Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC.
Complete scanning of the hereditary hemochromatosis gene (HFE) by use of denaturing HPLC.
复制标题
使用变性 HPLC 对遗传性血色素沉着病基因 (HFE) 进行完整扫描。
作者：
G. Gac;C. Mura;C. Férec
背景
4%~ 35%的遗传性血色素沉着症（HC）先证者为C282 Y或H63 D杂合子或同时缺乏这两种常见的HFE突变，最近已报道了15种新的HFE突变。我们评估了变性HPLC（DHPLC）用于筛查整个HFE编码区，并进一步确定具有不完整HFE基因型的HC先证者是否携带不常见突变。
方法
每个编码外显子的分析条件通过计算机解链曲线预测和实验解链曲线的组合来确定。为了测试扫描完整HFE编码区的准确度并优化DHPLC运行条件，用至少一个突变或一个多态性作为参考来研究每个解链结构域。我们检测了100个携带C282 Y、H63 D或S65 C突变的DNA样本和17个人工创建的阳性对照，这些阳性对照携带14个其他已知HFE突变中的1个或3个选定的多态性。
结果
可以在一个分析温度下对每个编码外显子1、2、4、5和6进行研究。编码外显子3显示更复杂的解链谱，需要两个分析温度。DHPLC检测所有已知的HFE突变以及三个选定的多态性。
结论
DHPLC可用于扫描HC先证者的HFE基因，其中至少一条染色体缺乏指定的突变。
BACKGROUND
Between 4% and 35% of hereditary hemochromatosis (HC) probands are C282Y or H63D heterozygotes or lack both of these two common HFE mutations, and 15 novel HFE mutations have been described recently. We evaluated denaturing HPLC (DHPLC) for screening of the whole HFE coding region and further defined whether HC probands with an incomplete HFE genotype carry uncommon mutations.
METHODS
Analytical conditions for each coding exon were determined by a combination of computer melting profile predictions and experimental melting curves. To test accuracy for scanning the complete HFE coding region and optimize DHPLC running conditions, each melting domain was investigated with at least one mutation or one polymorphism as reference. We tested 100 DNA samples harboring the C282Y, H63D, or S65C mutations and 17 artificially created positive controls that carried either 1 of the 14 other known HFE mutations or 3 selected polymorphisms.
RESULTS
Investigations on each of the coding exons 1, 2, 4, 5, and 6 could be performed at one analysis temperature. Coding exon 3 displayed a more complex melting profile and required two analysis temperatures. DHPLC detected all known HFE mutations as well as the three selected polymorphisms.
CONCLUSIONS
DHPLC can be used to scan the HFE gene in HC probands in whom at least one chromosome lacks an assigned mutation.
影响因子：
14.9
作者：
Liu, WG;Smith, DI;James, CD
通讯作者：
James, CD