Association Between Variation in the Actin-Binding Gene Caldesmon and Diabetic Nephropathy in Type 1 Diabetes

Diabetic nephropathy is the most common cause of end-stage renal failure in the western world (1). There is good evidence from epidemiological (2) and family (3–5) studies that a genetic predisposition to diabetic nephropathy exists, but as yet the causative genes remain elusive. To identify novel candidate genes for diabetic nephropathy, we previously employed suppression-subtractive hybridization and found >200 genes to be differentially expressed in mesangial cells exposed to a high extracellular glucose concentration (6,7). Prominent among the upregulated genes is a group coding for proteins that control regulation of the actin cytoskeleton. Both in vitro (8,9) and in vivo (9) studies suggest that there is disruption of the actin cytoskeletal structure in mesangial cells exposed to high glucose, and this is associated with glomerular hyporesponsiveness to vasopressor stimuli (8). Therefore, this may be partially responsible for the afferent arteriole vasodilation, intraglomerular hypertension, glomerular distention, and subsequent hyperfiltration seen in the early stages of diabetic nephropathy (10).

One of the upregulated actin-binding genes, caldesmon, is located on chromosome 7q35, a region previously linked to diabetic nephropathy in two independent family-based studies (11,12). Caldesmon is a cytoskeletal protein found in smooth muscle and modified smooth muscle cells, such as mesangial cells. It inhibits cell contractility (13) and promotes either assembly (14) or disassembly (13) of actin stress fibers. Phosphorylation of caldesmon is required for the formation of contractile rings during mitosis, and hence it may be involved in the regulation of cell proliferation (14). Furthermore, caldesmon may regulate exocytosis and hence matrix deposition (15). Thus, caldesmon is an excellent positional and biological candidate gene for diabetic nephropathy.

The caldesmon gene is large, comprising 16 exons and spanning over 180 kb of the genome (16). Screening for caldesmon gene variants included all exons, flanking intronic regions, the 5′- and 3′-untranslated regions (240 and 1001 bp in length, respectively), and 3 kb of promoter region upstream from the transcription start site (Fig. 1). We screened DNA samples from 30 type 1 diabetic patients from Northern Ireland (15 case and 15 control subjects, see research design and methods) for single nucleotide polymorphisms (SNPs) using WAVE (denaturing high-performance liquid chromatography) technology followed by DNA sequencing of samples for which the chromatograms differed from the wild type. In total, 12.5 kb were screened in each individual and a total of 20 SNPs identified, i.e., an average of 1 SNP for every 625 bp screened. Of these, 12 (60%) had an estimated minor allele frequency of >5% (Fig. 1A).

Based on their frequency and potential effect on gene function, three SNPs were initially tested for association with nephropathy in a case-control study, which used 393 patients from Northern Ireland with type 1 diabetes: 155 patients with nephropathy and 238 nonnephropathic control subjects (17). We selected variants with a minor allele frequency of at least 20% because these would provide >95% power to detect a susceptibility allele with an odds ratio (OR) of 2.0 at the 5% level of significance, given the size of the Northern Ireland samples. A −579A>G SNP in the promoter region may affect gene expression, and a +834T>C SNP in the 3′ untranslated region may alter stability of the mRNA; therefore, both SNPs may influence the intracellular mRNA levels available for translation into protein. A +569G>A SNP results in a histidine-to-arginine substitution at position 397 in exon 6b.

The ex6b+569G>A and the 3′-untranslated region +834T>C SNPs showed no evidence of association with nephropathy (data not shown). However, the G allele of the −579A>G promoter polymorphism was significantly associated with nephropathy (OR 1.38, 95% CI 1.02–1.86, P = 0.03) (Table 1). For all three polymorphisms, the genotype distributions in the patient groups were in Hardy-Weinberg equilibrium. There was limited linkage disequilibrium between these three SNPs (Table 2).

In order to replicate the association between the −579G allele and diabetic nephropathy in an independent sample, a further 213 patients with type 1 diabetes from the Republic of Ireland (95 patients with nephropathy and 118 nonnephropathic control subjects) were genotyped for the −579A>G SNP. An increase in the frequency of the −579G allele, similar in magnitude to that seen in the Northern Ireland samples, was observed in patients with nephropathy, although this finding did not reach significance (Table 1).

When the nephropathic and nonnephropathic patients were combined, neither the frequency of the G allele (43.9 vs. 43.2%) nor the distribution of the GG, AG, and AA genotypes (19.3, 49.1, and 31.6 vs. 21.1, 44.1, and 34.7%, respectively) was significantly different between the Northern Ireland and Republic of Ireland sample groups (P = 0.81 and 0.51, respectively). Therefore the data from the two samples were combined. This confirmed the association between the G allele and nephropathy (OR 1.35, 95% CI 1.07–1.71, P = 0.01) (Table 1).

To explore the possibility that the detected association with nephropathy was due to linkage disequilibrium between the −579A>G SNP and an adjacent unidentified causative SNP, a further 2 kb of upstream promoter and the first 2 kb of intron 1 were screened for polymorphisms using WAVE analysis in the same 30 individuals as before. A further eight SNPs with a minor allele frequency of >5% were identified (Fig. 1C). All of the common (>5% minor allele frequency) SNPs in this region were genotyped by DNA sequencing in 94 patients with type 1 diabetes (47 case and 47 control subjects) from Northern Ireland. The strong linkage disequilibrium between pairs of these four common SNPs confirms that they lie within a single haplotype block (SNPs 1–4; Table 2). From these data, we determined that within this block there were five common haplotypes, which could be identified using four haplotype-tagged SNPs (htSNPs) (Fig. 1D). The haplotype frequencies in the case and control subjects in the Northern Irish population were estimated and compared. This, however, was no more informative than the single SNP analysis (Table 3).

The −579A>G SNP is expected to cause the disruption of a ΔEF1 transcription factor–binding site and the creation of an AP2 binding site as predicted by Transfac (http://www.gene-regulation.com/pub/databases.html #transfac). This may affect expression of the caldesmon gene and hence provide a mechanism by which the −579G allele contributes to the development of diabetic nephropathy. Functional studies would be required to confirm this hypothesis.

In conclusion, we have found that a −579A>G polymorphism in the promoter region of the caldesmon gene is associated with diabetic nephropathy in the Northern Ireland population and that a similar trend was observed in an independent sample from the Republic of Ireland. While we cannot rule out that the observed association is due to linkage disequilibrium with another SNP in the caldesmon gene, we have suggested a mechanism by which this polymorphism may influence the pathogenesis of the disease. This association needs to be replicated in additional large case-control studies in other populations. The possibility that a variant in the caldesmon gene predisposes to nephropathy in type 1 diabetic patients should stimulate further investigation into the role of the actin cytoskeleton in the pathogenesis of diabetic nephropathy.