Allelic variation in the canine Cox-2 promoter causes hypermethylation of the canine Cox-2 promoter in clinical cases of renal dysplasia

Abstract

Background: Novel allelic variants in the promoter of the canine cyclooxygenase-2 (Cox-2) gene are associated with renal dysplasia (RD). These variants consist of either deletions of putative SP1 transcription factor-binding sites or insertions of tandem repeats of SP1-binding sites located in the CpG island just upstream of the ATG translation initiation site. The canine Cox-2 gene was studied because Cox-2-deficient mice have renal abnormalities and a pathology that is strikingly similar to RD in dogs.

Findings: The allelic variants were associated with hypermethylation of the Cox-2 promoter only in clinical cases of RD. The wild-type allele was never methylated, even in clinical cases that were heterozygous for a mutant allele. In cases that were biopsy-negative, the promoter remained unmethylated, regardless of the genotype. Methylated DNA was found in DNA from various adult tissues of dogs with clinical RD.

Conclusions: The mechanism of action of the allelic variation in the canine Cox-2 promoter most likely involves variation in the extent of epigenetic downregulation of this gene. This epigenetic downregulation must have occurred early in development because methylated Cox-2 promoter DNA sequences are found in various adult tissues.

Figure 1

Allelic variants in the cyclooxygenase-2 (Cox-2) promoter, just upstream of the ATG translation start site. Allelic variant 1 has DNA sequences that are deleted; these sequences are boxed with the number 1 below the box. Allelic variants 2 and 3 contain insertions. Allele 2 is underlined and not shaded. Allele 3 contains a further insertion of sequences at nucleotide -78 relative to the ATG start, and is a duplication of the inserted sequences in allele 2. Allelic variant 4 contains a deletion of 6 nucleotides (CCGCCG) and is boxed and marked with the number 4 below the box. The sequences of the complete promoter region and 5′ untranslated region are deposited in GenBank under accession number AY927786.1.

Figure 2

Representative results of methylation-specific PCR experiments with the dogs described in Table1. The sizes of the alleles are as follows: wild-type allele, 187 bp; Allele 1, 170 bp; Allele 2, 99 bp; Allele 3, 211 bp; Allele 4, 181 bp. Cases 1–5 are biopsy-positive dogs. Cases 6–8 are biopsy-negative dogs. U is the unmethylated DNA from the methylation-specific PCR, and M is the methylated DNA. The final two lanes represent negative controls (no DNA) for the methylation-specific PCR experiments. In the case of heterozygotes, a heteroduplex band (marked with an H) is presented above the actual alleles. wt, Wild-type.

Figure 3

Representative electropherograms from the DNA sequencing of bisulfite-converted DNA. (A) DNA that has not been converted with bisulfite (normal sequence). The primer set for this experiment was primer set 1 from reference [11]. (B) Wild-type allele showing that the CpG island of the Cox-2 promoter is unmethylated. (C) Electropherogram showing dense methylation of multiple alleles throughout the CpG island of the cyclooxygenase-2 (Cox-2) gene. (D) Electropherogram from Case number 2 (Shiz tzu) showing complete hypermethylation of the Cox-2 CpG island.