Copy number variations of cytochrome P450 genes in Kinh Vietnamese

A total of 154 unrelated healthy Kinh volunteers (93 females and 61 males) from Hanoi Medical University of Vietnam were recruited. All subjects were regarded as healthy according to their medical history and physical examination. The ethnic identity of volunteers was identified based on their personal documents with at least 3 generation of corresponding parental ancestry. The study purpose was explained to all individuals and written informed consent was obtained from each subject before sample collection. This project was approved by the Institutional Review Board (IRB) of the Institute of Genome Research, Vietnam Academy of Science and Technology. The study is in accordance with the STROBE statement [6]. For all subjects, 2 mL of peripheral blood was collected and preserved in EDTA containing tubes. Genomic DNA was subsequently extracted from the blood samples using E.Z.N.A Blood DNA Mini Kit (USA) according to manufacturer's instructions.

To identify deletions/duplications on CYP450 gene, MPLA was performed using the commercial SALSA MLPA P128-C1 Cytochrome P450 Probemix kit (MRC-Holland, Amsterdam, Netherlands) following the manufacturer's instructions. The CNVs data of CYP2D6 detected in these 154 subjects were separately reported [7]. In addition to CYP2D6, MLPA probemixs were specifically designed for 2–6 exons of other CYP450 genes, including CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, CYP3A4, and CYP3A5. Fifty nanograms of genomic DNA was denatured at 98°C for 5 min and cooled at 25°C. Denatured DNA were later hybridized with SALSA probemix at 60°C for 16–20 h. Subsequently, the annealed probes were ligated at 54°C for 15 min followed by heating at 98°C in 5 min. In the next step, complete ligation reactions were used for the PCR with thermocycle consisting of 35 cycles (95°C for 30 s, 60°C for 30 s and 72°C for 60 s), followed by incubation at 72°C for 20 min. The amplicons were separated by capillary gel electrophoresis and the collected data were analyzed using the Coffalyzer.net software. The copy number of subjects was determined by the final probe ratio distribution: 0 copy (0), 1 copy (0.4–0.65), 2 copies (0.8–1.2), 3 copies (1.3–1.65), and 4 copies (1.75–2.15).

Gene dosage of different samples was performed with relative quantification real-time PCR method. Real-time PCR reaction was performed using Luna Universal qPCR Master Mix (NEB) and RPPH1 (a reference gene with a single copy) primers used for quantitative analysis were referred to Ahani's study [8]. Additionally, primers were designed for specific detection of CYP1B1 (exons 1 and 2), CYP2E1 (exons 8 and 9) and CYP2C9 (exons 4 and 7) (Table 1). The reaction was performed in 96 wells plate with 10 μL volume in total, which included 10 ng genomic DNA, 1X Luna Universal qPCR Mastermix (NEB), 0.25 μL for each primer (10 pmole/μL) and Ultrapure Distilled Water (ThermoFisher Scientific). Subsequently, the covered plates were run on LightCycler 96 Instrument (Roche) with the thermocycle with denaturation at 95°C for 10 min, following by 45 cycles (95°C for 15 s and 60°C for 60 s). The copy number of targeted exon in comparison to reference gene was determined according to the following equation: ΔΔCt = [CtRPPH1(Reference sample) − Ct targeted exon (reference sample)] − [CtRPPH1(Unknown sample) − Ct targeted exon (Unknown sample)]. The relative copy number of the genes was later calculated following the ratio equation (2^−ΔΔCt).

Five primers were used in 2-step PCR method in order to detect CYP2A6 deletion allele as previously described by Oscarson et al. [9] (Table 2). Eight microliters of PCR II were aliquoted and subsequently analyzed on 1% agarose gel staining with ethidium bromide.

Inter-ethnic comparison of genes copy number frequency between Kinh Vietnamese and global populations were assessed by Chi square (c²) and Fisher exact test. All statistical analyses were 2-sided and P < 0.05 was considered as statistically significant.

CNVs of CYP450 genes were analyzed using MLPA with probes designed for 10 following genes except CYP2D6: CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5. Among 154 unrelated healthy individuals, CNVs were detected in only 4 genes including CYP1B1, CYP2A6, CYP2C9 and CYP2E1. Of these genes, deletions were identified in CYP2A6 and CYP2C9, duplications were found in CYP1B1, and only CYP2E1 with both duplications and deletions was identified. The representative MLPA data from samples harboring variable copy numbers are shown in Table 3. Additionally, the detail of CYP450 CNVs observed in studied subjects are shown in Figure 1.

Figure 1.

For CYP2A6, both homozygous and heterozygous deletions were detected in the studied subjects by MLPA with copy number probes designed for exons 1, 2, 3, and 5. Among 154 subjects, the most frequent genotype was Wt/Wt having 2 copies (80.52%), following by Wt/Del genotype having 1 copy (16,88%). The homozygous deletion of CYP2A6 with Del/Del genotype having 0 copy made up only 2.6% (Table 4). The frequency of CYP2A6 alleles ranged from 11% (CYP2A-6Del-0 copy) to 89% (CYP2A6Wt-1 copy) (Table 5). CYP2A6 duplication alleles (2 copies) were not identified in this study. Samples showing deletion alleles of CYP2A6 were randomly chosen for re-genotyping in order to confirm the MLPA results. The genotyping results were consistent with MLPA and indicated that deletions found in the studied subjects encompassed the entire CYP2A6 gene (Figure 2).

Figure 2.

For CYP1B1, MLPA data resulted in duplications in studied subjects with copy number probes specific for exons 1 and 3. Of 154 subjects, the homozygous wildtype was the most common genotype (98.05%). Meanwhile, heterozygous duplication of CYP1B1 (Wt/Dup) made up only 1.95% (Table 4). There were 2 CYP1B1 alleles identified with frequencies ranging from 1% (Dup-2 copies) to 99% (Wt-1 copy) (Table 5). Two real-time PCR assays were subsequently used in order to confirm the MLPA results with primers located in exon 1 and exon 2 of CYP1B1. The results of qPCR were 100% in agreement with those of MLPA data obtained (Figure 3A). Furthermore, no CYP1B1 deletion alleles were detected among the studied subjects.

Figure 3.

For CYP2C9, heterozygous deletion was detected in studied subjects by MLPA with copy number probes designed for exons 1, 7, 8(1), 8(2), and 9. In total of 154 subjects, 2 genotypes were observed with frequencies that varied from 1.95% (Wt/Del) to 98.05% (Wt/Wt) (Table 4). Two identified CYP2C9 alleles were Wt (1 copy) and Del (0 copy) accounting for 99% and 1%, respectively (Table 5). Two real-time PCR assays were used to confirm the MLPA results with primers located in exon 4 and exon 7 of CYP2C9. Both exons 4 and 7 of the gene showed the loss of CYP2C9 copy number, indicating the accuracy of MLPA (Figure 3B). Additionally, CYP2C9 duplication alleles were not determined in this study.

For CYP2E1, heterozygous duplications were detected in 3/154 subjects (1.95%) by MLPA with copy number probes designed for exons 5, 6, and 8. Additionally, heterozygous deletions were found in only 1/154 subjects (0.65%) (Table 4). Three observed CYP2E1 alleles consisted of Dup (2 copies), Wt (1 copy) and Del (0 copy), which made up 1%, 98.7%, and 0.3%, respectively (Table 5). Two real-time PCR assays were applied in order to verify the MLPA results using primers located in exon 8 and exon 9. Both exons of CYP2E1 showed the gain of copy number in 2 samples tested compared with reference sample. The loss of CYP2E1 copy number in tested samples was also in agreement with the data observed in MLPA (Figure 3C). Besides, no CYP2E1 deletion carriers were identified in this study.

We further compared the CNVs observed in CYP2A6 and CYP2E1 between Kinh Vietnamese and populations from different geographical areas in the world (Table 6). For CYP2A6, no significant difference in CYP2A6Dup allele (0%) was observed between the present study and other populations, except for Ashkenazi Jews (P < 0.05). In Kinh Vietnamese, frequency of CYP2A6Del was significantly higher than those found in other populations, including Asian. For CYP2E1, frequency of CYP2E1Dup was significantly lower compared with data observed in African-American and Hispanic. Both CYP2A6Wt and CYP2E1Wt allele frequency detected in the studied subjects were similar to those previously figured out in other populations.