Genetic analysis of cerebrotendinous xanthomatosis

1)CTX患者新突变的鉴定：脑腱黄瘤病(CTX)的潜在生化缺陷是缺乏甾醇27-羟基酶，导致胆甾醇在几个组织中积聚。我们调查了一位44岁的日本男性CTX患者。采用非RI-PCR-SSCP方法对CyP27基因进行分析。患者的异常迁移条带表明在外显子7内和/或其周围存在突变。通过直接测序分析，在CyP27的血红素配基结合区的397位密码子[GTG(Val)到GGT(Gly)]处发现了新的两个点突变。2)CyP27mRNA的核糖核酸酶保护分析：然后我们采用了核糖核酸酶保护分析，有望更特异和敏感地鉴定CYP27mRNA。(1)用聚合酶链式反应扩增了CYP27cDNA675-958片段，克隆到pBluescript II SK+载体上，获得了经Eco RI和Bam HI双酶切后的片段。(2)Eco RI di…线性化更多的食欲。~(32)P标记的反义RNA(CDNAs)是在存在~(32)&gt；P标记核苷酸的情况下，用T_3RNA聚合酶转录而成。(3)从人肝和人横纹肌肉瘤细胞中提取RNA。将~lt；32&gt；P标记的探针与15 mg的RNA杂交，未杂交区域用RNaseT1和RNaseA处理。(4)将样品加载到序列凝胶上。与DNA大小标记比较，确定了预期大小的条带。人肌动蛋白在肝脏和RD细胞中均有表达，而细胞色素P27在肝脏中仅有表达。3)CTX患者的治疗：动脉粥样硬化是CTX的主要症状之一，我们联合应用普伐他汀和CDCA治疗可以阻止疾病的进展，但对临床表现、黄瘤和电生理检查没有明显的影响。较少

1) Identification of novel mutation in CTX patient : The underlying biochemical defect of cerebrotendinous xanthomatosis (CTX) is a lack of sterol 27-hydroxylase, which leads to the accumulation of cholestanol in several tissues. We investigated a 44-year-old Japanese male CTX patient. Non-RI-PCR-SSCP method was applied to analyze the CYP 27 gene. Abnormally migrating bands of the patient indicated that a mutation exists within and/or around exon 7. Direct sequence analysis was conducted and new two point mutations at codon 397 [GTG (Val) to GGT (Gly)] at the heme ligand binding region of the CYP 27 were identified.2) RNase protection assay of CYP 27mRNA : Then we adopted an RNase protection assay which is expected to identify the CYP27 mRNA more specifically and sensitively. (1) CYP27cDNA (nt 675-958) was amplified by PCR and an Eco RI and Bam HI digested fragment of the CYP27cDNA was isolated and subcloned into the pBluescript II SK+vector. (2) The plasmid was linearized by Eco RI di … More gestion. ^<32>P-labeled anti-sense RNA (cDNA) was prepared by transcription of the DNA with T3 RNA polymerase with the exixtence of ^<32>P-labeled nucleotides. (3) RNA was extracted from human liver and human rhabdomyosarcoma cells. ^<32>P-labeled probe was hybridized with 15 mg of RNA.Unhybridized regions were digested by the RNase T1 and RNase A treatment. (4) Samples were loaded on the sequence gel. The bands of expected size were confirmed compared with the DNA size marker. Human g actin mRNA was detected in both liver and RD cells, but CYP27 mRNA was detected only in liver. For the purpose of CTX diagnosis, it is necessary to improve the sensitivity to detect mRNA using human fibroblast.3) Treatment of CTX patient : Atherosclerosis is one of the major symptoms in CTX.We applied pravastatin treatment together with CDCA administration The progression of disease was arrested, but dramatic effects on clinical manifestations, xanthoma, and electrophysiological findings could not be found after the treatment of these drug. Less