Contiguous deletion of the X-linked adrenoleukodystrophy gene (ABCD1) and DXS1357E: a novel neonatal phenotype similar to peroxisomal biogenesis disorders

Abstract

X-linked adrenoleukodystrophy (X-ALD) results from mutations in ABCD1. ABCD1 resides on Xq28 and encodes an integral peroxisomal membrane protein (ALD protein [ALDP]) that is of unknown function and that belongs to the ATP-binding cassette-transporter superfamily. Individuals with ABCD1 mutations accumulate very-long-chain fatty acids (VLCFA) (carbon length >22). Childhood cerebral X-ALD is the most devastating form of the disease. These children have the earliest onset (age 7.2 +/- 1.7 years) among the clinical phenotypes for ABCD1 mutations, but onset does not occur at <3 years of age. Individuals with either peroxisomal biogenesis disorders (PBD) or single-enzyme deficiencies (SED) in the peroxisomal beta-oxidation pathway--disorders such as acyl CoA oxidase deficiency and bifunctional protein deficiency--also accumulate VLCFA, but they present during the neonatal period. Until now, it has been possible to distinguish unequivocally between individuals with these autosomal recessively inherited syndromes and individuals with ABCD1 mutations, on the basis of the clinical presentation and measurement of other biochemical markers. We have identified three newborn boys who had clinical symptoms and initial biochemical results consistent with PBD or SED. In further study, however, we showed that they lacked ALDP, and we identified deletions that extended into the promoter region of ABCD1 and the neighboring gene, DXS1357E. Mutations in DXS1357E and the ABCD1 promoter region have not been described previously. We propose that the term "contiguous ABCD1 DXS1357E deletion syndrome" (CADDS) be used to identify this new contiguous-gene syndrome. The three patients with CADDS who are described here have important implications for genetic counseling, because individuals with CADDS may previously have been misdiagnosed as having an autosomal recessive PBD or SED

Figure 1

Peroxisomal proteins in cultured fibroblasts evaluated by immunocytochemical analysis. Fibroblasts were double-labeled for ALDP and catalase and were labeled separately for PMP70. A–D, ALDP, a protein encoded by ABCD1. Pt 1–Pt3 are immunonegative for this protein. Two-thirds of ABCD1 mutations result in immunonegative status (Watkins et al. Feigenbaum et al. 1996). E–H, Catalase, a peroxisomal matrix protein. Cells from Pt1, Pt2, and Pt3 and from a control subject have normal peroxisome size and number. In the control-subject panel, the catalase signal colocalizes with that seen for ALDP. I–L, PMP70, a peroxisomal membrane protein that belongs to the same ABC transporter subtype as does ALDP. Compared with those from the control subject, cell lines from Pt1, Pt2, and Pt3 have normal PMP70 localization. All cells were visualized at 1,000× magnification.

Figure 2

Large ABCD1 deletions that extend into the coding region of DXS1357E, in Pt1, Pt2, and Pt3. A, ABCD1, which spans ∼24 kb and has 10 exons (Sarde et al. 1994). Genomic DNA BamHI digestion was predicted to yield three ABCD1 fragments from Xq28. B, Results of Southern blot validation studies and analyses of patients. Validation studies were conducted to identify genomic fragments containing DNA from autosomal homologs (Eichler et al. ; Smith et al. 1999) that might cross-react with the full-length ABCD1 cDNA probe. DNA from wild-type mouse, from a mouse cell line harboring one human X chromosome (AHA11a [Dorman et al. 1978]), and from a human control subject (Ctl) were analyzed simultaneously. Four bands appeared for the AHA11a DNA, one corresponding to the band from wild-type mouse and three matching the predicted sizes (i.e., 10, 8.3, and 5.6 kb) of fragments for ABCD1 on Xq28 and aligning with bands in Ctl. The two additional bands in Ctl correspond to autosomal homologs that cross-react with the cDNA probe, since they did not appear in the mouse hybrid cell line harboring the human X chromosome. Pt1 and Pt3 lack the three ABCD1 bands corresponding to all 10 exons. In contrast, Pt2 lacks bands corresponding to exons 1–6 but retains the 8.3-kb band representing exons 6–10. Ex = exons. C, DNA map for Xq28, showing the location of four STS markers (gels A, B, D, and G), in relation to ABCD1 and surrounding genes (se the Entrez Nucleotide Web site of the National Center for Biotechnology Information [accession number U52111]). In addition, the location of ABCD1 exons 2 and 10 (gels E and F, respectively), the boundary between ABCD1 intron 5 and exon 6 (denoted by the asterisk [*]), and DXS1357E exon 5 (gel C) are shown. DXS1357E shares a CpG island and is in a head-to-head orientation with ABCD1 (Mosser et al. 1994). D, PCR primers for the markers shown were used for analysis in Pt1, Pt2, and Pt3 and in a male control subject (Ctl). These results are summarized in figure 3. E, Amplification of template for Xq28 and 16P11. Because of sequence homology between Xq28 and 16p11, the primers for reaction C (DXS1357E exon 5) amplify both templates. Compared with DNA from the control subject and Pt2, that from Pt1 and Pt3 yielded a small amount of product. When the amplicon from Pt1 and Pt3 was sequenced by use of primer CDM-1S (see the “Material and Methods” section), a 16p11-specific sequence was revealed, whereas the products from Pt2 and the control subject had DNA sequence specific to Xq28. Sequence specific to 16p11 was detectable only in the absence of the corresponding Xq28 region.

Figure 3

CADDS critical region, which lies between DXS1357E exon 5 and ABCD1 exon 6. The minimum and maximum extent of Xq28 deletion, on the basis of PCR and Southern blot analyses, is shown for each patient. The black segments denote DNA demonstrated to be absent. The horizontal dotted lines extending from the ends of each box denote regions that have yet to be excluded. An enlargement of the critical region that is missing in all three patients is shown. The smallest deletion occurs in Pt2 and spans 22.0 ⩾ 36.2 kb.