Wilson Anna M E, Schlade-Bartusiak Kamilla, Tison Jean-Luc, Macintyre Georgina, Cox Diane W
Department of Medical Genetics, University of Alberta, Edmonton, Canada.
Biochimie. 2009 Oct;91(10):1342-5. doi: 10.1016/j.biochi.2009.06.008. Epub 2009 Jun 21.
Wilson disease (WND) is an autosomal recessive condition that results in accumulation of copper in the liver and brain when a membrane bound copper transporter, ATP7B, is defective. ATP7B is expressed in hepatic, brain and kidney cells, and a defect can lead to liver, neurological and renal damage in WND patients. Presentation is variable with a broad range of age of onset and symptoms, and not all biochemical signs used in diagnosis are found in every patient. Therefore, diagnosis by mutation analysis is particularly important. To date, there are approximately 380 probable disease-causing variants in ATP7B, 33 of which are splice site variants that are predicted to affect splicing, based on their location. Few of these splice site variants have been analyzed in vivo. Some exonic variations also have the potential to affect splicing. The aim of this project was to use minigenes for transcript analysis. We have chosen exon 8 as our focus and have cloned a wild-type three-exon minigene into a mammalian expression vector. After transfection, extracted RNA was analyzed by reverse transcription PCR and accurate splicing was detected. This minigene will facilitate the analysis of the numerous potential splice variants identified in exon 8 of ATP7B, with the advantage that patient cell lines are not required for each variant.
威尔逊病（WND）是一种常染色体隐性疾病，当一种膜结合铜转运蛋白ATP7B存在缺陷时，会导致肝脏和大脑中铜的积累。ATP7B在肝细胞、脑细胞和肾细胞中表达，其缺陷可导致WND患者出现肝脏、神经和肾脏损伤。该病的表现多样，发病年龄和症状范围广泛，并非所有用于诊断的生化指标在每个患者中都能发现。因此，通过突变分析进行诊断尤为重要。迄今为止，ATP7B中大约有380种可能的致病变体，其中33种是剪接位点变体，根据其位置预测会影响剪接。这些剪接位点变体中很少有在体内进行分析的。一些外显子变异也有可能影响剪接。本项目的目的是使用小基因进行转录分析。我们选择了第8外显子作为重点，并将野生型三外显子小基因克隆到哺乳动物表达载体中。转染后，通过逆转录PCR分析提取的RNA，并检测到准确的剪接。这个小基因将有助于分析在ATP7B第8外显子中鉴定出的众多潜在剪接变体，其优点是每个变体不需要患者细胞系。
