A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time...

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.