We have previously shown evidence of linkage to diabetic nephropathy in African American families on chromosome 18q21.1-23 (Bowden et al. 2004
). This region has also been identified in genome wide linkage scans for diabetic nephropathy in other populations: Turkish, Pima Indian, European American, and American Indian families (Vardarli et al. 2002; Iyengar et al 2007
). Following studies have excluded the candidate gene ZNF236
, located at 18q22-23, from playing a role in diabetic nephropathy (Halama et al. 2003
), but have discovered that the carnosinase genes, CNDP1
, located at 18q22.3, influence diabetic nephropathy susceptibility (Janssen et al. 2005; Freedman et al. 2007; McDonough et al. 2009
). D18S880, a trinucleotide repeat in exon 2 of the CNDP1
gene, which encodes for a leucine repeat in the signal peptide of the preprotein, was associated with diabetic nephropathy in a small European population (Janssen et al. 2005
) and a European-American population (Freedman et al. 2007
). The association was seen in individuals who were homozygous for the shortest allelic form of the repeat (5L-5L); this genotype was significantly more common in the absence of diabetic nephropathy (Janssen et al. 2005, Freedman et al. 2007
). However, there was no association seen with D18S880 in African Americans (McDonough et al. 2009
). There were associations seen with other variants and haplotypes in the CNDP2
gene in African Americans. These results were associated with both risk and protection, and some were observed at low frequencies in the population (McDonough et al. 2009
). This suggests that there are other variants at the 18q21.1-18q23 locus that are contributing to DN risk in African Americans.
In order to refine the results of our previously published linkage peak (Bowden et al. 2004
), we increased our AA family population from 206 DN-affected sibling pairs to 270. We also increased the marker density in the region by adding an additional microsatellite marker, D18S880, and 48 SNPs. We examined the data using multiple methods of analysis. First, we used NPL analysis with two different marker sets (Micro Only and Micro+SNP). We saw a reduction in the LOD score using the combined marker set, however the LOD score remained at the same magnitude we had previously published (Bowden et al. 2004
) using just the microsatellite markers. In order to maximum our LOD scores, we performed OSA with age of diagnosis of ESRD and age of diagnosis of diabetes. This provided us with maximized LOD scores in pedigrees with earlier ages of onset of ESRD and earlier ages of onset of diabetes. Our significant maximized LOD scores ranged from 3.18 to 3.90 and from 80.6cM to 90.1cM.
We have looked at the data numerous ways in order to see if one model or one approach is better. Overall, we observe a wide range of results depending on the analysis method and the combination of markers used. This is consistent with the commonly accepted explanation of results from linkage mapping at this time: that there are multiple loci at the same region that are shared among and between families (Altshuler et al. 2008
). These results are clearly complex; however, there is continued evidence for linkage in this region after increasing the number of families and the number of markers.
We further investigated this region in African Americans by performing a dense SNP map. The dense SNP map increased our coverage to an average marker density of 6kb. These results were evaluated in two ways. First, we ranked SNPs by order of magnitude of association. Ten of the top 25 SNPs were in a gene. Second, we looked at genes that contained multiple associated SNPs. Using this method, we complied a list of 23 genes that contained one or more associated SNPs and 12 genes had one or more associated SNPs nearby (within 500kb).
After comparing the results between the two different SNP prioritization methods, two candidate genes stood out: NEDD4L
is a ubiquitin ligase located at 18q21. NEDD4L
has been previously shown to influence the regulation of renal sodium reabsorption (Manunta et al. 2008; Sile et al. 2008; Dunn et al. 2002
) and has been associated with essential hypertension in Caucasians and African Americans (Russo et al. 2005
), and in Han Chinese (Wen et al. 2008
). The Nedd4L protein is expressed in the distal nephron (Araki et al. 2008; Umemura et al. 2006
, also known as MESGIN
, is located at 18q21.33 (Scott et al. 1999
), is predominantly expressed in human mesangial cells, and is up regulated in IgA nephropathy (Miyata et al. 1998
). Two polymorphisms in the 3′UTR of SERPINB7
, 2093C and 2180T, were associated with IgA nephropathy in a Chinese population (Li et al. 2004
). These markers form the 2093C-2180T haplotype that was associated with a more severe form of IgA nephropathy in a Chinese population (Xia et al. 2006
), and poor renal survival in Korean IgA nephropathy patients (Lim et al. 2008
). Inagi et al. (2006)
created a severe diabetic nephropathy mouse model by overexpressing serpinb7, RAGE, and iNOS. There is also evidence that serpinb7 is unregulated in diabetic nephropathy, in turn inhibiting plasmin and MMP activities, leading to mesangial matrix accumulation (Ohtomo et al. 2008
We have looked at both of these genes in reference to diabetic nephropathy in our African American population. As we saw the most significant results in intron 1 of NEDD4L in the dense SNP map, we examined this gene from exon 1 to exon 3. We successfully genotyped 91 SNPs in this region; however, we only saw nominal association (P values = 0.025-0.0459) with five SNPs (data not shown). In addition, we successfully genotyped 26 SNPs across the SERPINB7 gene. We saw association with 6 SNPs throughout the gene (data not shown); however, these results require further validation. While we did not examine the entire genic region of NEDD4L, these results suggest that NEDD4L does not play a major role in diabetic nephropathy susceptibility in African Americans. Mutations in SERPINB7 may be involved in the mesangial matrix accumulation observed in diabetic nephropathy; however, this gene must be further investigated before any conclusion can be drawn on its relationship to diabetic nephropathy in African Americans.
Our study is not without limitations. One advantage of genome-wide linkage scans is the ability to identify regions where multiple, rare variants which lead to disease, are shared among and/or between families. These variants may not have been identified in our population based case-control dense SNP map follow-up study. In addition, due to our recruitment design, we are unable to distinguish if the linkage and associations we observed were with diabetes, nephropathy, or both.
Overall, we have performed a comprehensive evaluation of the 18q21-18q23 genomic region in African American. The results of the fine mapping performed in our African American T2DM-DN family population demonstrated continued evidence for linkage with the inclusion of additional subjects, and additional markers. The dense SNP map produced several candidate genes, including NEDD4L and SERPINB7, which warrant further investigation. Taken together, this suggests that there may be multiple loci in this region that affect diabetic nephropathy susceptibility in African Americans.