title: Further evidence for the association of MMP9 with nephropathy in type 2 diabetes and application of DNA pooling technology to candidate gene screening.
abstract: BACKGROUND
  Diabetic nephropathy is characterised by extracellular matrix (ECM) expansion, a key modulator of which is TGF-b1. Glucose-stimulated transcriptional activation of the TGF-b1 gene is an important component of the pathogenesis of nephropathy, following which latent TGF-b1 protein is synthesised. Matrix metalloproteinase 9 (MMP9) remodels the ECM and has been implicated in TGF-b1 activation. The ECM glycosaminoglycan hyaluronan (HA) influences TGF-b1 generation and can modulate its signal transduction activity; renal HA is synthesised by HA synthases HAS2 and HAS3.
  
  
  METHODS
  We report the first screening of the genes encoding HAS2 and HAS3 for sequence variants predisposing to nephropathy in UK type 2 diabetes patients, together with the MMP9 and TGF-b1 genes. Also for the first time, we used validated DNA pools to carry out association analyses of single nucleotide polymorphisms on nephropathic and non-nephropathic cohorts from a total of 199 type 2 diabetes patients, to increase the throughput and decrease the cost of genotype analysis.
  
  
  RESULTS
  None of the 23 single nucleotide polymorphisms analysed in DNA pools were found to be associated with diabetic nephropathy. However, genotyping of alleles at the MMP9 promoter microsatellite locus D20S838 in individual genomic DNA samples supported previous evidence of association between this locus and diabetic nephropathy.
  
  
  CONCLUSIONS
  The use of DNA pooling technology increased the throughput and decreased the cost of our association analysis of nephropathy in our type 2 diabetes sample, which demonstrated sufficient sensitivity to support previous positive findings of association with a microsatellite in the MMP9 promoter region.
authors.affiliation: Institute of Nephrology, School of Medicine, Cardiff University, Cardiff , Wales, UK.
authors.forename: Sunita
authors.initials: S
authors.lastname: Nair
authors.forename: Aled O
authors.initials: AO
authors.lastname: Phillips
authors.forename: Nadine
authors.initials: N
authors.lastname: Norton
authors.forename: Gillian
authors.initials: G
authors.lastname: Spurlock
authors.forename: Hywel J
authors.initials: HJ
authors.lastname: Williams
authors.forename: Kathrine J
authors.initials: KJ
authors.lastname: Craig
authors.forename: John D
authors.initials: JD
authors.lastname: Williams
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Williams
authors.forename: Timothy
authors.initials: T
authors.lastname: Bowen
mesh_terms: D000368:Aged; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D003928:Diabetic Nephropathies / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D014453:Glucuronosyltransferase / Q000235:genetics; D006801:Humans; D000076002:Hyaluronan Synthases; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics*; D018895:Microsatellite Repeats; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D053773:Transforming Growth Factor beta1 / Q000235:genetics
chemical_list: D053773:Transforming Growth Factor beta1; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; C105458:HAS3 protein, human; D000076002:Hyaluronan Synthases; D020780:Matrix Metalloproteinase 9