Polymorphisms in the Mn-SOD and EC-SOD Genes and Their Relationship to Diabetic Neuropathy in Type 1 Diabetes Mellitus

In this study, 88 healthy Russian donors selected at random (59 males and 29 females, mean age 28.3 ± 8.4 years, mean ± SEM) were examined. Healthy subjects had no autoimmune, cardiovascular, or other diseases. All study participants lived in Moscow or the Moscow region. Blood samples were collected in the Department of Endocrinology and Diabetology of the Russian Academy for Advanced Medical Studies. This study was approved by the Academy Review Board and was carried out in accordance with the principles of the second Helsinki Declaration. Informed consent was obtained from all subjects before participation in this study.

We studued a total of 166 genetically unrelated Russian patients affected with type 1 diabetes mellitus (97 males and 69 females, mean age 25.0 ± 13.4 years, mean duration of diabetes 9.6 ± 9.2 years). None of the patients studied were treated with antioxidants. Patients with causes of neuropathy other than diabetes (e. g. chronic alcohol abuse, drug-induced neuropathy), truncal neuropathy, and significant neurological diseases (e.g. Parkinson's disease, epilepsy, multiple sclerosis) were excluded from the association study. DN was diagnosed on the basis of symptomatic symmetrical distal neuropathy (reduced/absent ankle reflexes, reduced vibration, thermal, tactile, pin-prick, and/or position sensation) with one or more typical symptoms (burning pains or cramps, paresthesiae, numbness) of at least moderate severity in the feet. DN patients were also identified by measuring the motor conduction velocity of the peroneal nerve, with a velocity of <42 m/s (mean 31.6 ± 1.0 m/s) used to identify patients with DN. Accompanying retinopathy was diagnosed based on the presense of microaneurysms together with hard exudates and retinal hemorrhages, new vessel formation and/or fibrous retiniis proliferans Clinical nephropathy was diagnosed on basis of persistent proteinuria (urinary protein excretion rate > 0.3 g/24 h). The 166 patients tested were assigned to two groups according to the duration of diabetes. The groups were formed using principal of extreme phenotype and nonoverlapping selection criteria to minimize the masking effects of non-genetic factors. The first group consisted of 82 patients with diabetes of short duration (<3 years) and DN. The other group contsisted of the ramaining 84 diabetic patients, all of whom had diabetes of long duration (>10 years) and no neuropathy. The characteristics of the two groups are shown in Table 1.

DNA was extracted from whole human blood with phenol and chloroform [23]. Polymorphic regions were amplified by polymerase chain reaction (PCR) in 50 μl reaction mixture consisting of 67 mM Tris-HCl (pH 8.8), 16.7 mM ammonium chloride, 1.0 mM magnesium chloride, 0.1% Tween-20, 10% dimethyl sulfoxide, 0.2 mM of each dNTP, 5 pmol of each primer, 100 ng of genomic DNA, and 2.5 units of Taq polymerase (Biotekh, Russia). To amplify the polymorphic Ala(-9)Val region, we used primers SOD2-16F 5'-CCAGCAGGCAGCTGGCACCG-3' and SOD2-16R 5'-TCCAGGGCGCCGTAGTCGTAGG-3'. For the polymorphic Ile58Thr region of the Mn-SOD gene, PCR primers SOD2-58F 5'-AAGCTCCTCCCATTATCTAATAGC-3' and SOD2-58R 5'-TCAGTGCAGGCTGAAGAGAT-3' were used. PCR was carried out in a PHC-2 thermal cycler (Techne, UK) with 35 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C (Ile58Thr) or 60°C (Ala(-9)Val), and extension for 1 min at 72°C.

To detect Ala(-9)Val dimorphism, the PCR product was digested with BshTI. For the Ile58Thr polymorphism, the amplified fragment was digested with Eco32I (both enzymes from Fermentas, Lithuania). For digestion, 17 μl of PCR product, 2 μl of buffer O⁺/Tango™ (BshTI) or R⁺/Tango™ (Eco32I) (all buffers from Fermentas, Lithuania), and 1 μl of restriction endonuclease (1 unit/μl) were mixed and incubated for 12 h at 37°C. Digested DNA products were separated by electrophoresis in a 3% agarose gel with ethidium bromide or in an 8% polyacrylamide gel stained with silver [24].

The polymorphic region was amplified in a 50 μl reaction mixture consisting of 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM magnesium chloride, 0.1% Tween-20, 10% dimethyl sulfoxide, 0.2 mM of each dNTP, 5 pmol of each primer SOD3-213F 5'-GGCTGGCCTGCTGCGTGGTGG-3' and SOD3-213R 5'-CCTTGCACTCGCTCTCGCGCG-3', 100 ng of genomic DNA, and 2.5 units of Taq polymerase. The PCR cycling profile was as described above except that annealing was carried out for 1 min at 65°C the amplicon was cleaved with restriction endonuclease Eco52I (Fermentas, Lithuania) in Eco52I+ specific buffer according to the manufacturer's recommendations. Digested fragments were analyzed as described by electrophoresis for the genotyping of Mn-SOD.

Genotype frequencies were checked for deviation from Hardy-Weinberg equilibrium by the χ² and G-statistic tests, using the Rows and Columns program based on the Roff and Bentzen algorithm [25]. Genotype and allele frequencies in the groups studied were compared by Fisher's exact test The p value was corrected by multiplying it by the number of alleles (2) for each locus to obtain the p_c value. Odds ratios and 95% confidence interval (95% CI) were calculated to assess the strength of the relationship between the Mn-SOD or EC-SOD polymorphisms and DN.

The first adenosine residue from 3' end of the forward primer, SOD2-16F, is mismatched with genomic DNA. The original sequence, near the polymorphic site of the Mn-SOD gene, is 5'-TCCGGT-3' with the most variable 3' thymidine (Val) to cytidine (Ala) at position -9 [26]. Use of the mismatched primer, SOD2-16F, results in a 91-bp PCR product. This product may or may not contain a BshTI restriction site (5'-ACCGGT-3'), depending on the sequence. If the -9 codon is GTT (Val), than digestion with BshTI produces two DNA fragments, 17 and 74 bp in length. If codon -9 is GCT (Ala), the amplified product not digested with this enzyme (Fig. 1).

The Ala allele and the Ala/Val genotype were the most common in healthy subjects (Table 2). The observed level of heterozygosity was 53.4%. The observed genotype frequency was consistent with Hardy-Weinberg equilibrium (χ² = 2.0976 and G-statistic = 2.1149, p = 0.3930 ± 0.0154).

The frequencies of the Ala allele and the Ala/Ala genotype were significantly lower in patients with DN than in patients without DN whrereas the frequency of the Val allele and the homozygous Val/Val genotype was significantly higher in patients with DN (Table 3). This suggests that the Ala(-9)Val dimorphism in the Mn-SOD gene is associated with neuropathy in type 1 diabetes mellitus. The Ala allele (odds ratio = 0.48, 95% CI; 0.31–0.74) and the Ala/Ala genotype (odds ratio = 0.34, 95% CI; 0.17–0.66) are associated with lower risk of DN development in than the Val allele (odds ratio = 2.09, 95% CI; 1.35–3.24) and the Val/Val genotype (odds ratio = 5.10, 95% CI; 1.77–14.69).

The penultimate adenosine residue at 3' end of the reverse primer SOD2-58R used is not complemetary to the genomic sequence (5'-GATAGC-3'), creating an Eco32I restriction site (5'-GATATC-3') during amplification if codon 58 is ATA (Ile) [26]. In this case, a 140-bp PCR product is produced, which yields two fragments, 20-bp and 120-bp in size, on digestion with Eco32I. If codon 58 is ACA (Thr allele) the amplified product not cleaved by this enzyme.

The last guanosine residue at the 3' end of the reverse primer, SOD3-231R, displays a mismatch with the original sequence, 5'-CGGCGG-3', close to codon 213 of the EC-SOD gene. This resulted (depending on the sequence) in creation of an Eco52I restriction site (5'-CGGCCG-3') in the PCR product [19]. If codon 213 was CGG (Arg), a 104-bp PCR product was obtained that yielded two DNA fragments of 23 and 81 bp in size on digestion with Eco52I. In contrast, if codon 213 was GGG (the Gly allele), the 104-bp product was not digested with the enzyme (Fig. 2).

The Arg allele was 1.9 times more frequent than the Gly allele in healthy subjects. Arg/Arg homozygotes were 5.7 times more frequent than Gly/Gly homozygotes (Table 2). The observed level of heterozygosity was 54.6%. The observed genotype distribution was consisted with Hardy-Weinberg equilibrium (χ² = 1.9495 and G-statistic = 1.9614, p = 0.3980 ± 0.0155). No significant difference in allele and genotype distribution was found between DN patients and patients without DN (Table 4). Thus, Arg213Gly substitution in the EC-SOD gene is not associated with nerve lesions in type 1 diabetes mellitus.