Initial search and results
The initial literature search yielded 3,455 citations, 671 of which were genetic association studies investigating diabetic nephropathy in humans (Fig. 1). In these studies, we identified 34 reproduced genetic variants in 24 genes associated with diabetic nephropathy. Data on at least one of these 34 variants were found in 132 articles, representing 153 studies. Only three follow-up studies were included. All other studies were case–control studies. The maximum number of studies in an article was five. References of all articles and details of these studies are shown in the Electronic supplementary material (ESM) Tables 1 and 2. The 132 articles were published between 1994 and 2010. The number of cases included in these articles ranged from 4 to 1,572, and in a study from 4 to 656 cases. Of the 34 reproduced genetic variants, 21 genetic variants in or near 16 genes were significantly associated with diabetic nephropathy after random-effects meta-analysis (Fig. 2a). An overview of the pooled odds ratios of all reproduced variants in relation to diabetic nephropathy is shown in Fig. 2a, b. The odds ratios of the significant associations with diabetic nephropathy ranged between 0.48 and 0.78 for protective effects, and 1.12 to 1.70 for risk effects. Figure 3 contains an overview of the pooled odds ratios of all reproduced variants in relation to diabetic nephropathy among subgroups. Three reproduced variants were not significantly associated with diabetic nephropathy in the whole population after meta-analyses, but were associated in one subgroup: rs1799987 of CCR5 and rs741301 of ELMO1 in the Asian subgroup, and D18S880 of the CNDP1 gene in patients with type 2 diabetes mellitus. Details of analyses of all assessed genetic variants are provided in Table 1. Forest plots of all individual genetic variants and funnel plots for publication bias, as well as results of meta-regression for ethnicity are shown in the ESM (ESM Figs 1–36). If the meta-analysis revealed a positive association between a given genetic variant and diabetic nephropathy, and more than ten studies investigating that variant in relation to diabetic nephropathy were available, a meta-regression was performed. Only three genetic variants fitted the above-mentioned criteria (ACE rs179975, AKRB1 CA repeat Z−2, APOE E2/3/4). In these variants, metaregression showed that ethnicity did not explain the heterogeneity (ESM Figs 1–36).
Table 1 Details of reproduced variants after meta-analysis, including subgroups
Genetic variants involved in the renin–angiotensin system
A variant in ACE, rs179975, was the most studied polymorphism in diabetic nephropathy, with 42 studies resulting in a pooled odds ratio of 1.24 (95% CI 1.12–1.37). The association between the deletion of the rs179975 polymorphism and diabetic nephropathy was reproduced in all subgroups. In the meta-analysis, the rs179975 polymorphism was associated with diabetic nephropathy in type 1 diabetes mellitus (OR 1.13 [95% CI 1.04–1.23]), type 2 diabetes mellitus (OR 1.33 [95% CI 1.16–1.52]), ESRD (OR 1.39 [95% CI 1.21–1.60]), proteinuria (OR 1.20 [95% CI 1.07–1.36]) and in the Asian subgroup (OR 1.28 [95% CI 1.10–1.49]), but not in Europeans. Other variants in the renin–angiotensin system that were also widely studied and reproduced, such as the rs699 variant of AGT with 21 studies and the rs5186 polymorphism of AGTR1 with 15 studies, were not associated with diabetic nephropathy in the meta-analysis.
Genetic variants involved in the polyol pathway
The CA repeat and rs759853 in AKR1B1 were studied in 19 and 9 studies, respectively. The CA repeat has a Z−2 allele thought to lead to an increased risk of diabetic nephropathy and a Z+2 allele thought to have a protective effect. The Z+2 allele and Z−2 allele were both reproducibly associated with diabetic nephropathy, but only the Z−2 allele remained associated in a combined meta-analysis with a pooled odds ratio of 1.12 (95% CI 1.02–1.24). Although reproducibly associated with diabetic nephropathy in ‘type 1 diabetes mellitus’ and ‘European’ subgroups, Z−2 was not associated with diabetic nephropathy in the meta-analysis in these subgroups. The Z+2 allele was associated with diabetic nephropathy in the ‘type 1 diabetes mellitus’ and ‘European’ subgroups (OR 0.79 [95% CI 0.68–0.92] and OR 0.81 [95% CI 0.66–0.99], respectively). The T allele in SNP rs759853 was associated with risk of diabetic nephropathy in the meta-analysis (OR 1.40 [95% CI 1.13–1.74]) and in the subgroups ‘diabetic nephropathy due to type 1 diabetes mellitus’ and ‘Europeans’ (OR 1.58 [95% CI 1.01–2.46] and OR 1.45 [95% CI 1.07–1.97], respectively).
Genetic variants involved in lipid metabolism
Two variants in genes each coding for two different apolipoproteins are reproducibly associated with diabetic nephropathy and remained associated with diabetic nephropathy in the meta-analysis: E2, E3, E4 polymorphism of APOE and rs4420638 near APOC1. The E2 allele is thought to lead to an increased risk of diabetic nephropathy and the E4 allele is thought to have a protective effect. Both the E2 and the E4 allele were associated with diabetic nephropathy in the meta-analysis (OR 1.70 [95% CI 1.12–2.58] and OR 0.78 [95% CI 0.62–0.98] respectively). The E2 allele was also reproducibly associated with diabetic nephropathy in the subgroups ‘type 2 diabetes mellitus’, ‘Asians’ and ‘European/type 1 diabetes mellitus’ (all studies investigating Europeans had type 1 diabetes mellitus and vice versa), but only associated with diabetic nephropathy in the meta-analysis in the ‘type 2 diabetes mellitus’ and ‘Asian’ subgroups (OR 2.21 [95% CI 1.22–4.00] and OR 2.35 [95% CI 1.29–4.30], respectively). rs4420638 near the APOC1 gene was studied in two studies and was associated with diabetic nephropathy in the meta-analysis (OR 1.54 [95% CI 1.29–1.83]). Both studies contained type 1 diabetic nephropathy patients of European descent.
Genetic variants involved in inflammatory cytokines and angiogenesis
rs1799987 of the CCR5 (an inflammatory cytokine) gene was only associated with diabetic nephropathy in the Asian subgroup (OR 0.58 [95% CI 0.43–0.76]) consisting of four studies (n = 1,534), but not in the total group consisting of nine studies (n = 5,527). For the total group, funnel plot asymmetry was indicated by a significant Begg test.
Two genes involved in angiogenesis, VEGFA and EPO, each had a variant that was reproducibly associated with diabetic nephropathy. rs833061 of VEGFA was associated with diabetic nephropathy in the meta-analysis in two studies (n = 543) containing only type 1 diabetes mellitus patients of European origin (OR 0.48 [95% CI 0.37–0.61]). rs1617640 of EPO was associated with diabetic nephropathy (OR 0.67 [95% CI 0.60–0.76]) in three studies (n = 2,773), also in the subgroup with type 1 diabetes mellitus patients (OR 0.67 [95% CI 0.58–0.76]).
Genetic variants involved in oxidative stress
Five genetic variants in four genes thought to be related to oxidative stress were reproducibly associated with diabetic nephropathy. The 1/2 polymorphism of HP and rs1801282 of PPARG were not associated with diabetic nephropathy in the meta-analysis. For PPARG, funnel plot asymmetry was observed (p = 0.024) suggesting publication bias. The rs3138808 and the rs2070744 variants of NOS3 were associated with diabetic nephropathy in the meta-analysis (OR 1.31 [95% CI 1.02–1.67] and OR 1.39 [95% CI 1.09–1.78] respectively). The 5L allele of CNDP1 was associated with diabetic nephropathy only in the ‘type 2 diabetes mellitus’ subgroup (OR 0.77 [95% CI 0.61–0.97]).
Genetic variants in other pathways
rs17300539 of ADIPOQ, which is believed to mitigate vascular damage, was not associated with diabetic nephropathy in the meta-analysis. rs841853 of GLUT1 (also known as SLC2A1), coding for a glucose transporter, did not show an association with diabetic nephropathy in eight studies (OR 1.10 [95% CI 0.89–1.35]). rs1129456 of GREM1, which is involved in cell growth and differentiation, was associated with diabetic nephropathy (OR 1.53 [95% CI 1.25–1.89]) in two studies (n = 1799). rs3767140 of HSPG2, which is involved in maintenance of glomerular basement membrane electrostatic charge, was also associated with diabetic nephropathy in the meta-analysis (OR 0.72 [95% CI 0.59–0.87]), and additionally with diabetic nephropathy in Europeans with type 1 diabetes mellitus (OR 0.64 [95% CI 0.49–0.84]). rs13293564 of UNC13B, thought to be involved in apoptosis, was associated with diabetic nephropathy in four studies (OR 1.23 [95% CI 1.11–1.35]).
Genetic variants identified by genome-wide association studies
Of the 14 genetic variants found to be reproducibly associated with diabetic nephropathy from genome-wide association studies (GWAS), ten remained associated in the meta-analysis. rs2268388 of ACACB, rs11993333 of PVT1, rs39075 near CPVL and CHN2, and rs6492208 (not near a gene) were not associated with diabetic nephropathy in the meta-analysis. Another variant near ‘CPVL and CHN2’, rs39059, was associated with diabetic nephropathy in two studies (n = 1,705; OR 0.74 [95% CI 0.64–0.85]). rs741301 of ELMO1 was associated with diabetic nephropathy in Asians with type 2 diabetic nephropathy (OR 1.58 [95% CI 1.28–1.94]), but not in combination with a third study of European type 1 diabetes mellitus patients. rs451041 and rs739401 of CARS were associated with diabetic nephropathy in the meta-analysis (OR 1.37 [95% CI 1.21–1.54] and OR 1.32 [95% CI 1.15–1.51] respectively).
rs1888747 and rs10868025 of FRMD3 were associated with diabetic nephropathy (OR 0.74 [95% CI 0.65–0.83] and OR 0.72 [95% CI 0.64–0.81] respectively). Another four variants, rs1041466, rs1411766, rs7989848 and rs9521445, which do not lie near a known gene, were associated with diabetic nephropathy in the meta-analysis (OR 1.38 [95% CI 1.21–1.58], OR 1.36 [95% CI 1.20–1.54], OR 1.32 [95% CI 1.16–1.51] and OR 1.35 [95% CI 1.18–1.55] respectively). The variants in CARS, FRMD3, CPVL and CHN2, and the five variants not near genes were only investigated in European participants with type 1 diabetes mellitus.