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Copy number variations of cytochrome P450 genes in Kinh Vietnamese

Nhung Phuong Vu
Nhung Phuong Vu
, 
Ton Dang Nguyen
Ton Dang Nguyen
, 
Binh Huy Nguyen
Binh Huy Nguyen
, 
Duong Thuy Nguyen
Duong Thuy Nguyen
, 
Hai Van Nong
Hai Van Nong
 et 
Ha Hai Nguyen
Ha Hai Nguyen
   | 17 sept. 2023
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Article Category: Brief communication
Publié en ligne: 17 sept. 2023
Pages: 84 - 92
DOI: https://doi.org/10.2478/abm-2023-0048
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© 2023 Nhung Phuong Vu et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

Graphical illustration of CYP450 CNVs identified in current study. (A) Reference gen locus of CYP1B1 (up) and CYP1B1Dup allele (down). CYP1B1 located on chromosome 2 and represented by yellow boxes. (B) Reference gen locus on chromosome 19 of CYP2A6 (orange box) and pseudogene CYP2A7 (grey box) (up) and CYP2A6Del allele (down), downstream of exon 9 of CYP2A6 is denoted by the black boxes. Primers position and strategy used for CYP2A6 del allele detection are also shown. (C) Reference gene cluster on chromosome 10 including CYP2C9 (blue box), CYP2C8 (green box) (up), and CYP2C9 Del allele (down). (D) Reference gen locus of CYP2E1 (pink box) on chromosome 10 (up), CYP2E1Del allele (middle), and CYP2E1Dup allele with 2 copies of the gene (down). CNVs, copy number variations.
Graphical illustration of CYP450 CNVs identified in current study. (A) Reference gen locus of CYP1B1 (up) and CYP1B1Dup allele (down). CYP1B1 located on chromosome 2 and represented by yellow boxes. (B) Reference gen locus on chromosome 19 of CYP2A6 (orange box) and pseudogene CYP2A7 (grey box) (up) and CYP2A6Del allele (down), downstream of exon 9 of CYP2A6 is denoted by the black boxes. Primers position and strategy used for CYP2A6 del allele detection are also shown. (C) Reference gene cluster on chromosome 10 including CYP2C9 (blue box), CYP2C8 (green box) (up), and CYP2C9 Del allele (down). (D) Reference gen locus of CYP2E1 (pink box) on chromosome 10 (up), CYP2E1Del allele (middle), and CYP2E1Dup allele with 2 copies of the gene (down). CNVs, copy number variations.

Figure 2.

Copy number confirmation of CYP2A6Del. (A) Amplification of a part of CYP2A6 gene or hybrid CYP2A7/2A6 using primer pair 2AE7F/2A6R1 resulting in approximately 2 kb products. (B) Detection of CYP2A6Del allele, Wt: Wild-type allele, Del: Deletion allele. CYP2A6 genotype of tested samples were as follows: FVN48: Wt/Del, FVN74: Del/Del, FVN79: Wt/Wt.
Copy number confirmation of CYP2A6Del. (A) Amplification of a part of CYP2A6 gene or hybrid CYP2A7/2A6 using primer pair 2AE7F/2A6R1 resulting in approximately 2 kb products. (B) Detection of CYP2A6Del allele, Wt: Wild-type allele, Del: Deletion allele. CYP2A6 genotype of tested samples were as follows: FVN48: Wt/Del, FVN74: Del/Del, FVN79: Wt/Wt.

Figure 3.

Copy number confirmation of CYP1B1, CYP2C9, and CYP2E1. Primers for real-time PCR located in exon 1 and exon 2 of CYP1B1, exons 4 and 7 of CYP2C9, exons 8 and 9 of CYP2E1 were used to confirm CNVs detected by MLPA in these genes. REF: reference samples, in which MLPA showing 2 copies of gene of interest. (A) Heterozygous duplications of CYP1B1 detected in both FVN 96 and MVN 21. (B) Heterozygous deletions of CYP2C9 were found in both MVN 21 and MVN 23. (C) Copy number confirmation of CYP2E1 showed that heterozygous deletion was detected in FVN 73 while heterozygous duplications were identified in FVN 46, FVN 98, Kinh 712 and DB98. CNVs, copy number variations; MLPA, multiplex ligation-dependent probe amplification.
Copy number confirmation of CYP1B1, CYP2C9, and CYP2E1. Primers for real-time PCR located in exon 1 and exon 2 of CYP1B1, exons 4 and 7 of CYP2C9, exons 8 and 9 of CYP2E1 were used to confirm CNVs detected by MLPA in these genes. REF: reference samples, in which MLPA showing 2 copies of gene of interest. (A) Heterozygous duplications of CYP1B1 detected in both FVN 96 and MVN 21. (B) Heterozygous deletions of CYP2C9 were found in both MVN 21 and MVN 23. (C) Copy number confirmation of CYP2E1 showed that heterozygous deletion was detected in FVN 73 while heterozygous duplications were identified in FVN 46, FVN 98, Kinh 712 and DB98. CNVs, copy number variations; MLPA, multiplex ligation-dependent probe amplification.

Comparison of CYP450 CNVs frequency between Kinh Vietnamese and worldwide populations

Population N Alleles/copy number of CYP2A6 References
Dup (2) Wt (1) Del (0)
CYP2A6 copy number alleles frequency
Kinh Vietnamese 154 ND 0.89 0.11 Present study
Chinese 96 - 0.849 0.151 [9]
Finns 100 - 0.96 0.001***
Spaniards 100 - 0.965 0.005***
Korean 209 - 0.885 0.11 [10]
Japanese 92 - 0.799 0.201** [11]
African-American 105 0.01 0.952 0.038** [2]
Asian 102 0.015 0.902 0.083**
Caucasian 103 ND 0.985 0.015***
Hispanic 109 ND 0.986 0.014***
Ashkenazi Jews 123 0.02* 0.98 ND***
Colombian 123 0.004 0.976 0.02*** [12]

Primers sequence for real-time PCR detecting copy number of CYP1B1, CYP2C9, and CYP2E1

Gene-region Primer name Sequence (5′-3′)
CYP1B1 Exon 1 CYP1B1 E1F CTG CGACTCCAGTTGTGAGAG C
CYP1B1 E1R AGTCTCTTGGCGTCG TCAGTG
CYP1B1 Exon 2 CYP1B1 E2F CACAGCATGATGCGCAACTTC
CYP1B1 E2R CACTCATGACGTTGGCCA CG
CYP2C9 Exon 4 CYP2C9 E4F ATGCATGCCGAACTCTTTTT
CYP2C9 E4R2 AGGATGAAAGTGGGATCACAGG
CYP2C9 Exon 7 CYP2C9 E7F CACATTTGTGCATCTGTAACCA
CYP2C9 E7R3 CCGGTTTCTGCCAATCACACG
CYP2E1 Exon 8 CYP2E1 E8F GGCACAGTCGTAGTGCCAACTC
CYP2E1 E8R CTGCCTCTGATCTTTCTCACCTG
CYP2E1 Exon 9 CYP2E1 E9F TGGAGAAGGCCTGGCTCGCATG
CYP2E1 E9R GTTCAGGGTGTCCTCCACACAC

Primer used for genotyping the CYP2A6 deletion and duplication alleles [9]

Primer name Sequence (5′-3′)
2A E7F GRC CAA CAT GCC CTA CAT G
2A6R1 GCA CTT ATG TTT TGT GAG ACA TCA GAG ACA A
2A6E8F CAC TTC CTG AAT GAG
2A7E8F CAT TTC CTG GAT GAC
2A6R2 AAA ATG GGC ATG AAC GCC C

Final probe ratio from MLPA of representative samples carrying CYP450 copy number alterations

Samples CYP1B1 CYP2A6 CYP2C9 CYP2E1
Exon 2 Exon 3 Exon 1 Exon 2 Exon 3 Exon 5 Exon 1 Exon 7 Exon 8-1 Exon 8-2 Exon 9 Exon 5 Exon 6 Exon 8
FVN48 0.89 0.87 0.41 0.42 0.5 0.43 1.1 1.18 1.16 1.06 0.92 0.95 0.93 0.88
FVN74 0.89 0.85 0 0 0 0 1 1 1 0.96 0.99 1.02 0.94 1.11
MVN23 1.34 1.53 1.15 1.14 1.16 1.15 0.47 0.55 0.61 0.56 0.58 1.09 1.08 0.99
FVN73 1.08 0.99 1.05 1.07 1.04 1.08 1 0.99 0.98 0.92 0.96 0.51 0.51 0.47
FVN46 1.04 1.1 0.94 0.92 1.17 1.2 1.17 1.03 1.1 0.82 0.93 1.37 1.38 1.63
FVN98 0.86 0.86 0.97 0.98 1.16 0.91 1.06 0.96 1.02 1.28 1.11 1.36 1.37 1.37

Alleles frequency of CYP450 copy number variability

Allele/copies Gene/alleles frequency (n = 308)
CYP1B1 CYP2A6 CYP2C9 CYP2E1
Freq. 95% CI Freq. 95% CI Freq. 95% CI Freq. 95% CI
Dup (2) 0.010 0.000–0.010 - - - - 0.010 0.000–0.021
Wt (1) 0.990 0.980–1.000 0.890 0.852–0.927 0.990 0.980–1.000 0.987 0.974–1.000
Del (0) - - 0.110 0.073–0.148 0.010 0.000–0.021 0.003 0.000–0.010

Genotypes frequency of CYP450 copy number variability

Genotype Gene/genotypes frequency
CYP1B1 CYP2A6 CYP2C9 CYP2E1
N Freq. (%) N Freq. (%) N Freq. (%) N Freq. (%)
Wt/Del 0 0 26/154 16.88 3/154 1.95 1/154 0.65
Del/Del 0 0.00 4/154 2.60 0 0.00 0 0.00
Wt/Dup 3/154 1.95 0/154 0.00 0 0.00 3/154 1.95
Wt/Wt 151/154 98.05 124/154 80.52 151/154 98.05 150/54 97.40
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