Ophthalmology (Eye & Ear Hospital) - Theses
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ItemBiomarkers in diabetic retinopathy: genetic and proteomic profiling( 2014)Diabetic retinopathy (DR) is a common complication of diabetes mellitus and currently threatens the vision of over 100 million people worldwide. Blindness in diabetes is mostly preventable if sight threatening DR (STDR) is detected and treated early. However, the ophthalmic course in people diagnosed with diabetes can be difficult to predict, as risk indicators are not DR specific. This study adopted a multifactorial approach, assessing systemic circulating biomarkers and genetic markers, to investigate risk of blinding disease. The overriding aim of this thesis was to identify protein and genetic blood-based biomarkers that are consistently associated with severe STDR. METHOD: Samples were obtained from the Diabetes Management Project (DMP). The proteomic fraction of the thesis comprised 190 plasma samples from Type 2 diabetic patients, (n=60) STDR (proliferative DR (PDR) or severe non PDR (NPDR), (n=60) minimal or moderate NPDR, patients without DR (n=60) and (n=10) reference sample. Protein levels were analysed using iTRAQ conducted using mass spectrometry. To replicate this work a total of 70 samples with Type 2 diabetes was analysed for the candidate proteins using liquid chromatography/tandem mass spectrometry (LC/MS/MS) by high resolution multiple reaction monitoring (MRM- HR). The genetic aspects of the thesis utilised DNA extracted from the whole blood of patients from the DMP. Cases had STDR, and control participants had either no evidence of DR or only mild DR. Twenty-four single nucleotide polymorphisms (SNPs) were selected from previous genome-wide association studies (GWAS), eight VEGF and three EPO SNPs were selected from previous DR association studies, and rs4636297 has been implicated in-vivo to be associated with microvascular dysfunction. Samples were genotyped using the Sequenom iPLEX Gold chemistry on an Autoflex mass spectrometer. Association was tested using logistic regression analysis, adjusting for potential confounding variables. RESULTS: The most significant STDR protein biomarker candidates (p<0.0003) were; APOB100 (p<1.0 x 10-27), fibronectin (p<1.0 x 10-27), ceruloplasmin (p=5.34 x 10-12), gelsolin (p=9.51 x 10-12), C5 (p=4.71 x 10-8), ZAG (p=5.35 x 10-7), CD5L (p=6.57 x 10-6), protein AMBP (p=2.04 x 10-6) and ITIH1 (p=1.53 x 104) when compared to those without DR. These candidates were unable to be replicated by MRM-HR for STDR compared to without DR. Two loci from the GWAS replication were nominally associated with STDR: rs1073203 (OR=0.31, 95% CI 0.13-0.74, p=0.01 at the G allele and OR=0.24, 95% CI 0.09-0.63, p=0.003 in a dominate genotypic model), and rs4838605 (OR=1.64, 95% CI 1.02-2.65, p=0.04 in a co-dominant genotypic model). No statistically significant associations were found for VEGF and EPO polymorphisms with STDR to control participants. The miR-126-24A SNP variant was significantly associated with STDR: rs4636297 (OR=2.02, 95% CI 1.22-3.35, p=0.006 at the A allele after co-variable adjustment, and OR=2.00, 95% CI 1.19-3.38, p=0.009 in a co-dominant genotypic model). CONCLUSION: This thesis has shortlisted eight novel proteomic biomarkers, as well as one novel and two previously investigated genetic markers associated with STDR. Once replicated, we propose these biomarkers could help appropriate risk stratification of DR which would, in time allow for early diagnosis and rationalise screening programs.