A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum

Abstract

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

Figure 1

Localization of mutations causing PXE in ABCC6. A, The positions of mutations presented as either nucleotide changes or amino acid substitutions are indicated with respect to the exon/intron structure of ABCC6. The asterisk indicates mutations that have been described only by others. Boxes represent exons drawn approximately to scale. The number of every fifth exon is indicated. Unshaded boxes show exons encoding elements of NBD1 and NBD2. B, Horizontal lines indicate three large deletions involving some or all ABCC6 exons. The horizontal line with dashes depicts a large deletion involving all of ABCC6, with deletion breakpoints both upstream and downstream of ABCC6. C, The predicted topology of ABCC6 contains three transmembrane domains: TMD₀, TMD₁, and TMD₂, with five, six, and six membrane-spanning segments, respectively. NBD1 and NBD2 are also indicated. The various domains of ABCC6 are approximately aligned with the various exons encoding these regions.

Figure 2

Characterization of a homozygous deletion of ABCC6 exon 15 in a patient with PXE who was of Indian origin. A, PCR analysis of the deletion mutation. The amplification of a region corresponding to exons 14, 15, and 16 using primers 14a and 16m with genomic DNA from a control individual (lane 1) and from the Indian patient with PXE (lane 2) resulted in PCR products of 4.6 kb and 3.0 kb, respectively. Using primers flanking Alu repeats present in intron 14 and 15 (IVS14a and IVS15m), we obtained PCR products of 2.0 kb and 0.5 kb from the same control individual (lane 3) and the Indian patient with PXE (lane 4), indicating that a homozygous deletion of about 1.6 kb occurred in the subject with PXE. The position of the primers used to PCR-amplify exon 15 are shown in panel B. B, Schematic representation of the genomic region surrounding ABCC6 exon 15 in an unaffected individual (top) and in the Indian patient with PXE (bottom). The sequence of the junction of the deleted allele is also shown. C, The highest sequence homology of the two Alu Sx repeats within intron 14 and 15 was noted in a 12-bp region that contains the 1,583-bp deletion breakpoint (red). Nucleotide sequence denoted in green represents the 26-bp core sequences flanking the breakpoint domain in intron 14 and 15.

Figure 3

Haplotype analysis of a family with PXE of Indian ancestry originating from South Africa. A, The analysis was performed with 17 D16S polymorphic markers spanning ∼25 cM of 16p13.1. This analysis allowed the assignment of paternal and maternal chromosomes to the three offspring in this pedigree. Markers indicated in bold correspond to a stretch of 12 microsatellites found in a homozygous state and suggested consanguinity in this pedigree. B, The cosegregation of the deletion ABCC6del15 and the phenotype was verified using PCR amplification of genomic DNA from the family members. The deleted alleles generated a PCR product of 2,978 bp, whereas the PCR product derived from the normal allele was 4,561 bp. Individuals I-1, I-2, and II-1 carried both normal and mutant alleles, and both affected individuals, II-2 and II-3, displayed a homozygous deletion.