Type 2 diabetic nephropathy is the most common cause of end-stage renal disease in western Europe and the United States. Although patients with overt nephropathy generally experience greater cumulative glycemic exposure, the difference in glycemic control between patients developing nephropathy and to those who did not could not be demonstrated. This observation is consistent with the finding that factors other than glycemic control are involved in the development of nephropathy. Genetic factors which specifically increase the susceptibility to nephropathy in patients with diabetes have been proposed. A range of linkage, association, and gene expression studies have been performed for revealing the genetic background of diabetic nephropathy but were not yet successful in identifying mutations which could explain the development of diabetic nephropathy in the majority of diabetic patients. Because of relatively small case numbers of all studies being performed so far, conclusions from those studies are limited. With the development of better technologies for an affordable genomewide association study using thousands of markers, it might become possible to unravel the genetic susceptibility factors for diabetic nephropathy. Comparing the expression levels of thousands of genes in patients and controls may identify key players in the pathogenesis of diabetic nephropathy and targets for pharmacologic intervention in the future.
Because of relatively small case numbers of all studies being performed so far, conclusions from those studies are limited. With the development of better technologies for an affordable genome-wide association study using thousands of markers, it might become possible to unravel the genetic causes of diabetic nephropathy.
Linkage-and association-based approaches have been applied to attempt to unravel the genetic predisposition for complex diseases. However, studies often report contradictory results even when similar population backgrounds are investigated. Unrecognized population substructures could possibly explain these inconsistencies. In an apparently homogeneous German sample of 612 patients with type 2 diabetic and end-stage diabetic nephropathy and 214 healthy controls, we tested for hidden population substructures and their possible effects on association. Using a genetic vector space analysis of genotypes of 20 microsatellite markers, we identified four distinct subsets of cases and controls. The significance of these substructures was demonstrated by subsequent association analyses, using three genetic markers (UCSNP-43,-19,-63; intron 3 of the calpain-10 gene). In the undivided sample, we found no association between individual SNPs or any haplogenotypes (ie the genotype combination of two multilocus haplotypes) and type 2 diabetes. In contrast, when analyzing the four groups separately, we found that there was evidence for association of the common C allele of UCSNP-63 with the trait in the largest group (n ¼ 547 cases/101 controls; P ¼ 0.002). In this subset haplotype 112 was more frequent in controls than in cases (P ¼ 0.006; haplogenotype 112/121: odds ratio (OR) ¼ 0.27, 95% confidence intervals (CI) ¼ 0.13 -0.57), indicating a protective effect against the development of type 2 diabetes. Our study demonstrates that unconsidered population substructures (ethnicity-dependent factors) can severely bias association studies.
Complex disorders such as type 2 diabetes or coronary heart disease are of major public interest because they affect millions of individuals. To unravel the genetic background of those traits, genome-wide linkage scans with microsatellite markers have been performed, followed by association studies of single-nucleotide polymorphisms (SNPs) in the identified candidate regions. High-throughput SNP typing technologies such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (1 ), pyrosequencing (2 ), TaqMan-based allelic discrimination (3 ), and others (4 -8 ) can be used for that approach. However, many smaller and medium-throughput laboratories have no access to rapid, reliable, universal, and cost-effective SNP typing methods. The establishment of such a technique therefore has a high priority.We have developed a simple and inexpensive two-step method for SNP detection (PCR followed by modified restriction digestion), called double restriction mutagenesis primer PCR (DRMP-PCR). Our approach is based on the introduction of two restriction sites in one of the two PCR primers (mutagenesis reverse primer). This method appears to represent an improvement over older techniques such as PCR-primer-introduced restriction analysis (PIRA) (9 ) with respect to both specificity and reliability. In addition, we have generated a PERL-based computer program that provides the restriction enzymes and necessary mutagenesis sequences for primer design.Our method allows detection of SNPs by use of fluorescence scanning sequencers and automated automatic allele calling. The forward primer must therefore have a 5Ј fluorescence label (hexachloro-6-carboxyfluorescein, 6-carboxyfluorescein, or others) but does not undergo any additional changes. The 3Ј end of the reverse primer is positioned next to the SNP and forms a restriction site together with one of the two SNP alleles (Fig. 1A). In most cases, one or two bases of the 3Ј end of the primer must be changed to generate a restriction site. For prevention of mistyping events and evaluation of the completeness of cleavage, we introduced an additional identical restriction site near the 5Ј end of the reverse primer (5Ј control restriction site). These artificial restriction sites should be generated with as few sequence changes as possible.The restriction enzyme always recognizes the 5Ј control restriction site; thus, the entire product will be cut at least once. However, only one allele of the SNP undergoes an additional restriction at the 3Ј SNP restriction site. A third band would indicate undigested PCR product and can be easily recognized by its larger size (Fig. 1, A and B, in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/ content/vol50/issue12/). (B), primer sequences and recognition sequences of restriction enzymes used for the post-PCR procedure for UCSNP43. The mutagenesis nucleotides for generating the restriction sites in the reverse primers are in bold; the wild-type sequence...
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