Abstract
Disease-causing variants in ATP7A lead to two different phenotypes associated with copper deficiency; a lethal form called Menkes disease (MD), leading to early death, and a much milder form called occipital horn syndrome (OHS). Some investigators have proposed that an ATP7A transcript missing exon 10 leads to a partly active protein product resulting in the OHS phenotype. Here, we describe an individual with OHS, a biology professor, who survived until age 62 despite a splice site mutation, leading to skipping of exon 15. ATP7A transcripts missing exon 10, or exon 15 preserve the reading frame, but it is unknown if either of these alternative transcripts encode functional protein variants. We have investigated the molecular consequence of splice site mutations leading to skipping of exon 10 or exon 15 which have been identified in individuals with OHS, or MD. By comparing ATP7A expression in fibroblasts from three individuals with OHS (OHS-fibroblasts) to ATP7A expression in fibroblasts from two individuals with MD (MD-fibroblasts), we demonstrate that transcripts missing either exon 10 or exon 15 were present in similar amounts in OHS-fibroblasts and MD-fibroblasts. No ATP7A protein encoded from these transcripts could be detected in the OHS and MD fibroblast. These results, combined with the observation that constructs encoding ATP7A cDNA sequences missing either exon 10, or exon 15 were unable to complement the high iron requirement of the ccc2Δ yeast strain, provide evidence that neither a transcript missing exon 10 nor a transcript missing exon 15 results in functional ATP7A protein. In contrast, higher amounts of wild-type ATP7A transcript were present in the OHS-fibroblasts compared with the MD-fibroblasts. We found that the MD-fibroblasts contained between 0 and 0.5% of wild-type ATP7A transcript, whereas the OHS-fibroblasts contained between 3 and 5% wild-type transcripts compared with the control fibroblasts. In summary these results indicate that protein variants encoded by ATP7A transcripts missing either exon 10 or exon 15 are not functional and not responsible for the OHS phenotype. In contrast, expression of only 3-5% of wild-type transcript compared with the controls permits the OHS phenotype.
Highlights
ATP7A, an X-linked gene, encodes a copper-transporting ATPase that ensures the ATP-driven translocation of copper ions across cellular membranes (Skjørringe et al, 2017)
It was demonstrated that an ATP7A mRNA transcript without exon 10 encodes an ATP7A-protein variant, which is located in the endoplasmic reticulum instead of its correct location in the trans-Golgi network (TGN)
To evaluate if the occipital horn syndrome (OHS) phenotype, observed in patients P1, P2, and P3, could be explained by partial activity of the protein products encoded by transcripts missing exon 10 as proposed previously (Qi and Byers, 1998) or by transcripts missing exon 15, we here investigated the molecular effects of selected mutations affecting skipping of exon 10 and exon 15 in detail
Summary
ATP7A, an X-linked gene, encodes a copper-transporting ATPase that ensures the ATP-driven translocation of copper ions across cellular membranes (Skjørringe et al, 2017). Splice site mutations have been found frequently in these patients with OHS (Table 1) These splice site mutations can lead to skipping of one or several exons, and the resulting transcript can preserve or alter the reading frame. It was demonstrated that an ATP7A mRNA transcript without exon 10 encodes an ATP7A-protein variant, which is located in the endoplasmic reticulum instead of its correct location in the TGN. Exon 15 of ATP7A encodes for the CPC channel, and we have previously demonstrated (Skjørringe et al, 2017) that a mutation of this motif (p.Cys1000Arg) hampers copperdependent translocation, leading to permanent localization into the TGN. To evaluate if the OHS phenotype, observed in patients P1, P2, and P3, could be explained by partial activity of the protein products encoded by transcripts missing exon 10 as proposed previously (Qi and Byers, 1998) or by transcripts missing exon 15, we here investigated the molecular effects of selected mutations affecting skipping of exon 10 and exon 15 in detail
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