Genetic Copperdeficiency Diseases 31 Features of the Genetic Copper Deficiencies

There are three clinically distinct X-linked copper deficiency disorders known in humans: classical Menkes disease (MD), occipital horn syndrome (OHS), and mild Menkes disease. All three are very rare diseases, with an estimated frequency of between 1 per 100,000 and 300,000 live births (45). The rarity of these diseases, however, contrasts with the important role they continue to play in the understanding of copper homeostasis. Molecular analysis shows that all three genetic copper deficiencies result from mutations of ATP7A. A model proposing linking clinical phenotypes to the types of mutations in ATP7A has been recently proposed and is discussed later in more detail (46).

The clinical features of MD were first described in 1962 by John Menkes (47), and in 1972, David Danks realized that MD is a genetic copper-deficiency disorder, based on similar features found in copper-deficient sheep and pigs (48). Patients with MD are profoundly copper deficient and usually die by the age of 3-4 yr. As noted previously (see Fig. 1), some tissues of MD patients have higher

Fig. 4. (continued) and low plasma levels of the protein. Biliary excretion is blocked, so intracellular copper accumulates to very high levels, inducing cytoplasmic metallothioneins, which, as the cell ages, are ingested by lysosymes and the cell develops Cu-laden lysosomes. Eventually, the cell's copper storage is exceeded and it dies from copper toxicity, the excess copper is released into the circulation, depositing in extrahepatic tissues, such as the brain.

than normal amounts of copper. For example, the kidney and gut may contain more than 10-fold higher concentrations of copper compared with normal. In contrast the brain has only 2% of total-body copper in Menkes patients compared with normal of 35%, emphasizing the severity of the deficiency of copper in this organ (49). The phenomenon of copper accumulation in the presence of copper-deficiency symptoms is the result of normal cellular uptake of copper but reduced efflux (4). As noted previously, cells that accumulate copper are those normally involved in the transport of copper, such as the small-intestine enterocytes and cells of the blood-brain barrier (50), the proximal tubules of the kidney, and the placental cells of the affected fetus (51). The small amount of copper absorbed from the diet by the patients gradually accumulates in these peripheral tissues, but is unavailable to the rest of the body. The liver of Menkes patients has very low copper concentrations compared with normal, and this reflects the overall copper deficiency of the patient—the liver does not accumulate copper, as ATP7A is only expressed at a low level in this organ and copper efflux is achieved by ATP7B.

A major clinical feature, and suspected eventual cause of death, of patients with MD is profound neurological abnormalities in the brain. These neurological defects are possibly caused by the low activity of cytochrome-c oxidase, a copper-dependent enzyme in the electron-transport chain. Low activity of other copper-dependent enzymes in the brain such as superoxide dismutase, peptidylglycine-a-amidating monooxygenase, and dopamine-P-hydroxylase may also contribute to the abnormalities (14). MD patients also have connective tissue defects such as abnormalities of bone and weak vascular walls, resulting from reduced activity of lysyl oxidase. The connective tissue defects found in MD patients become the predominant features of the less severe condition, OHS. Lysyl oxidase is a copper-dependent enzyme that catalyzes the crosslinking of collagen and elastin, and the low activity of this enzyme is responsible for the aberrant connective tissue. Other clinical features of MD are caused by the low activity of specific cuproenzymes. Patients with MD are hypopigmented because of reduced activity of tyrosinase, a copper-dependent enzyme required for melanin synthesis. Hypothermia is also a feature of MD and is thought to be the result of low cyto-chrome-c oxidase activity. OHS patients have hyperelastic skin, arterial aneurisms, hernias, bladder diverticulae, and multiple skeletal abnormalities: Bony abnormalities of the occiput give rise to the name. Neurological abnormalities are not a feature of OHS although patients may be mildly mentally retarded. Another clinical phenotype, mild Menkes disease, has been described and a missense mutation in ATP7A has been identified in this patient (52).

Cultured cells from Menkes patients accumulate copper and this property is used for prenatal diagnosis (53,54). This excess copper results as a consequence of defective copper efflux from the cell (55). Figure 5 shows the pattern of copper distribution in a fibroblast from a Menkes patient (compare to Fig. 1, a normal fibroblast). Efflux of copper from the cell cannot occur, because this step requires both an active ATP7A and copper-induced trafficking of ATP7A. A role for ATP7A in copper efflux was strongly supported by the increased rate of copper efflux observed in cultured cell lines that overexpress the Menkes gene (56). Estimation of the rate of efflux of copper from cultured fetal cells is a more reliable method for prenatal diagnosis than simple cellular copper measurements (55). The absence of an active ATP7A in the TGN explains the low activity of lysyl oxidase secreted by fibroblasts from MD patients (57) because the Menkes Cu-ATPase is required to pump copper across the TGN membrane. Cultured fibroblasts from patients with OHS also accumulate excess copper (58) and have defective copper efflux (Camakaris, unpublished data).

3.2. Treatment of Menkes Disease

Menkes disease patients have been treated by daily injections of copper salts or complexes, such as copper histidine (59), but this treatment has not produced successful results in many patients (14). In responding patients, Cu-therapy prolongs survival and results in clinical improvement, but treatment must be commenced as soon as possible after birth, before significant brain damage has occurred (4,60,61). Even with early treatment, however, not all patients respond to therapy and Kaler has suggested that

hCTI

hCTI

Fig. 5. Copper transport in a fibroblast from a Menkes disease patient. When cells are cultured in media with normal copper concentration, copper entry into the cell is normal, but efflux is blocked because of the absent ATP7A activity. The measurement of copper concentration in the cell or the rate of efflux of copper can be used for prenatal diagnosis of Menkes disease. The absence of ATP7A also prevents the incorporation of copper into secreted cuproenzymes, such as lysyl oxidase.

Fig. 5. Copper transport in a fibroblast from a Menkes disease patient. When cells are cultured in media with normal copper concentration, copper entry into the cell is normal, but efflux is blocked because of the absent ATP7A activity. The measurement of copper concentration in the cell or the rate of efflux of copper can be used for prenatal diagnosis of Menkes disease. The absence of ATP7A also prevents the incorporation of copper into secreted cuproenzymes, such as lysyl oxidase.

the responding patients are those who retain some residual ATP7A activity (62). In patients who respond to therapy, the connective tissue abnormalities are not corrected and the patients acquire some of the clinical features of OHS (60,61).

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