ORIGINAL ARTICLESPhenotyping–genotyping of alternatively spliced genes in one step: study of CYP3A5*3 polymorphismBusi, Florent; Cresteil, ThierryAuthor Information ICSN – CNRS UPR2301, Gif-sur-Yvette, France Correspondence and requests for reprints to Florent Busi, ICSN – CNRS UPR2301, Avenue de la terrasse, 91198 Gif-sur-Yvette, France Tel/fax: +33 1698 23643; e-mail: [email protected] Received 24 January 2005 Accepted 8 March 2005 Pharmacogenetics and Genomics: June 2005 - Volume 15 - Issue 6 - p 433-439 Buy Abstract Alternative splicing is required to increase the mRNA diversity of many genes, but can also be responsible for the abnormal expression of genes. For example, the CYP3A5*3 defective allele is caused by a single nucleotide polymorphism in intron 3. This mutation activates a cryptic acceptor splice site, which leads to the insertion of an intronic sequence containing premature termination codons in the mature mRNA, and hence the very low CYP3A5 protein expression in 75% of the Caucasian population. In the present study, we propose a novel strategy based on the quantitative real-time polymerase chain reaction with SYBR Green I chemistry, followed by melting curve analysis, to demonstrate and quantify the amount of splice variant mRNA. Using oligonucleotides flanking the insertion site, two products with different size can be obtained, which can be resolved by melting curve analysis. The relative ratio of differently spliced RNA can be estimated at the plateau phase by using the peak height ratio. For the CYP3A5 gene, the genotype, the level of expression and the proportion of alternatively spliced products were determined in a single reaction without DNA sequencing. © 2005 Lippincott Williams & Wilkins, Inc.