Accuracy of genotyping using the TaqMan PCR assay for single nucleotide polymorphisms responsible for thiopurine sensitivity in Japanese patients with inflammatory bowel disease

Abstract

Abstract. Thiopurine drugs are the most common drugs used to maintain clinical remission in inflammatory bowel disease (IBD). Three single-nucleotide polymorphisms (SNPs), TPMT A719G (rs1142345), inosine triphosphate pyrophosphatase (ITPase) C94A (rs1127354) and multidrug resistance protein 4 MRP4 G2269A (rs3765534), have been reported to account for heightened sensitivity to thiopurine drugs in the Japanese population. We investigated the usefulness of the TaqMan ® PCR assay (Applied Biosystems) for the rapid detection of these SNPs to improve the safety of thiopurine therapy. We enrolled 44 healthy volunteers and 235 IBD patients. Genotyping of the SNPs was performed using Custom TaqMan SNP genotyping assays, direct sequencing and PCR-RFLP. Genotyping for MRP4 G2269A by the TaqMan PCR assay was successfully achieved in all samples. Comparison with our previous data using direct sequencing indicated one discordant result, and re-sequencing showed that the TaqMan PCR assay was correct. The overall accuracy of the TaqMan assay for MRP4 G2269A was 100%. The TaqMan PCR genotyping for TPMT A719G and ITPase C94A was successfully performed in all samples. The results of TPMT A719G by the TaqMan assay were identical with those of PCR-RFLP. In ITPase C94A, a comparison of the TaqMan assay and PCR-RFLP yielded 12 discordant results, and direct sequencing showed that the TaqMan PCR assay was correct. The allelic frequency determined by the TaqMan assay was 0.145 for MRP4 G2269A, 0.009 for TPMT A719G and 0.121 for ITPase C94A, respectively. In conclusion, the TaqMan ® PCR assay is useful for genotyping of SNPs responsible for thiopurine sensitivity in Japanese IBD patients.

Xenobiotic Sensor- and Metabolism-Related Gene Variants in Environmental Sensitivity-Related Illnesses: A Survey on the Italian Population

“…In this regard, most of the published genetic investigations on MCS patient, including our previous report on 110 Italian subjects [25], employed the PCR-RFLP technique which, lacking of the required sensitivity, often provides a misrepresentation of genotype distribution in the population, specifically with regard to the frequency of heterozygotes [46]. …”

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C)

“…Arguably, an assay with rapid turnaround time is advantageous since it will not delay the sometimes life-saving treatment [ 19 ]. On the other hand, to promote adoption of the test, the assay should be simple, objectively interpreted and have low consumable costs compared to traditional sequencing [ 4 ], with differentiation based on relative amplification efficiency [ 20 ] or by probe-based tests [ 21 , 22 , 23 ]. Additionally, the assay should be robust to variations caused by pre-analytical variables [ 24 ].…”

Influence of SLCO1B1 polymorphism on maintenance therapy for childhood leukemia

“…Since the variant allele frequency of TMPT *3C is very low (0.009), as previously reported, this polymorphism may be less valuable for Japanese patients.…”

“…DNA was extracted using Genomic DNA Isolation Kit (QIAamp DNA Blood Mini Kit, QIAamp DNA Blood Midi Kit: QIAGEN, Vealo, Netherlands) from 0.5–2 mL peripheral blood collected from patients in complete remission following the manufacturer's instructions. Polymorphisms of TPMT * 3C (c.719A > G, rs1142345), ITPA c.94C > A (rs1127354), MRP4 c.2269G > A (rs3765534), MTHFR c.677C > T (rs1801133), MTHFR c.1298 A > C (rs1801131), SLCO1B1 c.1865+4846 T > C (rs11045879), and SLCO1B1 c.521 T > C (rs4149056) were genotyped with the TaqMan PCR Genotyping Assays (ID: C_19567_20, C_27465000_10, C_27478235_20, C_1202883_20, C_850486_20, C_31106904_10, and C_30633906_10; Life Technologies, Foster City, CA, USA) following the manufacturer's instructions. The polymorphism of RFC1 c.80G > A (rs1051266) was determined using the TaqMan PCR method as previously described .…”