Background Currently, pharmacogenetic information is accumulating rapidly and is beginning to show consistent reproducible results for an increasing number of genetic markers for drug response. An increasing number of medical centres have acquired clinical genotyping facilities.
Purpose Among the first medical centres to implement pharmacogenetics there are many highly specialised care centres with complex patient populations. These patients may present some unexpected challenges as is exemplified by the following case description.
Materials and methods Patients undergoing a kidney transplantation in the Leiden University Medical Centre are pre-emptively genotyped for the CYP3A5*3(rs776746) and CYP3A5*6 (rs10264272) polymorphisms. A 4-ml blood sample of the patient n this report was sent to the pharmacogenetics laboratory of the hospital pharmacy. To minimise the risk of potential errors, clinical genotyping is performed in duplicate by two independent techniques; a commercially available pre-designed TaqMan assay (Life-Technologies, Nieuwerkerk a/d Ijssel, The Netherlands) and an pyrosequencing method developed and validated in-house. Genotype results obtained with the two techniques should agree 100% before results are authorised by the laboratory.
Results A 20-year-old female was admitted for a living related kidney transplant. Adequate tacrolimus exposure early after transplantation is essential to prevent acute rejection of the transplant. Tacrolimus is metabolised into active and inactive metabolites by CYP3A4 and CYP3A5. Patients carrying at least one copy of the CYP3A5*1 allele have been shown to require a significantly increased tacrolimus dose to attain therapeutic blood concentrations.
For this particular patient genotyping results from the two techniques were not in 100% concordance. One technique identified the patient as CYP3A5 *1/*3, the other CYP3A5 *3/*3. A second blood sample was genotyped but again showed conflicting results. Results also conflicted with results obtained with plasmid controls containing the SNPs of interest.
The attending nephrologist was consulted to discuss the results. It emerged that the patient had a history of allogeneic stem cell transplantation (allo-SCT), resulting in mixed haematopoietic chimerism (28% autologous, 72% donor).
We were interested in interrogating the patient’s germline DNA. After obtaining consent from the patient and the stem cell donor, saliva samples from both subjects were collected and genotyped for both CYP3A5 polymorphisms. The donor was autocalled CYP3A5*3/*3 and the patient CYP3A5*1/*3. Based on the genotyping results, the patient’s genotype was finally reported as CYP3A5*1/*3. This genotype is in line with the relatively low trough level (5.5 µg/L) and area-under-the-concentration-over-time-curve of 110 µg*hours/L achieved with a dose of 8 mg twice a day of tacrolimus.
Conclusions This case description demonstrates the challenging aspects of pharmacogenetic testing in an allo-SCT recipient and illustrates the importance of proper quality control mechanisms when performing pharmacogenetic testing. Furthermore, it is essential to consider the source of the DNA used to determine the genotype, especially in a population that includes patients receiving allo-SCT.
No conflict of interest.
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