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

Area of Science:

• Molecular Genetics
• Pharmacogenomics
• Biotechnology

Background:

• Thiopurine S-methyltransferase (TPMT) is crucial for metabolizing thiopurine drugs.
• Genetic variations in TPMT affect drug efficacy and toxicity, necessitating accurate genotyping.
• Existing reference materials may not adequately cover multiple TPMT polymorphisms simultaneously.

Purpose of the Study:

• To develop and validate a novel triple-compound heterozygous reference material (TCH-RM) for TPMT genotyping.
• To utilize gene synthesis technology for creating TCH-RM with specific wild-type and mutant TPMT alleles.
• To assess the stability and commutability of the synthesized TCH-RM.

Main Methods:

• Gene synthesis was employed to construct a polynucleotide chain with three wild-type and three mutant TPMT segments (238G>C, 460G>A, 719A>G).
• TCH-RM characteristics were validated using reverse hybridization, real-time PCR with hydrolysis probes, real-time PCR with melting temperature analysis, and DNA sequencing.
• TPMT genotyping was performed on 371 patients with autoimmune diseases treated with thiopurine drugs.

Main Results:

• The synthesized TCH-RM demonstrated triple heterozygous character and commutability across all tested methods.
• The TCH-RM remained stable after six months of storage at -80 °C.
• TPMT genotyping in patients revealed frequencies: TPMT*1 (97.5%), *3A (2.3%), *3C (0.1%), and *2 (0.1%).

Conclusions:

• Gene synthesis provides a universal platform for creating compound heterozygous reference materials for simultaneous analysis of multiple genetic polymorphisms.
• The developed TCH-RM is a stable, reliable tool for accurate TPMT genotyping in clinical diagnostics.
• This approach facilitates large-scale production of reference materials for various diagnostic techniques.