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Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside
Jason Saliba1, Ryan Zabriskie, Rajarshi Ghosh
1Departments of aMolecular and Human Genetics bBiochemistry and Molecular Biology cMolecular and Cellular Biology dHuman Genome Sequencing Center, Baylor College of Medicine eDepartment of Pediatrics, Texas Children's Cancer Center, Baylor College of Medicine fDepartment of Statistics, Rice University, Houston, Texas, USA.
Background:
Mutations or alterations in expression of the 5' nucleotidase gene family can lead to altered responses to treatment with nucleoside analogs. While investigating leukemia susceptibility genes, we discovered a very rare p.L254P NT5C1A missense variant in the substrate recognition motif. Given the paucity of cellular drug response data from the NT5C1A germline variation, we characterized p.L254P and eight rare variants of NT5C1A from genomic databases.
Materials And Methods:
Through lentiviral infection, we created HEK293 cell lines that stably overexpress wild-type NT5C1A, p.L254P, or eight NT5C1A variants reported in the National Heart Lung and Blood Institute Exome Variant Server (one truncating and seven missense). IC50 values were determined by cytotoxicity assays after exposure to chemotherapeutic nucleoside analogs (cladribine, gemcitabine, 5-fluorouracil). In addition, we used structure-based homology modeling to generate a three-dimensional model for the C-terminal region of NT5C1A.
Results:
The p.R180X (truncating), p.A214T, and p.L254P missense changes were the only variants that significantly impaired protein function across all nucleotide analogs tested (>5-fold difference vs. wild-type; P<0.05). Several of the remaining variants individually showed differential effects (both more and less resistant) across the analogs tested. The homology model provided a structural framework to understand the impact of NT5C1A mutants on catalysis and drug processing. The model predicted active site residues within NT5C1A motif III and we experimentally confirmed that p.K314 (not p.K320) is required for NT5C1A activity.
Conclusion:
We characterized germline variation and predicted protein structures of NT5C1A. Individual missense changes showed considerable variation in response to the different nucleoside analogs tested, which may impact patients' responses to treatment.
Insights
Rare NT5C1A variants impact drug response. Specific mutations like p.R180X, p.A214T, and p.L254P significantly alter protein function, affecting how cells process nucleoside analogs and potentially influencing patient treatment outcomes.
Area of Science:
- Genetics and Genomics
- Pharmacology
- Biochemistry
Background:
- Alterations in the 5' nucleotidase (NT5C1A) gene family can affect responses to nucleoside analog therapies.
- Rare NT5C1A variants, including the p.L254P missense variant, were identified during leukemia susceptibility gene investigations.
- Limited cellular drug response data exists for NT5C1A germline variations.
Purpose of the Study:
- To characterize the functional impact of the p.L254P NT5C1A variant and eight other rare NT5C1A variants.
- To assess how these NT5C1A variants affect cellular responses to common nucleoside analog chemotherapeutics.
- To generate a structural model of NT5C1A to understand variant effects on protein function.
Main Methods:
- Created HEK293 cell lines overexpressing wild-type NT5C1A, p.L254P, or eight other NT5C1A variants via lentiviral infection.
- Determined IC50 values using cytotoxicity assays after exposing cells to cladribine, gemcitabine, and 5-fluorouracil.
- Employed structure-based homology modeling to create a 3D model of the NT5C1A C-terminal region.
Main Results:
- The p.R180X (truncating), p.A214T, and p.L254P variants significantly impaired NT5C1A protein function across all tested nucleoside analogs (>5-fold difference, P<0.05).
- Other variants exhibited varied effects on drug resistance, with some increasing and others decreasing sensitivity.
- Homology modeling elucidated the impact of NT5C1A mutants on catalysis, identifying p.K314 as crucial for NT5C1A activity.
Conclusions:
- Germline variations in NT5C1A and their predicted protein structures were characterized.
- Individual NT5C1A missense changes demonstrated variable responses to different nucleoside analogs, suggesting potential impacts on patient treatment efficacy.
- The study provides a structural and functional basis for understanding NT5C1A variant effects on drug metabolism.
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