A novel frameshift mutation in exon 23 of ATP7A (MNK) results in occipital horn syndrome and not in Menkes disease

Abstract

Menkes disease and occipital horn syndrome (OHS) are allelic, X-linked recessive copper-deficiency disorders resulting from mutations in ATP7A, or MNK. Classic Menkes disease has a severe phenotype, with death in early childhood, whereas OHS has a milder phenotype, with, mainly, connective-tissue abnormalities. Data suggest that steady-state localization of ATP7A to the trans-Golgi network (TGN) is necessary for proper activity of lysyl oxidase, which is the predominant cuproenzyme whose activity is deficient in OHS and which is essential for maintenance of connective-tissue integrity. Recently, it was reported that ATP7A-transcript levels as low as 2%-5% of normal are sufficient to result in the milder phenotype, OHS, rather than the phenotype of Menkes disease. In contrast to previously reported cases of OHS, we describe a case of OHS in which, because of a frameshift mutation, no normal ATP7A is produced. Although abundant levels of mutant transcript are present, there are substantially reduced levels of the truncated protein, which lacks the key dileucine motif L1487L1488. It has been demonstrated that the dileucine motif L1487L1488 functions as an endocytic signal for ATP7A cycling between the TGN and the plasma membrane. The present report is the first to describe an ATP7A truncation that results in OHS rather than in Menkes disease. The data from the present report support the concepts that (1) OHS results from lower levels of functional ATP7A and (2) ATP7A does not require the dileucine motif to function in copper efflux.

Figure 1

Pedigree, consistent with X-linked inheritance, of the family with OHS. Family members included in this study were II-6, III-1, III-2, and III-3. Blackened squares indicate affected males, and unblackened squares indicate unaffected males. Circles with a dot indicate carrier females, and circles without a dot indicate either unaffected or untested females.

Figure 2

Frameshift mutation in ATP7A. The 5′ and 3′ UTRs of the ∼8.5-kb ATP7A transcript are shown atop a gray background, and the 4.5-kb coding region is shown without a background. In the lower portion of the figure, the coding sequence of wild-type (wt) ATP7A exon 23 is enlarged. The wild-type guanine triplet at positions 4497–4499 is shown atop a black background. The BslI (ccnnnnnnngg) restriction site is shown above the wild-type nucleotide sequence. Conceptual translation of wild-type (without background) and mutant (atop a gray background) ATP7A is illustrated below the nucleotide sequence. The dileucine motif L1487L1488 is underlined in wild-type ATP7A. Stop codons are indicated by asterisks (*).

Figure 3

Mutation analysis of the family with OHS. To generate 515-bp PCR products (upper panel), the exon 23 primer pair (table 2) and genomic DNAs isolated from lymphoblastoid cell lines from an unrelated normal control (wt), II-6, III-1, III-2, and III-3 were used. Digests in III-2, III-3, and in one allele of II-6 demonstrate loss of 312-bp and 119-bp BslI fragments (lower panel). The presence, in all digests, of the 84-bp fragment indicates complete digestion with BslI. The smallest band in the digest for II-6 is a primer band and is also present in the water blank.

Figure 4

Northern blot analysis of ATP7A-transcript levels. TRIZOL (Gibco) was used to isolate total RNA from lymphoblastoid cell lines from II-6, III-1, III-2, and III-3. mRNA was obtained using the PolyATtract mRNA Isolation System IV (Promega). A northern blot containing 2 μg of poly(A)⁺ from each family member was hybridized with a [³²P]-radiolabeled 1.2-kb cDNA probe, specific to exons 4–10 (nucleotides 1147–2335) of ATP7A. The primer sequences used to generate, via RT-PCR, the 1.2-kb product of normal lymphoblastoid cDNA were 5′- AATAGTGGCTGTATCACCGGG-3′ and 5′- GTACCAGCCTCCGAAAAACTG-3′. Subsequent hybridization with a [³²P]-radiolabeled β-actin probe (Clontech) demonstrated loading consistency of poly(A)⁺ RNA, in each lane (lower panel). Probes were radiolabeled by the rediprimeII Random Prime Labeling System (Amersham Pharmacia Biotech).

Figure 5

Western blot analysis of ATP7A levels. Protein from lymphoblastoid cell lines from CG, II-6, III-1, III-2, and III-3 was isolated according to a procedure described by Ambrosini and Mercer (1999). Fibroblast (Fb) protein from an unrelated normal control cell line was isolated according to a procedure described by Dierick et al. (1997). One hundred micrograms of protein per lane was fractionated through a 7.5% acrylamide gel and then was electroblotted onto a nitrocellulose membrane. This membrane was then hybridized with a 1:1,000 dilution of polyclonal α-ATP7A (upper panel), as described by Dierick et al. (1997); after several washes in Tris buffered saline solution plus Triton X-20, this membrane was incubated with a 1:2,000 dilution of horseradish peroxidase–conjugated donkey anti-rabbit antibodies. α-ATP7A hybridized to a 174-kD protein in fibroblast, consistent with ATP7A. Hybridization to a 174-kD protein in the fibroblast cell line confirms that the ATP7A antibody is hybridizing to the correct protein from lymphoblastoid cells. To demonstrate loading consistency, the blot was hybridized with a 1:10,000 dilution of tubulin-α Ab-2 antibody, which hybridized to a 57-kD protein. Ambrosini and Mercer (1999) have reported smearing of ATP7A isolated from lymphoblastoid cell lines and have suggested that it most likely was a result of protein glycosylation.

Figure 6

Indirect immunofluorescence of ATP7A in fixed fibroblasts. Immunocytochemistry was performed according to a procedure described by Dierick et al. (1997). Fibroblasts from II-1 (a), III-2 (b), and GM01981 (c) were incubated, for 1 h, with a 1:200 dilution of polyclonal α-ATP7A (Dierick et al. 1997). To visualize the hybridization of α-ATP7A, the fibroblasts were incubated with a 1:1,000 dilution of affinity-purified fluorescein isothiocyanate (H+L) (Vector Laboratories). Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole. Results were analyzed by a Zeiss Axioscope epifluorescence microscope and were documented by a Photometrics SenSys camera and Vysis Quips imaging software (Applied Imaging).