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. 2022 Nov 8:15:959-965.
doi: 10.2147/PGPM.S371709. eCollection 2022.

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis

Viktorija Igumnova  1   2 , Agnija Kivrane  1   2 , Anda Viksna  3   4 , Inga Norvaisa  4 , Renate Ranka  1   2
Affiliations

Next-Generation Sequencing and Bioinformatics-Based Protocol for the Full-Length CYP2E1 Gene Polymorphism Analysis

Viktorija Igumnova et al. Pharmgenomics Pers Med. .

Abstract

Introduction: 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.

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Conflict of interest statement

The authors declare that they have no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Graphical representation of the PCR fragment position in the reference CYP2E1 gene. The exons are indicated by green boxes. Names of the seven primer pairs used to generate the PCR fragments are indicated.
Figure 2
Figure 2
Visualization of PCR products of the seven primer pairs amplifying full-length CYP2E1 gene for the test samples (n=3) by agarose gel electrophoresis. (A) PCR products of the primer pairs Nr. 1, 2 and 3. (B) PCR products of the primer pair Nr. 4. (C) PCR products of the primer pairs Nr. 5, 6 and 7. Names of the primer pairs used to generate the PCR fragments are indicated. M – DNA molecular weight marker GeneRuler 1 kb DNA ladder (Thermo Fisher Scientific Baltics, UAB, Lithuania); size of the three reference bands (6000, 3000 and 1000 bp) is indicated; 30, 32 and 33 – test DNA samples.
See this image and copyright information in PMC

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