Lab Med Qual Assur.  2024 Mar;46(1):55-59. 10.15263/jlmqa.2024.46.1.55.

CYP3A5 rs776746 Genotyping by SYBR GreenBased Real-Time Multiplex Polymerase Chain Reaction Combined with Melting Curve Analysis Using Tm-Shift Method

• Park G

• KMID: 2554463
• DOI: http://doi.org/10.15263/jlmqa.2024.46.1.55

Abstract

Background
Cytochrome P450 3A subfamily members (CYP3A5) affects drug reactions and susceptibility to diseases. The homozygotic variant rs776746 (CYP3A5 *3/*3; NM_000777.5:c.219-237A>G) genotype is a nonexpressor of CYP3A5 and metabolizes CYP3A5 substrates more slowly than CYP3A5-expressor genotypes (CYP3A5*1/*1 and *1/*3). A rapid and reliable CYP3A5 genotyping is necessary to reduce the risk of adverse drug reactions.
Methods
A total of 101 randomly collected blood samples from hospitalized patients between May 2022 and December 2022 were used to evaluate this rapid CYP3A5 genotyping technique. One allele-nonspecific forward primer and two allele-specific reverse primers (a rs776746 G allele-specific primer with GC-rich sequences and a rs776746 A allele-specific primer without GCrich sequences) were used for CYP3A5 genotyping. We performed Sanger sequencing to confirm the results of real-time multiplex allele-specific polymerase chain reaction (PCR) and melting curve analysis.
Results
The duration of the analysis of the CYP3A5 genotyping, except DNA extraction, was approximately 1.5 hours. The mean melting temperatures were 79.1℃ and 75.1℃, which were characteristic of rs776746 G allelespecific amplicon and rs776746 A allele-specific amplicon, respectively. Frequencies of CYP3A5*1/*1, CYP3A5*1/*3, and CYP3A5*3/*3 were 7 (6.9%), 40 (39.6%), and 54 (53.5%) in 101 samples, respectively. The concordance rate between the real-time PCR combined with melting curve analysis and Sanger sequencing was 100%.
Conclusions
CYP3A5 genotyping by SYBR Green-based real-time multiplex PCR combined with melting curve analysis using three unlabeled primers including the melting temperature-shift primer in a single tube is a rapid and reliable technique for routine laboratory testing.

Keyword

CYP3A5; Genotyping; Real-time polymerase chain reaction