Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene
Validation of dye-binding/high-resolution thermal denaturation for the identification of mutations in the SLC22A5 gene
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DOI：
10.1002/humu.20137
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发表时间：
2005-01-01
期刊：
影响因子：
3.9
通讯作者：
Longo, N
中科院分区：
文献类型：
--
作者：
Dobrowolski, SE;McKinney, JT;Longo, N
原发性肉毒碱缺乏症是由于肉毒碱转运缺陷引起的脂肪酸氧化的常染色体隐性遗传疾病。这种疾病是由SLC 22 A5基因编码的OCTN 2肉毒碱转运蛋白突变引起的。在这里，我们验证染料结合/高分辨率热变性作为筛选程序，以确定该基因中的新突变。该程序基于在毛细管中用dsDNA结合染料LCGreen I通过PCR扩增DNA。然后在同一毛细管中通过高分辨率热变性分析PCR反应。对具有异常解链谱的样品进行测序。这项技术正确地确定了所有已知的患者谁是复合杂合子的不同突变的肉毒碱转运蛋白基因和约30%的纯合子患者。其余70%的纯合子患者通过第二次扩增鉴定，其中患者的DNA与正常对照的DNA混合。这个筛选系统正确地确定了8个新的突变和异常等位基因在6个新的家庭与原发性肉毒碱缺乏症。通过在中国仓鼠卵巢（CHO）细胞中的表达证实了所鉴定的错义突变（c.3G > T/p.M1I、c.695C>T/p.T232M和c.1403 C>G/p.T468R）的致病作用。这些结果扩大了原发性肉毒碱缺乏症的突变谱，并表明染料结合/高分辨率热变性作为一种理想的系统来筛选没有流行的分子改变的疾病中的突变。(C)2005 Wiley-Liss，Inc.
Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation resulting from defective carnitine transport. This disease is caused by mutations in the OCTN2 carnitine transporter encoded by the SLC22A5 gene. Here we validate dye-binding/high-resolution thermal denaturation as a screening procedure to identify novel mutations in this gene. This procedure is based on the amplification of DNA by PCR in capillaries with the dsDNA binding dye LCGreen I. The PCR reaction is then analyzed in the same capillary by high,resolution thermal denaturation. Samples with abnormal melting profiles are sequenced. This technique correctly identified all known patients who were compound heterozygotes for different mutations in the carnitine transporter gene and about 30% of homozygous patients. The remaining 70% of homozygous patients were identified by a second amplification, in which the patient's DNA was mixed with the DNA of a normal control. This screening system correctly identified eight novel mutations and both abnormal alleles in six new families with primary carnitine deficiency. The causative role of the missense mutations identified (c.3G > T/p.M1I, c.695C>T/p.T232M, and c.1403 C>G/p.T468R) was confirmed by expression in Chinese hamster ovary (CHO) cells. These results expand the mutational spectrum in primary carnitine deficiency and indicate dye-binding/high-resolution thermal denaturation as an ideal system to screen for mutations in diseases with no prevalent molecular alteration. (C) 2005 Wiley-Liss, Inc.