Pharmacogenomics and Personalized Medicine (Nov 2022)
Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis
Abstract
Viktorija Igumnova,1,2 Agnija Kivrane,1,2 Anda Viksna,3,4 Inga Norvaisa,4 Renate Ranka1,2 1Molecular Microbiology Group, Latvian Biomedical Research and Study Centre, Riga, Latvia; 2Department of Pharmaceutical Chemistry, Riga Stradins University, Riga, Latvia; 3Department of Infectology, Riga Stradins University, Riga, Latvia; 4Centre of Tuberculosis and Lung Diseases, Riga East University Hospital, Riga, LatviaCorrespondence: Renate Ranka, Molecular Microbiology Group, Latvian Biomedical Research and Study Centre, Ratsupites Street 1, k-1, Riga, Latvia, Tel +371 67808218, Email [email protected]: Pharmacogenetics studies provide clinically relevant information on the identified associations between genetic variants and individual variability in drug response, which, in turn, offers great promise for guiding personalized drug therapy and clinical trial design. However, there is a lack of information concerning the evidence-based clinical annotations of specific CYP2E1 genetic variants.Aim: To design and evaluate the next-generation sequencing-based method for full-length CYP2E1 gene polymorphism analysis.Materials and Methods: Seven gene-specific oligonucleotide primer pairs targeting overlapping CYP2E1 gene fragments spanning all nine gene exons with interleaving introns, untranslated (UTR) and intergenic regions were designed. Human DNA samples (n = 3) were used as a training set to check the primer performance and to optimize the PCR conditions. The effectiveness of the developed target amplification and sequencing protocol was evaluated using the test set comprising human DNA samples (n = 3) obtained from tuberculosis patients. Sequencing data analysis was performed on the Galaxy online-based platform.Results: The sequencing data quality was sufficient for the detection of genetic variants dispersed throughout the CYP2E1 gene with a high degree of confidence in fully covered regions achieving optimal reading depth of the targeted fragment with high base call accuracy.Conclusion: Developed protocol can be applied in subpopulation-level association studies to determine whether single nucleotide variants (SNVs) or variant combinations from multiple regions of the CYP2E1 gene are of clinical significance.Keywords: CYP2E1, cytochrome P450, next-generation sequencing, pharmacogenetics, single nucleotide variants
