Association of Genetic Polymorphisms with Age-Related Macular Degeneration Clinical and Visual Phenotypes and Response to Treatment AMD Pharmacogenetics and Metabolomics: Moving towards Personalized Medicine Milam Brantley, MD, PhD, Vanderbilt Eye Institute American Geriatrics Society Annual Meeting - 4 May 2013

Disclosures I have no financial disclosures. I will discuss the off-label use of bevacizumab.

Age-related macular degeneration Disease of central retina Blind spots, metamorphopsia Slow progression or rapid change Reading, TV, driving, recognizing faces

Wet Dry

Can dry AMD become wet? Dry Advanced Early Intermediate Wet

Treatment - intravitreal injections Anti-Vascular Endothelial Growth Factor (anti-VEGF) Multiple monthly treatments Can slow vision loss and even improve vision bevacizumab ranibizumab aflibercept

Jahnigen supported research

AMD Studies Genotype-phenotype associations Pharmacogenetics Oxidative stress quantification Metabolomics

Science March 2005 PNAS May 2005 Science 2005;308:419-21 Proc Natl Acad Sci 2005;102:7227-32

Complement Factor H Y402H Chromosome 1q32 Nucleotide change (1277): T to C Controls AMD TT 0.44 0.21 TC 0.45 0.47 CC 0.11 0.32 OR for AMD TT TC OR 2.5-4.6 CC OR 3.3-7.4 Edwards, Science 2005;308:421-4 Amino acid change: Tyrosine to Histidine Y402H

AMD-associated Genes Replicated in multiple studies CFH ARMS2/HTRA1 C2/BF C3 Developing evidence CFI TIMP3 LIPC Variable evidence VEGFA SERPING1 13

AMD genetics & genotype-phenotype associations

153 patients with NVAMD, looked at CFH Y402H 185 eyes with gradable lesions Classified lesions as Predominantly Classic or Occult Examined the % of PC lesions among genotypes CC – 47% PC TC – 37% PC TT – 17% PC OR=2.01 for each C allele Concluded C allele associated with PC in this cohort Am J Ophthalmol 2007;144:404-408.

Address conflicting literature 556 NVAMD cases, 256 controls 3 SERPING1 tSNPs Address conflicting literature Examined both allele frequency and genotypes for SNPs Br J Ophthalmol 2010;94:915-917.

SERPING1 Conclusions rs2511989 negatively associated with AMD rs1005510 positively associated with AMD

AMD Pharmacogenetics

86 pts rec’d bevacizumab monotherapy Treated as needed to inactivate lesion Followed for an average of >9 months Ophthalmology 2007;114:2168-2173. 19

Clinical phenotypes of AMD patients by CFH genotype Risk alleles 1 2 Characteristic TT TC CC p value Pre-treatment VA 20/248 20/206 0.86 Post-treatment VA 20/166 20/170 20/341 0.016 Eyes with improved VA (%) 5 (50.0) 31 (54.4) 2 (10.5) 0.004 * * * Patients with CFH CC genotype did significantly worse with bevacizumab treatment Brantley et al., Ophthalmology 2007;114:2168-2173.

Conclusion – Bevacizumab study Patients with CFH CC genotype faired worse with intravitreal bevacizumab

156 pts rec’d ranibizumab monotherapy Treated as needed to inactivate lesion Followed at least 9 months Br J Ophthalmol 2009;93:610-613. 22

VA outcomes and number of ranibizumab injections by CFH Y402H genotypes Risk alleles 1 2 TT TC CC p value Initial VA, mean logMAR (95% CI) 0.85 (0.68-1.02) 0.89 (0.73-1.05) 0.83 (0.67-0.99) Post-treatment VA at 6 months, mean logMAR (95% CI) 0.71 (0.57-0.84) 0.73 (0.63-0.84) 0.62 (0.50-0.74) 0.38 Post-treatment VA at 9 months, mean logMAR (95% CI) 0.75 (0.60-0.90) 0.78 (0.67-0.88) 0.70 Mean no of injections in 9 months (95% CI) 3.3 (2.87-3.78) 3.8 (3.54-4.16) 3.9 (3.51-4.36) 0.09 Lee et al., Br J Ophthalmol 2009;93:610-613.

Recurrent event analysis of interval between injections by CFH Y402H variant genotypes TC CC Hazard ratio (compared with TT genotype) 1.25 1.37 95% CI 0.94 to 1.67 1.01 to 1.87 p value 0.12 0.04 Lee et al., Br J Ophthalmol 2009;93:610-613.

Conclusions – ranibizumab study CFH CC genotype more likely to need ranibizumab injections

Oxidative Stress and AMD

Quest for AMD biomarkers Working hypothesis Certain biochemicals could identify those at risk for AMD Thiol redox Lipid peroxidation

Methods Blood drawn from 152 participants 77 AMD pts 75 controls

Plasma biomarker levels in AMD pts v. controls Cases (n =) Con. (mean ± SD) Controls Unadjusted p-value Adjusted p- value T-test Wilcoxon Rank Sum Cys (μM) 69 68 4.19 ± 1.45 4.11 ± 1.39 0.770 0.835 0.632 CySS (μM) 55.27 ± 11.21 50.66 ± 10.27 0.013 0.025 0.108 GSH (μM) 67 1.77 ± 0.65 1.89 ± 0.97 0.396 0.492 0.167 IsoP (ng/ml) 72 0.051 ± 0.034 0.059 ± 0.064 0.372 0.850 0.345 IsoF (ng/ml) 0.22 ± 0.38 0.15 ± 0.18 0.115 0.087 0.194 Brantley et al., Am J Ophthalmol 2012;153:460-467.

Conclusions CySS higher in AMD pts compared to controls prior to age adjustment

AMD Metabolomics

Moving toward Personalized Medicine in Ophthalmology Long-term goal: To develop personalized treatment regimens for patients with degenerative retinal diseases Approach: Determine metabolic and genetic profiles associated with disease and treatment response

Metabolic Profiling Metabolome – snapshot of individual’s physiological state Downstream of the genome and the proteome Liquid Chromatography-Mass Spectrometry (LC-MS) 3000-7000 unique metabolites

MWAS In a metabolome-wide association study (MWAS) of the 26 NVAMD patients and 19 controls, 94 unique metabolic features were identified that significantly differed between the two groups using FDR (q=0.05) This is a Manhattan plot of –log p for each metabolite expressed as a function of the m/z, with the significantly different features appearing above the broken line. 94 individual metabolites significantly different between AMD patients and controls (FDR = 0.05)

We performed correlation analysis on the 40 most consistently discriminant features. Seventeen features formed four correlation clusters, indicating that these metabolites move together and suggesting that these features may have a biological basis for association. We searched the Metlin metabolomics database for potential matches to these features. Interesting matches included a series di- and tripeptides which were elevated in NVAMD and bile acids and vitamin D metabolites which had lower levels in NVAMD.

Metabolites Associate with ARMS2 Genotypes T - risk allele To investigate metabologenetic relationships, we combined the metabolomic analysis with genotype data for the ARMS2 rs10490924 SNP. Using metabolomic data for the 26 NVAMD patients and 19 controls, we used OPLS-DA to identify features that contribute to separation of the ARMS2 GG genotype (no risk alleles) from the ARMS2 GT+TT genotype (1-2 risk alleles). We then overlaid the 94 FDR significant features that discriminated NVAMD from controls. This figure shows the loading plot showing the 26 metabolites that were associated with either ARMS2 GG (n=18, left) or ARMS2 GT+TT (n=8, right). Ovals represent metabolites that discriminate between the ARMS2 GG and GT+TT genotypes

Hierarchical Clustering Analysis (HCA) 44 AMD patients and 29 Controls 16 m/z features We then used Hierarchical Clustering Analysis (HCA) to show the relationship between participants and the 16 discriminatory metabolites. The analysis generated 14 clusters of individuals distributed into two major groupings: Group A, made up of 58% controls, and Group B, consisting of 78% AMD patients. Note Cluster 14 in Group A and Cluster 13 in Group B, both consist entirely of AMD patients, but are well separated by the cluster analysis.

Principal Component Analysis All 73 patients AMD Clusters 13 and 14 We performed principal component analysis of all 73 subjects using the 16 significant m/z features. This showed partial separation of controls and AMD patients. Clusters 13 and 14 from the HCA, all AMD patients, were largely separated from each other. These clusters were selected to investigate whether the metabolic profiles could be used to subclassify AMD patients. PCA of subjects present in Clusters 13 (n=8) and 14 (n=6) showed complete separation.

Manhattan plot for 2708 m/z features discriminating Clusters 13 and 14 We then used FDR to evaluate 2708 features for these 14 individuals in Clusters 13 & 14 to test for differences between these two clusters of AMD patients. A total of 335 features differed at a stringent q=0.01. This Manhattan plot shows that many m/z features had very significant p values. 335 m/z features differed at q=0.01

AMD Metabolomics Metabolites and metabolic pathways differ between AMD and control. Clinically indistinguishable AMD patients may be subcategorized based on their metabolic phenotype. These studies may improve understanding of AMD pathophysiology and may help reveal causes of variable disease progression and treatment response.

Personalized Ophthalmology DNA profile for all AMD patients at the VEI This information will help us: Choose specific treatment Establish frequency of follow-up Determine risk for relatives

Acknowledgments Vanderbilt Melissa Osborn Megan Parks Goodwin Burgess Emily Wade Samantha Williamson Christopher Estopinal Paul Sternberg Emory Dean Jones Youngja Park Karan Uppal Sky Lee Funding Jahnigen Award from AGS Reeves Foundation American Health Assistance Foundation International Retina Research Foundation Macula Society Vanderbilt Eye Institute RPB Core Grants