Background Cyclophosphamide, an immunosuppressant in patients with lupus nephritis, aims to prevent recurrence and is a steroid-sparing agent. The clinical response (efficacy and toxicity) of lupus nephritis patients receiving cyclophosphamide varied.1 Cyclophosphamide is a pro-drug metabolized by hydroxylation via the CYP2C19 and CYP2B6 enzymes and detoxification via the glutathione-S-transferase (GST) enzyme.2 The most common type of mutation that occurs is single nucleotide polymorphism (SNP). Several SNPs from previous studies associated with cyclophosphamide response, metabolism, and toxicity in patients with lupus nephritis, namely rs3957356 in the GSTA1, rs4244285 in the CYP2C19, rs7254579 and rs4802101 in the CYP2B6.3 Therefore, detecting and screening SNP genotypes in lupus nephritis patients can help analyze cyclophosphamide response variation. This study aims to develop a method for detecting the genotypes of the four target SNPs and several surrounding SNPs using PCR-Sanger sequencing.
Methods The gene polymorphism analysis method was optimized before taking patient samples at the hospital. PCR and Sanger sequencing methods are used for gene polymorphism analysis because they produce chromatograms with target amplicon base lengths and several SNPs that can be analyzed simultaneously.
Results The analysis results of the four target SNPs of the GSTA1, CYP2C19, and CYP2B6 (rs3957356, rs4244285, rs7254579, and rs4802101) showed one synonymous SNP and three SNPs (regulatory and intergenic). We have developed an SNP detection assay using four fragments from the GSTA1, CYP2C19, and CYP2B6 that can detect 15 SNPs simultaneously (figure 1). In addition, this method succeeded in distinguishing wild-type, heterozygous and homozygous genotypes. Furthermore, this method can be used to analyze GSTA1, CYP2C19, and CYP2B6 gene polymorphisms in lupus nephritis patients receiving cyclophosphamide therapy, especially in a population in West Java, Indonesia.
Conclusions PCR-sanger sequencing is a reliable, accurate, and simple method for determining SNP genotypes from blood samples.
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Alamilla Sanchez ME, Alcala-Salgado MA, Alonso-Bello CD, & Fonseca Gonzalez GT. Mechanism of Action and Efficacy of Immunosupressors in Lupus Nephritis. Int. J. Nephrol. Renovasc. Dis 2021;14:441–458.
Helsby, N. A., Yong, M., van Kan, M., de Zoysa, J. R. & Burns, K. E. The importance of both CYP2C19 and CYP2B6 germline variations in cyclophosphamide pharmacokinetics and clinical outcomes. Br. J. Clin. Pharmacol 2019;85:1925–1934.
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