Scanning mutations of the 5′UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography:: identification of new mutations
Scanning mutations of the 5′UTR regulatory sequence of L-ferritin by denaturing high-performance liquid chromatography:: identification of new mutations
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DOI：
10.1046/j.1365-2141.2003.04253.x
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发表时间：
2003-04-01
影响因子：
6.5
通讯作者：
Arosio, P
中科院分区：
文献类型：
--
作者：
Cremonesi, L;Paroni, R;Arosio, P
遗传性高铁蛋白血症白内障综合征是一种常染色体显性遗传性疾病，由铁蛋白 L 链 mRNA 中铁调节元件 (IRE) 的异质突变引起。这些突变很罕见，快速 DNA 扫描将有助于诊断。该研究的目的是比较两种快速 DNA 扫描技术的分析性能：变性高效液相色谱 (DHPLC) 和双梯度变性梯度凝胶电泳 (DG-DGGE)。我们分析了编码铁蛋白 L 链 mRNA 5' 非翻译侧翼区域的序列，其中包括 IRE 茎环结构。这两个系统在一次运行中明确识别了所有 12 个可接近的突变，包括困难的 C-G 颠换。 DHPLC 和 DG-DGGE 在 47 名不明原因高铁蛋白血症受试者的 DNA 样本中鉴定出七种异常模式；所有的 IRE 序列都有突变，其中包括两个以前未报道过的突变：C36G 和 A37G。对 HFE 基因分型受试者的 250 个 DNA 样本进行扫描，发现了四种新突变，均位于 IRE 结构之外：C10T、C16T、C90T 和 del-T156。我们得出的结论是，DHPLC 与 DG-DGGE 类似，可以在一次运行中检测到 l-铁蛋白 5'UTR 序列中的所有突变，并且各种突变发生在 IRE 结构之外。
Hereditary hyperferritinaemia cataract syndrome is an autosomal dominant disorder caused by heterogeneous mutations of the iron regulatory element (IRE) in the ferritin l-chain mRNA. The mutations are rare and fast DNA scanning would facilitate diagnosis. The aim of the study was to compare the analytical performances of two fast DNA scanning techniques: denaturing high-performance liquid chromatography (DHPLC) and double-gradient denaturing gradient gel electrophoresis (DG-DGGE). We analysed the sequence encoding the 5' untranslated flanking region of ferritin l-chain mRNA, which includes an IRE stem loop structure. The two systems unambiguously identified all the 12 accessible mutations in a single run, including the difficult C-G transversions. DHPLC and DG-DGGE identified seven abnormal patterns in DNA samples from 47 subjects with unexplained hyperferritinaemia; all had mutations in the IRE sequence, including two not reported before: C36G and A37G. The scanning of 250 DNA samples from subjects genotyped for HFE led to the identification of four new mutations, all outside the IRE structure: C10T, C16T, C90T and del-T156. We conclude that DHPLC, similar to DG-DGGE, detects all the mutations in the l-ferritin 5'UTR sequence in a single run, and that various mutations occur outside the IRE structure.