Copy number variations of cytochrome P450 genes in Kinh Vietnamese

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

Table 2.

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

Table 3.

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

Samples	*CYP1B1*		*CYP2A6*				*CYP2C9*					*CYP2E1*
Samples	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

[i] Final probes ratio indicated deletions (underline); final probes ratio indicated duplications (bold).

[ii] MLPA, multiplex ligation-dependent probe amplification.

Graphical illustration of *CYP450* CNVs identified in current study. **(A)** Reference gen locus of *CYP1B1* (up) and *CYP1B1*Dup 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 *CYP2A6*Del 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), *CYP2E1*Del allele (middle), and *CYP2E1*Dup allele with 2 copies of the gene (down). CNVs, copy number variations.

Table 4.

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

Table 5.

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

[i] CI, confidence interval; n, number of alleles.

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 *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.

Table 6.

Comparison of CYP450 CNVs frequency between Kinh Vietnamese and worldwide populations

Population	N	Alleles/copy number of CYP2A6			References
Population	N	Dup (2)	Wt (1)	Del (0)	References
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]

Population	N	Alleles/copy number of CYP2E1			References
Population	N	Dup (2)	Wt (1)	Del (0)	References
CYP2E1 copy number alleles frequency
Kinh Vietnamese	154	0.01	0.987	0.003	Present study
African-American	105	0.038^*	0.962	ND	[2]
Asian	102	ND	1	ND
Caucasian	103	0.029	0.971	ND
Hispanic	109	0.041^*	0.959	ND
Ashkenazi Jews	123	0.004	0.996	ND
Colombian	123	0.016	0.984	ND	[12]

* P < 0.05.

** P < 0.01.

*** P < 0.001.

CNVs, copy number variations; N, number of subjects; ND, not detected.

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DOI: https://doi.org/10.2478/abm-2023-0048 | Journal eISSN: 1875-855X | Journal ISSN: 1905-7415

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Language: English

Page range: 84 - 92

Published on: Sep 17, 2023

Published by: Chulalongkorn University

In partnership with: Paradigm Publishing Services

Keywords:

CNVs,

CYP450,

Related subjects:

Assistive professions, nursing,

Basic medical science,

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Clinical medicine,

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© 2023 Nhung Phuong Vu, Ton Dang Nguyen, Binh Huy Nguyen, Duong Thuy Nguyen, Hai Van Nong, Ha Hai Nguyen, published by Chulalongkorn University
This work is licensed under the Creative Commons Attribution 4.0 License.

Volume 17 (2023): Issue 2 (April 2023)

Copy number variations of cytochrome P450 genes in Kinh Vietnamese

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