Silent nucleotide substitution in the sterol 27-hydroxylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a cryptic 5′ splice site at the mutant codon in cerebrotendinous xanthomatosis patients
Silent nucleotide substitution in the sterol 27-hydroxylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a cryptic 5′ splice site at the mutant codon in cerebrotendinous xanthomatosis patients
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DOI：
10.1021/bi972940a
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发表时间：
1998-03-31
期刊：
影响因子：
2.9
通讯作者：
Seyama, Y
中科院分区：
文献类型：
--
作者：
Chen, W;Kubota, S;Seyama, Y
在两个脑腱黄瘤病(CTX)家系中发现了甾醇27-羟基酶基因(CyP27)的功能沉默核苷酸替换，这被证实导致了该基因的Pre-mRNA选择性剪接。对一个CTX家族患者的CyP27基因进行全长RT-PCR分析，发现了一条主带和一条额外的微弱条带。对克隆的RT-PCR产物进行序列分析，发现有3种：外显子2缺失、外显子2跳过、密码子112无G-T突变的全长cDNA(GGG(112)Gly到GGT(112)Gly)。基因组DNA序列分析发现，患者的CyP27基因只有一个碱基的改变：密码子112的第三位T取代了G，外显子2的3‘端上游13个碱基。无论有没有突变，构建的微基因通过激活突变密码子周围的一个隐蔽的5’剪接点，证实了这种沉默的突变导致了前mRNA的选择性剪接。在来自另一个CTX家族的两名患者中也发现了该突变，其372密码子上的A替换G的复合杂合子模式，这是我们小组先前报道的一个突变。这些结果阐明了CTX的一个新的分子基础，并表明了沉默的核苷酸替代对于前RNA剪接的意义。
A functionally silent nucleotide substitution of the sterol 27-hydroxylase gene (CYP 27), identified in two families with cerebrotendinous xanthomatosis (CTX), was confirmed to cause alternative pre-mRNA splicing of the gene. Full-length RT-PCR analysis of the CYP 27 gene in a patient from one of the CTX families revealed one major and an additional faint band. Sequence analysis of the cloned RT-PCR product showed three species of cDNA: 3' terminal 13 bp of exon 2 deleted cDNA, exon 2 skipped cDNA, and full-length cDNA with a functionally silent G to T mutation at codon 112 (GGG (112)Gly to GGT (112)Gly). Only a single base change was identified by genomic DNA sequence analysis of the CYP 27 gene in the patient: T replaced G at the third position of codon 112, 13 bp upstream from the 3' terminus of exon 2. Transfection of constructed minigenes, with or without the mutation, confirmed that this silent mutation resulted in alternative pre-mRNA splicing by activating a cryptic 5' splice site around the mutant codon. The mutation was also identified in two patients from another CTX family, with a compound heterozygous pattern of A for G substitution at codon 372, a mutation reported previously by our group. The results elucidate a novel molecular basis for the CTX and suggest the significance of a silent nucleotide substitution with regard to pre-RNA splicing.