Characterization and Development of Genomic DNA Quality Controls for Thrombophilia and Warfarin Sensitivity Testing

Background: Genetic tests for diagnosis of clotting disorders and monitoring their treatment are based on identification of single nucleotide polymorphisms (SNPs) that are related respectively to clotting and to drug metabolism crucial to the effectiveness of therapy. Thrombophilia, an inherited condition which predisposes to thromboembolism, is due in part to genetic factors, such as the presence of SNPs in the genes encoding clotting factors. Hyperhomocysteinemia, which poses an extremely elevated risk of thromboembolism to the patient, can be caused by certain mutations to the MTHFR (677CT, 1298AC) gene; genotyping all genes involved in thrombophilia concurrently can offer a more complete picture of the genetic component of thrombophilic risk. Warfarin is the most frequently used oral anticoagulant for treatment and prevention of thromboembolism and requires careful adjustment of therapeutic dose and close monitoring because of its narrow therapeutic index and wide individual variability in response and therapeutic dose. Genetic variations in two genes, CYP2C9 and vitamin K epoxide reductase (VKORC1), have been shown to have a profound effect on warfarin sensitivity. Using genetic tests for identification of these variants clinicians to better estimate the appropriate starting dose for therapy, which may improve drug safety. Currently a number of test systems detect these SNPs. Use of a well-characterized independent quality control sample is important to monitor the accuracy and reproducibility of these tests. We have developed multi-analyte quality controls that contain major SNP variants for both thrombophilia MTHFR and warfarin sensitivity.

Methods: PBMCs from multiple donors were screened for mutations for MTHFR for Thrombophilia and for CYP2C9 and VKORC1 for warfarin sensitivity. Mutations were identified using the Third Wave Technologies INVADER® and Autogenomics INFINITI^™ platforms; and ABI TaqMan® SNP assay. All mutations were confirmed by sequencing on an ABI 3130 instrument.

Results: PBMC samples were screened for CYP2C9, VKORC1, and MTHFR mutations. The mutation frequency for CYP2C9*2, CYP2C9*3 and VKORC1 1639GA variants was 28%, 7% and 63% respectively. 100% genotyping concordance was observed in all testing. In addition, heterozygotes for additional VKORC1 SNPs 5808TG, 6484CT, 6853GC, 7566CT, and 9041GA were present in one sample bearing VKORC -1639GA. The mutation frequency for MTHFR 677CT and 1298AC was 17% wt and 29% het for both SNPs. The identification of heterozygotes in several SNPs allowed development of multi-analyte controls for both genes. Data showing reproducibility and accuracy in SNP call rate on various test platforms will be presented.

Conclusion: We have developed multi-analyte controls for thrombophilia and warfarin sensitivity that can monitor extraction, amplification and detection, and evaluated their performance with several test methods.