Compound heterozygosity for a recurrent 16.5-kb Alu-mediated deletion mutation and single-base-pair substitutions in the ABCC6 gene results in pseudoxanthoma elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality. Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed. Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations. In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern. In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans. The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide. The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently. Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.

Figure 1

Pedigree of family 1 with PXE. The proband (II-5 [arrow]) and her older brother (II-2) were clinically affected. Haplotype analysis with 12 microsatellite markers (left side) spanning ∼9 cM of 16p13.1 allowed assignment of phase of a deletion mutation (D) and a nonsense mutation (R1164X), as indicated at the bottom of the haplotypes.

Figure 2

Identification of a nonsense mutation in ABCC6 in family 1 with PXE. A, Results of automated sequencing of the proband’s PCR product, which revealed an apparently homozygous single-base-pair substitution, 3490C→T (bottom), in comparison with her mother’s sequence (top), which showed an apparently homozygous normal sequence, 3490C. Sequencing of PCR products of III-7 and III-9 (see fig. 1) showed heterozygous 3490C/T (not shown). The C→T transition changed the codon for Arg (CGA) to a stop codon (TGA). B, The C→T substitution abolished an AciI restriction site. Digestion of the 327-bp PCR product (uncut [lane U] resulted, in the case of the normal allele, in 239- and 88-bp fragments. Digestions with AciI confirmed that the proband, II-5, is homozygous for the mutant allele, that her mother, I-5, is homozygous for the wild-type allele, and that two of the proband’s children, III-7 and III-9, are heterozygous carriers, whereas another son, III-8, is apparently homozygous for the normal sequence.

Figure 3

Characterization of the 16.5-kb deletion mutation in ABCC6 in PXE. A, Schematic representation of the 3′ end of ABCC6, including exons 22–31. Positions of HindIII restriction enzyme sites (H) are indicated. Note that the deletion mutation extends from intron 22 to intron 29. The positions of the three primers used for PCR detection of the deletion mutation are indicated (circled numbers) B, Southern analysis of HindIII digests of DNA isolated from the probands in the four families with PXE (families 1–4 [lanes 1–4, respectively]) and from four healthy unrelated controls (lanes 5–8). The hybridization was performed with a cDNA probe extending from exon 27 to exon 31. Note the presence of two constant bands, of 11.9 and 3.7 kb, whereas the individuals with PXE demonstrate an additional, 8.8-kb band. C, PCR analysis of the deletion mutation in patients with PXE. Three PCR primers at positions shown in panel A and employed in the same PCR reaction were used for amplification of genomic DNA either from patients with PXE (lanes 1–4) or from unrelated healthy controls (lanes 5–8). In control individuals, amplification with primers 2 and 3 results in a 552-bp band (wt), whereas, in case of the heterozygous deletion mutation, primers 1 and 3 result in an additional band, of 652 bp (del).

Figure 4

Schematic structure of the MRP6 protein, consisting of three TMSDs (TMSD1–TMSD3) and two NBFs (NBF1 and NBF2) (A) and the repetitive sequences surrounding the breakpoints in ABCC6 (B). A, 16.5-kb genomic deletion, resulting in out-of-frame elimination of 1,213 bp from the ABCC6 mRNA corresponding to exons 23–29. At the protein level, deletion of these exons is predicted to result in elimination of most of the sequences within TMSD3 as well as of the entire NBF2. A downstream PTC for translation, resulting in truncation of the polypeptide at amino acid position 1463, is indicated by a white arrow. B, Abundance of repetitive sequences within introns 22 and 29. Alu and other interspersed repetitive elements are denoted by black arrows along the intronic sequences, and the types of these elements are identified the arrows. The white arrowheads denote the positions of simple repeats, and the asterisks denote the positions of the AluSx repeats containing the deletion breakpoints (top). Note the overall sequence homology in AluSx repeats, including a 16-bp sequence containing the deletion breakpoints (yellow), within introns 22 and 29 (bottom). The boldface type represents continuation of the DNA strands after the deletion event.