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)

Abstract

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

Genetic variants in promoter regions associated with type 2 diabetes mellitus: A large‐scale meta‐analysis and subgroup analysis

“…At the same time, studies which used other methods (mainly PCR‐RFLP) than Taqman assays or sequencing assays as genotype detection had significant association between the risk alleles (rs266729 in ADIPOQ and rs2144908 in HNF4A) and T2D. Sequencing has been regarded as “golden standard” in sequence variations determination, while in this study, none of five studies using sequencing assays has significant association between G allele of rs266729 and T2D.…”

Effective long-term solution to therapeutic remission in Inflammatory Bowel Disease: Role of Azathioprine

“…3C allele to be most common in the Asian population[1]. Studies in Nigeria and Libya have shown majority of the African population to have the *3C allele, a finding supported in a study on TPMT activity in African-Americans byHon et al (1999) [8,51,52].…”

“…Thiopurine methyltransferase is encoded by the TPMT gene which is located on chromosome 6 [1]. This gene exhibits an autosomal codominant genetic polymorphism which can lead to an absent or low level of TPMT activity in individuals who are heterozygous or homozygous for this genetic polymorphism [8].…”

“…[61]. None the less, in 2003, the FDA made it compulsory to add that the status of TPMT be determined prior to dosing, to the AZA package insert [1].…”

“…Azathioprine (AZA) is an immunosuppressant drug that was first produced in 1957 and is included in the World Health Organization's (WHO) List of Essential Medicines. AZA is a purine analogue which interrupts the synthesis of purine ribonucleotides guanine and adenine, causing mis-incorporation of bases and preventing deoxyribonucleic acid (DNA) repair mechanisms [1][2][3]. It has a most notable effect on fast dividing cells such as T-lymphocytes; at low doses AZA works as an anti-inflammatory while at high doses it has immunosuppressant and cytotoxic characteristics [4,5].…”