Forbes J R, Cox D W
Department of Medical Genetics, 8-39 Medical Sciences Building, University of Alberta, Edmonton, Canada.
Am J Hum Genet. 1998 Dec;63(6):1663-74. doi: 10.1086/302163.
Wilson disease is an autosomal recessive disorder of copper transport that causes hepatic and/or neurological disease resulting from copper accumulation in the liver and brain. The protein defective in this disorder is a putative copper-transporting P-type ATPase, ATP7B. More than 100 mutations have been identified in the ATP7B gene of patients with Wilson disease. To determine the effect of Wilson disease missense mutations on ATP7B function, we have developed a yeast complementation assay based on the ability of ATP7B to complement the high-affinity iron-uptake deficiency of the yeast mutant ccc2. We characterized missense mutations found in the predicted membrane-spanning segments of ATP7B. Ten mutations have been made in the ATP7B cDNA by site-directed mutagenesis: five Wilson disease missense mutations, two mutations originally classified as possible disease-causing mutations, two putative ATP7B normal variants, and mutation of the cysteine-proline-cysteine (CPC) motif conserved in heavy-metal-transporting P-type ATPases. All seven putative Wilson disease mutants tested were able to at least partially complement ccc2 mutant yeast, indicating that they retain some ability to transport copper. One mutation was a temperature-sensitive mutation that was able to complement ccc2 mutant yeast at 30 degreesC but was unable to complement at 37 degreesC. Mutation of the CPC motif resulted in a nonfunctional protein, which demonstrates that this motif is essential for copper transport by ATP7B. Of the two putative ATP7B normal variants tested, one resulted in a nonfunctional protein, which suggests that it is a disease-causing mutation.
威尔逊病是一种常染色体隐性铜转运障碍疾病，因铜在肝脏和大脑中蓄积而导致肝脏和/或神经系统疾病。该疾病中存在缺陷的蛋白质是一种假定的铜转运P型ATP酶，即ATP7B。在威尔逊病患者的ATP7B基因中已鉴定出100多种突变。为了确定威尔逊病错义突变对ATP7B功能的影响，我们基于ATP7B补充酵母突变体ccc2高亲和力铁摄取缺陷的能力，开发了一种酵母互补测定法。我们对在ATP7B预测跨膜区段中发现的错义突变进行了表征。通过定点诱变在ATP7B cDNA中产生了10种突变：5种威尔逊病错义突变、2种最初归类为可能致病突变的突变、2种假定的ATP7B正常变体，以及重金属转运P型ATP酶中保守的半胱氨酸-脯氨酸-半胱氨酸（CPC）基序的突变。所测试的所有7种假定的威尔逊病突变体都能够至少部分补充ccc2突变酵母，表明它们保留了一定的铜转运能力。一种突变是温度敏感型突变，它能够在30℃时补充ccc2突变酵母，但在37℃时无法补充。CPC基序的突变导致产生无功能的蛋白质，这表明该基序对于ATP7B的铜转运至关重要。在所测试的2种假定的ATP7B正常变体中，有一种导致产生无功能的蛋白质，这表明它是一种致病突变。
