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Non-synonymous single nucleotide alterations found in the CYP2C8 gene result in reduced in vitro paclitaxel
1Project Team for Pharmacogenetics, National Institute of Health Sciences, Tokyo, Japan.
Abstract:
By sequencing genomic DNA from 73 established cell lines derived from Japanese individuals, we detected 9 single nucleotide polymorphisms (SNPs) in the CYP2C8 gene. Of them, 3 exonic SNPs resulted in amino acid alterations (g416a, R139K; a1196g, K399R; c1210g, P404A). The first two alterations were detected concurrently in one cell line and thought to be the same as CYP2C8*3. To examine the effects of these amino acid alterations on CYP2C8 function, wild-type and four types of variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R and P404A) were transfected into Hep G2 cells and their paclitaxel 6a-hydroxylase activities were determined in vitro. Km values were not significantly different from that of the wild-type in any of the variants studied. The variant R139K/K399R showed reduced values for Vmax and clearance (Vmax/Km) similar to those of its single variant, R139K. The variant P404A also showed a significantly lowered clearance due to reduced level of protein expression. These results suggest that not only the double variant (R139K/K399R, CYP2C8*3) but also our novel variant P404A in the CYP2C8 gene are less efficient in paclitaxel metabolism.
Insights
Researchers identified novel genetic variations in the CYP2C8 gene, impacting paclitaxel metabolism. These findings reveal that specific CYP2C8 variants, including a new one, reduce the efficiency of processing this important chemotherapy drug.
Area of Science:
- Pharmacogenomics
- Molecular Biology
- Drug Metabolism
Background:
- The CYP2C8 gene encodes a crucial enzyme involved in drug metabolism.
- Genetic variations (polymorphisms) in CYP2C8 can alter its function and affect drug efficacy and toxicity.
- Understanding these variations is vital for personalized medicine, particularly for drugs like paclitaxel.
Purpose of the Study:
- To identify and characterize single nucleotide polymorphisms (SNPs) in the CYP2C8 gene within a Japanese population.
- To investigate the functional impact of identified CYP2C8 variants on paclitaxel metabolism.
- To evaluate the efficiency of novel and known CYP2C8 variants in metabolizing paclitaxel.
Main Methods:
- Genomic DNA sequencing of 73 Japanese cell lines to detect CYP2C8 SNPs.
- Site-directed mutagenesis to create variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R, P404A).
- Transfection of wild-type and variant CYP2C8 constructs into Hep G2 cells and in vitro determination of paclitaxel 6a-hydroxylase activity.
Main Results:
- Nine SNPs were identified in the CYP2C8 gene, with three resulting in amino acid alterations: R139K, K399R, and P404A.
- The double variant R139K/K399R (CYP2C8*3) exhibited reduced Vmax and clearance, similar to the single R139K variant.
- The novel P404A variant demonstrated significantly lowered clearance due to reduced protein expression levels.
Conclusions:
- The study identified novel functional variants in the CYP2C8 gene, including P404A.
- Both the known CYP2C8*3 variant and the novel P404A variant exhibit reduced efficiency in paclitaxel metabolism.
- These findings highlight the importance of CYP2C8 genetic variations in influencing paclitaxel pharmacokinetics and suggest potential implications for patient response to this chemotherapy agent.