Translational read-through of a nonsense mutation in ATP7A impacts treatment outcome in Menkes disease

Abstract

Protein translation ends when a stop codon in a gene's messenger RNA transcript enters the ribosomal A site. Mutations that create premature stop codons (nonsense mutations) typically cause premature translation termination. An alternative outcome, read-through translation (or nonsense suppression), is well known in prokaryotic, viral, and yeast genes but has not been clearly documented in humans except in the context of pharmacological manipulations. Here, we identify and characterize native read-through of a nonsense mutation (R201X) in the human copper transport gene, ATP7A. Western blotting, in vitro expression analyses, immunohistochemistry, and yeast complementation assays using cultured fibroblasts from a classic Menkes disease patient all indicated small amounts of native ATP7A(R201X) read-through and were associated with a dramatic clinical response to early copper treatment.

Figures

Figure 1. Molecular and Radiologic findings in Patient with Menkes disease

A. Upper panel: Axial flair magnetic resonance image of the patient’s brain at 3 years of age shows normal volume and myelination (hypointense signal). (b) Lateral skull radiograph at age 11.5 years shows occipital exostoses (arrowhead). B. Sequence of cDNA illustrating a cytosine (C) to thymine (T) transition producing the R201X mutation in ATP7A.

Figure 2. Model of the ATP7A Copper Transporter

The R201X mutation is located between the second and third copper-binding motifs. The amino- and carboxy-terminal antibody binding locations are noted (orange color).

Figure 3. Evidence of Translational Read-through of R201X in ATP7A

A. Western blots of fibroblast microsomal protein from a normal (WT) control male and Menkes disease patient with the R201X mutation show that the full length (178 kDa) gene product is detected with an amino-terminal antibody generated against amino acids 146–160 of ATP7A (left hand panel), as well as with a carboxy-terminal antibody against residues 1479–1500 of ATP7A (right hand panel). The quantity is reduced in the R201X cells (note asterisks). The C-terminal antibody gives a stronger signal than the N-terminal antibody. B. Confocal microscopy of fibroblasts from normal (WT) control (top panels) and R201X patient (bottom panels) stained with N-terminal and C-terminal antibodies to ATP7A, and with Texas-Red labeled anti-rabbit IgG secondary antibody alone, as a negative control. The normal cells show perinuclear staining consistent with trans-Golgi localization, a pattern also seen in R201X cells at much lower intensity. The C-terminal antibody gives a stronger signal than the N-terminal antibody.

Figure 4. Yeast Complementation Documents Functional Copper Transport Associated with R201X

A. On copper/iron-deficient media, the copper transport mutant ccc2Δ fails to grow, whereas the WT strain grows normally. Transformation of ccc2Δ with the R201X mutant allele restores growth although less robustly than when transformed with the wild type ATP7A. B. Timed growth assays estimate R201X growth in the range of 5–10% compared to wild type (WT). Diamond = wild type, Square = R201X, Triangle = ccc2Δ.

Figure 5. In Vitro Expression Analyses of 201X Indicate Full-length and Truncated ATP7A Peptides

A. Reporter constructs for 201X and 201R in the expression vector pTR-UF11. CMV = cytomegalovirus enhancer; CBA = chicken ß-actin promoter; ATP7A = cDNA sequence encoding the initial 249 amino acids of ATP7A; polyA = polyadenylation sequence. B. Western blots of HEK-293 protein following transient expression detect the full length peptide (28 kDa) using a N-terminal ATP7A antibody with both constructs, as well as a smaller form (22 kDa) corresponding in size to the prematurely terminated peptide (200 amino acids) from the mutant construct.