Ahmed Saeed Ibrahim. M.D., Ph. D. Researcher of Ophthalmology National Research Center -Cairo

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1 Association of Single Nucleotide olymorphism in -46and +45 of Vascular Endothelial Growth Factor Gene with the Development of roliferative Diabetic Retinopathy in Type 2 Diabetes Mellitus Ahmed Saeed Ibrahim M.D., h. D. Researcher of Ophthalmology National Research Center -Cairo Diabetes mellitus (DM) : -A group of metabolic diseases - chronic hyperglycemia with Disturbance of carbohydrate, fat and protein metabolism. Resulting from defects in insulin secretion (type 1 DM) or insulin action (type 2 DM), or both. Blindness due to DM : increased prevalence of Retinopathy, Cataract, Glaucoma Two significant forms of sight-threatening retinopathy roliferative diabetic retinopathy (DR) Diabetic maculopathy 1

2 DM Diagnostic criteria : (ADA,213) -Hemoglobin A1c ( Hb A1c) 6.5% -Fasting plasma glucose (FG) 126 mg/dl- at least 8 hrs fasting. OR - Symptoms of hyperglycemia and a random plasma glucose 2 mg/dl OR - 2 hours plasma glucose 2 mg/dl (11.1 mmol/l) during an oral glucose tolerance test - Symptoms of hyperglycemia: polyuria, polydipsia, weight loss, sometimes with polyphagia, and blurred vision, impairment of growth and susceptibility to certain infections. DR is characterized by: 1- New vessels on the optic disc (NVD) on or within 1,5 μm (1 standard disc diameter) from the optic nerve head - It is considered sever if it is more than 1/3 disc diameter 2- New vessels elsewhere on the retina (NVE) along the course of the major vessels, pre-retinal hemorrhage (RH), vitreous hemorrhage, and/or fibrous tissue proliferation. (Gordon and Klintworth, 211) (Stewart, et al., 29) 2

3 - Often accompanied by extensive hemorrhage and fibrovascular proliferation and retinal traction. -NVs may affect the anterior segment, iris (rubiosis iridis) and the angle of the anterior chamber (AC) in DR. Vascular endothelial growth factor (VEGF) plays a basic role in the retinal microvascular complications in patients with DM - Stimulates proliferation, migration and tube formation with angiogenic growth of new blood vessels Diabetes Control and Complications Trial Research Group The Duration Of Diabetes the development and progression strongest predictor of retinopathy Improving control of blood glucose reduced the risk of the development of retinopathy in diabetic patients. Blood pressure: Elevations in blood pressure plays a role in the pathogenesis of diabetic retinopathy. 3

4 These factors alone do not explain the development of DR : Absent with poor glycaemic control even over a long period of time of DM ( even more than 2 years). It develops after a short period of DM despite good glycemic control Errera et al., 27; -634G SN in the 5 -untranslated region VEGF gene, an increased risk of developing DR, (in a case-control study in a Brazilian population of European ancestry). * Nakamura et al., 29; The AA genotype at 2578 C/A SN is associated with DR Indian populations, while 634C>G SN in VEGF gene is not associated with DR 4

5 The Aim Of the Work: To determine whether single nucleotide polymorphism in -46 T>C and +45 C>G in the VEGF gene are associated with the development of DR in type 2 DM patients, in Egyptian population. 5

6 A Case-control Study. GROU CONTROL GROU 35 diabetic patients diagnosed as having DR, duration of diabetes (< 15 years). 35 diabetic patients free of diabetic retinopathy, duration of diabetes (> 2 years). Matching as age, sex, race, body mass index, triglyceride and total cholesterol serum aspect. Informed consent forms, approved from Medical Ethics Committee in The National Research Center, will be signed by all atients & controls after they will be informed by the purpose of the opthalmological examinations laboratory analysis & and investigations in this study. 6

7 Major Eligibility Criteria 1- Diagnosis of diabetes mellitus according to the basis of the ADA, (211), criteria, with no overlapping disease. 2- Diagnosis of DR signs, including new vessels on or within one disc diameter (DD) of the disc (NVD), new vessels elsewhere (NVE), vitreous hemorrhage, fibrovascular tissue proliferation and rubeosis iridis for the cases group. 3- DR free subjects for the control group: upon the clinical ophthalmological examination and investigations. Exclusion Criteria Other causes of ocular neo-vascularization Branch retinal vein occlusion Ocular ischemic syndrome Familial exudative viteroretinopathy Encircling buckle Retinal vasculitis. Intermediate uveitis. Other diseases associated with VEGF gene -46 T/C and +45 C/G SNs, other than DR including: prostate cancer, endometriosis, rheumatoid arthritis. 7

8 Methodology Complete ophthalmological examination - Best corrected visual acuity. - Slit-lamp biomicroscopic examination of ant. segment and gonioscopy using Goldmann three miror. - Tonometery using erkins tonometer -Fundus examination using non-contact fundus lenses with slitlamp and indirect ophthalmoscope. - Fundus angiography (FFA) using a digital fundus camera DNA Analysis Genomic DNA is extracted from the peripheral blood using DNA extraction kit. Genotyping for the -46 T>C and +45 C>G SNs were performed using real time CR. Other parameters - Duration of diabetes - HbA1c - body mass index (BMI) - Systolic and diastolic blood pressure the serum aspect of total cholesterol, triglyceride obtained from medical record closest to the day of eye examination. 8

9 -According to the severity of the DR, the diabetic cases with DR were classified into two subgroups: - Early DR, 2 patients, (57%), and - High risk DR subgroup ( 15 patients, (43%) ), based on Early Treatment Diabetic Retinopathy Study Research Group, (1992). roliferative diabetic retinopathy Early DR High-risk DR Characteristics New vessels definitely present Characteristics not met for more severe DR. One or more of the following: - NVD greater than standard photo 1 A* (i.e., 1/3 disc area). - Any NVDs with vitreous or preretinal hemorrhage - NV elsewhere on the retina (NVE) greater than 1/2 disc area with vitreous or preretinal hemorrhage 9

10 DR (Early) Comparison between both groups as regard general data Age (years) BMI DM duration(years ) Hypertension duration(years ) Gender Male Females (57.1%) 15(42.9%) (57.1%) 15(42.9%) t # <. 1 HS <.5 S 95% CI

11 Comparison between both groups as regard blood pressure. t 95% CI Systloic blood pressure (mmhg) Diastolic blood pressure (mmhg) <. 1 HS Comparison between both groups as regard laboratory data HBA1c Fasting blood sugar (mg/dl) Total cholesterol (mg/dl) Triglycerides (mg/dl) High density lipoproteins (mg/dl) Low density lipoproteins (mg/dl) Very low density lipoproteins (mg/dl) TC/HDL LDL/HDL Creatinin (mg/dl) Albumin /creatinin t % CI

12 -46 T>C SN Comparison between both study groups as regarding genotyping -46 T>C SN of VEGF gene. TT CT CC 15 (42.9%) 2 (57.1%) 5 (14.3%) 25 (71.4%) 5 (14.3%) X 2 16 <.1 HS TT CT CC Comparison between both study groups as regard the distribution of C and T allele of -46 T>C SNs of VEGF gene. C allele T allele 55 (78.6%) 15 (21.4%) 35 (5%) 35 (5%) X2 12 <. 1 HS 95% CI OR

13 + 45 C>G SNs Comparison between the cases and control groups as regarding the genotype of VEGF gene + 45 C>G SNs. CC GC GG 5(14.3%) 15(42.9%) 15(42.9%) 5(14.3%) 25(71.4%) 5(14.3%) X2 7.5 <.5 S CC GC GG Comparison between both groups as regarding the frequency of G and C allele of +45 C>G SNs. X2 95% CI OR G allele C allele 45 (64%) 25 (36%) 35 (5%) 35 (5%)

14 Comparison between both groups as regarding combined genotyping of -46 T>C SNs and +45 C>G SNs, respectively. TT/CC CT/GC CC/CC CC/GG 15 (42.9%) 5 (14.3%) 15 (42.9%) 5 (14.3%) 25 (71.4%) 5 (14.3%) X <.1 HS TT/CC CT/GC CC/CC CC/GG Comparison between genotypes combination of -46 T>C SNs and +45 C>G SNs, respectively, among DR subgroups. CT/GC CC/CC CC/GG DR degree Early High 15 (75%) 5 (33.3%) 5 (25%) 1 (66.7%) X2 21 <.1 HS Early DR High risk DR CT/GC CC/CC CC/GG 14

15 Comparison between DR subgroups in the distribution of -46 T>C SNs genotypes. TT CT CC DR subgroup Early High risk 15(75%) 5(25%) 15(1%) <.1 HS Early DR High risk DR TT CT CC Comparison between DR subgroup in the distribution of genotypes of VEGF gene +45 C>G SNs. CC GC GG DR subgruop Early 15(75%) 5(25%) High risk 5 (33.3%) 1 (66.7%) <.1 HS Early DR High risk DR CC GC GG 15

16 Distribution at +45 C>G SNs and Gene, -46T>C SNs of VEGF -46T>C SNs CC genotype at was prevalent in the cases group CT and TT genotype at -46T>C SNs prevalent in the control group. +45C>G SNs. GG genotype at was prevalent in the cases group, GC genotype was prevalent in the control group. More-over, the -46T>C SNs CC genotype together with +45C>G SNs GG genotype at were prevalent in "the patients with high risk DR. In conclusion, we showed for the first time in an Egyptian diabetic patients that -46T>C SNs (CC genotype ) and +45C>G SNs (GG genotype) of VEGF gene were associated, independently, with proliferative diabetic retinopathy in patients with type 2 DM. The potential value of identifying a genetic predisposition to proliferative diabetic retinopathy would be an advantage of early therapeutic intervention and could reduce the progression of the DR, although additional large scale studies must be performed in the future to clarify these issues. 16

17 17

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