Dinucleotide repeat polymorphism of matrix metalloproteinase-9 gene is associated with diabetic nephropathy

Background. Although genetic susceptibility has been proposed as an importa nt factor for the development and progression of diabetic nephropathy. the definitive gene has not been identified. To identify the genetic marker for diabetic nephropathy, we examined the association between the (A-C)n dinuc leotide repeat polymorphism upstream of the matrix metalloproteinase-9 (MMP -9) gene and diabetic nephropathy in a group of Japanese patients with type 2 diabetes. Methods. Patients were divided into three groups based on their urinary alb umin excretion rate (AER) and the stage of diabetic retinopathy as follows: uncomplicated group (U), normal albuminuria (AER < 20 mug/min) without pro liferative retinopathy and with the duration of diabetes more than 20 years (N = 32). microalbuminuria group (M), 20 less than or equal to AER < 200 m ug/min (N 155), overt nephropathy group (O), AER greater than or equal to 2 00 mug/min (N = 63). The region containing the dinucleotide repeat upstream of MMP-9 gene was amplified by polymerase chain reaction (PCR). The amplif ied products were analyzed with 7% formamide/urea acrylamide gel electropho resis. The promoter constructs of the MMP-9 gene were transfected with the CMV-beta -galactosidase construct into 293 cells using the lipo,some method . Twenty-four hours after transfection, cells were harvested, and luciferas e and P-galactosidase activities were measured. Results. Nine alleles of the dinucleotide repeat polymorphism (17 to 25 rep eats) were identified, and the frequency of each allele in diabetic subject s was not different from that in nondiabetic controls. The frequency of the allele containing 21 repeats (A21) was most abundant (42.4% in control and 45.6% in diabetic subjects), followed by the allele With 23 repeats (A23; 35.4% in control and 27.6% in diabetic subjects). The A21 allele was less f requent in M and O than U (O, 38.9%, M, 45.5%; U. 59.3%, chi (2) = 7.18; P < 0.05, O vs. U), while the frequency of the alleles other than A21 was not different among each group. The calculated odds ratio for nephropathy in t he noncarrier, heterozygote, or homozygote of A21 allele was 3.38, 1.97, an d 0.2, respectively. Furthermore, the promoter assay for the MMP-9 gene rev ealed that the A21 allele had a higher promoter activity compared with othe r alleles. No significant correlation was observed between serum MMP-9 conc entrations and the MMP-9 gene polymorphism. Conclusion. These results indicate that the patients with A21 allele of the MMP-9 gene may be protected from the development and progression of diabet ic nephropathy. Thus, the microsatellite polymorphism upstream of the MMP-9 gene could be a useful genetic marker for diabetic nephropathy.