Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications

Abstract

Significant morbidity and mortality among patients with diabetes mellitus result largely from a greatly increased incidence of microvascular complications. Proliferative diabetic retinopathy (PDR) and end stage renal disease (ESRD) are two of the most common and severe microvascular complications of diabetes. A high concordance exists in the development of PDR and ESRD in diabetic patients, as well as strong familial aggregation of these complications, suggesting a common underlying genetic mechanism. However, the precise gene(s) and genetic variant(s) involved remain largely unknown. Erythropoietin (EPO) is a potent angiogenic factor observed in the diabetic human and mouse eye. By a combination of case-control association and functional studies, we demonstrate that the T allele of SNP rs1617640 in the promoter of the EPO gene is significantly associated with PDR and ESRD in three European-American cohorts [Utah: P = 1.91 x 10(-3); Genetics of Kidneys in Diabetes (GoKinD) Study: P = 2.66 x 10(-8); and Boston: P = 2.1 x 10(-2)]. The EPO concentration in human vitreous body was 7.5-fold higher in normal subjects with the TT risk genotype than in those with the GG genotype. Computational analysis suggests that the risk allele (T) of rs1617640 creates a matrix match with the EVI1/MEL1 or AP1 binding site, accounting for an observed 25-fold enhancement of luciferase reporter expression as compared with the G allele. These results suggest that rs1617640 in the EPO promoter is significantly associated with PDR and ESRD. This study identifies a disease risk-associated gene and potential pathway mediating severe diabetic microvascular complications.

Fig. 1.

Genetic association. (A) Negative log P values (y axis) from association analyses for 11 SNPs in the chromosome 7q22 region harboring the EPO gene (see also Table S3). The violet squares represent all SNPs around rs1617640 from the Utah T2D cohort. The green square represents rs1617640 from the Boston T1D cohort. The pink square represents rs1617640 from the Utah T2D cohort. The light blue square represents rs1617640 from the GoKinD patients who have retinopathy and nephropathy but have not progressed into ESRD. The red square represents rs1617640 from the GoKinD patients who have both PDR and ESRD. The black square represents rs1617640 from the GoKinD cohort. (B) Genomic structure and locations of genes between base pairs 99.94 and 100.04 million (National Center for Biotechnology Information build 35). (C) Pairwise D′ and R² Haploview (38) plots for SNPs in chromosome 7 in the region around the EPO gene when Utah T2D case and control data are used.

Fig. 2.

Comparison of EPO protein level in the vitreous body of nondiabetic individuals with TT genotype and GG genotype by an ELISA. Five TT and five GG samples were used and each sample was assayed three times. The mean ± SD is given for each genotype. Significance was examined by using SPSS's independent sample t test.

Fig. 3.

Effects of the rs1617640 variants on luciferase reporter expression in cultured HEK 293 cells. pGL3 luciferase reporter recombinant plasmids containing an EPO promoter sequence with the risk allele T (T construct) or wild-type G allele (G construct) at SNP rs1617640 were transfected into HEK 293 cells. Additionally, pGL3 luciferase reporter recombinant plasmids containing mutated alleles C or A, or the deletion of this nucleotide (DEL) and a shorter promoter sequence without rs1617640 (short), and the pGL3-Basic vector without insert (Negative) were transfected into HEK 293 cells. Renilla luciferase plasmid pTK-RL was cotransfected with each construct as an internal control for normalization. Normalized luciferase activity was measured in 12 independent experiments. The mean ± SD is given for each construct. Significance was examined by using SPSS's independent sample t test.

Fig. 4.

Real-time RT-PCR semiquantitative analysis of EPO mRNA levels. (A) EPO mRNA levels derived from kidneys of three db/db mice and two normal littermate controls at 2 months of age. (B) EPO mRNA levels derived from retina of five mice with oxygen-induced retinal neovascularization (OIR model) and four control mice at postnatal day 17. Each RT-PCR was assayed in triplicate. Significance was examined by using SPSS's independent samples t test.