GWAS of Diabetic Nephropathy: Is the GENIE out of the Bottle?

Diabetic nephropathy (DN) is associated with excess morbidity and mortality, in both type 1 (T1D) and type 2 (T2D) diabetic patients. Despite intensification of treatment, DN remains a growing problem worldwide [1–6]. In 2009, treatment of diabetic end stage renal disease patients accounted for approximately 40% of the US$43 billion expended for dialysis treatment in the United States New management and treatment approaches are desperately needed and defining the genetic architecture regulating DN would accelerate their development. The landmark study by Seaquist et al. in 1989 [7] showed strong familial aggregation of DN and spurred the search for genetic risk variants associated with DN. However, family-based linkage and candidate gene analyses as well as the initial genome-wide association studies (GWAS), performed in single studies with limited power, showed inconsistent results in both T1D and T2D patients [8]. In this issue of PLOS Genetics, the GENIE consortium presents results of the largest DN GWAS meta-analysis performed to date. The discovery phase included 6,691 T1D patients from three cohorts, and SNPs with p,10 were moved forward into a replication analysis that included an additional 5,156 T1D patients in nine cohorts ascertained for nephropathy phenotypes [9]. Generally accepted phenotype definitions were used to identify DN cases (macroalbuminuria or end stage renal disease [ESRD] due to DN) and diabetic control individuals without nephropathy (diabetes duration of at least 10 years with normal albumin excretion). The combined metaanalysis for DN showed, disappointingly, no genomewide signals, although an intronic SNP in ERBB4 (chromosome 2) showed a consistent protective effect across cohorts (OR 0.66, p = 2.1610). Intriguingly, ERBB4 encodes a member of the EGF receptor tyrosine kinase family and modulates kidney tubule proliferation and polarity during nephrogenesis [10]. However, the DN definition essentially mixes two traits, each with distinct underlying pathomechanisms: ESRD as the extreme form of reduced kidney function (glomerular filtration rate, GFR), and macroalbuminuria reflecting severe glomerular filtration barrier dysfunction. Since these two traits have distinct genetic underpinnings [11–16], the authors refined their DN case definition to include only diabetic ESRD patients, which were contrasted with all other diabetic individuals regardless of albumin excretion level. Using these phenotypic criteria, the combined meta-analysis of discovery and replication cohorts identified genome-wide significant signals in an intron in the AFF3 gene, and an intergenic locus between RGMA and MCTP2 on chromosome 15. However, as the authors correctly point out, enthusiasm for AFF3, a transcriptional activator, should be tempered. This locus appears driven by two cohorts and technically did not replicate (p = 0.25 in stage 2 replication), although the effect direction was consistent across studies. The authors argue that power of the replication sample was limited for the alternative case definition due to the low number of ESRD cases (n = 363 versus n = 3,465 controls). The authors further support the association of AFF3 with diabetic ESRD by providing experimental evidence that AFF3 expression levels mediate TGF-b-1–driven fibrosis in an epithelial cell culture model. TGF-b-1 has consistently been implicated in the pathogenesis of fibrosis in DN, and these data provide a plausible function for AFF3 in profibrotic pathways that characterize progressive diabetic kidney disease. However, the lack of significant association in replication analysis calls for independent confirmation of this locus in other studies before its implications for DN mechanisms can be drawn. So—does this publication really let the ‘‘GENIE’’ for DN gene discovery out of the bottle, discovering at last the definitive ‘‘DN gene(s)’’—or is this merely wishful thinking? It is sobering that this largest and longawaited GWAS of T1D DN fails to provide unassailable statistical genetic evidence for associated variants, especially when compared to the success of GWAS in identifying convincing loci associated with other kidney diseases such as idiopathic membranous and IgA nephropathy, ANCAassociated nephropathy, or nondiabetic ESRD in African Americans [17–22]. We believe that the definition of DN may lie at the crux of the overall disappointing reproducibility of genetic DN studies. In contrast to kidney diseases where diagnosis is based on a kidney biopsy (e.g., IgA nephropathy, membranous nephropathy) or imaging studies (e.g., ADPKD), the diagnosis of DN is almost always made using clinical criteria and not by histology. The clinical diagnosis uses phenotypic parameters derived from the typical course of DN: after many years of diabetes duration with normal GFR and absent albuminuria, DN onset is marked by mildly elevated albuminuria (also termed microalbuminuria), frequently with increased GFR (Figure 1). Subsequently, DN is characterised by overlap-