BACKGROUND: Polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are considered the major genetic factors modulating warfarin dose requirement. As a result, a rapid and economic method for genotyping patients for predicting warfarin dose could be useful in clinical applications. METHODS: In this study, we developed and validated a melting temperature (T(m))-shift genotyping assay for detection of VKORC1 C1173T and CYP2C9 A1075C polymorphisms by using SYBR Green-based multiplex allele-specific PCR and consequent melting curve analysis. In this T(m)-shift genotyping assay, a GC-tail was incorporated into one of the two allele-specific primers to increase the melting temperature of PCR product, such that both alleles can be differentially detected simultaneously at their respective melting curve patterns. In total, 100 randomly selected samples of both VKORC1 and CYP2C9 genes from each genotype group determined by melting curve were validated by direct DNA sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. RESULTS: There was 100% concordance between the results of DNA sequencing and T(m)-shift genotyping, and 99% concordance between the results of DHPLC assay and T(m)-shift genotyping. CONCLUSIONS: This allele-specific PCR assay is convenient, inexpensive and will be useful for VKORC1 and CYP2C9 genotyping in a clinic laboratory.
BACKGROUND: Polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are considered the major genetic factors modulating warfarin dose requirement. As a result, a rapid and economic method for genotyping patients for predicting warfarin dose could be useful in clinical applications. METHODS: In this study, we developed and validated a melting temperature (T(m))-shift genotyping assay for detection of VKORC1C1173T and CYP2C9A1075C polymorphisms by using SYBR Green-based multiplex allele-specific PCR and consequent melting curve analysis. In this T(m)-shift genotyping assay, a GC-tail was incorporated into one of the two allele-specific primers to increase the melting temperature of PCR product, such that both alleles can be differentially detected simultaneously at their respective melting curve patterns. In total, 100 randomly selected samples of both VKORC1 and CYP2C9 genes from each genotype group determined by melting curve were validated by direct DNA sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. RESULTS: There was 100% concordance between the results of DNA sequencing and T(m)-shift genotyping, and 99% concordance between the results of DHPLC assay and T(m)-shift genotyping. CONCLUSIONS: This allele-specific PCR assay is convenient, inexpensive and will be useful for VKORC1 and CYP2C9 genotyping in a clinic laboratory.
Authors: Sherif M M Ekladious; Marianne Samir M Issac; Sahar Abd El-Atty Sharaf; Hazem S Abou-Youssef Journal: Mol Diagn Ther Date: 2013-12 Impact factor: 4.074