Preparing Triple-Compound Heterozygous Control Material for Molecular Diagnostics of <i>TPMT</i> Allelic Variants

Beránek, Martin; Drastíková, M.; Bureš, J.; Palička, V.

doi:10.14712/fb2015061030091

Folia Biologica > Archive > 2015, Vol. 61 > Issue 3 > Preparing Triple-Compound Heterozygous…

Fol. Biol. 2015, 61, 91-96

https://doi.org/10.14712/fb2015061030091

Preparing Triple-Compound Heterozygous Control Material for Molecular Diagnostics of TPMT Allelic Variants

Martin Beránek^1,2, M. Drastíková², J. Bureš³, V. Palička²

¹Faculty of Pharmacy in Hradec Králové, Charles University in Prague and University Hospital Hradec Králové, Czech Republic
²Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine in Hradec Králové, Charles University in Prague and University Hospital Hradec Králové, Czech Republic
³2nd Department of Internal Medicine, Faculty of Medicine in Hradec Králové, Charles University in Prague and University Hospital Hradec Králové, Czech Republic

Received March 2015
Accepted April 2015

The aim of the study is to present a novel approach for preparing triple-compound heterozygous reference material (TCH-RM) for thiopurine S-methyltransferase (TPMT) genotyping by using the gene synthesis technology. The polynucleotide chain we prepared consisted of three wild-type and three mutant segments corresponding to the TPMT 238G>C, 460G>A, and 719A>G polymorphic sites. TCH-RM characteristics were assessed via four methods: reverse hybridization, real-time PCR with hydrolysis probes, real-time PCR followed by subsequent melting temperature analysis, and DNA sequencing. Consequently, we investigated the TPMT genotype of 371 patients suffering from autoimmune diseases requiring immunosuppressive therapy with thiopurine drugs, mostly inflammatory bowel disease. All methods confirmed the triple heterozygous character and commutability of TCH-RM. In evaluating its stability we obtained very comparable data before and after six months of storage at -80 °C. The determined genotypes were as follows: 352 wild-type subjects (94.8 %), 17 TPMT*3A heterozygotes (460G>A and 719A>G, 4.6 %), one patient heterozygous for the TPMT*2 allele (238G>C, 0.3 %), and one TPMT*3C heterozygote (719A>G, 0.3 %). The frequencies of TPMT*1, *3A, *3C, and *2 in the patients were 97.5 %, 2.3 %, 0.1 %, and 0.1 %, respectively. Assembling segments of synthetic DNA into long polynucleotide chains is a universal way of obtaining compound heterozygous material for performing any simultaneous analysis of polymorphic sites in the human genome. The batches are manufactured with a perfect concentration match of wildtype and mutant fragments, and can be made in large quantities for most diagnostic techniques.