Evidence that translation reinitiation leads to a partially functional Menkes protein containing two copper-binding sites

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Menkes disease (MD) is an X-linked recessive disorder of copper metabolism. It is caused by mutations in the ATP7A gene encoding a copper-translocating P-type ATPase, which contains six N-terminal copper-binding sites (CBS1-CBS6). Most patients die in early childhood. We investigated the functional effect of a large frameshift deletion in ATP7A (including exons 3 and 4) identified in a patient with MD with unexpectedly mild symptoms and long survival. The mutated transcript, ATP7A(Delta ex3+ex4), contains a premature termination codon after 46 codons. Although such transcripts are generally degraded by nonsense-mediated mRNA decay (NMD), it was established by real-time PCR quantification that the ATP7A(Delta ex3+ex4) transcript was protected from degradation. A combination of in vitro translation, recombinant expression, and immunocytochemical analysis provided evidence that the ATP7A(Delta ex3+ex4) transcript was protected from degradation because of reinitiation of protein translation. Our findings suggest that reinitiation takes place at two downstream internal codons. The putative N-terminally truncated proteins contain only CBS5 and CBS6. Cellular localization and copper-dependent trafficking of the major part of endogenous and recombinant ATP7A(Delta ex3+ex4) proteins were similar to the wild-type ATP7A protein. Furthermore, the ATP7A(Delta ex3+ex4) cDNA was able to rescue a yeast strain lacking the homologous gene, CCC2. In summary, we propose that reinitiation of the NMD-resistant ATP7A(Delta ex3+ex4) transcript leads to the synthesis of N-terminally truncated and at-least-partially functional Menkes proteins missing CBS1-CBS4. This finding--that a mutation that would have been assumed to be null is not--highlights the need to examine the biochemical phenotype of patients to deduce the efficacy of copper therapy.
Original languageEnglish
JournalAmerican Journal of Human Genetics
Volume79
Issue number2
Pages (from-to)214-29
Number of pages15
ISSN0002-9297
DOIs
Publication statusPublished - 2006

Bibliographical note

Keywords: Adenosine Triphosphatases; Adolescent; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; CHO Cells; Cation Transport Proteins; Cell-Free System; Cells, Cultured; Child; Child, Preschool; Copper; Cricetinae; Fibroblasts; Frameshift Mutation; Humans; Infant; Male; Menkes Kinky Hair Syndrome; Molecular Sequence Data; Peptide Chain Initiation, Translational; Sequence Deletion; Survivors

ID: 9067396