Study design and sample size
A cross-sectional, observational study was undertaken in a sample of type I diabetic patients who attend diabetes polyclinic of RIO’s hospital, Giza, Egypt, between October 2012 and December 2016 and who were willing to participate. The sampling procedure consisted of randomly selecting 2 days each week (Sunday and Wednesday) and recruiting all the diabetic patients (type I) who attended on those days to be the study population. A comprehensive data was collected from patients with type I diabetes with the aim of identifying genetic and environmental risk factors for diabetic complications. In the first visit, a detailed history followed by baseline blood samples for fasting blood sugar (FBS) and glycosylated hemoglobin (HBA1c) were obtained to confirm DM. Once confirmed, all patients were referred to the Ophthalmic Department of the diabetic polyclinic. Written informed consents were obtained from all subjects in accordance with the principles of the Declaration of Helsinki. The protocol of the study was approved by the RIO Ethics Committee; a venous blood sample was collected from each subject after the consent form had been signed.
Participants diagnosed to have diabetes mellitus without diabetic retinopathy were included in this study.
Participants diagnosed to have diabetes mellitus with diabetic retinopathy were also included.
Participants with known other systemic diseases which could manifest as retinal pathological lesions such as hypertensive retinopathy.
Participants with very hazy ocular media which obscure the ocular fundus.
Participants not accepting the informed consent.
Two hundred and sixty-six type I diabetic patients were studied. (108 males, 158 females) aged 41.7 + 12.5 (mean ± SD) years. They were diabetic for > 5 years.
All patients in our study were subjected to:
Full medical examination
Full ophthalmic examination
Full personal, family, and medical history include a standardized questionnaire for cardiovascular disease. The following parameters were collected: gender, age of onset of diabetes, and duration of diabetes.
Full medical examination:
Cardiovascular examination and ECG.
Blood pressure was measured twice in a sitting position using a mercury sphygmomanometer and after rest of at least 10 minutes.
Investigations for other diabetic complications as neuropathy and nephropathy.
Monthly follow-up of patients with uncontrolled blood sugar levels.
Complete ophthalmological examination including:
Absolute visual acuity
Intraocular pressure using slit lamp applanation tonometer or air buff tonometer
Pin torch external eye examination to screen for extra ocular abnormalities
Anterior segment biomicroscopic examination
Fundus examination using slit lamp biomicroscopy after full dilation of the pupil using cyclopentolate 1% eye drop
Fluorescein fundus angiography (FFA) using fundus camera (TRC50EX) and intravenous injection of 5 ml 10% sodium fluorescein solution
The pictures were studied to categorize the different phenotypes of DR according to FFA as being mentioned in the phenotypes classification of DR (A.B.C). Also a report on the state of macular area was done for the presence or absence of edema
Sample collection: venous blood samples (10 ml) were withdrawn from all subjects and emptied on:
Sterile ethylene diamine-tetra-acetate “EDTA” vacutainer (2 ml) tubes used for DNA extraction and HbA1c. HbA1c was extracted using the Lobona system. DNA was extracted from blood samples and stored at – 30 °C till time of assay
Sterile fluoride vacutainer tube for blood sugar (2 ml)
Sterile plain vacutainer tube (6 ml) was centrifuged, and the serum was stored at – 20 °C to measure the rest of the parameters. Lipid profile [total cholesterol, high density lipoprotein (HDL-cholesterol), low density lipoprotein (LDL-cholesterol) and triglycerides] using regular commercial kits. Urea and creatinine using commercial kits and Glycation end products (GEP) using immune-sorbent assay (ELISA) kits
HbA1c was extracted using the Lobona system (American Diabetes Association 2010)
Lipid profile, total cholesterol, high-density lipoprotein (HDL-cholesterol), low-density lipoprotein (LDL-Cholesterol), and triglycerides using regular commercial kits
Glycation end products using immune-sorbent assay (ELISA) kits (Onarato et al. 2000)
One hundred and ninety patients were examined at the ophthalmic clinic for diabetic patients; 133 were diagnosed as having DR based on the ophthalmologist’s examination. Also, a group of 57 diabetic patients without DR was taken as a control group. All were subjected to molecular genetic analysis to evaluate its association with two selected candidate genes proposed to be related to DR pathogenesis. We investigate the association between genotypes of two polymorphisms of sorbitol dehydrogenase gene (SDH) in the promoter region: a C/G transversion located at _1214 position (the C_1214G polymorphism) and a G/C transversion at _888 position (the G_888C polymorphism).
Full medical history includes name, age, sex, family history, parental consanguinity and complaints; also, the family pedigree was constructed, and a complete medical genetic examination was done for all participants.
Blood samples, 2 ml peripheral blood on ethylene diamine tetra acetic acid (EDTA) tubes, were collected from all participants.
Extraction of DNA was done and stored at – 30 °C.
Allele-specific PCR for C˗1214G polymorphism
The genotypes of the C˗1214G polymorphism were determined by using the following primers:
The two allele-specific:
C variant 5′-TGTTGCCCAGGCTGGTGTTC-3′
G variant 5′-TGTTGCCCAGGCTGGTGTTG-3′
The two control primers:
Thermo-Scientific kit was used for the PCR reactions. This includes the polymerase enzyme Taq DNA polymerase, dNTPs, and PCR Green buffer. Where each 20 μl of the PCR reaction contained Genomic DNA 6 ng (3 μl), 2.5 μl of 10× PCR buffer, 2.5 μl dNTPs of dNTP mixture stock (2 mM), 2 μl of 10.0 pmol forward primer, 2 μl of 10.0 pmol reverse primer, Taq polymerase 2.5 U (0.5 μl), and PCR water nuclease free 7.5 μl
PCR was carried out on a Biometra™ Thermal Cycler, (Model: TProfessional Basic) using the following conditions:
Initial denaturation step at 95 °C for 1 min.
Denaturation at 95 °C for 30 s.
Annealing temperature at 62 °C for 30 s.
Extension for 30 s at 72 °C.
Stages 2–4 carried out 35 times.
The final extension step was performed at 72 °C for 5 min.
PCR products were separated onto a 2% agarose gel.
PCR was carried out on a Biometra™ Thermal Cycler, (TProfessional Basic) using the following conditions table (IV): for G-888C polymorphism, each 20 μl of the PCR reaction contained:
1. Genomic DNA 6 ng (2.0 ng/μl equal to 3μl).
2. 2.5μl of 10× PCR buffer.
3. 2.5μl dNTPs of dNTPs mixture stock (2 mM).
4. 2μl of 10.0 pmol forward primer.
5. 2μl of 10.0 pmol reverse primer.
6. Taq polymerase 2.5 U (0.5μl).
7. PCR water nuclease free 7.5μl
Forward Primer 5′CGCCCGGCCTCATGTCTTTT-3′
Reverse Primer 5′TTGGGGTGGGGAATGTGAGG-3′
Each exon of interest (in the studied genes) was amplified using the suitable pair of primers via conventional Polymerase Chain Reaction (PCR) technique.
The PCR amplicons for each exon of interest were digested using the suitable restriction enzyme according to the Restriction Fragment length Polymorphism (RFLP) technique.
Horizontal agarose gel electrophoresis technique was used to detect the genotype(s) for each PCR product according to the size of DNA fragments after digestion.
The bands were detected via gel documentation system.
Excel (2016) was employed to perform the statistical analysis of the results.
All data was analyzed using the statistical package for social studies software (SPSS version 21). Descriptive analysis was conducted with the X2 test for categorical variables. Fisher’s exact test is a statistical significance test used in the analysis of contingency tables where sample sizes are small as opposed to the chi-square (X2) test that can be used with larger samples. Normally distributed variables were presented as the mean (± SD) and non-normally distributed variables expressed as median (inter-quartile range).
Frequencies and percentages were calculated for all the qualitative data including gender, age group, and type of DR. logistic regression analyses, providing odds ratios (OR), their 95% confidence intervals (CI), p values, and Wald’s chi-square estimates, were performed. P values < 0.05 were accepted as indicating statistical significance. In this study, the allele frequencies for each variant in patients and controls were tested for agreement with expectations using Fisher’s exact test as a sort of chi-square (X2) test (P ≤ 0.05).