Allelic dropout in long QT syndrome genetic testing: A possible mechanism underlying false-negative results
Allelic dropout in long QT syndrome genetic testing: A possible mechanism underlying false-negative results
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DOI：
10.1016/j.hrthm.2006.03.016
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发表时间：
2006-07-01
期刊：
影响因子：
5.5
通讯作者：
Ackerman, Michael J.
中科院分区：
文献类型：
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作者：
Tester, David J.;Cronk, Lisa B.;Ackerman, Michael J.
背景在过去的十年里，先天性长QT综合征（LQTS）的基因检测一直在研究实验室进行。大约75%的LQTS临床概率高的患者在五个LQTS引起的心脏通道基因中有一个突变。其余基因分型阴性病例的可能解释包括LQTS模拟物、新的LQTS致病基因、已知基因的未探索区域，目的本研究的目的是探讨等位基因缺失作为一种潜在的假阳性的可能机制的可能性。方法对目前许多研究实验室使用的已发表的引物进行综合分析，对60个已翻译的分析KCNQ 1（LQT 1）、KCNH 2（LQT 2）、SCN 5A（LQT 3）、KCNE 1（LQT 5）和KCNE 2（LQT 6）基因外显子中常见内含子单核苷酸多态性（SNP）的存在。重复突变分析，引物/扩增子重新设计后，使用聚合酶链反应，变性高效液相色谱，DNA测序，进行了一个队列的541个连续的，无关的患者转介LQTS genetictesting.Results常见（>1%的次要等位基因频率）内含子SNPs被发现内的引物序列的60个翻译外显子。在重新设计引物以消除等位基因缺失的可能性后，发现4个先前基因型阴性的索引病例具有引起LQTS的突变：外显子15的R591 H-KCNQ 1和R594 Q-KCNQ 1，以及2个不相关病例的E229 X-KCNH 2。重复检查这两个扩增子在400个参考等位基因没有确定这些或任何额外的氨基酸variants.Conclusion等位基因脱落继发于内含子SNP引物错配防止发现LQTS引起的突变在4例。考虑到许多LQTS基因检测研究实验室已经使用了这些引物，报告基因型阴性的患者可能会受益于重新检查那些容易因引物破坏SNP而导致等位基因缺失的区域，特别是KCNQ 1中的外显子15和KCNH 2中的外显子4。
Background Genetic testing for congenital Long QT syndrome (LQTS) has been performed in research Laboratories for the past decade. Approximately 75% of patients with high clinical probability for LQTS have a mutation in one of five LQTS-causing cardiac channel genes. Possible explanations for the remaining genotypenegative cases include LQTS mimickers, novel LQTS-causing genes, unexplored regions of the known genes, and genetic testing detection failures.Objectives The purpose of this study was to explore the possibility of allelic dropout as a possible mechanism underlying false-negative test results.Methods The published primers currently used by many research laboratories to conduct a comprehensive analysis of the 60 translated exons in the KCNQ1 (LQT1), KCNH2 (LQT2), SCN5A (LQT3), KCNE1 (LQT5), and KCNE2 (LQT6) genes were analyzed for the presence of common intronic single nucleotide polymorphisms (SNPs). Repeat mutational analysis, following primer/amplicon redesign using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, was performed on a cohort of 541 consecutive, unrelated patients referred for LQTS genetic testing.Results Common (>1% minor allele frequency) intronic SNPs were discovered within the primer sequences of five of 60 translated exons. Following primer redesign to eliminate the possibility of allelic dropout, four previously genotype-negative index cases were found to possess LQTS-causing mutations: R591H-KCNQ1 and R594Q-KCNQ1 for exon 15 and E229X-KCNH2 in two unrelated cases. Repeat examination of these two amplicons in 400 reference alleles did not identify these or any additional amino acid variants.Conclusion Allelic dropout secondary to intronic SNP-primer mismatch prevented the discovery of LQTS-causing mutations in four cases. Considering that many LQTS genetic testing research laboratories have used these primers, patients who reportedly are genotype negative may benefit from re-examination of those regions susceptible to allelic dropout due to primer-disrupting SNPs, particularly exon 15 in KCNQ1 and exon 4 in KCNH2.