Michael A. Grassi

Genome-wide Meta-analysis for Severe Diabetic Retinopathy

Diabetic retinopathy is a leading cause of blindness. The purpose of this study is to identify novel genetic loci associated with the sight threatening complications of diabetic retinopathy. We performed a meta-analysis of genome-wide association data for severe diabetic retinopathy as defined by diabetic macular edema or proliferative diabetic retinopathy in unrelated cases ascertained from two large, type I diabetic cohorts: the Genetics of Kidney in Diabetes (GoKinD) and the Epidemiology of Diabetes Intervention and Control Trial (EDIC) studies. Controls were other diabetic subjects in the cohort. A combined total of 2829 subjects (973 cases, 1856 controls) were studied on 2 543 887 single nucleotide polymorphisms (SNPs). Subjects with nephropathy were excluded in a sub-analysis of 281 severe retinopathy cases. We also performed an association analysis of 1390 copy number variations (CNVs) using tag SNPs. No associations were significant at a genome-wide level after correcting for multiple measures. The meta-analysis did identify several associations that can be pursued in future replication studies, including an intergenic SNP, rs476141, on chromosome 1 (P-value 1.2 × 10(-7)). The most interesting signal from the CNV analysis came from the sub-group analysis without nephropathy subjects and is rs10521145 (P-value 3.4 × 10(-6)) in the intron of CCDC101, a histone acetyltransferase. This SNP tags the copy number region CNVR6685.1 on chromosome 16 at 28.5 Mb, a gain/loss site. In summary, this study nominates several novel genetic loci associated with the sight-threatening complications of diabetic retinopathy and anticipates future large-scale consortium-based validation studies.

Vision-related quality of life in patients with diabetic macular oedema.

Aims: The aim of this study was to determine the impact of diabetic macular oedema (DME) on the quality of life (QOL) in patients with type 2 diabetes mellitus. Methods: The study was a prospective, consecutive, non-comparative case series. An observational study evaluated the quality of vision and vision-specific QOL using the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25). Mean VFQ-25 subscale scores in type 2 diabetic study patients were compared with mean VFQ-25 subscale score in groups of patients with type 1 diabetic retinopathy (T1DR) and varying degrees of age-related macular degeneration (ARMD), glaucoma and cataracts and in reference populations. Results: Thirty-three patients completed the NEI VFQ-25. The mean age of the study population was 64 years. When performing a comparison of those patients with DME versus those with isolated T1DR we found that for the general health subscale, the DME versus T1DR group means were 42±4.4 versus 61±1.0 respectively. The DME versus T1DR quality of vision categorical mean scores were 69±4.1 versus 93±3.9. The DME versus T1DR VR-QOL categorical mean scores were 62±5.0 versus 93±1.0. The DME group was significantly worse in each of these three categories compared with the T1DR group (p<0.01). An additional analysis was performed to examine the differences in VR-QOL in the DME group versus varying common ocular diseases, including age-related macular degeneration (ARMD), glaucoma, cataracts and disease-free reference groups. The mean values of VFQ-25 subscale in the DME group were significantly lower then the glaucoma group in ten of 12 subscales, the cataract group in 11 of 12 subscales, and the reference group in 12 of 12 subscales. However, the mean values of VFQ-25 subscale in the DME group were only significantly different from the ARMD group in three of 12 subscales. Conclusions: Type 2 diabetes patients with macular oedema experience a decreased VR-QOL compared with type 1 diabetic patients with diabetic retinopathy, glaucoma or cataracts. However, VR-QOL in type 2 diabetic patients with macular oedema was similar to those individuals with ARMD.

Toxoplasmosis-associated neovascular lesions treated successfully with ranibizumab and antiparasitic therapy

Joseph D. Benevento, MD1, Rama D. Jager, MD, MBA1, A. Gwendolyn Noble, MD, PhD2, Paul Latkany, MD1,3, William F. Mieler, MD1, Mari Sautter, BA1, Sanford Meyers, MD1, Marilyn Mets, MD2, Michael A. Grassi, MD1, Peter Rabiah, MD1, Kennneth Boyer, MD4, Charles Swisher, MD2, and Rima McLeod, MD1,* other members of the Toxoplasmosis Study Group† 1University of Chicago Pritzker School of Medicine, Chicago, IL 2Children’s Memorial Hospital, Chicago, IL 3St. Luke’s Roosevelt Hospital, The New York Eye & Ear Infirmary, New York, NY 4Rush University Medical Center, Chicago, IL

Replication analysis for severe diabetic retinopathy

PURPOSE The purpose of this study is to attempt to replicate the top single nucleotide polymorphism (SNP) associations from a previous genome-wide association study (GWAS) for the sight-threatening complications of diabetic retinopathy in an independent cohort of diabetic subjects from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR). METHODS This study included 469 type 1 diabetic, Caucasian subjects from WESDR. Cases (n = 208) were defined by prior laser treatment for either proliferative diabetic retinopathy or diabetic macular edema. Controls (n = 261) were all other subjects in the cohort. Three hundred eighty-nine SNPs were tested for association using the Illumina GoldenGate custom array. A retinopathy-only subanalysis was conducted in 437 subjects by removing those with end-stage renal disease. Evaluation for association between cases and controls was conducted by using chi-square tests. A combined analysis incorporated the results from WESDR with the prior GWAS. RESULTS No associations were significant at a genome-wide level. The analysis did identify SNPs that can be pursued in future replication studies. The top association was at rs4865047, an intronic SNP, in the gene CEP135 (P value 2.06 × 10(-5)). The top association from the subanalysis was at rs1902491 (P value 2.81 × 10(-5)), a SNP that sits upstream of the gene NPY2R. CONCLUSIONS This study nominates several novel genetic loci that may be associated with severe diabetic retinopathy. In order to confirm these findings, replication and extension in additional cohorts will be necessary as susceptibility alleles for diabetic retinopathy appear to be of modest effect.

Towards applying text mining and natural language processing for biomedical ontology acquisition

The use of text mining and natural language processing can extend into the realm of knowledge acquisition and management for biomedical applications. In this paper, we describe how we implemented natural language processing and text mining techniques on the transcribed verbal descriptions from retinal experts of biomedical disease features. The feature-attribute pairs generated were then incorporated within a user interface for a collaborative ontology development tool. This tool, IDOCS, is being used in the biomedical domain to help retinal specialists reach a consensus on a common ontology for describing age-related macular degeneration (AMD). We compare the use of traditional text mining and natural language processing techniques with that of a retinal specialists analysis and discuss how we might integrate these techniques for future biomedical ontology and user interface development.

Mitochondrial Variant G4132A is Associated with Familial Non-Arteritic Anterior Ischemic Optic Neuropathy in One Large Pedigree

Objective: To identify the genetic factors associated with familial non-arteritic anterior ischemic optic neuropathy (NA-AION) in a large pedigree. Methods: Eleven family members of a single pedigree, including six affected with NA-AION, underwent detailed clinical examinations. The mitochondrial DNA of the proband was sequenced in its entirety in search of disease-causing mutations associated with NA-AION in the pedigree. A control panel comprising 1488 patients suspected of having Leber hereditary optic neuropathy (LHON) and 97 general-population control subjects was screened for the mitochondrial sequence variant identified in the family. Results: Affected family members were all male and exhibited classic features of NA-AION. Their mean age was 50.2 ± 5.0 years. A total of 23 sequence variations were detected in the mitochondrial genome of the proband, including one novel sequence variation (G4132A, Ala276Thr) in the NADH dehydrogenase subunit 1 gene (ND1). The G4132A mitochondrial variant was detected in six members of a single pedigree with NA-AION. The G4132A variation was not observed in any of the 1585 subjects in the control panel. Moreover, this variant was not identified in over 2469 ethnically diverse individuals previously evaluated through the Human Mitochondrial Genome Database. None of the three major mutations associated with LHON (G3460A, G11778A, T14484C) were identified in the family. Conclusion: The G4132A mitochondrial variation is associated with familial NA-AION in our pedigree.

Fas ligand-Fas signaling participates in light-induced apoptotic death in photoreceptor cells.

PURPOSE We investigated the function of Fas in photoreceptors. METHODS Postmortem human eyes and mouse-derived photoreceptor cells (661W) were examined for Fas expression by in situ hybridization and immunofluorescence. 661W cells were treated with FasL or Fas agonistic antibody, or exposed to light with/without pharmacological manipulation of Fas signaling, followed by apoptosis detection by TUNEL, immunofluorescence and fluorescence activated cell scanning (FACS). Fractionated cellular extracts were used to detect protein expression or protein phosphorylation after immunoprecipitation by Western blot. RESULTS Fas was expressed in the photoreceptor layer of human retina. Fas and a cleaved form of FasL were found on the cell surface of 661W cells. Treatment with FasL or Fas agonistic antibody induced apoptosis in 661W cells. Blocking the activity of FasL or administration of caspase-8 inhibitor z-IETD inhibited light-induced apoptosis. However, it simultaneously caused induction of necroptosis, which could be blocked by the receptor-interacting protein 1 (RIP1) inhibitor, necrostatin-1. Light exposure in the presence of z-IETD caused hyper-phosphorylation of RIP1. Light exposure did not elevate the expression of Fas, FasL, or the Fas-associated death domain adaptor protein (FADD). Cells or conditioned medium after light exposure induced apoptosis in dark-adapted cells, which could be attenuated by blockade of Fas. CONCLUSIONS Fas has a pro-apoptotic role in photoreceptors. Under light stress, soluble and membrane-bound FasL can bind to Fas, inducing apoptosis via a paracrine mechanism. Although blocking Fas signaling inhibits apoptosis, it does not improve the overall photoreceptor survival due to a compensatory activation of necroptosis. Hence, prevention of photoreceptor loss from retinal photo-oxidative stress should target Fas and RIP1.

Validity of Self-Report in Type 1 Diabetic Subjects for Laser Treatment of Retinopathy

PURPOSE This study sought to determine the validity of self-report of prior panretinal photocoagulation (PRP) and focal photocoagulation (FP) compared with fundus photography. DESIGN Prospective cohort study. PARTICIPANTS One thousand three hundred sixty-three type 1 diabetic subjects from the Epidemiology of Diabetes Interventions and Complications (EDIC) study, a subset of the 1441 subjects originally enrolled in the multicenter Diabetes Control and Complications Trial. METHODS At each annual visit, subjects were asked by EDIC staff whether they had undergone PRP, FP, or both since the last completed annual clinic visit. Fundus photographs were collected from one quarter of the cohort each year and from the entire cohort at EDIC years 4 and 10. Photographs were graded for the presence and extent of PRP and FP. Seventeen years of subject reporting and photograph grading of PRP and FP were compared in EDIC subjects. MAIN OUTCOME MEASURES The κ, sensitivity, specificity, and positive and negative predictive values were calculated for subject-reported PRP and FP. Factors influencing subject misreporting were investigated. RESULTS For subject reporting, 1244 (96%) of 1296 subjects with gradable photographs accurately reported whether they had a history of PRP in one or both eyes, and 1259 (97.5%) of 1291 with valid photographs correctly reported their history of FP. For PRP and FP, sensitivities were 90.4% and 74.0%, respectively; specificities were 96.0% and 98.8%, respectively; positive predictive values were 75.9% and 80.3%, respectively; negative predictive values were 98.9% and 98.4%, respectively; and κ values were 0.80 and 0.76, respectively. Risk factors associated with misreporting included prior laser for diabetic retinopathy and prior ocular surgery (each P<0.04). CONCLUSIONS For subjects with type 1 diabetes, in the absence of a clinical examination or fundus photographs, subject self-report could be a reliable tool in a well-monitored study for assessing laser treatment type in diabetic retinopathy.

Cytochrome P450 2C Epoxygenases Mediate Photochemical Stress-induced Death of Photoreceptors

Background: New pharmacological entities are actively sought for treatment of inherited and age-related retinal degenerative diseases. Results: Sulfaphenazole, a selective inhibitor of human cytochrome P450 (CYP) 2C9 enzyme, was identified as a novel cytoprotective agent against light-induced death of photoreceptors. Conclusion: Cytochrome P450 CYP2C epoxygenases mediate photochemical stress-induced death of photoreceptors. Significance: The CYP monooxygenase system is a risk factor for retinal photodamage. Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids.

Genetic Variation Is the Major Determinant of Individual Differences in Leukocyte Endothelial Adhesion

Objective To determine the genetic contribution to leukocyte endothelial adhesion. Methods Leukocyte endothelial adhesion was assessed through a novel cell-based assay using human lymphoblastoid cell lines. A high-throughput screening method was developed to evaluate the inter-individual variability in leukocyte endothelial adhesion using lymphoblastoid cell lines derived from different donors. To assess heritability, ninety-two lymphoblastoid cell lines derived from twenty-three monozygotic twin pairs and twenty-three sibling pairs were compared. These lymphoblastoid cell lines were plated with the endothelial cell line EA.hy926 and labeled with Calcein AM dye. Fluorescence was assessed to determine endothelial cell adhesion to each lymphoblastoid cell line. Intra-pair similarity was determined for monozygotic twins and siblings using Pearson pairwise correlation coefficients. Results A leukocyte endothelial adhesion assay for lymphoblastoid cell lines was developed and optimized (CV = 8.68, Z′-factor = 0.67, SNR = 18.41). A higher adhesion correlation was found between the twins than that between the siblings. Intra-pair similarity for leukocyte endothelial adhesion in monozygotic twins was 0.60 compared to 0.25 in the siblings. The extent to which these differences are attributable to underlying genetic factors was quantified and the heritability of leukocyte endothelial adhesion was calculated to be 69.66% (p-value<0.0001). Conclusions There is a heritable component to leukocyte endothelial adhesion. Underlying genetic predisposition plays a significant role in inter-individual variability of leukocyte endothelial adhesion.