Genetic analysis of cerebrotendinous xanthomatosis

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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