Rapid diagnosis of MEN213 using unlabeled probe melting analysis and the LightCycler 480 instrument
Rapid diagnosis of MEN213 using unlabeled probe melting analysis and the LightCycler 480 instrument
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DOI：
10.2353/jmoldx.2008.070111
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发表时间：
2008-03-01
影响因子：
4.1
通讯作者：
Wittwer, Carl T.
中科院分区：
文献类型：
--
作者：
Margraf, Rebecca L.;Mao, Rong;Wittwer, Carl T.
多发性内分泌瘤2B型（MEN 2B）是一种常染色体显性遗传的癌症综合征。MEN 2B患者发生甲状腺髓样癌的风险很高，建议在6个月大时进行预防性甲状腺切除术。基因检测可以在癌症进展之前识别MEN 2B患者。两个RET原癌基因突变，在外显子15的密码子883（GCT>TTT）和外显子16的密码子918（ATG>ACG），占MEN 2B病例的98%以上。开发了使用未标记的探针和LightCycler 480仪器的测定来对这两种常见的MEN 2B RET突变进行基因分型。使用不对称聚合酶链反应增加ssDNA产物，然后对未标记的探针/ssDNA扩增子双链体进行熔解分析。可用的样品是已知RET基因型的患者DNA或人工模板。密码子883杂合突变的分析表明Δ T-m为5.70 +/- 0.11 ℃，而密码子918杂合突变产生的Δ T-m为-5.72 +/- 0.11 ℃。具有靶向RET突变基因型的样品被准确地检测到，并且使用这些探针很容易与其他五个报道的序列变化区分开。因此，使用未标记探针和LightCycler 480的MEN 2B诊断是一种快速的闭管方法，比测序耗时更少，成本更低。该测定证明了用于鉴定引起MEN 2B的RET突变的100%特异性和灵敏度。
Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant, inherited cancer syndrome. MEN2B patients have a high risk of developing medullary thyroid carcinoma, and prophylactic thyroidectomy is recommended by 6 months of age. Genetic testing can identify MEN2B patients before cancer progression. Two RET proto-oncogene mutations, in exon 15 at codon 883 (GCT>TTT) and in exon 16 at codon 918 (ATG>ACG), account for more than 98% of MEN2B cases. An assay using unlabeled probes and the LightCycler 480 instrument was developed to genotype these two common MEN2B RET mutations. Asymmetric polymerase chain reaction was used to increase ssDNA products followed by melting analysis of the unlabeled probe/ssDNA amplicon duplex. The available samples were either patient DNA of known RET genotype or artificial templates. Analysis of the codon 883 heterozygous mutation demonstrated a Delta T-m of 5.70 +/- 0.11 degrees C, while the codon 918 heterozygous mutation generated a Delta T-m of -5.72 +/- 0.11 degrees C. Samples with the targeted RET mutation genotypes were accurately detected and easily distinguishable from five other reported sequence changes using these probes. Thus, MEN2B diagnosis using unlabeled probes and the LightCycler 480 is a rapid, closed-tube method that is less time consuming and less expensive than sequencing. This assay demonstrates 100% specificity and sensitivity for the identification of RET mutations causative of MEN2B.