretreatment and posttreatment VA according to complement factor H (CFH) genotype. *The difference in posttreatment VA between patients with the CFH CC genotype and those with the CFH TC or TT genotype was significant (P ⫽ 0.016). B, Pretreatment and posttreatment VA according to LOC387715 genotype. logMAR ⫽ logarithm of the minimum angle of resolution.
were similar among the 3 LOC387715 genotypes. The percentage of predominantly classic lesions was not significantly different among the LOC387715 genotypes (P ⫽ 0.07). The LOC387715 TT genotype had the largest mean GLD (P ⫽ 0.012) and disc area
In our study population, we identified a significant association between CFH Y402H genotype and response to treatment for neovascular AMD with bevacizumab. Although a small number of reports have correlated AMD clinical phenotypes with CFH Y402H genotype,21–23 and one study showed a possible association between response to photodynamic therapy and CFH genotype,22 there are no available data on response to VEGF inhibitors and genotype. The overall frequency of the CFH Y402H C allele in our population (55.2%) is consistent with previous reports. We found no significant difference in the percentage of eyes in each CFH genotype group with predominantly classic lesions. This contrasts with our and other earlier reports21–23 that found a higher percentage of predominantly classic lesions in the CFH CC group, and may be related to the
Table 3. Clinical Phenotypes of Age-related Macular Degeneration Patients by LOC387715 Genotype Characteristic
TT
GT
GG
P
Total number of patients (%) Mean age Female (%) Eyes with predominantly classic lesions (% of gradable lesions) GLD of lesions (m) Disc area Follow-up period (mos) Mean treatments/month Pretreatment VA Posttreatment VA Eyes with improved VA (%)
15 (17.4) 77.4 66.7 3/14 (21.4)
38 (44.2) 81.1 60.5 17/31 (54.9)
33 (38.4) 79.4 66.7 11/32 (34.3)
0.32 0.85 0.07
4603 7.92 10.33 0.332 20/288 20/344 6 (40.0)
2768 3.06 9.03 0.306 20/196 20/166 17 (44.7)
2909 3.37 9.21 0.302 20/197 20/191 15 (45.5)
0.012 0.021 0.20 0.76 0.51 0.18 0.94
GLD ⫽ greatest linear dimension; VA ⫽ visual acuity.
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Ophthalmology Volume 114, Number 12, December 2007 smaller size of our patient cohort in the present study. Interestingly, patients with no high-risk CFH Y402H alleles (CFH TT) had the largest mean neovascular lesion size as measured by GLD and disc area, consistent with our previous study.23 This suggests that, in this population, the presence of CFH Y402H high-risk alleles does not correlate with increased choroidal neovascular lesion size. Visual acuity at presentation was not significantly different among the CFH genotypes. Mean posttreatment VA improved for both the CFH TT (⫹1.7 lines) and TC (⫹0.8 lines) genotypes, but fell (⫺2.2 lines) for the CC genotype. The fact that post-bevacizumab VA was significantly worse in the CFH CC genotype than for the CFH TC or TT genotypes (P ⫽ 0.016) suggests that genetic factors may play a critical role in the efficacy of therapeutic interventions for exudative AMD. One possibility is that patients with the CFH CC genotype have critical alterations in local inflammatory mediators owing to an abundance of the variant CFH protein, and that additional or more frequent bevacizumab therapy is required to achieve VA improvement. Alternatively, it may be that bevacizumab is simply not as effective in patients with the CFH CC genotype, and that more intensive treatment will not be beneficial. A prospective study evaluating response to standardized treatment regimens according to CFH genotype would help to distinguish between these possibilities. The overall frequency of the LOC387715 high-risk T allele in our population was 39.5%, consistent with previous reports. There was no significant difference in the percentage of predominantly classic lesions among the LOC387715 genotypes. Patients with two copies of the LOC387715 risk allele (LOC387715 TT) had a significantly larger mean lesion size as measured by GLD and disc area (P ⫽ 0.01). This is interesting and suggests that the LOC387715 A69S variant, in contrast to the CFH Y402H variant, could be associated with anatomic or metabolic changes that lead to the development of larger neovascular lesions. Lesion type could potentially play a role; the LOC387715 TT group had the highest percentage of occult lesions (78.6%; P ⫽ 0.07), which can often be larger than predominantly classic lesions at the time of discovery. Initial VA was somewhat worse in the LOC387715 TT group compared with the other groups, and again, this difference may be due to the larger lesion size in this group. Mean posttreatment VA improved for both the LOC387715 GG (⫹0.1 lines) and GT (⫹0.7 lines) genotypes, but fell (⫺0.8 lines) for the LOC387715 TT genotype. This difference in response to treatment, however, did not reach statistical significance. The percentage of patients in each LOC387715 genotype group whose VA improved was also similar. This retrospective study was limited by the size of the patient cohort; additional genotype–phenotype correlations might be detected with a larger sample size. Despite this, we did identify significant differences in lesion size among the CFH and LOC387715 genotypes and, most strikingly, in the response of the CFH CC genotype to intravitreal bevacizumab. We assessed the interaction of the CFH Y402H and LOC387715 A69S variants and found no relationship between the total number of risk alleles and posttreatment
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VA. From this we believe that, in this sample, the CFH Y402H variant influences posttreatment VA, but the LOC387715 A69S variant does not. Further investigation with a larger patient cohort may allow for a better evaluation of such gene interactions and their relationship to treatment response. In summary, we have evaluated AMD clinical phenotypic characteristics and response to treatment with intravitreal bevacizumab with respect to the CFH Y402H and LOC387715 A69S genotypes. We found a relationship between choroidal neovascular lesion size and CFH and LOC387715 genotypes. Most important, we found that the response to intravitreal bevacizumab was significantly worse for the CFH Y402H CC genotype compared with the TC and CC genotypes. Further investigation of this potential pharmacogenetic effect is warranted. Acknowledgments. The authors thank Michael Plotzke for statistical assistance.
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complement factor H to disease risk. Hum Mol Genet 2005; 14:3227–36. Schmidt S, Hauser MA, Scott WK, et al. Cigarette smoking strongly modifies the association of LOC387715 and agerelated macular degeneration. Am J Hum Genet 2006;78: 852– 64. Fisher SA, Rivera A, Fritsche LG, et al. Assessment of the contribution of CFH and chromosome 10q26 AMD susceptibility loci in a Russian population isolate. Br J Ophthalmol 2007;91:576 – 8. Tanimoto S, Tamura H, Ue T, et al. A polymorphism of LOC387715 is associated with age-related macular degeneration. Neurosci Lett 2007;414:71– 4. Ross RJ, Bojanowski CM, Wang JJ, et al. The LOC387715 polymorphism and age-related macular degeneration: replication in three case-control samples. Invest Ophthalmol Vis Sci 2007;48:1128 –32. Francis PJ, George S, Schultz DW, et al. The LOC387715 gene, smoking, body mass index, environmental associations with advanced age-related macular degeneration. Hum Hered 2007;63:212– 8. DeWan A, Liu M, Hartman S, et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 2006;314:989 –92. Yang Z, Camp NJ, Sun H, et al. A variant of the HTRA1 gene increases susceptibility to age-related macular degeneration. Science 2006;314:992–3. Cameron DJ, Yang Z, Gibbs D, et al. HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration. Cell Cycle 2007;6: 1122–5. Yoshida T, DeWan A, Zhang H, et al. HTRA1 promoter polymorphism predisposes Japanese to age-related macular degeneration. Mol Vis 2007;13:545– 8. Avery RL, Pieramici DJ, Rabena MD, et al. Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology 2006;113:363–72. Spaide RF, Laud K, Fine HF, et al. Intravitreal bevacizumab treatment of choroidal neovascularization secondary to agerelated macular degeneration. Retina 2006;26:383–90. Rich RM, Rosenfeld PJ, Puliafito CA, et al. Short-term safety and efficacy of intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Retina 2006;26:495– 511. Costa RA, Jorge R, Calucci D, et al. Intravitreal bevacizumab for choroidal neovascularization caused by AMD (IBeNA study): results of a phase 1 dose-escalation study. Invest Ophthalmol Vis Sci 2006;47:4569 –78. Emerson MV, Lauer AK, Flaxel CJ, et al. Intravitreal bevacizumab (Avastin) treatment of age-related macular degeneration. Retina 2007;27:439 – 44. Iturralde D, Spaide RF, Meyerle CB, et al. Intravitreal bevacizumab (Avastin) treatment of macular edema in central retinal vein occlusion: a short-term study. Retina 2006;26: 279 – 84. Avery RL, Pearlman J, Pieramici DJ, et al. Intravitreal bevacizumab (Avastin) in the treatment of proliferative diabetic retinopathy. Ophthalmology 2006;113:1695–705. Yamamoto I, Rogers AH, Reichel E, et al. Intravitreal bevacizumab (Avastin) as treatment for subfoveal choroidal neovascularisation secondary to pathological myopia. Br J Ophthalmol 2007;91:157– 60.
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Age-related macular degeneration (AMD) is the most common irreversible cause of severe vision loss throughout the world in individuals ⬎50 years old. Advanced AMD includes geographic atrophy, characterized by extensive loss of the choriocapillaris and overlying retinal pigment epithelium, and exudative (neovascular) AMD, characterized by Originally received: June 8, 2007. Final revision: September 12, 2007. Accepted: September 12, 2007. Manuscript no. 2007-771. 1 Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri. 2 Barnes Retina Institute, St. Louis, Missouri. Presented at: Association of Research in Vision and Ophthalmology meeting, May 2007, Fort Lauderdale, Florida. Supported by the National Eye Institute, Bethesda, Maryland (grant no. EY012284, NEI Core Grant 5 P30 EY02687), and a grant from Research to Prevent Blindness, New York, New York, to the Department
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© 2007 by the American Academy of Ophthalmology Published by Elsevier Inc.
invasion of blood vessels into subretinal spaces. Along with dietary and environmental risk factors for AMD, heredity is a primary contributor to AMD susceptibility.1 Recently, a coding variation in the complement factor H (CFH) gene on chromosome 1q32 was found to be strongly associated with AMD.2–5 The predicted tyrosine-to-histidine substitution at amino acid position 402 (Y402H) in the CFH protein is the result of a T-to-C transition at nucleotide position 1277 in exon-9 of the gene. Additional reports have confirmed this association in numerous populations of Ophthalmology and Visual Sciences, Washington University School of Medicine. No conflicting relationship exists for any author. Correspondence to Milam A. Brantley, Jr, MD, PhD, Department of Ophthalmology and Visual Sciences/Campus Box 8096, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail: [email protected]. ISSN 0161-6420/07/$–see front matter doi:10.1016/j.ophtha.2007.09.008
Brantley et al 䡠 CFH and LOC387715 Genotypes and Response to Intravitreal Bevacizumab throughout the world,6 –23 although this relationship seems to be absent in the Japanese population.24 –26 The CFH Y402H polymorphism has been associated with both exudative8,15 and advanced atrophic10 AMD, as well as AMD progression,20 but limited data are available evaluating AMD phenotypes with regard to CFH genotype.21–23 Linkage studies have identified a region within chromosome 10q26 as a second major locus contributing to AMD pathogenesis. In initial reports,27,28 a single nucleotide polymorphism (rs10490924) within the hypothetical gene LOC387715 was found to confer an increased risk for development of AMD. Further studies have supported the association of this alanine-to-serine substitution (A69S) with AMD,17,18,20,29 –33 and have suggested that cigarette smoking may influence susceptibility to AMD conferred by LOC387715.29 This two-exon gene encodes a distinct 107-amino acid protein with no matches in public protein or protein motif databases.27 LOC387715 appears to be a phylogenetically recent gene with conservation restricted to the primate lineage.27 Expression of the mRNA is abundant in placental tissue and has been found in human retina. This locus has been renamed ARMS2 (age-related maculopathy susceptibility gene 2), but biochemical data still are necessary to confirm that this putative protein plays a role in the pathogenesis of AMD. A recent genome-wide association study in patients from the United States and Hong Kong identified another single nucleotide polymorphism (rs11200638), located approximately 5.5 kilobases downstream of the LOC387715 rs10490924 single nucleotide polymorphism in the putative GC-rich promoter region of the HTRA1 gene, which codes for a serine protease.34,35 Additional studies have linked HTRA1 with AMD,36,37 but it remains unclear if either of the polymorphisms in the 10q26 region (rs10490924 and rs11200638) is causally associated with AMD. The addition of antivascular endothelial growth factor (anti-VEGF) therapy to the treatment armamentarium for choroidal neovascularization has revolutionized treatment of exudative AMD. Bevacizumab (Avastin, Genentech, South San Francisco, CA) is a recombinant, humanized monoclonal anti-VEGF antibody that binds all VEGF isoforms and exerts its neutralizing effect by inhibiting the VEGF–receptor interaction, thus blocking both increased vascular permeability and angiogenesis. The drug is approved by the United States Food and Drug Administration for intravenous use for metastatic colorectal cancer. Bevacizumab has been administered off label for the treatment of neovascular AMD38 – 42 and other retinal vascular conditions43– 45 with encouraging results. Genotype–phenotype correlation studies have suggested that the AMD-associated CFH genetic variant may play a role in the type or size of neovascular complex that forms in exudative AMD.21–23 The purpose of this study was investigate whether there is an association between response to treatment for neovascular AMD with bevacizumab and the CFH Y402H variant. For comparison, we also evaluated one of the chromosome 10q26 polymorphic loci, LOC387715 A69S, for potential relationships to treatment response.
Materials and Methods Patients, Clinical Examination, and Treatment This retrospective cohort study was approved by the Washington University Human Research Protection Office and the Barnes Retina Institute Study Center. Research adhered to the tenets of the Declaration of Helsinki and was conducted in accordance with Health Insurance Portability and Accountability Act regulations. All participants were enrolled from the clinical offices of the Barnes Retina Institute and signed written informed consent before participation. Mouthwash samples for genotyping were collected from 86 Caucasian patients with a diagnosis of exudative AMD who were undergoing treatment for an active neovascular lesion with intravitreal bevacizumab. Only eyes that had received no previous therapeutic intervention for AMD were enrolled in the study. For each patient, an intravitreal injection of 1.25 mg bevacizumab was performed at the initial presentation of an active choroidal neovascular complex, and subsequent injections were performed at 6-week intervals until there was no longer evidence of active neovascularization. Each patient was followed for a minimum of 6 months for inclusion in the study. The AMD phenotypes were characterized by clinical examination, including dilated fundus examination, fundus photography, and fluorescein angiography. Fluorescein angiograms obtained at initial presentation of active choroidal neovascular lesions were classified as either predominantly classic (⬎50% classic, a definable vascular complex appearing early in the angiogram followed by late leakage), minimally classic (⬍50% classic), or occult (leakage appearing only late in the angiogram). Angiograms were read independently by two retina specialists (MAB and AT) masked to patients’ genotype (intergrader ⫽ 0.92). Any discrepancies in lesion classification were openly adjudicated. Fluorescein angiograms were obtained digitally with a Zeiss fundus camera and imaging software (OIS, Sacramento, CA). Snellen visual acuity (VA) was recorded in a standardized manner for all patients at initial presentation and follow-up visits. For all calculations and comparisons, Snellen acuities were converted to log minimal angle of resolution values. These values were converted back to Snellen acuities for reporting the results.
DNA Preparation and Genotyping Participants provided buccal tissue samples by expectorating into 50-ml Falcon conical tubes (BD Biosciences, Franklin Lakes, NJ) after vigorously rinsing for 30 seconds with ⬃20 ml Scope mouthwash (Procter & Gamble, Cincinnati, OH). Genomic DNA was prepared from buccal cells using the Puregene mouthwash kit (Gentra Systems, Minneapolis, MN) and quantified by absorbance at 260 nm (GeneQuant pro, GE Healthcare, Waukesha, WI). Exon-9 of CFH was amplified by polymerase chain reaction using AmpliTaq Gold Universal PCR Master Mix (Applied Biosystems, Foster City, CA) and gene-specific primers located in intron-8 (5=-ctttgttagtaactttagttcgtcttcag) and intron-9 (5=-acaaggtgacataaacattttgcc). Similarly, exon-1 of LOC387715 was amplified using primers located in the 5=-untranslated region (5=-tgagtgagatggcagctgg) and intron-1 (5=-tccagctattcaaccagagg). For restriction fragment length analysis, CFH amplicons (443 base pairs) and LOC387715 amplicons (547 base pairs) were digested with Hsp92 II (Promega, Madison, WI) and Pvu II (New England Biolabs, Ipswich, MA), respectively, according to the manufacturers’ instructions, then visualized on 2.5% agarose-gels stained with SYBR Gold (Molecular Probes, Eugene, OR).
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Ophthalmology Volume 114, Number 12, December 2007 Table 1. Demographics and Age-related Macular Degeneration (AMD) Phenotypes of Study Participants Variable
Value
Number of patients Mean age (yr) Female Exudative AMD in contralateral eye (%) Predominantly classic lesions (% of gradable lesions) Mean GLD Mean disc area Mean number of treatments Mean follow-up (mos) Treatments/month
86 79.8 64.0% 23 (26.7) 31/77 (40.3) 3104 3.90 2.85 9.33 0.305
GLD ⫽ greatest linear dimension.
Data Analysis Descriptive statistics for all demographic and clinical variables were calculated and comparisons made using the t test for means with continuous data (e.g., age, VA) and the chi-square test for categorical data (e.g., gender). Comparisons between genotypes were adjusted for multiple comparisons using the Bonferroni approach. The association between genotype and VA was assessed using generalized linear modeling techniques, with VA expressed in log minimal angle of resolution units. The posttreatment VA was modeled after adjustment for pretreatment acuity. Univariate analysis was conducted and any risk factor for change in VA with an association at the Pⱕ0.10 level was included in the final multivariate model. Genotype was included in the model with a dummy variable for the homozygous candidate allele (CC for CFH, TT for LOC387715) with the P-value associated with the coefficient indicating if posttreatment VA was influenced by the candidate allele (Pⱕ0.05 indicating a significant association).
Results Eighty-six patients with exudative macular degeneration undergoing treatment with intravitreal bevacizumab in one eye were enrolled in the study. Demographic information and characterization of AMD phenotypes are presented in Table 1. Neovascularization
in the study eye was the first evidence of exudative AMD in 73.3% of patients. Subjects received a mean of 2.8 injections over a mean follow-up period of 9.3 months. Table 2 displays the distribution of demographic and clinical phenotype data for the AMD patients according to CFH Y402H genotype. The high-risk CFH CC genotype was seen in 22.1% of AMD patients, and the overall frequency of the C allele in this population was 55.2%. Mean age and gender distributions were similar among the 3 CFH genotypes. The percentage of predominantly classic lesions did not differ among the CFH genotypes. Choroidal neovascular lesions in patients with he CFH TT genotype had the largest mean greatest linear dimension (GLD; P ⫽ 0.02) and disc area (P ⫽ 0.03) on fluorescein angiograms obtained at the time of initial presentation (n ⫽ 77). There was no difference in the mean follow-up period (P ⫽ 0.17) or mean number of treatments per months of follow-up (P ⫽ 0.82) among the CFH genotypes. We examined the pretreatment and posttreatment VA for all eyes in the study by CFH genotype (Fig 1). Mean pretreatment VA for the CFH CC genotype (n ⫽ 19) and for the CFH TC genotype (n ⫽ 57) was 20/206 (Snellen acuity converted from log minimal angle of resolution). The mean pretreatment VA (20/248) for the CFH TT genotype (n ⫽ 10) was somewhat worse than this, but this difference was not significant (P ⫽ 0.86). After treatment, mean VA for the CFH TC genotype (20/170) and the CFH TT genotype (20/166) improved substantially, whereas the mean posttreatment VA for the CFH CC group fell to 20/341. Adjusting for age, pretreatment VA, and lesion size, we found that the mean posttreatment VA for the CFH CC genotype was significantly worse than mean posttreatment VA of the TC and TT genotypes (P ⫽ 0.016), indicating that bevacizumab was much less effective as measured by VA in patients with the CFH CC genotype than in patients with the CFH TC or TT genotypes. In fact, although a majority (53.7%) of patients in the CFH TC and TT groups showed improvement in VA with bevacizumab treatment, only 10.5% of CFH CC patients demonstrated VA improvement (P ⫽ 0.004). Comparing patients with known exudative AMD in the contralateral eye at the time of study enrollment to those with a new diagnosis of exudative AMD, we found no effect of contralateral eye status on posttreatment VA. Table 3 displays the distribution of demographic and clinical phenotype data for the AMD patients according to LOC387715 A69S genotype. The high-risk LOC387715 TT genotype was seen in 17.4% of AMD patients and the overall frequency of the T allele in this population was 39.5%. Mean age and gender distributions
Table 2. Clinical Phenotypes of Age-related Macular Degeneration Patients by Complement Factor H Genotype Characteristic
CC
TC
TT
P
Total number of patients (%) Mean age Female (%) Eyes with predominantly classic lesions (% of gradable lesions) GLD of lesions (m) Disc area Follow-up period (mos) Mean treatments/month Pretreatment VA Posttreatment VA Eyes with improved VA (%)
19 (22.1) 78.2 52.6 6/16 (37.5)
57 (66.3) 79.9 64.9 22/52 (42.3)
10 (11.6) 82.0 80.0 3/9 (33.3)
0.35 0.27 0.94
3528 5.00 8.68 0.27 20/206 20/341 2 (10.5)
2718 2.88 9.79 0.32 20/206 20/170 31 (54.4)
4414 7.56 7.90 0.28 20/248 20/166 5 (50.0)
0.02 0.03 0.17 0.82 0.86 0.016 0.004
GLD ⫽ greatest linear dimension; VA ⫽ visual acuity.
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Brantley et al 䡠 CFH and LOC387715 Genotypes and Response to Intravitreal Bevacizumab (P ⫽ 0.021) on angiograms obtained at the time of initial presentation (n ⫽ 77). There was no difference in the mean follow-up period (P ⫽ 0.20) or mean number of treatments per month of follow-up (P ⫽ 0.76) among the LOC387715 genotypes. Mean pretreatment VA for the LOC387715 GG genotype (n ⫽ 33) was 20/197 and for the GT genotype (n ⫽ 38), 20/196. The mean pretreatment VA (20/288) for the LOC387715 TT genotype (n ⫽ 15) was worse than this, but this difference was not significant (P ⫽ 0.51). Mean posttreatment VA for the LOC387715 GG group (20/191) and the GT group (20/166) improved, whereas the mean posttreatment VA for the TT group fell to 20/344. However, when adjusting for age, pretreatment VA, and lesion size, we found that the mean posttreatment VA for the LOC387715 TT genotype was not significantly different than for those without the TT genotype (P ⫽ 0.18). A similar percentage of patients in each LOC387715 genotype group demonstrated improvement in VA with treatment (P ⫽ 0.94). As with the CFH genotypes, we found no effect of contralateral eye status on posttreatment VA for LOC387715. We examined the combination of CFH and LOC387715 genotypes to determine if the total number of risk alleles (CFH “C” and LOC387715 “T”) was associated with a difference in posttreatment VA. Evaluating 3 groups (0 –1 risk allele, 2 risk alleles, or 3– 4 risk alleles), we found no relationship between the number of risk alleles and posttreatment VA.
Discussion
Figure 1. Graphs demonstrating visual acuity (VA) before (e) and after () treatment with intravitreal bevacizumab. A, Pretreatment and posttreatment VA according to complement factor H (CFH) genotype. *The difference in posttreatment VA between patients with the CFH CC genotype and those with the CFH TC or TT genotype was significant (P ⫽ 0.016). B, Pretreatment and posttreatment VA according to LOC387715 genotype. logMAR ⫽ logarithm of the minimum angle of resolution.
were similar among the 3 LOC387715 genotypes. The percentage of predominantly classic lesions was not significantly different among the LOC387715 genotypes (P ⫽ 0.07). The LOC387715 TT genotype had the largest mean GLD (P ⫽ 0.012) and disc area
In our study population, we identified a significant association between CFH Y402H genotype and response to treatment for neovascular AMD with bevacizumab. Although a small number of reports have correlated AMD clinical phenotypes with CFH Y402H genotype,21–23 and one study showed a possible association between response to photodynamic therapy and CFH genotype,22 there are no available data on response to VEGF inhibitors and genotype. The overall frequency of the CFH Y402H C allele in our population (55.2%) is consistent with previous reports. We found no significant difference in the percentage of eyes in each CFH genotype group with predominantly classic lesions. This contrasts with our and other earlier reports21–23 that found a higher percentage of predominantly classic lesions in the CFH CC group, and may be related to the
Table 3. Clinical Phenotypes of Age-related Macular Degeneration Patients by LOC387715 Genotype Characteristic
TT
GT
GG
P
Total number of patients (%) Mean age Female (%) Eyes with predominantly classic lesions (% of gradable lesions) GLD of lesions (m) Disc area Follow-up period (mos) Mean treatments/month Pretreatment VA Posttreatment VA Eyes with improved VA (%)
15 (17.4) 77.4 66.7 3/14 (21.4)
38 (44.2) 81.1 60.5 17/31 (54.9)
33 (38.4) 79.4 66.7 11/32 (34.3)
0.32 0.85 0.07
4603 7.92 10.33 0.332 20/288 20/344 6 (40.0)
2768 3.06 9.03 0.306 20/196 20/166 17 (44.7)
2909 3.37 9.21 0.302 20/197 20/191 15 (45.5)
0.012 0.021 0.20 0.76 0.51 0.18 0.94
GLD ⫽ greatest linear dimension; VA ⫽ visual acuity.
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Ophthalmology Volume 114, Number 12, December 2007 smaller size of our patient cohort in the present study. Interestingly, patients with no high-risk CFH Y402H alleles (CFH TT) had the largest mean neovascular lesion size as measured by GLD and disc area, consistent with our previous study.23 This suggests that, in this population, the presence of CFH Y402H high-risk alleles does not correlate with increased choroidal neovascular lesion size. Visual acuity at presentation was not significantly different among the CFH genotypes. Mean posttreatment VA improved for both the CFH TT (⫹1.7 lines) and TC (⫹0.8 lines) genotypes, but fell (⫺2.2 lines) for the CC genotype. The fact that post-bevacizumab VA was significantly worse in the CFH CC genotype than for the CFH TC or TT genotypes (P ⫽ 0.016) suggests that genetic factors may play a critical role in the efficacy of therapeutic interventions for exudative AMD. One possibility is that patients with the CFH CC genotype have critical alterations in local inflammatory mediators owing to an abundance of the variant CFH protein, and that additional or more frequent bevacizumab therapy is required to achieve VA improvement. Alternatively, it may be that bevacizumab is simply not as effective in patients with the CFH CC genotype, and that more intensive treatment will not be beneficial. A prospective study evaluating response to standardized treatment regimens according to CFH genotype would help to distinguish between these possibilities. The overall frequency of the LOC387715 high-risk T allele in our population was 39.5%, consistent with previous reports. There was no significant difference in the percentage of predominantly classic lesions among the LOC387715 genotypes. Patients with two copies of the LOC387715 risk allele (LOC387715 TT) had a significantly larger mean lesion size as measured by GLD and disc area (P ⫽ 0.01). This is interesting and suggests that the LOC387715 A69S variant, in contrast to the CFH Y402H variant, could be associated with anatomic or metabolic changes that lead to the development of larger neovascular lesions. Lesion type could potentially play a role; the LOC387715 TT group had the highest percentage of occult lesions (78.6%; P ⫽ 0.07), which can often be larger than predominantly classic lesions at the time of discovery. Initial VA was somewhat worse in the LOC387715 TT group compared with the other groups, and again, this difference may be due to the larger lesion size in this group. Mean posttreatment VA improved for both the LOC387715 GG (⫹0.1 lines) and GT (⫹0.7 lines) genotypes, but fell (⫺0.8 lines) for the LOC387715 TT genotype. This difference in response to treatment, however, did not reach statistical significance. The percentage of patients in each LOC387715 genotype group whose VA improved was also similar. This retrospective study was limited by the size of the patient cohort; additional genotype–phenotype correlations might be detected with a larger sample size. Despite this, we did identify significant differences in lesion size among the CFH and LOC387715 genotypes and, most strikingly, in the response of the CFH CC genotype to intravitreal bevacizumab. We assessed the interaction of the CFH Y402H and LOC387715 A69S variants and found no relationship between the total number of risk alleles and posttreatment
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VA. From this we believe that, in this sample, the CFH Y402H variant influences posttreatment VA, but the LOC387715 A69S variant does not. Further investigation with a larger patient cohort may allow for a better evaluation of such gene interactions and their relationship to treatment response. In summary, we have evaluated AMD clinical phenotypic characteristics and response to treatment with intravitreal bevacizumab with respect to the CFH Y402H and LOC387715 A69S genotypes. We found a relationship between choroidal neovascular lesion size and CFH and LOC387715 genotypes. Most important, we found that the response to intravitreal bevacizumab was significantly worse for the CFH Y402H CC genotype compared with the TC and CC genotypes. Further investigation of this potential pharmacogenetic effect is warranted. Acknowledgments. The authors thank Michael Plotzke for statistical assistance.
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