Sample information and PCR amplification of human CYP2D6 gene
A total of 75 SR cases of P. vivax were enrolled, including 71 cases of one suspected relapsed event, 2 cases of two suspected relapse events, and two case of three suspected relapsed events. The geographic information of 75 NR cases is listed in Table 1. The male-female ratio was 2.66:1, and the proportions of cases imported from Myanmar, Africa countries, Laos and Yunnan were 96.7% (145/150), 1.3% (2/150), 0.7% (1/150) and 1.3% (2/150), respectively. The odds ratio of relapsed was calculated in different age groups and genders. Statistical significance was determined in age groups of 5-20 years (OR = 0.380; 95% CI: 0.986~0.146) and 21-60 years (OR = 2.471:95% CI: 5.417~1.127). No significant correlation between other age groups or gender and the relapsed of vivax malaria was found.
Table 1. Information of 150 vivax malaria cases for amplification of CYP2D6 gene exon1-9 fragments
|
Variable
|
Total (n, F%)
|
SR group (n, F%)
|
NR group (n, F%)
|
Total
|
150(100)
|
75(50)
|
75(50)
|
Gender
|
|
|
|
Male
|
109(72.7)
|
58(77.3)
|
51(68.0)
|
Female
|
41(27.3)
|
17(22.7)
|
24(32.0)
|
Age (in years)
|
|
|
|
0-4
|
4(2.7)
|
3(4.0)
|
1(1.3)
|
5-20
|
23(15.3)
|
7(9.3)
|
16(21.4)
|
21-60
|
114(76.0)
|
63(84.0)
|
51(68.0)
|
above 60
|
9(6.0)
|
2(2.7)
|
7(9.3)
|
Malaria relapse
|
|
|
|
1 episode
|
71(47.4)
|
71(94.6)
|
--
|
2 episodes
|
2(1.3)
|
2(2.7)
|
--
|
3 episodes
|
2(1.3)
|
2(2.7)
|
--
|
Infection sourcea
|
|
|
|
Myanmar
|
145(96.7)
|
70(93.3)
|
75(100.0)
|
Africa
|
2(1.3)
|
2(2.7)
|
--
|
Laos
|
1(0.7)
|
1(1.3)
|
--
|
Yunnan indigenous
|
2(1.3)
|
2(2.7)
|
--
|
n: number of cases; F: Frequency; a Identified by epidemiological investigation
|
PCR amplification of two segments of human CYP2D6 gene, including exons1-4 and exons5-9, was performed by using 156 blood samples of relapsed cases and 75 blood samples of non-relapsed cases. The amplification products showed clear bands at >2000bp, which were considered as the target bands .
Polymorphism analysis of CYP2D6 gene coding region
Locus polymorphism of CDS chain
The PCR amplification products of CYP2D6 gene from the SR group and NR group were trimmed, and the CDS chains containing the complete exon1-9 (total length = 1491bp) were obtained from all samples. A total of 150 CDS chains (Genbank accession number: MT339044-MT339193) were selected from the 75 SR cases and 75 NR cases, and were then compared with wild-type sequence (NC: 000022.11). Base substitutions at 12 loci, such as c.31 and c.100, were determined (Table 2). All the mutation loci were found in Genbank and the corresponding ID, was regarded as result normal, 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 two groups differences in mutation allele frequency at the 12 SNPs (Table 2). In the SR group, the mutation allele frequency was the highest in c.1457 G>C (80.7%,121/150), followed by c. 408 G>C (73.3%,110/150). In the NR group, on the contrary, the mutation allele frequency was the highest in c. 408 G>C (80.0%,120/150), followed by c. 1457 G>C (74.7%,112/150). Regarding the two mutation alleles, a non- significant trend (x2=1.863 and x2=1.557,P > 0.05) for difference in frequency distribution among groups SR and NR was detected. However, the frequency differences of the other two mutation alleles c. 100C>T and c. 886C>T between the two groups were statistically significant. The variant c. 100C>T allele showed lower frequency in SR group (x2=9.039,P < 0.05). But the variant c. 886C>T allele showed higher frequency in SR group (x2=8.165,P < 0.05).
Table 2. Polymorphism and mutation allele frequency comparison of SR group and NR group in the coding domain of CYP2D6 Genes from
1st aa to 497th aa
|
SR group
|
|
NR group
|
|
Pc
|
|
SNP ID in Genbank
|
Loci
|
Codon changea
|
Amino acid change
|
No.(n=150,%b)
|
|
Loci
|
Codon changea
|
Amino acid change
|
No. (n=150,%b)
|
|
c.31
|
GTG>ATG
|
V11M
|
1(0.7)
|
|
--
|
--
|
--
|
--
|
|
--
|
|
rs769258
|
c.100
|
CCA>TCA
|
P34S
|
66(44.0)
|
|
c.100
|
CCA>TCA
|
P34S
|
92(61.3)
|
|
0.003d(S)
|
|
rs1065852
|
c.271
|
CTG>ATG
|
L91M
|
4(2.7)
|
|
c.271
|
CTG>TTG
|
L91L
|
2(1.3)
|
|
0.680d(NS)
|
|
rs28371703
|
c.281
|
CAC>CGC
|
H94R
|
4(2.7)
|
|
--
|
--
|
--
|
--
|
|
--
|
|
rs28371704
|
c.294
|
ACC>ACG
|
T98T
|
4(2.7)
|
|
c.294
|
ACC>ACG
|
T98T
|
1(0.7)
|
|
0.367d(NS)
|
|
rs28371705
|
c.297
|
GCC>GCT
|
A99A
|
1(0.7)
|
|
--
|
--
|
--
|
--
|
|
--
|
|
rs200269944
|
c.336
|
TTC>TTT
|
F112F
|
61(40.7)
|
|
c.336
|
TTC>TTT
|
F112F
|
76(50.7)
|
|
0.082d(NS)
|
|
rs1081003
|
c.408
|
GTG>GTC
|
V136V
|
110(73.3)
|
|
c.408
|
GTG>GTC
|
V136V
|
120(80.0)
|
|
0.172d(NS)
|
|
rs1058164
|
c.505
|
GGT>AGT
|
G169S
|
2(1.3)
|
|
--
|
--
|
--
|
--
|
|
--
|
|
rs5030865
|
--
|
--
|
--
|
--
|
|
c.801
|
CCC>CCA
|
P267P
|
2(1.3)
|
|
--
|
|
rs28371718
|
c.886
|
CGC>TGC
|
R296C
|
29(19.3)
|
|
c.886
|
CGC>TGC
|
R296C
|
12(8.0)
|
|
0.004d(S)
|
|
rs16947
|
c.1457
|
AGC>ACC
|
S486T
|
121(80.7)
|
|
c.1457
|
AGC>ACC
|
S486T
|
112(74.7)
|
|
0.212d(NS)
|
|
rs1135840
|
a DNA base highlighted in bold indicates the occurrence of SNP; n: number of chromosomes; b:Detection frequency, denominator is 150; cAnalysis of mutation
allele frequency difference between two groups; dChi-square test; NS: not significant; S: significant (P<0.05).
|
Polymorphism of haplotypes
A total of 24 CYP2D6 haplotypes (Hap_1~Hap_24) were identified amongst the 150 vivax malaria samples; 17 haplotypes were observed in the sequences of SR group (π=0.0015, He= 0.8191). 15 haplotypes were observed in the sequences of NR group (π = 0.0014 and He = 0.8065). 8 haplotypes, including Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_14 and Hap_17, were found in both SR and NR group. Haplotype and frequency were shown in Fig. 1. Hap_3 was wild-type sequence, and the rest were mutant sequence. Hap_2 accounted for the largest proportion of 36.6% (55/150), followed by 15.3% for Hap_3 (23/150), 8.6% for Hap_4 (13/150), 6.6% for Hap_5 (10/150), 8.0% for Hap_6 (12/150), 6.6% for Hap_7 (10/150) and 4.6% for Hap_21 (7/150). The proportions of Hap_1, Hap_8, Hap_9, Hap_10, Hap_12, Hap_13, Hap_15, Hap_16, Hap_18, Hap_19, Hap_20, Hap_22, Hap_23, and Hap_24 were as low as 0.7% (1/150). The proportions of Hap_11, Hap_14 and Hap_17 haplotypes were 1.3% (2/150).
Fig 1. 9 exons are indicated by numbered boxes with DNA polymorphisms indicated on top. Predicted amino acid changes are indicated below; no change is synonymous mutation. SR: Suspected relapsed group; NR: Non-relapsed group.
Polymorphism of diplotypes
The results of allelic form recognition to mutation loci showed that the CYP2D6 gene in 150 cases could be defined as 33 diplotypes (Table 3). Among them, only one diplotype (A_l) was non-mutation homozygous in 12 SNPs, the other 32 diplotypes showed mutant heterozygous at least one locus (A_21). In the 32 mutant diplotypes, the allelic form of 96 mutation loci accumulated in 12 loci were mutant homozygous accounted for 72.9% (70/96). Although there was no regularity in the number of mutation loci between the SR group and the NR group, the diplotype A_17 showed mutations at as many as 7 loci (c. 100, c. 271, c. 281, c. 294, c. 297, c. 408 and c. 1457), and the diplotype came from a patient suspected of relapsed vivax malaria for 3 times. In addition, The diplotypes A_10 and A_12 showed mutant homozygous at c.408, c.1457 and c.408, c.886, c.1457, respectively, and that the frequency of SR group was significantly higher than that of NR group (Fisher's Exact Test,P < 0.05) (Table 3). But the frequency of diplotype A_30 in the NR group was significantly higher than that in the SR group (Fisher's Exact Test,P < 0.05) (Table 3).
Table 3 Analysis the diplotypes in SR group and NR group
|
Haplotypes
|
Diplotypes
|
Mutation loci and allelic forma
|
|
Case groups
|
Pb
|
c.31
|
c.100
|
c.271
|
c.281
|
c.294
|
c.297
|
c.336
|
c.408
|
c.505
|
c.801
|
c.886
|
c.1457
|
|
SR group
No. (n=75, F/%)
|
NR group No. (n=75, F/%)
|
|
NC_000022.11
|
--
|
G/G
|
C/C
|
C/C
|
A/A
|
C/C
|
C/C
|
C/C
|
G/G
|
G/G
|
C/C
|
C/C
|
G/G
|
|
--
|
--
|
--
|
Hap_3
|
A_1
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
|
8(10.7)
|
15(20.0)
|
0.113c(NS)
|
Hap_1
|
A_2
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/A
|
-/-
|
T/T
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_2
|
A_3
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
27(36.0)
|
24(32.0)
|
0.605c(NS)
|
A_4
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
0
|
1(1.3)
|
--
|
A_5
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
-/C
|
-/-
|
-/-
|
-/-
|
-/C
|
|
0
|
1(1.3)
|
--
|
A_6
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
-/-
|
-/C
|
|
1(1.3)
|
1(1.3)
|
--
|
Hap_4
|
A_7
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
0
|
2(2.7)
|
0.497d(NS)
|
A_8
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
2(2.7)
|
6(8.0)
|
0.276c(NS)
|
A_9
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
-/C
|
|
0
|
3(4.0)
|
0.245d(NS)
|
Hap_5
|
A_10
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
8(10.7)
|
1(1.3)
|
0.039c(S)
|
A_11
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
-/C
|
|
0
|
1(1.3)
|
--
|
Hap_6
|
A_12
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
T/T
|
C/C
|
|
8(10.7)
|
0
|
0.003d(S)
|
A_13
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/T
|
C/C
|
|
2(2.7)
|
2(2.7)
|
--
|
Hap_7
|
A_14
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
|
4(5.4)
|
1(1.3)
|
0.363c(NS)
|
A_15
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/C
|
|
3(4.0)
|
2(2.7)
|
--
|
Hap_8
|
A_16
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
T/T
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_9
|
A_17
|
-/-
|
T/T
|
A/A
|
G/G
|
G/G
|
-/T
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_10
|
A_18
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_11
|
A_19
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
-/C
|
|
2(2.7)
|
0
|
0.497d(NS)
|
Hap_12
|
A_20
|
-/-
|
T/T
|
A/A
|
G/G
|
G/G
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_13
|
A_21
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
T/T
|
C/C
|
|
1(1.3)
|
0
|
--
|
Hap_14
|
A_22
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
-/-
|
|
1(1.3)
|
1(1.3)
|
--
|
Hap_15
|
A_23
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/T
|
-/-
|
|
1(1.3)
|
0
|
--
|
Hap_16
|
A_24
|
-/A
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/C
|
|
1(1.3)
|
0
|
--
|
Hap_17
|
A_25
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
|
0
|
1(1.3)
|
--
|
A_26
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
|
1(1.3)
|
0
|
--
|
Hap_18
|
A_27
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
-/-
|
-/-
|
|
0
|
1(1.3)
|
--
|
Hap_19
|
A_28
|
-/-
|
-/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/-
|
-/-
|
|
0
|
1(1.3)
|
--
|
Hap_20
|
A_29
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
A/A
|
T/T
|
C/C
|
|
0
|
1(1.3)
|
--
|
Hap_21
|
A_30
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
C/C
|
-/-
|
-/-
|
-/T
|
C/C
|
|
0
|
7(9.4)
|
0.007d(S)
|
Hap_22
|
A_31
|
-/-
|
-/-
|
T/T
|
-/-
|
-/G
|
-/-
|
T/T
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
|
0
|
1(1.3)
|
--
|
Hap_23
|
A_32
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
-/C
|
-/-
|
-/-
|
-/T
|
-/C
|
|
0
|
1(1.3)
|
--
|
Hap_24
|
A_33
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
-/-
|
T/T
|
C/C
|
-/-
|
-/-
|
-/-
|
C/C
|
|
0
|
1(1.3)
|
--
|
a DNA base highlighted in bold indicates the occurrence of SNP, non-mutation is indicated by lines; n: number of cases; F: Frequency; bAnalysis of haplotype frequency difference between two groups; cChi-square test; dFisher's Exact Test; NS: not significant; S: significant (P<0.05).
|