Apolipoprotein E gene polymorphism is not a strong risk factor for diabetic nephropathy and retinopathy in Type I diabetes: case-control study

Background The gene encoding apolipoprotein E (APOE) has been proposed as a candidate gene for vascular complications in Type I diabetes. This study aimed to investigate the influence of three-allelic variations in the APOE gene for the development of diabetic retinopathy and nephropathy. Results Neither APOE alleles frequencies or APOE genotypes frequencies differed between Type I diabetic groups either with or without nephropathy. Similar results were found for patients with and without diabetic retinopathy. Conclusions APOE gene polymorphism does not determine genetic susceptibility for the development of diabetic retinopathy in Type I diabetes patients. Association between APOE gene polymorphism and diabetic nephropathy may be weak or moderate, but not strong.


Background
Familial and epidemiological studies have indicated that there is a strong genetic component in the aetiology of diabetic nephropathy in Type I diabetes patients [1,2]. Apolipoprotein E (apoE) was discovered as a plasma protein involved in the metabolism of lipoproteins. Recently, the apolipoprotein E (APOE) gene has been suggested to be a risk factor for the development of micro-and macrovascular complications in diabetic patients.
The APOE gene is polymorphic. There are three common alleles, E2, E3, and E4, which code for three major isoforms, resulting in six common genotypes [3,4]. Individuals with apoE2 have higher triglyceride levels and are associated with lower cholesterol compared to individuals with apoE3. Individuals with apoE4 often have elevated plasma cholesterol levels. There is an increased prevalence of cardiovascular disease and particularly Alzheimer's disease [5,6,7].
Apolipoprotein E polymorphism may influence the metabolism of lipoproteins in diabetic patients. Several recent studies have suggested that this polymorphism may be associated with a genetic predisposition for diabetic nephropathy [8,9,10,11]. Thus, APOE is an important candidate gene for the development of microvascular complications in Type I diabetes patients.
The aim of this study was to investigate the influence of APOE gene polymorphism in the development of diabetic nephropathy and retinopathy in Type I diabetes patients.

Patients
In this study, all patients were Russians with unrelated Type I diabetes. All diabetic patients for the case-control study were recruited from St.Petersburg Diabetological Centres and had participated in the program "Diabetes mellitus" (which included monitoring of trends and examinations of the determinants of vascular complications in Type I diabetes) carried out in St. Petersburg, Russia since 1997.
The diabetic patients included in this study satisfied the World Health Organization criteria for diabetes mellitus [12]. Glycated haemoglobin (HbAlc) was measured by ion exchange chromatography [13]. At entry the patients had HbAlc levels of 5.7 to 16.8%.
The Type I diabetes groups consisted of 74 subjects with nephropathy and 92 subjects without. The study covered 76 Type I diabetes patients with diabetic retinopathy and 96 patients without. Some subjects had more than one type of diabetic complication, and consequently were analysed in more than one group.
For Type I diabetes patients, all the information was examined for evidence of nondiabetic renal disease. Then questionnaires, medical records, and measurements of albumin excretion were used to determine whether they had diabetic nephropathy. Those patients were considered to have diabetic nephropathy if they were receiving treatment for renal disease and either suffered from persisting proteinuria, or had persisting high albuminuria. Urinary albumin excretion was measured by nephelometry [14]. Nephropathy was defined as a persistent urinary albumin excretion of more than 20 µg/min, or 30 mg/24 h in two out of three consecutive urinalyses. Indi-viduals with no history of nephropathy and no albumin excretion were considered free of nephropathy.
Fundus ophthalmoscopy and angiofluorography were used to diagnose retinopathy. The diabetic retinopathy group consisted of those showing retinal change, while Type I diabetes control group were those who showed no signs of retinopathy.

DNA genotyping
Blood was collected from each individual and stored in ethylenediaminetetraacetic acid (EDTA) tubes at -20°C. Genomic DNA was obtained from lysed white blood cells by phenol-chloroform extraction. The APOE gene polymorphism was detected by polymerase chain reaction (PCR). After denaturation step at 95°C for 5 minutes followed by 30 cycle of denaturation at 92°C (1 min), annealing at 64°C (1 min), extension at 72°C (1.5 min) and a final extension at 72°C (5 min) using a MiniCycler (MJ Research, Watertown, MA, USA). Genotypes were determined by Hin61 (Fermentas, Vilnius, Lithuania) digestion of a 244-base pair PCR-amplified fragment spanning the two polymorphic sites. The digested DNA fragments were separated using migration on 12% polyacrylamide gels and visualized under UV illumination af- 10.5 ± 2.2 9.8 ± 1.9 10.6 ± 1.7 9.9 ± 1.7 Data are means ± SD ter ethidium bromide staining [16]. The codominant alleles E2, E3, and E4 determine the six APOE genotypes.

Statistical testing
All data are presented as means ± SD. Groups were compared using the Student's t test for comparison of two groups for quantitative variables. Allele frequency among control subjects and case subjects were compared using standard Χ 2 tests. The difference in genotype fre-quencies between the groups was tested by Fisher's exact test. A value of P < 0.05 was considered significant.

Results
Clinical characteristics by study group are shown in Table 1. No significant difference was seen between groups with vascular complications (nephropathy and retinopathy) and those without, no matter the age, diabetes duration, age at diabetes onset, and mean HbAlc. The frequency of each genotype in each group conformed to the Hardy -Weinberg equilibrium.
The distribution of the APOE genotypes is shown in Table 2. There was no significant difference in APOE genotype and allele frequency between nephropathic and normoalbuminuric diabetic patients. In this study, no association was found between diabetic retinopathy and APOE polymorphisms. APOE allele frequencies for males and females were similar within groups and in different groups.

Discussion
Diabetic nephropathy and diabetic retinopathy are the two most important microangiopathic complications in Type I diabetic patients. Many of the environmental factors that influence the risk of vascular disease are known, but genetic components of the risk for diabetic microangiopathy are poorly understood. Several [10,11] were larger than in this study. With increased sample size comes the possibility to demonstrate an association. But the larger the necessary and sufficient minimal sample size, the weaker the association demonstrated.
We also analysed the association of the APOE gene polymorphism with diabetic retinopathy in the present study. No significant differences were found between groups with and without this vascular complication. A similar result has been shown by Tarnow et al.

Conclusions
The present study found no evidence for a role of the APOE gene polymorphism in genetic susceptibility for the development of diabetic retinopathy in Type I diabetes patients. Association between APOE gene polymorphism and diabetic nephropathy may be weak or moderate, but not strong.