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LP-110 Development of GSTA1, CYP2C19, and CYP2B6 gene polymorphism detection methods on the response of cyclophosphamide therapy for lupus nephritis patients
  1. Yen Yen Ari Indrawijaya1,2,
  2. Maria Immaculata Iwo3,
  3. Aluicia Anita Artarini4 and
  4. Laniyati Hamijoyo5,6
  1. 1School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesha, 10 Bandung, 40132, West Java, Indonesia
  2. 2Department of Pharmacy, Faculty of Medicine and Health Sciences, Maulana Malik Ibrahim Malang State Islamic University, Indonesia
  3. 3Department of Pharmacology-Clinical Pharmacy, School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesha, 10 Bandung, 40132, West Java, Indonesia
  4. 4Laboratory of Pharmaceutical Biotechnology, School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesha, 10 Bandung, 40132, West Java, Indonesia
  5. 5Department of Internal Medicine, University of Padjajaran, Hasan Sadikin Hospital Bandung, Indonesia
  6. 6Immunology Study Center, Faculty of Medicine, University of Padjajaran Bandung, Indonesia


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.

Abstract LP-110 Figure 1

Chromatogram depicting four genotypes SNP target and adjacent SNP simultaneously


  1. Mok CC, et al. The Asia-Pacific League of Associations for Rheumatology consensus statements on the management of systemic lupus erythematosus. Lancet Rheumatol 2021;3:e517–e531.

  2. 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.

  3. 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.

  • SNP detection
  • Cyclophosphamide
  • Lupus Nephritis

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