A remote cis-acting variant at 3q links glomerular NCK1 to diabetic nephropathy

PLoS One. 2013;8(2):e56414. doi: 10.1371/journal.pone.0056414. Epub 2013 Feb 18.

Abstract

We have previously reported genetic association of a single nucleotide polymorphism (SNP), rs1866813, at 3q locus with increased risk of diabetic nephropathy (DN). The SNP is located approximately 70 kb downstream of a cluster of four genes. This raises a question how the remote noncoding polymorphism affects the risk of DN. In this study, we tested a long-range regulatory potential of this variant by a series of experiments. In a luciferase assay, two alleles of the SNP showed differential effects on the luciferase activity in transfected cells in vitro. Using transgenic zebrafish, we further demonstrated in vivo that two alleles of the SNP differentially regulated GFP expression in zebrafish podocytes. Immunofluorescence staining and Western blotting verified that only Nck1 of the four nearby genes was predominantly expressed in mouse glomeruli as well as in podocytes. Furthermore, genotypes of the SNP rs1866813 were correlated with NCK1 expression in immortalized lymphocytes from diabetic patients. The risk allele was associated with increased NCK1 expression compared to the non-risk allele, consistent with the results of the reporter-based studies. Interestingly, differential expression of glomerular Nck1 between mouse strains carrying the nephropathy-prone 129/Sv allele and nephropathy-resistant C57BL/6 allele was also observed. Our results show that the DN-associated SNP rs1866813 is a remote cis-acting variant differentially regulating glomerular NCK1 expression. This finding implicates an important role for glomerular NCK1 in DN pathogenesis under hyperglycemia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics*
  • Animals
  • Animals, Genetically Modified
  • Chromosomes, Human, Pair 3*
  • Diabetic Nephropathies / genetics*
  • Gene Expression
  • Gene Expression Regulation
  • Gene Order
  • Genes, Reporter
  • Genetic Variation*
  • Genotype
  • Humans
  • Kidney Glomerulus / metabolism*
  • Kidney Glomerulus / pathology
  • Mice
  • Oncogene Proteins / genetics*
  • Polymorphism, Single Nucleotide
  • Regulatory Sequences, Nucleic Acid*
  • Zebrafish

Substances

  • Adaptor Proteins, Signal Transducing
  • Nck protein
  • Oncogene Proteins

Grant support

This work was supported in part by grants from Foundations of Knut and Alice Wallenberg (http://www.wallenberg.com), Söderberg (http://www.ragnarsoderbergsstiftelse.se) Hedlund (http://www.hedlundsstiftelse.se), the Swedish Medical Research Council (http://www.vr.se) and the Swedish Foundation for Strategic Research (http://www.stratresearch.se). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.