Menkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations to the Menkes (MNK) gene. We identified three novel mutations of the MNK gene in three unrelated Japanese patients with classical Menkes disease by analyzing reversetranscriptase polymerase chain reaction products and genomic DNA of the MNK gene. Firstly, an insertional mutation was found, 1173 ins A, which led to a premature termination and resulted in a very immature Menkes protein. Secondly, we found a point mutation, T2763G, resulting in a leucine-to-arginine conversion, which we predicted would cause a change in the secondary structure of the Menkes protein. Finally, we identified a splicing mutation, 2317+5G>C, which resulted in the skipping of both exons 8 and 9 or exon 9 only, and led to a truncation of the protein.Each of these mutations is hypothesized to destroy copperATPase-mediated copper transport. We propose that each of these mutations in the MNK gene plays a causative role in the disease.
An isoleucine/valine polymorphism was observed at codon 1464 of the ATP7A gene, which is thought to encode a copper transporting adenosine triphosphatase (ATPase). The frequency of Val1464 was estimated to be 5.7% in the Japanese population. This polymorphism may be useful in genetic studies of Menkes disease.
Ornithine transcarbamylase (OTC) is located in the mitochondrial matrix of the liver and small intestine and catalyzes the second step of the urea cycle. OTC deficiency (OTCD) is an X-linked inborn error of metabolism and causes hyperammonemia. We reported in 1992 the A152V and G195R mutations in patients with OTCD. These mutant OTC cDNAs were prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The wild-type and mutant cDNAs were transiently expressed in COS-7 cells. The wild-type cDNA gave an OTC activity of 1180Ϯ47␣ nmol/min per mg protein. The OTC activities of the A152V and the G195R mutants were 3.7% and 2.5% of that of wild-type, respectively. Immunoblot analysis showed that the quantities of OTC proteins in the A152V and G195R mutants were 29% and 12% of that of wildtype, respectively. In pulse-labeling and pulse-chase experiments, the precursor form of OTC was synthesized and processed to the mature form. The A152V mutant OTC was processed to the mature form as rapidly as the wild-type precursor. However, the processed, mature form of the mutant OTC was rapidly degraded, presumably in the mitochondrial matrix. These results indicate that OTCD with the A152V mutation is due both to rapid degradation of the processed, mature form, and to a lower specific activity of the remaining protein.
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