Prediction of The CYP2D6 Enzymatic Activity Based on Investigating of The CYP2D6 Genotypes Around The Vivax Malaria Patients in Yunnan Province, China

doi:10.21203/rs.3.rs-714877/v1

Background

To preliminary explore CYP2D6 enzyme activity in patients with recurrent vivax malaria in Yunnan Province by analyzing mutational polymorphism in the coding region of CYP2D6 gene.

Methods

Blood samples were collected from vivax malaria patients with suspected relapse (SR) malaria and non-relapsed (NR) in Yunnan Province. The DNA fragments containing 9 exons region of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were compiled by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape.

The allelic forms of CYP2D6 gene were identified according to the criteria prescribed by "Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database" and the CYP2D6 enzyme activity was predicted based on diploid genotype.

Results

A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). High-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group (x² = 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. *10 allele was the most frequent among these genotypes (0.423). Among the 10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group (x² = 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group (x² = 10.050, P < 0.05), and genotype *4/*4 was detected only in the SR group.

Conclusion

In the patients receiving primaquine dosage in Yunnan Province, both rs1135840 single nucleotide polymorphism and *10 allele form was the common in the coding region CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse patients.

Infectious Diseases

Yunnan province

Vivax malaria pateint

CYP2D6

Genotype

Enzyme activity

Prediction

So far, primaquine is still the only reliable drug to eliminate the hypnozoites of P. vivax [1]. However, the production of its active ingredient, 5-hydroxy-primaquine, relies on the catalysis of CYP2D isoenzymes in human hepatocytes [2–4]. CYP2D6 is the only monooxygenase in the P450 family that cannot be induced by the environment. CYP2D6 is characterized by low capacity, high catalytic efficiency, and complex heterogeneity; more than 30% of commonly used clinical drugs are catalyzed by CYP2D6 [4–6]. Previous study has revealed the association between CYP2D6 gene polymorphism and the ineffective potency of primaquine in the radical cure of vivax malaria [7–8]. Evidence revealed the notably high frequency of rs1065852 and rs1135840 locus mutations with vivax malaria patients relapse in Brazilian recurrent [9].

In the relapsed vivax malaria cases of Papua New Guinea, CYP2D6 enzyme activity was mostly deficient star alleles *3, *4, *5, *6, *7, and *8, which indicates the phenotype of Poor metabolizer (PM) [10]. Still, the impaired degree of CYP2D6 enzyme activity induced by non-functional star alleles (*3), homozygous diploid (*3/*3) [11], as well as the dose-effect relationship between the copy number and the relapses occurring vivax malaria patients [7–8] remains to be clarified.

Recently, the World Health Organization (WHO) classified the impaired CYP2D6 function as one of the four risk factors for primaquine therapy, in addition to G6PD deficiency, pregnancy and/or lactation, and infancy [1, 12–13]. WHO further advocated the personalized dosage of primaquine therapy for the eradicative treatment of vivax malaria based on the acquired understanding of patients’ CYP2D6 genetic polymorphism and heterogeneity of enzyme activity [13–14].

Molecular epidemiological investigation on G6PD deficiency in vivax malaria patients has been initiated in Yunnan province [15–16] to carry out the polymorphism analysis of the full coding region of G6PD gene, revealing that G > T (rs72554665) missense mutation at c.1376 locus could be used as the molecular marker for indicating G6PD deficiency. It is also found that vivax malaria patients with genotype *4/*4 of CYP2D6 poor enzyme activity were prone to the failure of primaquine radical treatment for vivax malaria[yellow], suggesting that the influence of biological factors may cause obstacles to effectively block the transmission of vivax malaria and effectively elimination of the malaria infection source in Yunnan Province. The current study further analyzed CYP2D6 genotype and its enzyme activity from increased vivax malaria patients in Yunnan province who underwent primaquine treatment in recent years, in order to systematically understand the adverse factors during employing the primaquine for eradicative treatment of vivax malariai in Yunnan Province.

Study subjects and blood sampling

Blood samples were collected from vivax malaria patients whose symptoms were diagnosed and confirmed by both optical microscopy and Plasmodium genetic testing in the Yunnan Provincial Malaria Diagnostic Reference Laboratory (YPMDRL).

The subjects of vivax malaria were subdivided into suspected-relapse group (SR group) and non-relapse group (NR group). In the NR group, the subjects had not experienced relapsed symptom after being clinically cured by "eight-day chloroquine/primaquine therapy" from 2018 to 2020 (NR). In the SR group, the patients were re-diagnosed as P. vivax infection 28 days after being clinically cured by "eight-day chloroquine/primaquine therapy" from 2014 to 2020. Moreover, the infected strains paired between previous and of relapse episodes were verified to be clones with the same gene sequences, that both pvcsp gene and pvmsp-1 gene fragment have a 100% similarity and without any variable sites in every a gene sequence from one paired samples.

0.6 ml of venous blood was collected from every subject, and was then stored in a dried tube at -80 ℃ before the DNA extraction. Dried blood spots (DBS) sampling was used to help with genetic analysis.

Extraction of human genomic DNA and PCR amplification of CYP2D6 gene fragments

Dried blood spots (DBS) method was used on the filter paper dried blood stain (diameter = 5mm). QIAgen Mini Kit (DNA Mini Kit, QIAamp, Germany) was used to extract human genomic DNA from the blood stain, according to the instruction of the manufacturer. The DNA samples were stored at -20°C for the subsequent assay.

Reference sequence (ID: NC_000022.11) was used as the template to design the PCR primers and to determine the reaction conditions for CYP2D6 gene, according to the methods described in previous studies [17–19]. The coding regions covering exons 1–4 and exons 5–9 were amplified by segmentation. It is predicted that the amplification regions contain from 42131088 to 42128678 nt and from 42126035 to 42128422 nt and amply products with length of 2411 bp and 2388 bp,respectively. The amplification products were sequenced by Shanghai Meiji Biomedical Technology Co. Ltd by using the Sanger method.

Analysis of CYP2D6 gene coding region polymorphism and prediction of enzyme activity

The sequencing results were collated by using DNAStar 11.0 and BioEdit 7.2.5, and the first CDS chain of CYP2D6 gene of each sample, known as maternal CDS chain, was obtained by splicing method, according to the protocol described in the literature [19]. The maternal CDS chain was used to derive the paternal CDS chain by replacing the bases of the unread double allelic heterozygous sites [20], which were identified by searching the DNA sequencing peak chromatograms. DnaSP 6.11.01 software was used to identify site-specific mutations and haplotypes in the CDS chain, to calculate the nucleic acid diversity index (π) and Expected heterozygosity (He), which were aligned with referent sequence and determined the ID No. of each mutation on the NCBI platform (https://www.ncbi. nlm.nih.gov/snp/). GenVisR package in R (version: 4.0.2) was used to visualize the mutational waterfall plots of the haplotypes of the loci in the allelic form.

The haplotypes (allelic forms) of the CDS chain of CYP2D6 gene were determined in accordance with the criteria of the "Human Cytochrome P450 Allele Nomenclature Committee Database" (NM_000106.5) [21] and published literatures [22, 19]. The haplotypes at the sub-allelic level. For the haplotypes devoid of the criteria for the allelic forms, they were customized as "* + a single lowercase letter" [19].

The genotype of each sample was presented in the diploid form consisting of maternal and paternal CDS chains, such as *10/*39, *1/*10 and so forth. The diploid allelic form of the genotype was no longer refined to the sub-allelic level, as the sub-allelic forms should be merged in the first place. For example, *10.001 and *10.002 could be combined into the allelic form *10 to count the frequency.

For the genotypes with standard nomenclature, the CYP2D6 enzyme activity was predicted by calculating the genotype score based on diploid genotypes, following to the method described in previous literatures [23–25]. The genotype score is the sum of the two allelic form scores. For instance, a score of 1.0 for genotype *10/*39 is the sum of 0.5 score for allelic form *10 and 0.5 score for allelic form *39. In the event of genotype score being 0.0, the enzyme activity is absent and hence poor metabolizer (PM). The genotype score ranging from 0.5 to 1.0, 1.5 to 2.0, and > 2.0 [7] indicates intermediate metabolizer (IM), normal metabolizer (NM), and ultrarapid metabolize (UM), respectively [17–18, 25–28]. Meanwhile, the online tool PROVEAN (http://provean.jcvi.org/index.php) was used to predict the function of CYP2D6 enzyme protein of different genotypes as deleterious (DT) or Neutral (NT).

Statistics

The frequency of mutation site was presented by the ratio of the number of base substitutions to the total number of CDS chains. Each study subject has two CDS chains, including maternal chain and paternal chain. Chi-square test (x²) was used to analyze the differences in the locus mutations and genotypes between the two groups. P < 0.05 was regarded to indicate statistical significance.

Sample information and PCR amplification of CYP2D6 gene fragment

A total of 345 blood samples were collected from the vivax malaria patients for the subsequent PCR amplification of human genomic DNA, which covers exon1-4 and exon5-9 regions of CYP2D6 gene. PCR amplication produces showed the target fragment more than 2,000 bp in length. After collating the sequencing results of PCR amplification products, all of 320 maternal CDS chains of CYP2D6 gene (1491 bp in length) were obtained in 345 subject blood samples (92.8%, 320/345), including 63 SR cases and 257 NR cases.

The blood samples of obtained 320 maternal CDS chains were collected from the vivax malaria patients diagnosed and distributed in the following these prefectures of Yunnan province: Yingjiang (180 cases), Mangshi (20 cases), Longchuan (8 cases), Ruili (16 cases), Tengchong (39 cases), Baoshan (20 cases), Lianghe (3 cases), Mengla (1 case), Menglian (1 case), Diqing (1 case), Nujiang (1 case), Jinping (4 cases), Yuanyang (2 cases), Linxiang (11 cases), Canyuan (9 cases), Dali (2 cases) and Kunming (2 cases).

Cds Chain Polymorphism And Allelic Forms Of Cyp2d6 Gene

The polymorphism of 640 CDS chains both 320 maternal ones and 320 paternal ones of CYP2D6 genes revealed that base substitutions (G>A, C>T, C>A.T, A>G, C>G, C>T, C>T, G>C, G>A, C>A, C>T and G>C) at 12 loci, including c.31, c.100, c.271, c.281, c.294, c.297, c.336, c.408, c.505, c.801, c.886, and c.1457. Most of these mutation sites have been registered as single nucleotide polymorphism (SNP) loci (Table 1). Of these loci, c.31 (rs769258), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.505 (rs5030865), c.886 (rs16947) and c.1457 (rs1135840) were non-synonymous mutation. On the amino acid peptide chain, the 11th, 34th, 91th, 94th, 169th, 296th, and 486th amino acids exhibited V/M, P/S, L/M, H/R, G/S, R/C, and S/T variants (Table 1).

The highest frequency of locus mutation was observed at c.1457 (70.3%, rs1135840), followed and the minor allele frequency (MAF) was 0.698 for c.408 (rs1058164) (Table 1). Loci mutation at c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), and c.505 (rs5030865) were detected only in the SR group. Meanwhile, mutation at c.408 (rs1058164) and c.1457 (rs1135840) showed significantly higher frequency in the SR group (x^2 = 4.468, 5.889, P < 0.05), as opposed to the NR group (Table 1). Mutant homozygotes were mainly composed of four loci, including c.408 (rs1058164), c.1457 (rs1135840), c.336 (rs1081003), and c.100 (rs1065852) (Table 1).

These 640 CDS chains could be subdivided into 23 haplotypes (from Hap_1 to Hap_23, π = 0.002, He = 0.772) (Fig. 1). Among them, the non-mutant Hap_3 accounted for 26.6% (170/640). In contrast, Hap_1, Hap_2, and Hap_4 to Hap_23 were mutant, which accounted for 0.2% ( 1/640), 36.9% (236/640) and 9.1% (58/640), 4.5% (29/640), 10.2% (65/640), 0.6% (4/640), 0.8% (5/640), 0.2% (1/640), 0.3% (2/640), 1.1% (7/640), 0.5% (3/ 640), 3.6% (23/640), 0.9% (6/640), 0.2% (1/640), 0.2% (1/640), 0.2% (1/640), 1.3% (8/640), 1.3% (8/640), 0.5% (3/640), 0.3% (2/640), 0.6% (4/640), 0.2% (1/640), and 0.3% (2/640), respectively.

Of the 23 haplotypes, Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_8, Hap_11, Hap_12, Hap_14, and Hap_20 were known haplotypes and could be identified as *10.002, *1.001, *10.001, * 39.001, *2.001, *39.002, *2.002, *2.004, *4.001, *1.011, and *2.025 in the respective allelic forms. The remaining 12 haplotypes were presented in the allelic form of *m ~*x (Fig. 1).

When compared with the non-mutated sequence (Hap_3), Hap_9 showed the most complicated sequence composition, exhibiting mutations sites at c.100, c.271, c.281, c.294, c.297, c.408, c.1457 and so forth. Hap_14 showed the simplest sequence composition, with mutation detected only at c.408 only (Fig. 1). On the other hand, the exon 3 was the region with the highest mutation frequency,73.9% (17/23) in 23 haplotypes, but the heat of mutation in exon 9, exon2, exon 1, exon 6 and exon 5 decreased gradually (Fig. 1), showing the loci mutation frequency of 65.2% (15/23), 47.8% (11/23), 43.5% (10/23), 39.1% (9/23) and 4.5% (1/23), respectively (Fig. 1). In contrast, no locus mutations could be determined in exon 4, exon 7 and exon 8.

Table 1. The polymorphism and diploid of CDS mutation loci in CYP2D6 gene of vivax malaria cases in Yunnan Province
Loci	SNP ID ^a	Codon change	Amino acid change	Frequency ^b	Diploid				Different type of cases
Loci	SNP ID ^a	Codon change	Amino acid change	Frequency ^b	No. loci	NMHO ^c (CR) ^d	MHE ^e (CR) ^d	MHO ^f (CR) ^d	No. SR(TPR)^g	No. NR (TPR)^g	P
c.31	rs769258	GTG>ATG	V11M	0.002	1	319 (99.7)	1 (0.3)	0 (0.0)	1 (1.6)	0 (0.0)	0.197
c.100	rs1065852	CCA>TCA	P34S	0.527	337	87 (27.2)	129 (40.3)	104 (32.5)	43 (68.3)	190 (73.9)	0.364
c.271	rs28371703	CTG>ATG	L91M	0.006	4	318 (99.4)	0 (0.0)	2 (0.6)	2 (3.2)	0 (0.0)	0.038 ^h
c.271	rs28371703	CTG>TTG	L91L	0.003	2	319 (99.7)	0 (0.0)	1 (0.3)	0 (0.0)	1 (0.4)	1
c.281	rs28371704	CAC>CGC	H94R	0.006	4	318 (99.4)	0 (0.0)	2 (0.6)	2 (3.2)	0 (0.0)	0.038 ^h
c.294	rs28371705	ACC>ACG	T98T	0.006	4	318 (99.4)	0 (0.0)	2 (0.6)	2 (3.2)	0 (0.0)	0.038 ^h
c.297	rs200269944	GCC>GCT	A99A	0.002	1	319 (99.7)	1 (0.3)	0 (0.0)	1 (3.2)	0 (0.0)	0.197
c.336	rs1081003	TTC>TTT	F112F	0.552	353	93 (29.1)	101 (31.5)	126 (39.4)	39 (61.9)	188 (73.2)	0.078
c.408	rs1058164	GTG>GTC	V136V	0.698	447	54 (16.9)	85 (26.6)	181 (56.5)	58 (92.1)	208 (80.9)	0.035 ^h
c.505	rs5030865	GGT>AGT	G169S	0.003	2	318 (99.4)	2 (0.6)	0 (0.0)	2 (3.2)	0 (0.0)	0.038 ^h
c.801	rs28371718	CCC>CCA	P267P	0.002	1	319 (99.7)	1 (0.3)	0 (0.0)	0 (0.0)	1 (0.4)	1
c.886	rs16947	CGC>TGC	R296C	0.178	114	220 (68.8)	86 (26.9)	14 (4.4)	24 (38.1)	76 (29.6)	0.191
c.1457	rs1135840	AGC>ACC	S486T	0.703	450	46 (14.4)	98 (30.6)	176 (55.0)	60 (95.2)	214 (83.3)	0.015 ^h
Note: a. The SNP ID was determined by searching on NCBI platform; b. The denominator was 640, which is the total number of 320 maternal CDS chains and 320 paternal CDS chains; c. Constituent ratio, the denominator was 320; d. NMHO is the acronym for Not mutation homozygote; e. MHE is the acronym for Mutation heterozygote; f. MHO is the acronym for Mutation homozygote; g. TPR is the acronym for Test positive rate; h. Chi-square test (significant level at P < 0.05);

Cyp2d6 Genotypes And Enzyme Activity

A total number of 34 CYP2D6 genotypes were obtained from the blood sample of 320 vivax malaria patients. Among them, genotype *1/*1, *2/*2, *1/*2, *2/*10, *1/*10, *10/*10, *10/*39, *39/*39, *2/*39, and *4/*4 s belong to the named genotypes (Table 2), and are suitable for assignments of allelic form and prediction of CYP2D6 enzyme activity. These variants jointly accounted for 84.1% in the study population (269/ 320). In all of genotypes, *10/*10 had the most frequent mutant (26.9%, 86/320), the remaining genotype *1/*10* being 18.1%(58/320)and *1/*1 being 13.1% (43/320)(Table 2).These genotypes account for 15.9% (51/320) were not predicted for the CYP2D6 activity, owing to the presence of at least one undocumented allelic form.

Among ten documented genotypes, the most common genotype *10/*10 predicted into two CYP2D6 enzyme activity of IM and NT, respectively, based on the Genotype method and PVOVEAN method. In spite of the different classify in predicted enzyme activity, the detection rate between SR and NR groups was not statistically significant (x²)=0.115, P > 0.05) (Table 2). The other genotypes with frequent mutation included *1/*10, *1/*1 and *2/*10 (10.0%, 32/320), which were predicted as NM and NT by both genetic method and PVOVEAN methods. The frequency of these three allelic variants was all significantly higher in the NR group (x²)= 3.911, 7.103 and 4.060, P < 0.05) than in the SR group (Table 2). In contrast, genotypes *10/*39 (2.8%, 9/320) manifesting impaired enzyme activity (IM and DT) were predicted by both two methods, with its frequency being significantly higher in the SR group (x²)= 10.050, P < 0.05), as compared with the NR group (Table 2). Genotype *4/*4 that indicates deficient enzyme activity (PM and DT) was only found in the SR group (Table 2).

Table 2

Prediction of CYP2D6 enzyme activity based on genotyping of CDS chain of vivax malaria patients in Yunnan Province
Genotypes	No. (CR)^a	Genotype mothed		PROVEAN mothed			Different type of cases
Genotypes	No. (CR)^a	Value	Classify	Score	Cutoff	Function	No. SR(TPR)^b	No. NR (TPR) ^b	P
1/1	43 (13.4)	2	NM	2.000	-2.5	NT	2 (3.2)	41 (16.0)	0.008 ^c
2/2	10 (3.1)	2	NM	-0.887	-2.5	NT	4(6.3)	6(2.3)	0.216
1/2	22 (6.9)	2	NM	-0.773	-2.5	NT	7(11.1)	15(5.8)	0.228
2/10	32 (10.0)	1.5	NM	-1.568	-2.5	NT	2(3.2)	30(11.7)	0.044 ^c
1/10	58 (18.1)	1.5	NM	-2.341	-2.5	NT	6(9.5)	52(20.2)	0.048 ^c
10/10	86 (26.9)	1.0	IM	-1.671	-2.5	NT	18(28.6)	68(26.5)	0.735
10/39	9 (2.8)	1.0	IM	-2.666	-2.5	DT	6(9.5)	3(1.2)	0.002 ^c
39/39	3 (0.9)	1.0	IM	1.350	-2.5	NT	2(3.2)	1(0.4)	0.100
2/39	5 (1.6)	1.5	NM	-1.098	-2.5	NT	3(4.8)	2(0.8)	0.054
4/4	1 (0.3)	0	PM	-9.431	-2.5	DT	1(1.6)	0(0.0)	0.197
Others ^d	51 (15.9)	-	-	-	-	-	12(19.0)	39(15.2)	0.452
Total	320						63	257
Note: a. CR is acronym for constituent ratio; b. TPR is acronym for Test positive rate; c. Chi-square test (significant level at P < 0.05); d. The diploids with at least one undocumented allele (m, n, o, p, q,r, s ,t, u, v, w and x).

Located in the non-telomeric region of the long arm of human chromosome 22, CYP2D6 gene has 4,383 bp in length and is composed of 8–9 exons and 7–8 introns. The CDS chain of CYP2D6 has 1,338-1,491 bp to encode 446–497 amino acids. To be more specific, CYP2D6 gene with 9 exons has a codon sequence encoding 51 amino acids sequence of "NVFLARYGPAWREQRRFSVSTLRNLGLGKKSLEQWVTEEA

ACLCAAFANHSGC" inserted in the downstream of exon 2, compared with that has 8 exons [29]. The CYP2D6 genes analyzed in this study all have 9 exons, and as high as 92.8% of the maternal CDS chains were obtained. Such a high efficiency of PCR amplification of CYP2D6 gene could be attributable to the absence of Poly-T/A structure in the CYP2D6 gene and the lower degree of DNA polymerase slippage on the template to facilitate the extension of DNA chain.

To date, as many as 300 allelic and sub-allelic variants of CYP2D6 gene have been identified by the researcher [21]. Although the 12 locus mutations detected in this study at the CYP2D6 locus, such as c.31 (rs769259), c.100 (rs1065852), c.886 (rs16947), and c.1457 (rs1135840), were all validated SNPs, up to 52.2% (12/23) of the star allelic variants by combination [29] have not been documented or assigned [24–27]. This could hinder the real classify of CYP2D6 enzyme activities based on the genotype score of biallelic mutation for prediction. The insensitivity in the previous confirmation criteria of allele [21–22] to the combination of mutant loci obtained by whole gene sequencing is the most probable reason, in addition to the lack of accuracy due to the inclusion of intron SNPs in CYP2D6 gene allele identification.

Intriguingly, low-frequency mutant loci, such as c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), and c.505 (rs5030865), were present only in samples of SR group (Table 1). This study observed that genotype *4.001 and diploid *4/*4 indicate null CYP2D6 enzyme function (PM), a conclusion consistent with the findings of Jason et al [7], who validated *4 allele signifies severely impaired CYP2D6 enzyme activity. Thusly, the rational suggestion has been put forward those mutations at rs28371703, rs28371704, rs28371705 as well as other loci may be applied as the molecular markers of impaired CYP2D6 enzyme activity in clinical practice. Mutant variant rs16947, which was validated as damaged CYP2D6 enzyme activity in previous study [30], showed a frequency of merely 17.8% in the present study. However, the allelic form *2 and its homozygous diploid allele *2/*2 did not display preferential distribution in the SR group or the NR group, let alone presaging the phenotype of abnormal enzyme activity (Table 2).

This could be attributed to the rigorous scoring and classification of CYP2D6 enzyme activity, which could not reflect the subtle disparities in the genetic structure.

In contrast, the PROVEAN method, by taking every amino acid substitution into the CYP2D6 peptide chain, preserves the polymorphic information of the gene structure in its prediction results. Furthermore, in this study, the damaging allelic form *10 was detected in 57.8% (185/320) of the samples [31–32], and exhibited the highest frequency (0.423) in the samples. This is consistent with the previous conclusion that *10 allele is more widely distributed in Southeast Asian populations [33], yet no findings could corroborate the notable preferential distribution of genotype *10/*10 in the SR group or the NR groups. In contrast, genotype *10/*39 indicates both Intermediate Metabolizer (IM) and Deleterious (DT), and was commonly observed in the SR population. Such finding suggests the potential of *39 allele to be used as a molecular marker indicating the impairment of CYP2D6 enzyme activity.

The radical cure of vivax malaria is a critically important intervention to prevent the retransmission and repeated attack of malaria [33]. To our knowledge, this study reveals the CYP2D6 gene allelic polymorphism and its distribution in the primaquine- exposed population in Yunnan Province for the first time, and the experimental approaches are developed and introduced to press forward the accurate screening of population suitable for primaquine therapy in Yunnan Province. Nevertheless, this study is not devoid of certain shortcomings. First, the determination of the recurrence of vivax malaria was somewhat flawed, as the Plasmodium reservoir in bone marrow again into peripheral blood [34–35] or the resistance of Plasmodium to primaquine [36], despite of the effort that has been made to validate the genetic consistency of the infected strains of patients with recurrent vivax malaria. Second, the effect of gene copy number on the prediction of CYP2D6 enzyme activity was not ruled out. It was shown that once the normal allele of CYP2D6 gene activity has more than two copies, CYP2D6 enzyme activity will be change from the phenotype of normal metabolizer into ultra-metabolizer [37–38]. Third, SNPs or single nucleotide variant (SNV) in the intron region of CYP2D6 gene were not identified nor applied to define the allelic form, hence more undocumented allelic forms of CYP2D6 gene require further validation. In the next stage, our research team will refine and optimize the experimental protocol by making constant adjustment.

In this study, only 12 mutation loci including c.31, c.100, c.271, c.281, c.294, c.297, c.336, c.408, c.505, c.801, c.886, and c.1457 were detected in Yunnan Province vivax malaria patients, which is consistent with the findings of previous small sample. In the patients receiving primaquine dosage in Yunnan Province, both rs1135840 single nucleotide polymorphism and *10 allele form was the common in CYP2D6 gene coding region. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse patients. Genotype *10/*39 could be suitable for the indicating of impaired CYP2D6 enzyme activity in this population to be treated with primaquine.

WHO: World Health Organization

CYP2D6: Cytochrome P450, family 2, subfamily D, polypeptide 6

G6PD: Glucose-6-Phosphate Dehydrogenase

CDS: Coding DNA sequence

PCR: Polymerase chain reaction

YPMDRL: Yunnan Province Malaria Diagnosis Referent Laboratory

DBS: Dried blood spots

pvcsp: Plasmodium vivax circumsporozoite surface protein

pvmsp-1: Plasmodium vivax merozoite surface protein 1

Hap: Haplotype

π: Diversity index

He: Expected heterozygosity

CI: Confidence interval

SNP: Single nucleotide polymorphism

SNV: Single nucleotide variant

Ethics approval and consent to participate

The design and protocol of the study was approved by the Yunnan Institute of Parasitic Diseases and Ethical Committee (Ethical Approval No.: 2019 Yunnan Ethics Auditing No. 5). The purpose of the study was explained to the subjects or their relatives, genetic testing was performed on stored blood samples obtained as part of the routine diagnostic work-up of patients with fever who were suspected to have malaria.

Consent for publication

All authors provided their consent for the publication of this report.

Availability of data and materials

Not applicable.

Competing interests

The authors declare no conflicted interest regarding the publication of the present manuscript.

Funding

The current study was funded by National Science Foundation, China (Nos. 81960579, 81660559).

Author information

Affiliations

Yunnan Institute of Parasitic Diseases Control, Yunnan Provincial Key Laboratory of Vector-Borne Diseases Control and Research, Yunnan Centre of Malaria Research, Pu’er, 665000, China

Ying Dong, Yan Deng, Yanchun Xu, Mengni Chen, Yan Liu & Canglin Zhang

School of Basic Medical Sciences, Dali University, Dali, 667000, China

Herong Huang

Contributions

Ying Dong wrote the manuscript, and was responsible for the study design, coordinated the project, and statistical analysis; HH undertook the genetic testing for Plasmodium and human gene; Yan Deng, YX, MC, YL and CZ performed the collection of blood samples and microscopy examination. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ying Dong.

Acknowledgements

Our sincere gratitude goest to the provided initial diagnosis information on malaria cases and their blood samples for the Centers for Disease Control and Prevention in prefetures and counties such as Dehong, Baoshan, Kunming, Pu’er, Lincang, Dali, Nujiang, Lijiang, Xishuangbanna, Yuxi, Chuxiong, Honghe, Zhaotong, Diqing, Qujing, and Whenshan.

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Prediction of The CYP2D6 Enzymatic Activity Based on Investigating of The CYP2D6 Genotypes Around The Vivax Malaria Patients in Yunnan Province, China

Status:

Journal Publication

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Abstract

Figures

Background

Materials And Methods

Study subjects and blood sampling

Statistics

Results

Sample information and PCR amplification of CYP2D6 gene fragment

Cds Chain Polymorphism And Allelic Forms Of Cyp2d6 Gene

Cyp2d6 Genotypes And Enzyme Activity

Discussion

Conclusions

Abbreviations

Declarations

References

Status:

Journal Publication

Version 1