Identification of ABCC6 pseudogenes on human chromosome 16p: implications for mutation detection in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE), a heritable disorder affecting the skin, ey es, and the cardiovascular system. has recently been linked to mutations in the ABCC6 gene on chromosome 16p13.1. The original mutation detection stra tegy employed by us consisted of the amplification of each exon of the ABCC 6 gene with primer pairs placed on the flanking introns, followed by hetero duplex scanning and direct nucleotide sequencing. However, this approach su ggested the presence of multiple copies of the 5'-region of the gene when t otal genomic DNA was used as a template. In this study, we have identified two pseudogenes containing sequences highly homologous to the 5'-end of ABC C6. First, by the use of allele-specific polymerase chain reaction (PCR), t wo bacterial artificial chromosome (BAC) clones containing a putative pseud ogene of ABCC6. designated as ABCC6-psi1, were isolated from the human BAC library. Sequence analysis of ABCC6-psi1 revealed it to be a truncated copy of ABCC6, which contains the upstream region and exon 1 through intron 9 o f the gene. Secondly, a homology search of a high-throughput sequence datab ase revealed the presence of another truncated copy of ABCC6, which was des ignated as ABCC6-psi2, and which was shown to harbor upstream sequences and a segment spanning exon 1 through intron 4 of ABCC6. In addition to severa l nucleotide differences in the Ranking introns and the upstream region, bo th pseudogenes contain several nucleotide changes in the exonic sequences, including stop codon mutations, which complicate mutation analysis in patie nts with PXE. Nucleotide differences in flanking introns between these two pseudogenes and ABCC6 allowed us to design allele-specific primers that eli minated the amplification of both pseudogene sequences by PCR and provided reliable amplification of ABCC6-specific sequences only. The use of allele- specific PCR has revealed, thus far, two novel 5'-end PXE mutations, 179de1 9 and T364R in exons 2 and 9, respectively, and several polymorphisms withi n the upstream region and exons 1-9 of ABCC6. These strategies facilitate c omprehensive analysis of ABCC6 for mutations in PXE.