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Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups

¹ Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Tuebingen, Germany; ² University of Ghana Medical School, Accra, Ghana; ³ Department of Pharmacology and Pharmacogenomics Research Center, Inje University College of Medicine, Busan Paik Hospital, Busan, Korea; ⁴ Department of Clinical Pharmacology, University Hospital Tuebingen, Tuebingen, Germany.

^aAddress correspondence to this author at: Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstrasse 112, D-70376 Stuttgart, Germany. Fax +49 711 85 92 95; e-mail matthias.schwab{at}ikp-stuttgart.de.

Background: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted.

Methods: We used MALDI-TOF mass spectrometry (MS) based on Sequenom iPLEX® technology to develop novel multiplex assays for comprehensive testing of TPMT. Two assays, a 15-plex and a 7-plex assay, consisting of multiplex PCR, shrimp alkaline phosphatase treatment, primer extension, and MALDI-TOF MS analysis, allow detection of all currently known functionally relevant 24 TPMT alleles (TPMT*2 to *18, *20 to *23). Previously identified variant DNA samples and newly constructed synthetic templates were used as quality controls.

Results: Assay evaluation performed on a panel of 586 genomic DNA samples previously genotyped by other methods (denaturing HPLC, sequencing) resulted in 100% agreement. Analyses of the distribution of TPMT alleles in 116 samples from a Ghanaian population revealed a TPMT*8 allele frequency of 3.4%. In a Korean population of 118 unrelated individuals, we found a TPMT*6 allele frequency of 1.3%.

Conclusions: The newly developed multiplex MALDI-TOF MS assay allows efficient genotyping for all currently known functional TPMT variants. To achieve the most accurate prediction of TPMT phenotype, molecular diagnosis of TPMT should include all these variants.

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