Clinical outcomes in Menkes disease patients with a copper-responsive ATP7A mutation, G727R

Abstract

Menkes disease is a fatal neurodegenerative disorder of infancy caused by defects in an X-linked copper transport gene, ATP7A. Evidence from a recent clinical trial indicates that favorable response to early treatment of this disorder with copper injections involves mutations that retain some copper transport capacity. In three unrelated infants, we identified the same mutation, G727R, in the second transmembrane segment of ATP7A that complemented a Saccharomyces cerevisiae copper transport mutant, consistent with partial copper transport activity. Quantitative reverse transcription-polymerase chain reaction studies showed approximately normal levels of ATP7A(G727R) transcript in two patients' fibroblasts compared to wild-type controls, but Western blot analyses showed markedly reduced quantities of ATP7A, suggesting post-translational degradation. We confirmed the latter by comparing degradation rates of mutant and wild-type ATP7A via cyclohexamide treatment of cultured fibroblasts; half-life of the G727R mutant was 2.9h and for the wild-type, 11.4h. We also documented a X-box binding protein 1 splice variant in G727R cells-known to be associated with the cellular misfolded protein response. Patient A, diagnosed 6 months of age, began treatment at 228days (7.6 months) of age. At his current age (2.5 years), his overall neurodevelopment remains at a 2- to 4-month level. In contrast, patient B and patient C were diagnosed in the neonatal period, began treatment within 25 days of age, and show near normal neurodevelopment at their current ages, 3years (patient B), and 7 months (patient C). The poor clinical outcome in patient A with the same missense mutation as patient A and patient B with near normal oucomes, confirms the importance of early medical intervention in Menkes disease and highlights the critical potential benefit of newborn screening for this disorder.

Figures

Figure 1. G727R Mutation in ATP7A

Automated sequence of genomic DNA from patient B shows a guanine to adenine transition at nucleotide 2279 (asterisk) in exon 10 of ATP7A that changes the wild type triplet codon from GGA to AGA, predicting substitution of arginine for glycine at amino acid residue 727. The identical alteration was detected in patient A and patient C.

Figure 2. Western Analysis of CCC2 Deletion Constructs Confirms Expression of ATP7A in Transformed Strains

Western blot of protein extracts from S. cerevisiae constructs, using a carboxy-terminal antibody to ATP7A, indicates expression of the expected 178 kDa protein in CCC2 deletion strains transformed with wild-type ATP7A and the G727R allele, and not in a mock-transformed strain (arrow). All samples showed considerable nonspecific binding in this assay.

Figure 3. G727R Mutant Allele Rescues CCC2 Deletion in Copper/Iron-Limited Medium

Four different S. cerevisiae constructs were studied in the yeast complementation assay, clockwise, from top: CCC2 deletion strain transformed with wild-type ATP7A, CCC2 deletion strain mock-transformed with an empty vector, CCC2 deletion strain without transformation, and the CCC2 deletion strain transformed with the mutant ATP7A allele G727R. Yeast strains were plated on four different media. All strains grew on yeast peptone dextrose (YPD) media (panel A), copper-sufficient (panel B) and iron-sufficient media (panel C), whereas only the wild-type ATP7A and G727R mutant grew on copper/iron-limited media, indicating complementation of the CCC2 deletion strain (panel D).

Figure 4. Timed growth Assay

Timed growth in copper/iron-limited medium curve for CCC2 deletion mock-transformed with pYes6/CT vector (CCC2), and transformed with pYes6/CT vector harboring either the mutant G727R allele or Wild-type ATP7A allele. Data points represent average OD600 absorbance during log-phase growth. Mean growth (triplicate measures) mediated by the G727R allele was 57% of Wild-type.

Figure 5. Steady State ATP7AG727R mRNA Expression Compared to Wild-type ATP7A

Quantitative RT-PCR using fibroblasts from patients A and B indicate that levels of the ATP7AG727R mutant transcript (normalized to β-actin transcript) approximate that of Wild-type ATP7A in three normal control fibroblast cell lines.

Figure 6. Steady State Protein Levels Produced by ATP7AG727R Compared to Wild-type ATP7A

In contrast to mRNA transcript levels (Fig. 5), Western blot analysis of whole cell protein homogenates from fibroblasts of patient A and patient B indicates that the ATP7A protein level is significantly decreased in cells with ATP7AG727R compared to levels of wild-type ATP7A in two normal control fibroblast lines (arrow). As a negative control and to illustrate specificity of the carboxy-terminal ATP7A antibody used, we loaded fibroblast protein from a previously studied Menkes disease patient with deletion of ATP7A exons 20–23 [19].

Figure 7. XBP1 Splicing Pattern Suggests Cellular Misfolded Protein Response Associated with G727R

Reverse transcriptase-polymerase chain reaction (RT-PCR) patterns for XBP1 in patient A, patient B, and three normal fibroblast cell lines (WT-1, GM5659, WT-2) reveal relative prominence of an alternatively spliced transcript (arrow) in the two patients’ cells, consistent with the misfolded protein response [27]. The two smaller bands are fragments from the properly spliced XBP1 transcript. Densitometric quantitation indicated that the alternatively spliced fragment was 15% (patient A) and 19% (patient B) of total XBP1 transcript, compared to 6%, 9%, and 4%, respectively in the three control cell lines. L = ladder of molecular weight standards.

Figure 8. ATP7AG727R is Degraded More Rapidly Than Wild-type ATP7A

Western blot analysis indicate that the quantity of G727R mutant protein (patient A) is lower after 60 minutes incubation with cyclohexamide, an inhibitor of protein synthesis, relative to wild-type ATP7A (cell line GM5659). Densitometric quantitation indicated that 79% of ATP7AG727R remained after cyclohexamide treatment, versus 94% of wild-type ATP7A (arrow). Calculated half-lives for the G727R and wild-type forms of ATP7A are 2.9 hr and 11.4 hr, respectively.

Figure 9. Brain Magnetic Resonance Imaging

Axial FLAIR brain images of patient A and patient B at 20 and 24 months of age, respectively. The brain for patient A shows marked cortical atrophy and hypomyelination, the latter denoted by an increase in the intensity of the periventricular signal, whereas patient B shows normal brain volume and slightly delayed myelination.