Yu Shihui, Kielt Matthew, Stegner Andrew L, Kibiryeva Nataliya, Bittel Douglas C, Cooley Linda D
Department of Pathology, Children's Mercy Hospitals and Clinics, and University of Missouri-Kansas City School of Medicine, Kansas City, Missouri, USA.
Genet Test Mol Biomarkers. 2009 Dec;13(6):751-60. doi: 10.1089/gtmb.2009.0056.
The American College of Medical Genetics guidelines for microarray analysis for constitutional cytogenetic abnormalities require abnormal or ambiguous results from microarray-based comparative genomic hybridization (aCGH) analysis be confirmed by an alternative method. We employed quantitative real-time polymerase chain reaction (qPCR) technology using SYBR Green I reagents for confirmation of 93 abnormal aCGH results (50 deletions and 43 duplications) and 54 parental samples. A novel qPCR protocol using DNA sequences coding for X-linked lethal diseases in males for designing reference primers was established. Of the 81 sets of test primers used for confirmation of 93 abnormal copy number variants (CNVs) in 80 patients, 71 sets worked after the initial primer design (88%), 9 sets were redesigned once, and 1 set twice because of poor amplification. Fifty-four parental samples were tested using 33 sets of test primers to follow up 34 CNVs in 30 patients. Nineteen CNVs were confirmed as inherited, 13 were negative in both parents, and 2 were inconclusive due to a negative result in a single parent. The qPCR assessment clarified aCGH results in two cases and corrected a fluorescence in situ hybridization result in one case. Our data illustrate that qPCR methodology using SYBR Green I reagents is accurate, highly sensitive, specific, rapid, and cost-effective for verification of chromosomal imbalances detected by aCGH in the clinical setting.
美国医学遗传学学会关于先天性细胞遗传学异常的微阵列分析指南要求，基于微阵列的比较基因组杂交（aCGH）分析得出的异常或模糊结果需通过另一种方法进行确认。我们采用使用SYBR Green I试剂的定量实时聚合酶链反应（qPCR）技术，对93个aCGH异常结果（50个缺失和43个重复）以及54个亲代样本进行确认。建立了一种新的qPCR方案，该方案利用编码男性X连锁致死性疾病的DNA序列设计参考引物。在用于确认80例患者中93个异常拷贝数变异（CNV）的81组检测引物中，初始引物设计后71组有效（88%），9组重新设计了一次，1组因扩增效果差重新设计了两次。使用33组检测引物对54个亲代样本进行检测，以追踪30例患者中的34个CNV。19个CNV被确认为遗传性的，13个在双亲中均为阴性，2个因单亲结果为阴性而无法确定。qPCR评估在两例中明确了aCGH结果，在一例中纠正了荧光原位杂交结果。我们的数据表明，在临床环境中，使用SYBR Green I试剂的qPCR方法对于验证aCGH检测到的染色体失衡准确、高度灵敏、特异、快速且具有成本效益。
