(CA)n Dinucleotide repeat polymorphism at the 5′‐end of the aldose reductase gene is not associated with microangiopathy in Caucasians with long‐term diabetes mellitus 1

Sir, Initial evaluations of a CA dinucleotide repeat polymorphic marker of the ALR2‐gene suggested that the CA23 (Z‐2) allele confers an increased risk of microvascular diabetic complications, whereas the CA25 (Z+2) allele was a marker of protection [1–3]. However, recent studies in type 1 [4] and in type 2 diabetic patients [5,6] did not find an association of the ALR2 polymorphism with diabetic nephropathy. We compared the frequency of the ALR2 polymorphism with microvascular and macrovascular complications in a large population of German type 1 diabetic patients. In a blinded cross‐sectional study the ALR2 polymorphism was initially analysed in 274 German type 1 diabetic patients born in South Germany. Patients with a duration of diabetes <10 years (patients with diabetic complications) and <20 years (patients without diabetic complications), respectively, were excluded. Thus, of the 274 patients initially approached 174 and 122 patients were evaluated for microvascular and macrovascular complications, respectively. Patients were considered to have diabetic nephropathy if albumin concentration was higher than 20 mg/l in the morning urine sample on at least two out of three consecutive occasions over 12 months. Diabetic nephropathy was always associated with diabetic retinopathy. The diagnosis of non‐proliferative or proliferative diabetic retinopathy was established using fundoscopy by an ophthalmologist after dilatation of the pupils. Coronary heart disease (CHD) was diagnosed when cardiovascular disease was found by stress ECG, or when there was a history of documented myocardial infarction or of persistent angina pectoris, respectively. PCR was performed as described by Ko et al. [1]. Aliquots (4 μl) of amplified DNA were electrophoresed on a 5% formamide–urea gel together with cloned and sequenced reference PCR‐products (PCRR2.1 vector, Invitrogen, Carlsbad, CA, USA). Frequency differences of ALR2 alleles between groups were tested using 2×2 contingency tables and the χ2 test or the two‐tailed Fisher's exact test. All nine alleles found by electrophoresis were confirmed by sequencing. When comparing patients with either retinopathy only or with nephropathy to patients without these microvascular complications no significant differences in the frequency of the ALR 2 alleles (Table 1) were observed. Patients with CHD were older (55.3 years vs 47.4 years), but did otherwise, especially in respect to disease duration and incidence of nephropathy, not differ from patient without CHD. The Z+2 allele was not found in any patient with the diagnosis of CHD (14.1% in patients without CHD, Table 1). However, following Bonferroni correction for multiple testing this difference was not significant. An association between the ALR polymorphism and nephropathy has been only reported in type 1 diabetic patients [2,3], while no association was found in type 2 diabetic patients [5,6]. However, our results are in line with a recent large study in Caucasian type 1 patient [4], which did not find an association of the ALR polymorphism and diabetic nephropathy, and questioned that this locus has a significant role in the pathogenic aetiology of diabetic nephropathy. Conversely, the association of the ALR polymorphism with diabetic retinopathy has been more consistent, and has been found either in patients with early onset [1] or early stages [7,8] of diabetic retinopathy. Here we report that after an average diabetes duration of 24.1 years (SD 9.8 years) no difference in the frequency of the ALR polymorphism in patients with and without diabetic retinopathy is seen. Background diabetic retinopathy is found in almost all patients after 15 years of diabetes, and proliferative retinopathy increases at a rate of 3% per year after an initial lag period [9]. Therefore, differences in the ALR activity might be important only during the early stages of diabetic retinopathy, whereas at later stages other factors might be more important for the disease progression. In the present study the Z+2 allele was not found in patients with CHD. This would be compatible with a protective role of this allele, but confirmation in a larger cohort is required. Contribution of the polyol pathway to diabetic macroangiopathy and diabetic heart disease has been suggested [10], but evidence for a genetic basis has been lacking so far. Interestingly, the gene coding for the constitutively expressed endothelial NO synthase has been localised to the same chromosomal region (7q35‐7q36) as ALR2 [11]. In conclusion, we did not find an association between the ALR2 polymorphism and diabetic retino‐ or nephropathy in type 1 diabetic patients. A tentative association between the ALR polymorphism and CHD was found in type 1 diabetic patients, requiring confirmation in an independent patient cohort. Frequency (percentage) of 5′‐ALR2 microsatellite alleles in type 1 diabetic patients without diabetic complications after 20 years of diabetes mellitus, or with diabetic complications after 10 years of diabetes mellitus *After diabetes duration for at least 20 years (because some patients had microangiopathy, but not CHD, after diabetes duration <20 years, the group size in the microangiopathy and CHD comparison is not identical). †Frequency in patients with CHD vs patients without CHD, χ2=6.071, P=0.0137, P corrected=NS. Frequency (percentage) of 5′‐ALR2 microsatellite alleles in type 1 diabetic patients without diabetic complications after 20 years of diabetes mellitus, or with diabetic complications after 10 years of diabetes mellitus *After diabetes duration for at least 20 years (because some patients had microangiopathy, but not CHD, after diabetes duration <20 years, the group size in the microangiopathy and CHD comparison is not identical). †Frequency in patients with CHD vs patients without CHD, χ2=6.071, P=0.0137, P corrected=NS. This work was in part supported by grants from the Deutsche Forschungsgemeinschaft (BI and PPN). PPN performed this work during the tenure of a Schilling‐professorship.