A copper treatable Menkes disease mutation associated with defective trafficking of a functional Menkes copper ATPase.

Copper dependency in humans is most dramatically illustrated in Menkes disease, an X linked recessive copper deficiency disorder that is generally lethal in early childhood. 2 Menkes disease is caused by mutations in a transmembrane copper transporting P type ATPase, MNK (or ATP7A), which is expressed in virtually all non-hepatic tissues. Studies using cultured cells suggest that MNK is located in the trans-Golgi network (TGN), where it transports copper to copper dependent enzymes synthesised within secretory compartments. In addition to this biosynthetic role, MNK functions in the efflux of excess copper from cells via a process of copper stimulated trafficking to the plasma membrane. 9 Copper export via MNK from intestinal enterocytes is essential for supplying the blood with dietary copper. Similarly, MNK mediated copper export from the capillary endothelium of the blood brain barrier is thought to supply copper to the central nervous system. In Menkes patients, these processes are defective resulting in a range of symptoms attributable to deficiencies in copper dependent metabolism. These include neurological degeneration, mental retardation, seizures, arterial and bone abnormalities, hypothermia, and hypopigmentation. Classical Menkes disease rapidly progresses and is generally lethal during early childhood, although milder variants of the disease exist. 11 The treatment of Menkes disease involves parenteral injections of copper-histidine, which in the most successful cases reduces neurological defects and prolongs life expectancy. 12 13 This copper replacement therapy bypasses the intestinal blockage of dietary copper absorption and increases circulating copper levels. However, to prevent the onset of neurological symptoms in Menkes patients, copper must be delivered across the endothelial cells of the blood brain barrier to supply copper to the central nervous system. Within the central nervous system, copper transport into secretory compartments of neurones and other cells to supply copper to cuproenzymes, such as dopamine β hydroxylase and peptidylglycine α amidating mono-oxygenase, may also be important for the successful treatment of Menkes disease. The clinical outcome of copper replacement therapy for the small number of reported human cases ranges from poor to favourable, 16 with no definitive biochemical explanation for this variability. To begin to understand the contribution of the Menkes genotype to the success of copper replacement therapy, we characterised the biochemical and cell biological defect associated with a MNK mutation previously identified in a successfully treated patient. This mutation involves a splice site mutation resulting in the deletion of exon 8, which encodes a small region between the sixth copper binding site and the first membrane spanning domain of the MNK protein. In this study, we assessed the copper induced trafficking and copper transport function of this exon 8 skipped MNK protein. MATERIALS AND METHODS Reagents and cell culture All chemicals were purchased from Sigma. The immortalised Menkes fibroblast cell line, Me32a, lacks detectable MNK protein owing to an early frameshift mutation in MNK, as described previously. The primary Menkes patient fibroblast cell line, GM13672, and control primary fibroblasts, GM00302 and GM00970, were obtained from the Coriell Genetic Cell Repository. The GM13672 cell line was originally derived from the affected cousin of patient III.6 (patient 1433 in Das et al. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum and 100 U/ml penicillin and streptomycin in a 5% CO2, 37°C incubator.