Genetic Polymorphism of Cyp2d6 Gene Coding Region and Association Between Them With Vivax Malaria Relapse in Yunnan Province, China

doi:10.21203/rs.3.rs-77457/v1

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Genetic Polymorphism of Cyp2d6 Gene Coding Region and Association Between Them With Vivax Malaria Relapse in Yunnan Province, China

https://doi.org/10.21203/rs.3.rs-77457/v1

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published 20 Mar, 2021

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Background

Primaquine is one of two medications able to effectively eliminate the hypnozoites of P. vivax, and therefore has been recommended by World Health Organization (WHO) as the anti-relapsing drug for vivax malaria patients. However, the reduced CYP2D6 enzyme activity caused by gene mutations has been considered to result in the failure of primaquine relapse vivax malaria. Based on the analysis of CYP2D6 gene polymorphisms in vivax malaria cases the current study aims to determine the association between human CYP2D6 polymorphism and the relapse of vivax malaria.

Methods

Blood samples of vivax malaria cases who received "chloroquine/primaquine eight-day therapy" from 2014 to 2018 in Yunnan Province were collected. The suspected relapsed cases were determined by epidemiology and gene sequence alignment. Human genomic DNA was extracted from the blood samples, nine exons of CYP2D6 gene were amplified by polymerase chain reaction (PCR) and PCR products were then sequenced. The coding DNA sequence (CDS) of CYP2D6 gene was obtained by splicing and aligning with the non-mutation reference sequence. The mutation loci, haplotypes (star alleles) and genotypes of CYP2D6 gene were inferred and their association with vivax malaria relapse were analyzed.

Results

A total of 120 blood samples from 45 suspected relapsed cases of vivax malaria and 75 non-relapsed cases were collected and two nested-PCR products (2411bp and 2388bp) containing exon1-4 and exon5-9, were obtained from 119 samples. 119 CDS chains (1491bp length) were formed by splicing and combining sequencing sequences, A total of 12 mutation loci were identified in 238 CDS chains (including 119 chains from direct sequencing and another 119 chains identified from sister chromosome). Among them, the mutation locus at c.886 has the closest relationship with the relapse of vivax malaria (OR=2.167, 95%CI: 1.104~4.252, P<0.05). There were 23 haplotypes (Hap_1~Hap_23) to be identified and be defined as 23 alleles of CYP2D6 gene, Among them, the 5 star alleles (*1, *2, *4, *10 and *39) were confirmed by comparison, and the CYP2D6*10 allele accounted for the most (45.4%, 108/238). the frequency of CYP2D6*2 allele in the SR group was significantly higher than that in the NR group (P<0.05). However, among the defined 24 genotypes, there were 8 genotypes (*4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10) appeared only in the SR group.

Conclusion

Among the numerous mutations of CYP2D6 gene，CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutation at c. 886 locus is most closely related to the relapse of vivax malaria. In addition, there was the genotype *4/*4 with null CYP2D6 enzyme function. These results suggest that Yunnan Province should pay attention to the risk of reduced CYP2D6 enzyme activity on the radical cure therapeutic effect of primaquine

Infectious Diseases

Plasmodium vivax

Relapse

CYP2D6 gene

Coding region

Primaquine

Allele

Genotype

In recent years, the global malaria epidemic has gradually scaled down, yet the death tolls of malaria remain high, and the number of malaria cases in certain areas has rebounded [1]. Meanwhile, multiple technological limitations pose daunting challenges to the elimination of malaria, such as the difficulty in identifying low-density Plasmodium infection [2], the population expansion of drug resistant Plasmodium [3-4], and few new drugs in the antimalarial pipeline [5]. Although Chinese local cases of malaria (without a travel history to malaria epidemic areas outside China within the last 30 days) have not been reported since 2016 [6], China still needs to cope with a large number of imported malaria cases every year. Located in southwest China bordering Myanmar, Laos and other Southeast Asian countries (Supplement 1), Yunnan Province has experienced large-scale malaria epidemic and was once the hardest-hit area of malaria [7]. Yunnan Province is still facing a great risk of imported malaria. And from 2011 to 2018, 50.7% of the imported malaria cases in Yunnan Province were caused by P. vivax [8]. Therefore, the elimination of malaria in Yunnan Province calls for the strict enforcement radical cure treatment to block the transmission of P. vivax.

Primaquine has proven to be effective in eradicating the hypnozoites of P. vivax, and the World Health Organization (WHO) recommends it for the treatment of relapsed malaria (and since 2018, the newly approved tafenoquine) [9]. The Cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6) enzyme in human hepatocyte metabolizes primaquineinto5-hydroxy-primaquine, the component thought to be responsible for killing P. vivax hypnozoites [10-12]. CYP2D6, also known as debrisoquine4-hydroxylase, is an isozyme belonging to the Cytochrome P450 (CYP450) superfamily and an important phase I drug-metabolizing enzyme in the humans. CYP2D6has low capacity and high affinity. Even though CYP2D6 only accounts for 4% of the total P450 enzyme protein in the liver, it is involved with the metabolism of ~30% of commonly used drugs, including primaquine [12]. Studies have found that the heterogeneity of CYP2D6 activity is predominantly governed by its genetic variation [13], and that decreased CYP2D6 isoenzyme activity caused by genetic polymorphisms would obstruct the generation of 5-hydroxy-primaquine, making large doses or repeated use of primaquine necessary to compensate for the declined efficacy of primaquine. Meanwhile, primaquine can cause life threatening hemolysis in patients with Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency, subsequently inflicting dual hazards, including low efficacy of anti-relapse and acute hemolysis, on the patients [9,14-15]. So the patient's G6PD genotype should be identified by care workers prior to receiving anti-relapse treatment of vivax malaria and based on our and others’ findings, it should be considered to test for CYP2D6 genotype and predicted enzyme activity as well [14,16-17].

CYP2D6 enzyme activity is measured mainly through phenotype observation and genotype analysis prediction. Compared with phenotype measurement, prediction method based on CYP2D6 genotype is characterized by the following advantages [18-19]. Firstly, genotypes do not change throughout one's life, and are not affected by environment or physical factors [19-20]. Secondly, genotyping only requires one specimen and can be completed before treatment [21]. In addition, using sequencing to determine the combination form of CYP2D6 alleles is a technique that can be easily obtained at present. Therefore, the use of genotyping method can create more stable results in predicting the phenotype of CYP2D6 enzyme activity [18,22-26]. The prediction is based on the locus status of CYP2D6 gene [13], or the allelic combination form of genotype [27-28]. However, more than 130 CYP2D6 alleles have been defined by the Cytochrome P450 Nomenclature Committee at https://www.pharmvar.org/gene/CYP2D6. The prediction of CYP2D6 enzyme activity by performing genotype analysis needs to be further optimized to enhance accuracy and systematisms [29].

Gene mutations of enzymes related to drug metabolism not only affect the clinical consequences of drug metabolism, but can even lead to individual adverse event [13]. Therefore, understanding the specific CYP2D6 genetic polymorphism in different individuals is the prerequisite to ensure radical treatment of vivax malaria based on personalized primaquine dose [24]. Despite the frequent use of primaquine in malaria endemic areas of Yunnan Province, the systematic survey about genetic polymorphisms of CYP2D6 has been not conducted. In this study, based on the gene sequence method the polymorphism investigation of CYP2D6 gene from vivax malaria cases and the association between human CYP2D6 polymorphisms and relapse of vivax malaria were explored in order to effectively assess the risk of using primaquine to stop malaria transmission or eliminate the source of P. vivax in Yunnan Province.

Ethics statement

The study was approved by the Yunnan Institute of Parasitic Diseases and Ethical Committee. 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. Although there was no risk and the data processing after sample collection was done anonymously, informed consent was obtained.

Subjects and blood samples

The subjects were diagnosed cases of P. vivax in Yunnan Province. Mono-infection of P. vivax was confirmed by both microscopy examination and Plasmodium 18S rRNA gene detection (PCR) by Yunnan Province Malaria Diagnosis Referent Laboratory (YPMDRL). All vivax malaria cases were treated by oral Chloroquine therapy (total 1550 mg) during spending three days, followed by an 8-day course of primaquine (22.5 mg/day). The subjects include two groups of cases: (1) Suspected relapsed cases of vivax malaria (SR group). The SR cases were re-diagnosed as P. vivax infection within 28–180 days after initial diagnosis and who has not been exposed to malaria transmission again between two clinical malaria outbreaks through epidemiological investigation [30]. Besides, the cases were confirmed to be infected by the same P. vivax isolates, when the alignment results of sequencing sequences of csp (circumsporozoite surface protein) and msp-1 (merozoite surface protein 1) gene showed not variation, both Variable sites and He being 0, between isolates from initial and recurrence vivax malaria cases. The SR cases were confirmed from the vivax malaria cases diagnosed in Yunnan Province from 2014 to 2018. (2) Non-relapsed cases of vivax malaria (NR group). The NR cases were selected by simple random sampling from the 143 cases of vivax malaria diagnosed in 2018, who were followed up for one year and had no recurrent history of P. vivax. The infection sources of P. vivax were confirmed according to epidemiological investigation in all the subjects: those without a travel history to epidemic areas outside Yunnan Province within the last 30 days before the onset of malaria were defined as local cases; those have history of travelling to epidemic regions, such as Myanmar and Africa, were regarded as imported cases.

The blood samples of all the subjects were the rest part of the routine diagnostic work-up provided by the Centers for Disease Control and Prevention in Yunnan Province. The patient's dried blood spots (DBS) samples for genetic analysis were derived from 0.6 ml of venous blood, which were then put in a drying tube and stored at -80 ℃ before DNA extraction.

Extraction of human genomic DNA

Three dried blood spots (diameter = 5 mm) were made, and human genomic DNA was extracted according to the manufacturer’s instructions of the QIAgen Mini Kit (Germany, QIAamp Company's DNA Mini Kit), according to the manufacture’s instruction. The extracted DNA was stored at -20 ℃.

PCR amplification of CYP2D6 gene fragment

The primers of CYP2D6 for polymerase chain reaction (PCR) amplification were designed by using GenBank (https://www.ncbi.nlm.nih.gov/gene/), reference sequence (ID：NC_000022.11) was used as template, following the reaction condition in previous studies [23,31]. The primers for first-round nested PCR covering the coding region of CYP2D6 gene exons1~4 were: 5'-CCAGTGACAGATAAGGGTGC-3' and 5'-GACGTGGATAGGAGGTACAGAG-3'; the primers for second-round nested PCR were: 5'-GGTGACTTCTCCGACCAGG-3' and 5'-TTCCCAAACCCATCTATGC-3'. The final amplification region was 42131088~42128678, and the expected fragment length of the product was 2411 bp. The primers that cover exons 5~9 of CYP2D6 gene were: 5'-GCCGACTGAGCCCTGGGAGGTAGGTA-3' and 5'-GCTGGGGCCTGAGACTT-3. The amplification area was 42126035~42128422, and the amplification products had an expected fragment length of 2388 bp. For all the PCR reaction systems, we used 2.6μl DNA template, 14.0μl 2 × Taq PCR hybrid system (QIAGEN, Germany), 0.7μl upstream primer (20umol / L), 0.7μl downstream primers (20umol / L). The total volume was adjusted to 25.0μl with ddH₂O. PCR reaction conditions were clarified as follows: 92 to 94 ° C for 2-5 mins; 92 to 94 ° C for 10-30s, 50 to 56 ° C for 15-30s, 68 to 72 ° C for 2 to 3 min, 35 cycles; 68 to 72 ° C for 7 mins. The triplicated parallel repetition was adopted for each PCR reaction. The amplified products were observed using 1.5% agarose gel electrophoresis. The positive amplification products were sent to Shanghai Meiji Biomedical Technology Co, Ltd. for sequencing by using the dideoxy chain-termination method. Only the sequences that showed identical results in at least two tests were used for subsequent analysis.

Mutation analysis of the coding region in CYP2D6 gene

The sequencing results were aligned by using DNAStar v5.10 and BioEdit v7.0.9.0 software. The obtained all DNA sequences were assessed by using the Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) in the NCBI platform. Sequences with identifications equals 100% and Query cover above 99% were considered as CYP2D6 gene sequence of human, this chain was identified as the first DNA sequence of each sample in this study. The second DNA sequence from sister chromosome was based on the first one, which supplements the base pairs that have been shown in the sequencing peak but not been read by the sequencer [32]. The DNA sequence of exon1~9 region of CYP2D6 gene was determined and spliced into the CDS of CYP2D6 gene in the order from exon1 to exon9.MEGA v5.04 software was used to align the CDSs with the non-mutation sequence to confirm the loci of missense mutation and synonymous mutation. The defined mutation loci were queried for the corresponding ID in Genbank. The diploid of each mutation loci was identified by checking the sequencing peak maps (Supplement 2). DnaSP v6.11.01 software was used to identify haplotypes of all CDSs, and calculate the parameters of CDSs diversity index (π) and expected heterozygosity (He). Every haplotype of CDSs chain was given the allele type according to the criteria from Human Cytochrome P450 Allele Nomenclature Committee Database (NM_000106.5) [33]. Those Haplotypes not matched with known CYP2D6 alleles were grouped into the “other” category when calculating odds ratio. The genotype of each sample was composed of the sister CDS chains. The all suballeles under every star (*) allele were combined when calculating the frequency of allele or genotype.

SPSS software (version 21.0; IBN; Chicago; IL) was used to conduct chi-square test on the frequency of genotypes and mutation loci co-existed in SR and NR cases. The odds ratio (OR) value of genotypes and mutation loci with statistical significance were calculated by comparing to the relapse rate of P. vivax. The significance level was set as P< 0.05; OR ˂ 1 indicates that the haplotype is a protective factor, otherwise a risk factor.

Sample information and PCR amplification of human CYP2D6 gene

A total of 45 SR vivax malaria cases were confirmed by epidemiology and gene sequence alignment (Supplement 3), including 44 cases with one suspected relapsed event, 1 case with three suspected relapsed events. The geographic information of 45 SR cases and 75 NR cases is listed in Table 1. The male-female ratio was 3:1, and the proportions of cases imported and infected from Myanmar, Africa countries and Yunnan were 97.5% (117/120), 0.8% (1/120) and 1.7% (2/120), respectively. Most of the patients presented only one suspected relapsed episode (97.8%)

Table 1. Information of 120 vivax malaria cases for amplification of CYP2D6 gene exon1-9 fragments
Variable	Total (n, F%)	SR group (n, F%)	NR group (n, F%)
Total	120(100.0)	45(37.5)	75(62.5)
Gender
Male	90(75.0)	32(71.1)	51(68.0)
Female	30(25.0)	13(28.9)	24(32.0)
Age (in years)
0-4	3(2.5)	2(4.4)	1(1.3)
5-20	19(15.8)	3(6.7)	16(21.4)
21-60	89(74.2)	38(84.5)	51(68.0)
above 60	9(7.5)	2(4.4)	7(9.3)
Malaria relapse
1 episode	44(36.7)	44(97.8)	--
3 episodes	1(0.8)	1(2.2)	--
Infection source^a
Myanmar	117(97.5)	42(93.4)	75(100.0)
Africa	1(0.8)	1(2.2)	--
Yunnan indigenous	2(1.7)	2(4.4)	--
Note: n: number of cases; F: Frequency; ^aIdentified by epidemiological investigation

PCR amplification of two segments of human CYP2D6 gene, including exons1-4 and exons5-9, was performed by using 45 blood samples of SR cases and 75 blood samples of NR cases. The amplification products showed clear bands at ＞2000bp, which were considered as the target bands (Supplement 4). CYP2D6 gene fragments of 75 NR cases and 44 SR cases were successfully amplified, the DNA extract from one SR case did not amplify the PCR product of CYP2D6 gene.

Locus polymorphism of CDS chains and their association with vivax malaria relapse

A total of 119 PCR amplification products of CYP2D6 gene were sequenced. The 119 DNA sequencing sequences were trimmed as the first CDS chains (Genbank accession number: MT339075-MT339193) containing the complete exon1-9 (total length = 1491bp) from all samples. Another 119 sister CDS chains were deduced from the sequencing peak maps. Base substitutions at 12 loci, such as c.31 and c.100, were determined by comparing with non-mutation sequence (NC: 000022.11) in. 238 CDS chains (Table 2). All the mutation loci were given the corresponding ID from Genbank and no new mutation loci were found. The proportions of third-base and first-base substitution in the codon triplet were 41.7% (5/12), and the proportion of second-base substitution was 16.6% (2/12). 7 missense mutation loci and 5 synonymous mutation loci were determined. Of these mutation loci, the SR cases accounted for 91.7% (11/12), whereas the mutation loci of NR cases accounted for 66.7% (8/12) (Table 2).

Comparison of the different mutation allele at the 12 single nucleotide polymorphisms (SNPs) in two groups differences (Table2). In the SR group, the frequency of mutation allele was the highest at c.1457 G>C (85.2%，75/88), and c. 408 G>C (85.2%, 75/88). In the NR group, the highest frequency was at c. 408 G>C (76.0%，114/150), followed by c. 1457 G>C (72.0 %, 108/150). Eight mutation loci found both in SR group and NR group were selected to analyze the association between loci mutation and vivax malaria relapse. Among them, only the association of c.886 (OR=2.167; 95% CI: 1.104~4.252) showed statistical significance (P ˂0.05). Hence, it is suggested that the mutation at c.886 locus could increase the relapse risk of 2.167-fold higher than other SNPs. The remaining 7 mutation loci of c.100, c.271, c.294, c.336, c.408, c.886, c.1457 may not be associated with the relapse of vivax malaria (OR=0.664-3.524, P>0.05) (Table 2).

Table 2. Polymorphism of CDS mutation in CYP2D6 gene between different vivax malaria cases
SR group				NR group					OR^c	95%CI		P	SNP ID in Genbank
Loci	Codon changea	Amino acid change	No. (n=88，%^b1)	Loci	Codon changea	Amino acid change	No. (n=150，%^b2)		OR^c	Upper limit	Low limit	P	SNP ID in Genbank
c.31	GTG＞ATG	V11M	1(0.7)	--	--	--		--	--	--	--	--	rs769258
c.100	CCA＞TCA	P34S	47(53.4)	c.100	CCA＞TCA	P34S		95(63.3)	0.664	0.389	1.133	0.132^d(NS)	rs1065852
c.271	CTG＞ATG	L91M	4(4.5)	c.271	CTG＞TTG	L91L		2(1.3)	3.524	0.632	19.647	0.272^d(NS)	rs28371703
c.281	CAC＞CGC	H94R	4(4.5)	--	--	--		--	--	--	--	--	rs28371704
c.294	ACC＞ACG	T98T	4(4.5)	c.294	ACC＞ACG	T98T		1(0.7)	7.095	0.780	64.521	0.122^d(NS)	rs28371705
c.297	GCC＞GCT	A99A	1(1.4)	--	--	--		--	--	--	--	--	rs200269944
c.336	TTC＞TTT	F112F	42(47.7)	c.336	TTC＞TTT	F112F		81(54.0)	0.778	0.459	1.318	0.350^d(NS)	rs1081003
c.408	GTG＞GTC	V136V	75(85.2)	c.408	GTG＞GTC	V136V		114(76.0)	1.822	0.907	3.661	0.089^d(NS)	rs1058164
c.505	GGT＞AGT	G169S	2(2.3)	--	--	--		--	--	--	--	--	rs5030865
--	--	--	--	c.801	CCC＞CCA	P267P		2(1.3)	--	--	--	--	rs28371718
c.886	CGC＞TGC	R296C	22(23.9)	c.886	CGC＞TGC	R296C		20(13.3)	2.167	1.104	4.252	0.023^d(S)	rs16947
c.1457	AGC＞ACC	S486T	75(85.2)	c.1457	AGC＞ACC	S486T		108(72.0)	1.416	0.819	2.447	0.212^d(NS)	rs1135840
Note: ^aDNA base highlighted in bold indicates the occurrence of SNP; n: number of chromosomes; ^b1Detection frequency, denominator is 88; ^b2Detection frequency, denominator is 150; ^cThe OR value of mutation loci were calculated by comparing to the relapse rate of P. vivax; ^dChi-square test; NS: not significant(P>0.05); S: significant(P<0.05).

Polymorphism of haplotypes

A total of 23 haplotypes (Hap_1～Hap_23) were identified amongst the 238 CDSs of CYP2D6 gene. There were 9 haplotypes only observed in the sequences of SR group (π=0.0015, He= 0.821) and 6 haplotypes only found in the sequences of NR group (π = 0.0014 and He = 0.760). These 8 haplotypes, including Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_13 and Hap_14, were found in both SR and NR group. The compositions of every haplotype were shown in Fig1. Hap_3 did not contain mutation loci, and the rest haplotypes contained different mutation loci.

Alleles /genotypes and their association with vivax malaria relapse

Among the 23 haplotypes, 11 haplotypes were defined as known 11 suballeles, such as *1.001,*1.011,*2.001,*2.004,etc., under 5 star alleles(*1, *2, *4, *10 and *39), and the other 12 haplotypes without alleles criteria were customized as from *m to *x. CYP2D6*10 alleles were the most frequent accounting for 45.4% (108/238), next *1 alleles for 19.7% (47/238) and *39 alleles for 11.8% (28/238) (Table 3). In addition, although CYP2D6*10 allele was the most common allele both in SR group and NR group, which accounted for 38.6% (34/88) and 49.3% (74/150), respectively, but the difference was not statistically significant (x²= 2.560, P> 0.05) (Table 3). Different from the above result, the frequency of CYP2D6*1 allele in the NR group was significantly higher than in the SR group (x²=6.193, P<0.05) and the frequency of CYP2D6*2 allele in the SR group was significantly higher in the NR group (x²=16.177, P<0.05) (Table 3).

In terms of genotypes, a total of 32 genotypes defined at the suballele level had be merged into 24 genotypes at star allele level (Supplement 5). Among 24 genotypes, the frequency of *10/*10 genotype was the highest in both the SR group and the NR group, 27.3% (12/44) and 32.0% (24/75) (Supplement 5), respectively. Moreover, the 8 genotypes of *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n and *v/*10 only appeared in the SR group, in which genotypes the *4/*4 was found from one case with three episodes of vivax malaria. The association between 9 genotypes both in SR group and NR group and relapse vivax malaria was analyzed, including *1/*1, *2/*2, *1/*2, *1/*10, *10/*10, *10/*39, *39/*39, *10/*s and *39/*s. The OR value did not show statistically significant (P> 0.05) (Table 3). It is suggested that the difference of CYP2D6 genotypes in this study had no effect on the relapse of vivax malaria.

Table 3 Analysis of the association between alleles and genotypes of CYP2D6 gene and relapse of vivax malaria
a. Alleles
Alleles	Total No (n1=238, F/%)		Case groups					x²		P
			SR group No. (n1=88, F/%)		NR group No. (n1=150, F/%)
*1	47(19.7)		10(11.4)		37(24.7)			6.193		0.013^b(S)
*2	17(7.1)		14(15.9)		3(2.0)			16.177		0.000^b(S)
*4	3(1.3)		3(3.4)		0			2.802		0.094^b(NS)
*10	108(45.4)		34(38.6)		74(49.3)			2.560		0.110^b(NS)
*39	28(11.8)		15(17.0)		13(8.7)			3.751		0.053^b(NS)
*s^a	20(8.4)		5(5.7)		15(10.0)			1.344		0.246^b(NS)
Other^a	15(6.3)		7(8.0)		8(5.3)			1.766		0.184^b(NS)
b. Genotypes
Genotypes	Case groups			OR^c		95%CI			P
	SR group No. (n2=44, F/%)	NR group No. (n2=75, F/%)				Upper limit	Low limit
1/1	1(2.3)	11(14.7)		0.135		0.017	1.087		0.064^b(NS)
2/2	4(9.1)	1(1.3)		7.400		0.800	68.463		0.118^b(NS)
1/2	4(9.1)	1(1.3)		7.400		0.800	68.463		0.118^b(NS)
1/10	2(4.5)	12(16.0)		0.250		0.050	1.174		0.115^b(NS)
10/10	12(27.3)	24(32.0)		0.797		0.350	1.873		0.588^b(NS)
10/s^a	1(2.3)	1(1.3)		1.721		0.105	28.220		1.000^b(NS)
10/39	5(11.4)	3(4.0)		3.077		0.698	13.563		0.242^b(NS)
39/39	2(4.5)	1(1.3)		3.524		0.310	40.032		0.636^b(NS)
39/s^a	2(4.5)	7(9.3)		0.463		0.092	2.332		0.552^b(NS)
Note: n1=number of chromosomes; n2: number of cases; F: Frequency; Other:m, n, o, p, q,r,t, u, v, w and x; ^aThey could not meet the allele inclusion criteria provided by the Allele Nomenclature Committee[33]; ^bChi-square test; NS: not significant (P>0.05); ^cThe OR value of genotypes were calculated by comparing to the relapse rate of P. vivax*; S: significant (P<0.05); NS: not significant (P＞0.05)

Located on chromosome 22q13.1, CYP2D6 gene (4383bp in length) consists of 8~9 exons and 7~8 introns. Its CDS is 1338~1491bp in length and encodes 446～497 amino acids. The heterogeneity of CYP2D6 monooxygenase activity varies from complete dysfunction to ultra-rapid enzyme metabolism, depending on the various genotypes of CYP2D6 [14]. Among the 150 identified alleles, multiple mutations such as CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*6 are considered to represent null CYP2D6 enzyme activity [31,34].

To our knowledge, the current study is the first one that attempts to analyze mutation polymorphism in the whole coding region of human CYP2D6 gene to Yunnan Province, thereby verifying its possible association with thwarted CYP2D6 enzyme activity in relapsed vivax malaria patients after primaquine treatment. In this study, the mutation at c. 886 locus of CYP2D6 gene was calculated to increase the vivax malaria relapse risk of 2.167-fold (P＜0.05). Combined with the observation by Wang et al. [39] and Gaedigk et al. [40], that the base substitution at c. 886 locus will lead to the decrease of CYP2D6 enzyme activity. It is inferred that one of the reasons for the relapse of vivax malaria may be relate to the decrease of CYP2D6 enzyme activity caused at the c.886.mutation of CYP2D6 gene in Yunnan Province

Among the 23 alleles defined from 23 haplotypes in all the CDSs chains, the proportion of CYP2D6*10 reached as high as 45.4% (108/238). The composition of CYP2D6 gene alleles is the same as that found by Qian et al. [32] in Chinese Han population, also consistent with previous studies which reported that CYP2D6*10 allele is most frequently detected in Asian populations [31, 35-36]. Although the frequencies of CYP2D6*10 allele defined from SR group and NR group did show significant different, but the frequency of CYP2D6*10 allele was the highest in both groups, and CYP2D6*10 has been an allele leading to the decrease of CYP2D6 enzyme activity. It is suggested that the danger. of vivax malaria infection reservoir should be ignored under wildly existing CYP2D6*10 allele which results in for primaquine not to effectively kill P. vivax hypnozoite of vivax malaria cases in Yunnan Province.

On the other hand, the CYP2D*2 allele was more common in the suspected relapsed patients in this study. This result is not in line with that of Brasil et al. [30], which reported that the detected frequency of CYP2D*2 in the relapsed patients was significantly higher in the non-relapsed patients. Such very different results might be attributed to the different subjects chosen between two studies, the subjects in this study were vivax malaria cases who mainly settled in the traditional malaria endemic areas in Yunnan Province, and the long-term malaria epidemic is thought to result in positive screening on human defective genes [41-43]. Moreover, in this study all of alleles were identified only based on the mutations of CYP2D6 gene exon regions, but it is still different from the allelic recognition including intron mutations [33,44]. Therefore, the homogeneity of CYP2D6*2 allele in the two studies may be the reason why the results between research display different. Although the CYP2D6*2 allele is generally considered to be an allele of CYP2D6 normal enzyme activity, the multiple mutation loci making up CYP2D6*2 contain the mutation at c. 886 locus which has the effect of reducing the activity of CYP2D6 enzyme [39-40]. Therefore, this study may further support the complexity of CYP2D6*2 allele effect on CYP2D6 enzyme activity change [39-40,44-45].

As expected, there were eight genotypes to be found only in the SP group, such as genotype *4/*4, *2/*39, etc., rather than in all vivax malaria cases in this study, and the association between different genotypes of CYP2D6 gene and the relapse of vivax malaria was not found, too. However, in view of the reality, that the genotype *10/*10 still accounted for the largest proportion (30.3%) in the study sample, and found the non-functional genotype (*4/*4) [33] only in SR group. It is once again suggested that Yunnan Province should not ignore the risk of decline in therapeutic efficacy of primaquine caused by the decrease of CYP2D6 enzyme activity in malaria endemic areas. In addition, the amplification failure case in this study needs to be further verified whether the really existing the full gene deletion (*5/*5) [33], or not the other factors resulting in the failure of PCR amplification for CYP2D6 gene.

However, this study has been some limitations. Firstly, the sample size is not large enough, and the detected low-frequency alleles (*4, *m-*x, Supplement 5) of CYP2D6 genes cannot be effectively analyzed. Secondly, only the possibility of P. vivax re-infection was excluded in identification of the relapsed vivax malaria patients, but other confounding factors which may also influence on the killing effect for the P. vivax hypnozoites under using primaquine, such as no excluding out the effect from primaquine-resistance Plasmodium, the quality of primaquine, and the exposure experience to other inhibitors in the CYP2D6 enzyme activity. Therefore, this study could not infer the causal relationship between the recurrence of vivax malaria and the mutation of CYP2D6 gene. Finally, although the whole coding region polymorphism of CYP2D6 gene was detected and analyzed in this study, but still lacked the research on the others factors, such as the mutation in intron region and the copy number of CYP2D6 gene, which has been considered to also disturb on the activity of CYP2D6 enzyme. So the activity of CYP2D6 cannot be indirectly predicted through CYP2D6 genotype in this study. In view of this deficiency, the research team will introduce multiplex ligation-dependent probe amplification (MLPA) and other methods to improve the accurate of identification between different human CYP2D6 genotypes and the effect of prediction for CYP2D6 enzyme activity in future, finally in order to provide a molecular support for the safe and effective application of primaquine in Yunnan Province.

In conclusion, this study preliminarily reveals the polymorphisms of CYP2D6 gene coding region and the associate between them with vivax malaria relapse in the population exposed to primaquine in Yunnan Province. It makes up for the lack about risk assessment when primaquine is used in key malaria endemic areas in China, and provides the following findings: (1) CYP2D6*10 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. (2) The mutation at c. 886 locus of CYP2D6 gene is most closely related to the relapse of vivax malaria (OR=2.167, P<0.05), and the frequency of CYP2D6*2 allele containing this mutation locus was significantly higher in SR (P<0.05). (3) The *4/*4 genotype with null CYP2D6 enzyme function were only found in the SR cases. These results suggest that it could exist the risk that the radical cure effect on vivax malaria cases may be reduced under using primaquine due to the damage of CYP2D6 enzyme activity in Yunnan Province.

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

DOT directly observed therapy

CDC Center for Disease Control

DBS Dried blood spots

NR non-relapsed cases of vivax malaria

SR suspected relapsed cases of vivax malaria

csp circumsporozoite surface protein

msp-1 merozoite surface protein 1

Hap Haplotype

π diversity index

He expected heterozygosity

OR odds ratio

CI confidence interval

MPLA Multiplex ligation-dependent probe amplification

SNP single nucleotide polymorphism

Acknowledgements

We appreciate the support provided by the Centers for Disease Control and Prevention in states/cities and counties such as Dehong, Baoshan, Kunming, Pu’er, Lincang, Dali, Nujiang, Lijiang, Xishuangbanna, Yuxi, Chuxiong, Honghe, Zhaotong, Diqing, Qujing, and Whenshan.

Authors’ contributions

Herong Huang was responsible for the gene testing, study design, statistics and analysis of the data, and wrote the manuscript; Ying Dong performed coordination of all project, study design, analysis of the data and the manuscript guidance and revision. Shuping Liu participated in the study design; Yan Liu, Yan Deng, Yanchun Xu, Mengni Chen, Canglin Zhang performed the collection blood samples and microscopy examination. All authors read and approved the final draft of the manuscript.

Funding

This study was supported by National Science Foundation, China (No.81660559, 81960579).

Availability of data and material

Not applicable.

Ethics approval and consent to participate

The study was approved by the Yunnan Institute of Parasitic Diseases and Ethical Committee. Genetic testing were performed on stored blood samples obtained as part of the routine diagnostic work-up of patients with fever who were suspected to have malaria. Although there was no risk and the data processing after sample collection was done anonymously, informed consent was obtained. Database access will be restricted by password, and Yunnan Institute Parasitic Diseases will not allow retrieving and saving the personal identification information into the project database. It is committed not to provide information about the patient to any person unrelated to the study.

Consent for publication

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

Competing interests

The authors declare that they have no competing interests.

Author details

¹School of Basic Medical Sciences, Dali University, Dali 667000, China. ²Yunnan 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

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Genetic Polymorphism of Cyp2d6 Gene Coding Region and Association Between Them With Vivax Malaria Relapse in Yunnan Province, China

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