Identification of diabetic retinopathy genes through a genome-wide association study among Mexican-Americans from Starr County, Texas

To identify genetic susceptibility loci for severe diabetic retinopathy, 286 Mexican-Americans with type 2 diabetes from Starr County, Texas completed detailed physical and ophthalmologic examinations including fundus photography for diabetic retinopathy grading. 103 individuals with moderate-to-severe non-proliferative diabetic retinopathy or proliferative diabetic retinopathy were defined as cases for this study. DNA samples extracted from study subjects were genotyped using the Affymetrix GeneChip® Human Mapping 100K Set, which includes 116,204 single nucleotide polymorphisms (SNPs) across the whole genome. Single-marker allelic tests and 2- to 8-SNP sliding-window Haplotype Trend Regression implemented in HelixTreeTM were first performed with these direct genotypes to identify genes/regions contributing to the risk of severe diabetic retinopathy. An additional 1,885,781 HapMap Phase II SNPs were imputed from the direct genotypes to expand the genomic coverage for a more detailed exploration of genetic susceptibility to diabetic retinopathy. The average estimated allelic dosage and imputed genotypes with the highest posterior probabilities were subsequently analyzed for associations using logistic regression and Fisher's Exact allelic tests, respectively. To move beyond these SNP-based approaches, 104,572 directly genotyped and 333,375 well-imputed SNPs were used to construct genetic distance matrices based on 262 retinopathy candidate genes and their 112 related biological pathways. Multivariate distance matrix regression was then used to test hypotheses with genes and pathways as the units of inference in the context of susceptibility to diabetic retinopathy. This study provides a framework for genome-wide association analyses, and implicated several genes involved in the regulation of oxidative stress, inflammatory processes, histidine metabolism, and pancreatic cancer pathways associated with severe diabetic retinopathy. Many of these loci have not previously been implicated in either diabetic retinopathy or diabetes. In summary, CDC73, IL12RB2, and SULF1 had the best evidence as candidates to influence diabetic retinopathy, possibly through novel biological mechanisms related to VEGF-mediated signaling pathway or inflammatory processes. While this study uncovered some genes for diabetic retinopathy, a comprehensive picture of the genetic architecture of diabetic retinopathy has not yet been achieved. Once fully understood, the genetics and biology of diabetic retinopathy will contribute to better strategies for diagnosis, treatment and prevention of this disease.^

Multimodal Imaging of Cotton Wool Spots in Branch Retinal Vein Occlusion.

PURPOSE: To describe and follow cotton wool spots (CWS) in branch retinal vein occlusion (BRVO) using multimodal imaging. METHODS: In this prospective cohort study including 24 patients with new-onset BRVO, CWS were described and analyzed in color fundus photography (CF), spectral domain optical coherence tomography (SD-OCT), infrared (IR) and fluorescein angiography (FA) every 3 months for 3 years. The CWS area on SD-OCT and CF was evaluated using OCT-Tool-Kit software: CWS were marked in each single OCT B-scan and the software calculated the area by interpolation. RESULTS: 29 central CWS lesions were found. 100% of these CWS were visible on SD-OCT, 100% on FA and 86.2% on IR imaging, but only 65.5% on CF imaging. CWS were visible for 12.4 ± 7.5 months on SD-OCT, for 4.4 ± 3 months and 4.3 ± 3.4 months on CF and on IR, respectively, and for 17.5 ± 7.1 months on FA. The evaluated CWS area on SD-OCT was larger than on CF (0.26 ± 0.17 mm(2) vs. 0.13 ± 0.1 mm(2), p < 0.0001). The CWS area on SD-OCT and surrounding pathology such as intraretinal cysts, avascular zones and intraretinal hemorrhage were predictive for how long CWS remained visible (r(2) = 0.497, p < 0.002). CONCLUSIONS: The lifetime and presentation of CWS in BRVO seem comparable to other diseases. SD-OCT shows a higher sensitivity for detecting CWS compared to CF. The duration of visibility of CWS varies among different image modalities and depends on the surrounding pathology and the CWS size.

Acute, posterior multifocal placoid pigment epitheliopathy: a case of 11 recurrences over 15 years

PURPOSE: To report the case of a patient with recurrent, acute posterior multifocal placoid pigment epitheliopathy. To the best of our knowledge, this is the longest documented course with the greatest number of recurrences reported. METHODS: Observational case report of one patient. A 27-year-old otherwise healthy male patient presented with recurrence of new scotomata over 15 years. Fundus photography, fluorescein angiography, indocyanine green angiography, fundus autofluorescence, and optical coherence tomography documented his clinical course. RESULTS: Over the course of 15 years, the patient developed 11 symptomatic (5 imaging-documented) recurrences of acute, posterior multifocal placoid pigment epitheliopathy affecting both eyes. Each episode manifested with new subjective scotomata and new lesions noted on imaging. Symptoms mostly resolved after each episode, and visual outcome remained excellent (20/20 in the right eye and 20/25 left eye at the last follow-up). CONCLUSION: Although typically monophasic, acute posterior multifocal placoid pigment epitheliopathy can rarely present with a recurrent course over a prolonged period of time and should be considered as a diagnosis in patients presenting with recurrent visual symptoms and new placoid lesions on imaging. In recurrent cases, visual recovery may still remain excellent.

Visual field and structural alterations in age-related macular degeneration

The thesis investigated progression of the central 10° visual field with structural changes at the macula in a cross-section of patients with varying degrees of agerelated macular degeneration (AMD). The relationships between structure and function were investigated for both standard and short-wavelength automated perimetry (SWAP). Factors known to influence the measure of visual field progression were considered, including the accuracy of the refractive correction on SWAP thresholds and the learning effect. Techniques of assessing the structure to function relationships between fundus images and the visual field were developed with computer programming and evaluated for repeatability. Drusen quantification of fundus photographs and retro-mode scanning laser ophthalmoscopic images was performed. Visual field progression was related to structural changes derived from both manual and automated methods. Principal Findings: • Visual field sensitivity declined with advancing stage of AMD. SWAP showed greater sensitivity to progressive changes than standard perimetry. • Defects were confined to the central 5°. SWAP defects occurred at similar locations but were deeper and wider than corresponding standard perimetry defects. • The central field became less uniform as severity of AMD increased. SWAP visual field indices of focal loss were of more importance when detecting early change in AMD, than indices of diffuse loss. • The decline in visual field sensitivity over stage of severity of AMD was not uniform, whereas a linear relationship was found between the automated measure of drusen area and visual field parameters. • Perimetry exhibited a stronger relationship with drusen area than other measures of visual function. • Overcorrection of the refraction for the working distance in SWAP should be avoided in subjects with insufficient accommodative facility. • The perimetric learning effect in the 10° field did not differ significantly between normal subjects and AMD patients. • Subretinal deposits appeared more numerous in retro-mode imaging than in fundus photography.

The effect of nutritional supplementation on visual function

The aim of this research was to determine the effect of a lutein-based nutritional supplemented on measures of visual function in normal and ARMD-affected eyes. Thirty participants were recruited to the ARMD cohort (aged between 55 and 82 years, mean ± SD: 69.2 ± 7.8) and 46 were recruited into the normal cohort (aged between 22 and 73 years, mean ± SD: 50.0 ± 15.9). Outcome measures were distance (DVA) and near (NVA) visual acuity, contrast sensitivity (CS), photostress recovery time measured with the Eger Macular Stressometer (EMS), central visual function assessed with the Macular Mapping test (MMT), and fundus photography. Reliability studies were carried out for the EMS and the MMT. A change of 14 s is required to indicate a clinically significant change in EMS time, and a change of 14 MMT points is required to indicate a clinically significant change in MMT score. Sample sizes were sufficient for the trial to have 80% power to detect a significant clinical effect at the 5% significance level for all outcome measures in the normal cohort, and for CS in the ARMD cohort. The study demonstrated that a nutritional supplement containing 6mg lutein, 750 mg vitamin A, 250 mg vitamin C, 34 mg vitamin E, 10 mg zinc, and 0.5 mg copper had no effect on the outcome measures over nine or 18 months in normal or ARMD affected participants. The finding that nine months of antioxidant supplementation, in this case, has no significant effect on CS in ARMD-affected participants adds to the literature, and contrasts with previous RCTs, the AREDS and the LAST. This project has added to the debate about the use of nutritional supplementation prior to the onset of ARMD.

Continuous retinal vessel diameter m easurements: the future in retinal vessel assessment?

PURPOSE. To establish an alternative method, sequential and diameter response analysis (SDRA), to determine dynamic retinal vessel responses and their time course in serial stimulation compared with the established method of averaged diameter responses and standard static assessment. METHODS. SDRA focuses on individual time and diameter responses, taking into account the fluctuation in baseline diameter, providing improved insight into reaction patterns when compared with established methods as delivered by retinal vessel analyzer (RVA) software. SDRA patterns were developed with measurements from 78 healthy nonsmokers and subsequently validated in a group of 21 otherwise healthy smokers. Fundus photography and retinal vessel responses were assessed by RVA, intraocular pressure by contact tonometry, and blood pressure by sphygmomanometry. RESULTS. Compared with the RVA software method, SDRA demonstrated a marked difference in retinal vessel responses to flickering light (P 0.05). As a validation of that finding, SDRA showed a strong relation between baseline retinal vessel diameter and subsequent dilatory response in both healthy subjects and smokers (P 0.001). The RVA software was unable to detect this difference or to find a difference in retinal vessel arteriovenous ratio between smokers and nonsmokers (P 0.243). However, SDRA revealed that smokers’ vessels showed both an increased level of arterial baseline diameter fluctuation before flicker stimulation (P 0.005) and an increased stiffness of retinal arterioles (P 0.035) compared with those in nonsmokers. These differences were unrelated to intraocular pressure or systemic blood pressure. CONCLUSIONS. SDRA shows promise as a tool for the assessment of vessel physiology. Further studies are needed to explore its application in patients with vascular diseases.

Pain response and follow-up of patients undergoing panretinal laser photocoagulation with reduced exposure times

Purpose - We performed a study of laser panretinal photocoagulation in 20 patients with proliferative retinopathy. We compared short exposure, high-energy laser settings with conventional settings, using a 532?nm, frequency doubled, Neodymium–Yag laser and assessed the patients in terms of pain experienced and effectiveness of treatment. Methods - Twenty patients having panretinal photocoagulation for the first time underwent random allocation to treatment of the superior and inferior hemi-retina. Treatment A used ‘conventional’ parameters: exposure time 0.1?s, power sufficient to produce a visible grey-white burns, spot size 300?µm. The other hemi- retina was treated with treatment B using exposure 0.02?s, 300?µm and sufficient power to have similar endpoint. All patients were asked to evaluate severity of pain on a visual analogue scale. (0=no pain, 10=most severe pain). All patients were masked as to the type of treatment and the order of carrying out the treatment on each patient was randomised. Patients underwent fundus photography and were followed up for 6–45 months. Results - Seventeen patients had proliferative diabetic retinopathy, two had ischaemic central retinal vein occlusion and one had ocular ischaemic syndrome. The mean response to treatment A was 5.11, compared to 1.40 treatment B, on the visual analogue scale, which was statistically significant (P=0.001). All patients preferred treatment B. Further treatments, if required, were performed with treatment B parameters and long-term follow-up has shown no evidence of undertreatment. Conclusions - Shortening exposure time of retinal laser is significantly less painful but equally effective as conventional parameters.

Drusen detection in retro-mode imaging by a scanning laser ophthalmoscope

Purpose: The Nidek F-10 is a scanning laser ophthalmoscope that is capable of a novel fundus imaging technique, so-called ‘retro-mode’ imaging. The standard method of imaging drusen in age-related macular degeneration (AMD) is by fundus photography. The aim of the study was to assess drusen quantification using retro-mode imaging. Methods: Stereoscopic fundus photographs and retro-mode images were captured in 31 eyes of 20 patients with varying stages of AMD. Two experienced masked retinal graders independently assessed images for the number and size of drusen, using purpose-designed software. Drusen were further assessed in a subset of eight patients using optical coherence tomography (OCT) imaging. Results: Drusen observed by fundus photography (mean 33.5) were significantly fewer in number than subretinal deposits seen in retro-mode (mean 81.6; p < 0.001). The predominant deposit diameter was on average 5 µm smaller in retro-mode imaging than in fundus photography (p = 0.004). Agreement between graders for both types of imaging was substantial for number of deposits (weighted ? = 0.69) and moderate for size of deposits (weighted ? = 0.42). Retro-mode deposits corresponded to drusen on OCT imaging in all eight patients. Conclusion: The subretinal deposits detected by retro-mode imaging were consistent with the appearance of drusen on OCT imaging; however, a larger longitudinal study would be required to confirm this finding. Retro-mode imaging detected significantly more deposits than conventional colour fundus photography. Retro-mode imaging provides a rapid non-invasive technique, useful in monitoring subtle changes and progression of AMD, which may be useful in monitoring the response of drusen to future therapeutic interventions.

Retinal drusen detection using the Nidek F-10 SLO in retro-mode

Presentaton Purpose:We conducted a small study to assess the novel, retro - mode imaging technique of the NIDEK F-10 scanning laser ophthalmoscope, for detecting and quantifying retinal drusen. Methods:Fundus photographs of 4 eyes of 2 patients taken in retro-mode on the Nidek F-10 SLO were graded independently by 6,experienced, masked fundus graders for the presence of retinal drusen , and compared to stereo colour fundus photographs taken with a Topcon TRC-50DX camera. Results:The mean number of retinal drusen detected in retro mode was 142.96+/- 60.8, range 63-265, and on colour fundus photography mean of 66.6+/-32.6, range 26-177. All observers independently detected approximately twice as many drusen on retro-mode than colour fundus photography (p<0.0001, Student’s paired t-test) . The statistical significance of interobserver variation in drusen detection was p=0.07 on colour fundus photography , and p=0.02 on retro mode ( ANOVA) . Conclusions:The retro-mode of the F-10 camera uses infrared laser and an aperture with a modified central stop, with the aperture deviated laterally from the confocal light path. This forms a pseudo -3D image which is a new means of detecting abnomalites in the deeper retinal layers. Retro-mode imaging of retinal drusen using the F-10 Nidek SLO is a highly sensitive technique for detecting and quantifying retinal drusen , and detected twice as many drusen than colour fundus photography. This small pilot study suggests that this novel type of imaging may have a role in the future detection and analysis of retinal drusen, a field that is likely to become increasingly important in future AMD prevention studies.

Rapid access wet AmD referrals using teleophthalmology and a central retinal grading centre

Introduction: We have adapted the existing , optometry diabetic retinopathy screening pathway and software , so that it can be used for wet AMD fast track referral. Purpose: To compare the conventional, optometry wet AMD fast track referral service using FAX transmission, with a teleophthalmology service using colour fundus photography transmitted to a central retinal grading centre. Method: 40 optometry practices involved in diabetic retinopathy screening were enrolled and had modified computer software installed. Referrals were made by conventional fast track FAX to the macular clinic, and patients were photographed by the optometrist and images transmitted to a central grading centre Results of the two pathways were compared in terms of 1)speed of diagnosis and 2)sensitivity and specificity of diagnosis of wet AMD. Results: Over a ten month period, 62 consecutive patients were referred. The mean time for conventional pathway was 20.8 days (range 3-34),and for new teleophthalmology pathway was 6.9 days (range 1-13). Sensitivity of technician grading of images was 96%, Specificity 53%, and consultant ophthalmologist was sensitivity 96%, specificiity 87%. The technician showed a learning effect with specificity increasing from 30.7% for first 31 patient cohort, to 70.6% for the second cohort. One patient had images that could not be graded. Conclusion: Rapid referral of wet AMD cases by optometrists using modified diabetic retinopathy screening software, allows fast and accurate diagnosis, and may reduce unnecessary referrals. Retinal grading technicians can be trained to grade wet AMD images.

A randomised controlled trial investigating the effect of nutritional supplementation on visual function in normal, and age-related macular disease affected eyes:design and methodology [ISRCTN78467674]

Background: Age-related macular disease is the leading cause of blind registration in the developed world. One aetiological hypothesis involves oxidation, and the intrinsic vulnerability of the retina to damage via this process. This has prompted interest in the role of antioxidants, particularly the carotenoids lutein and zeaxanthin, in the prevention and treatment of this eye disease. Methods: The aim of this randomised controlled trial is to determine the effect of a nutritional supplement containing lutein, vitamins A, C and E, zinc, and copper on measures of visual function in people with and without age-related macular disease. Outcome measures are distance and near visual acuity, contrast sensitivity, colour vision, macular visual field, glare recovery, and fundus photography. Randomisation is achieved via a random number generator, and masking achieved by third party coding of the active and placebo containers. Data collection will take place at nine and 18 months, and statistical analysis will employ Student's t test. Discussion: A paucity of treatment modalities for age-related macular disease has prompted research into the development of prevention strategies. A positive effect on normals may be indicative of a role of nutritional supplementation in preventing or delaying onset of the condition. An observed benefit in the age-related macular disease group may indicate a potential role of supplementation in prevention of progression, or even a degree reversal of the visual effects caused by this condition.

Improving the description of the retinal vasculature and patient history taking for monitoring systemic hypertension

Systemic hypertension is an important public health concern. If optometrists are to perform a more active role in the detection and monitoring of high blood pressure (BP), there is a need to improve the consistency of describing the retinal vasculature and to assess patient's ability to correctly report the diagnosis of hypertension, its control and medication. One hundred and one patients aged >40 years were dilated and had fundus photography performed. BP was measured and a self-reported history of general health and current medication was compared with the records of their general practitioner (GP). The status of the retinal vasculature was quantified using a numeric scale by five clinicians and this was compared to the same evaluation performed with the aid of a basic pictorial grading scale. Image analysis was used to objectively measure the artery-to-vein (A/V) ratio and arterial reflex. Arteriolar tortuosity and calibre changes were found to be the most sensitive retinal signs of high BP. Using the grading scale to describe the retinal vasculature significantly improved inter- and intra-observer repeatability. Almost half the patients examined were on medication for high BP or cardiovascular disease. Patients' ability to give their complete medical history was poor, as was their ability to recall what medication they had been prescribed. GPs indicated it was useful to receive details of their patient's BP when it was >140/90 mmHg. The use of improved description of the retinal vasculature and stronger links between optometrists and GPs may enhance future patient care. © 2001 The College of Optometrists. Published by Elsevier Science Ltd. All rights reserved.

Retinal arteriolar diameter response to flicker light provocation - A useful marker for risk stratification in cardiovascular disease?

Purpose: To to evaluate the benefit of bilinear and linear fitting to characterize the retinal vessel dilation to flicker light stimulation for the purpose of risk stratification in cardiovascular disease. Methods: Forty-five patients (15 with coronary artery disease (CAD), 15 with Diabetes Mellitus (DM) and 15 with CAD and DM) all underwent contact tonometry, digital blood pressure measurement, fundus photography, retinal vessel oximetry, static retinal vessel analysis and continous retinal diameter assessment using the retinal vessel analyser (and flicker light provocation). In addition we measured blood glucose (HbA1c) and keratinin levels in DM patients. Results: With increased severity of cardiovascular disease a more linear reaction profile of retinal arteriolar diameter to flicker light provocation can be observed. Conclusion: Absolute values of vessel dilation provide only limited information on the state of retinal arteriolar dilatory response to flicker light. The approach of bilinear fitting takes into account the immediate response to flicker light provocation as well as the maintained dilatory capacity during prolonged stimulation. Individuals with cardiovascular disease however show a largely linear reaction profile indicating an impairment of the initial rapid dilatory response as usually observed in healty individuals

Development of Multi-modal and Super-resolved Retinal Imaging Systems

Advancements in retinal imaging technologies have drastically improved the quality of eye care in the past couple decades. Scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) are two examples of critical imaging modalities for the diagnosis of retinal pathologies. However current-generation SLO and OCT systems have limitations in diagnostic capability due to the following factors: the use of bulky tabletop systems, monochromatic imaging, and resolution degradation due to ocular aberrations and diffraction.

Bulky tabletop SLO and OCT systems are incapable of imaging patients that are supine, under anesthesia, or otherwise unable to maintain the required posture and fixation. Monochromatic SLO and OCT imaging prevents the identification of various color-specific diagnostic markers visible with color fundus photography like those of neovascular age-related macular degeneration. Resolution degradation due to ocular aberrations and diffraction has prevented the imaging of photoreceptors close to the fovea without the use of adaptive optics (AO), which require bulky and expensive components that limit the potential for widespread clinical use.

In this dissertation, techniques for extending the diagnostic capability of SLO and OCT systems are developed. These techniques include design strategies for miniaturizing and combining SLO and OCT to permit multi-modal, lightweight handheld probes to extend high quality retinal imaging to pediatric eye care. In addition, a method for extending true color retinal imaging to SLO to enable high-contrast, depth-resolved, high-fidelity color fundus imaging is demonstrated using a supercontinuum light source. Finally, the development and combination of SLO with a super-resolution confocal microscopy technique known as optical photon reassignment (OPRA) is demonstrated to enable high-resolution imaging of retinal photoreceptors without the use of adaptive optics.

Digital Photography in the Diagnosis and Follow-up of Ocular Diseases

Purpose: The aim of the present study was to develop and test new digital imaging equipment and methods for diagnosis and follow-up of ocular diseases. Methods: The whole material comprised 398 subjects (469 examined eyes), including 241 patients with melanocytic choroidal tumours, 56 patients with melanocytic iris tumours, 42 patients with diabetes, a 52-year old patient with chronic phase of VKH disease, a 30-year old patient with an old blunt eye injury, and 57 normal healthy subjects. Digital 50° (Topcon TRC 50 IA) and 45° (Canon CR6-45NM) fundus cameras, a new handheld digital colour videocamera for eye examinations (MediTell), a new subtraction method using the Topcon Image Net Program (Topcon corporation, Tokyo, Japan), a new method for digital IRT imaging of the iris we developed, and Zeiss photoslitlamp with a digital camera body were used for digital imaging. Results: Digital 50° red-free imaging had a sensitivity of 97.7% and two-field 45° and 50° colour imaging a sensitivity of 88.9-94%. The specificity of the digital 45°-50° imaging modalities was 98.9-100% versus the reference standard and ungradeable images that were 1.2-1.6%. By using the handheld digital colour video camera only, the optic disc and central fundus located inside 20° from the fovea could be recorded with a sensitivity of 6.9% for detection of at least mild NPDR when compared with the reference standard. Comparative use of digital colour, red-free, and red light imaging showed 85.7% sensitivity, 99% specificity, and 98.2 % exact agreement versus the reference standard in differentiation of small choroidal melanoma from pseudomelanoma. The new subtraction method showed growth in four of 94 melanocytic tumours (4.3%) during a mean ±SD follow-up of 23 ± 11 months. The new digital IRT imaging of the iris showed the sphincter muscle and radial contraction folds of Schwalbe in the pupillary zone and radial structural folds of Schwalbe and circular contraction furrows in the ciliary zone of the iris. The 52-year-old patient with a chronic phase of VKH disease showed extensive atrophy and occasional pigment clumps in the iris stroma, detachment of the ciliary body with severe ocular hypotony, and shallow retinal detachment of the posterior pole in both eyes. Infrared transillumination imaging and fluorescein angiographic findings of the iris showed that IR translucence (p=0.53), complete masking of fluorescence (p=0.69), presence of disorganized vessels (p=0.32), and fluorescein leakage (p=1.0) at the site of the lesion did not differentiate an iris nevus from a melanoma. Conclusions: Digital 50° red-free and two-field 50° or 45° colour imaging were suitable for DR screening, whereas the handheld digital video camera did not fulfill the needs of DR screening. Comparative use of digital colour, red-free and red light imaging was a suitable method in the differentiation of small choroidal melanoma from different pseudomelanomas. The subtraction method may reveal early growth of the melanocytic choroidal tumours. Digital IRT imaging may be used to study changes of the stroma and posterior surface of the iris in various diseases of the uvea. It contributed to the revealment of iris atrophy and serous detachment of the ciliary body with ocular hypotony together with the shallow retinal detachment of the posterior pole as new findings of the chronic phase of VKH disease. Infrared translucence and angiographic findings are useful in differential diagnosis of melanocytic iris tumours, but they cannot be used to determine if the lesion is benign or malignant.