Sample information and PCR amplification of human CYP2D6 gene
A total of 75 relapsed cases of P. vivax were enrolled. Among them 71 cases, 2 cases and 2 cases had one, two and three relapse events, respectively. The basic information of 75 non-relapsed cases is listed in Table 1. The mean age of the 150 cases was 33 years, male-female ratio was 2.66:1, and the proportions of vivax infections in Myanmar, Africa, Laos and Yunnan were 96.7% (145/150), 1.3% (2/150), 0.7% (1/150) and 1.3% (2/150), respectively.
Table 1. Information of 150 vivax malaria cases with successful amplified CYP2D6 gene exon1-9 fragments
|
Years information
|
2014
|
2015
|
2016
|
2017
|
2018
|
Total
|
Number
|
18
|
27
|
20
|
10
|
75
|
150
|
1.Relapse times
|
|
|
|
|
|
|
0
|
0
|
0
|
0
|
0
|
75
|
75
|
1
|
17
|
25
|
19
|
10
|
0
|
71
|
2
|
0
|
1
|
1
|
0
|
0
|
2
|
3
|
1
|
1
|
0
|
0
|
0
|
2
|
2.Agea
|
|
|
|
|
|
|
Maximum
|
62
|
52
|
56
|
62
|
69
|
60
|
minimum
|
21
|
7
|
4
|
2
|
1
|
7
|
Average
|
36
|
35
|
29
|
33
|
33
|
33
|
3.Genderb
|
|
|
|
|
|
|
Male
|
16
|
22
|
14
|
6
|
51
|
109
|
Female
|
2
|
5
|
6
|
4
|
24
|
41
|
4.Infection source c
|
|
|
|
|
|
|
Myanmar
|
16
|
26
|
18
|
10
|
75
|
145
|
Africa
|
1
|
0
|
1
|
0
|
0
|
2
|
Laos
|
0
|
1
|
0
|
0
|
0
|
1
|
Yunnan indigenous
|
1
|
0
|
1
|
0
|
0
|
2
|
a Years old, b Numbers, c Identified by epidemiological investigation
|
156 blood samples of relapsed cases in total were collected, and PCR amplification of two segments of human CYP2D6 gene including exons1-4 and exons5-9 was performed, along with 75 blood samples of non-relapsed cases. The amplification products went through electrophoretic and the clear target bands were observed at 2411 bp and 2388 bp, respectively (Fig.1).
Fig.1 PCR products of CYP2D6 gene fragment. a M: DNA marker; lane 1: blank control of PCR for the first round; lane 2: blank control of PCR for the second round; lane 3-6: The amplification products of exon 1-4 in CYP2D6 gene. b M: DNA marker; lane 1: blank control of PCR; lane 2-5: The amplification products of exon 5-9 in CYP2D6 gene;
Polymorphism analysis of CYP2D6 gene coding region
Locus polymorphism of CDS chain
The PCR amplification products of CYP2D6 gene from the relapsed cases and non-relapsed cases were trimmed, and the CDS chains containing complete exon1-9 (total length = 1491bp) were obtained from all samples. A total of 150 CDS chains were selected from each of the 75 relapsed case and 75 non-relapsed cases, and were compared with wild-type sequence (NC:000022.11). Base replacement at 12 loci, including c.31 and c100 was found (Table 2). The proportion of the third and the first base replacement of the codon triplet was 41.7% (5/12), and the proportion of the second base replacement was 16.6% (2/12). 7 missense mutation loci and 5 synonymous mutation loci were determined. 91.7% (11/12) mutated loci were found in the sequence of relapsed cases, and 66.7% (8/12) mutated loci were determined in the sequence of non-relapsed cases (Table 2).
Table 2. Comparison of loci polymorphisms in 1staa-497thaa coding region of CYP2D6 gene
|
Relapse cases
|
Non-relapse cases
|
Orders
|
Loci
|
BS
|
Codon change
|
Variation
|
|
Orders
|
Loci
|
BS
|
Codon change
|
Variation
|
1
|
c.31
|
G>A
|
GTG>ATG
|
V11M
|
|
1
|
--
|
--
|
--
|
--
|
2
|
c.100
|
C>T
|
CCA>TCA
|
P34S
|
|
2
|
c.100
|
C>T
|
CCA>TCA
|
P34S
|
3
|
c.271
|
C>A
|
CTG>ATG
|
L91M
|
|
3
|
c.271
|
C>T
|
CTG>TTG
|
L91L
|
4
|
c.281
|
A>G
|
CAC>CGC
|
H94R
|
|
4
|
--
|
--
|
--
|
--
|
5
|
c.294
|
C>G
|
ACC>ACG
|
T98T
|
|
5
|
c.294
|
C>G
|
ACC>ACG
|
T98T
|
6
|
c.297
|
C>T
|
GCC>GCT
|
A99A
|
|
6
|
--
|
--
|
--
|
--
|
7
|
c.336
|
C>T
|
TTC>TTT
|
F112F
|
|
7
|
c.336
|
C>T
|
TTC>TTT
|
F112F
|
8
|
c.408
|
G>C
|
GTC>GTG
|
V136V
|
|
8
|
c.408
|
G>C
|
GTC>GTG
|
V136V
|
9
|
c.505
|
G>A
|
GGT>AGT
|
G169S
|
|
9
|
--
|
--
|
--
|
--
|
10
|
--
|
--
|
--
|
--
|
|
10
|
c.801
|
C>A
|
CCC>CCA
|
P267P
|
11
|
c.886
|
C>T
|
CGC>TGC
|
R296C
|
|
11
|
c.886
|
C>T
|
CGC>TGC
|
R296C
|
12
|
c.1457
|
G>C
|
AGC>ACC
|
S486T
|
|
12
|
c.1457
|
G>C
|
AGC>ACC
|
S486T
|
Abbreviations: BS, Base substitution
|
|
|
|
|
|
|
|
|
|
|
|
|
Polymorphism of haplotypes
150 CDS chains of CYP2D6 gene were clustered into 24 haplotypes(Hap_1~Hap_24), as shown in Fig. 2.Among them, Hap_3 is wild-type sequence, and all the rest are 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) and4.6% 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).Haplotypes with one locus mutation included Hap_7, Hap_14, and Hap_15.The two mutations were Hap_5, Hap_11, Hap_16 and Hap_17.The three loci mutation were Hap_4, Hap_6, Hap_10, Hap_18, Hap_19, Hap_22, and Hap_24.The four loci mutation were Hap_1, Hap_2, Hap_8, Hap_20, Hap_21, and Hap_23.Hap_13 showed five loci mutations;Hap_12 had six loci mutations;Hap_9 had seven loci mutations. There are 17 haplotypes in the sequence of relapsed patients, π and He equal to 0.0015 and 0.8191, respectively. There were 15 haplotypes in the sequence of non-relapsed patients (π = 0.0014 and He = 0.8065).There are eight haplotypes in the sequence of relapsed and non-relapsed patients, including Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_14 and Hap_17(Fig. 2).
Fig2.The 9 exons are expressed by numbered boxes with DNA polymorphisms indicated on the top. The predicted amino acid changes are indicated below. No synonymous mutation was found. R represented Relapsed cases, while N indicates Non-relapsed cases.
Haplotypes of CYP2D6 gene coding region and their association with relapse
The odds ratio of genotype to relapse was calculated by selecting six haplotype sequences with sequence number > 1 that simultaneously existed in relapsed patients and non-relapsed patients and could be used for analysis (Table 3).Among them, the OR values of Hap_4 and Hap_6 were 0.159 (95% CI: 0.746 ~ 0.034) and 5.615 (95% CI: 26.577 ~ 1.186), respectively. These were the only two haplotypes that showed statistical significance (P ˂0.05), which demonstrated that Hap_4 indicates reduced lapse risk. By contrast, the risk of relapse for Hap_6 was about 5.615-fold higher than that of other haplotypes. The OR values of Hap_2, Hap_3, Hap_5 and Hap_7 showed no statistical significance (P>0.05) (Table 3).
Table 3. Analysis of the association between haplotypes and relapse
|
Hap
|
Relapse cases(n)
|
Non-relapse cases(n)
|
OR
|
95%CI
|
P
|
Upper limit
|
Low limit
|
Hap_2
|
28
|
27
|
1.059
|
2.058
|
0.545
|
0.865a(NS)
|
Hap_3
|
8
|
15
|
0.478
|
1.206
|
1.189
|
0.113a(NS)
|
Hap_4
|
2
|
11
|
0.159
|
0.746
|
0.034
|
0.009a(S)
|
Hap_5
|
8
|
2
|
4.358
|
21.258
|
0.894
|
0.050a(NS)
|
Hap_6
|
10
|
2
|
5.615
|
26.577
|
1.186
|
0.016a(S)
|
Hap_7
|
7
|
3
|
2.471
|
9.944
|
0.614
|
0.190a(NS)
|
Abbreviations: n, number of cases; NS, not significant; S, significant. a Chi-square test
|
Enzyme activity prediction of significant haplotypes and their diploids
Hap_6 and Hap_4 were verified to exert effect on relapse based on the prediction of enzyme activity. Among them, there were 12 CDS chains in Hap_6 (Table 4) from 10 relapsed patients and 2 non-relapsed patients. The diploids in relapsed cases presented 4 genotypes, including *1/*2, *2/*2, *2/*41 and *1/*41. Among the 4 scoring loci of each genotype, including c.408, c.886, c.1023+36 and c.1457, mutation alleles were found only in the diploid loci of c.886 and c.1023+36. Regardless of the diploid of c.886 being mutant heterozygosity (C/T) or mutant homozygosity (T/T), as long as it co-exists with the mutant heterozygosity of c.1023+36, the genotype score of CYP2D6 cannot reach the full score of 2.0. In this case, the score is 1.5. The enzyme activity of CYP2D6 was as “NM” in accordance with the score (Table 4). Two genotypes of *1/*2 and *1/*41 were found in the non-relapsed cases. By contrast, *1/*41 was also determined in the relapsed cases and reached the genotype score of 1.5. It was predicted to be at "NM" level in terms of enzyme activity, along with the *1/*2 genotype showing mutant heterozygous (C/T) only at c.886 and its genotype score reaching the full score of 2.0 (Table 4).
Table 4. Prediction ofHap-6and Hap-4 and their enzyme activities
|
A. Hap_6
|
Genotypes
|
Relapse times
|
No. of samples
|
Scored loci and their diploid
|
|
Caudle's method
|
c.408
|
c.886
|
c.1023+36a
|
c.1457
|
|
Score of diploid
|
Degree
|
NC_000022.11
|
--
|
--
|
G/G
|
C/C
|
G/G
|
G/G
|
|
2
|
NM
|
*1/*2
|
0
|
1
|
C/C
|
C/T
|
G/G
|
C/C
|
|
2
|
NM
|
*1/*41
|
0
|
1
|
C/C
|
C/T
|
G/A
|
C/C
|
|
1.5
|
NM
|
*1/*2
|
1
|
1
|
C/C
|
C/T
|
G/G
|
C/C
|
|
2
|
NM
|
*2/*2
|
1
|
5
|
C/C
|
T/T
|
G/G
|
C/C
|
|
2
|
NM
|
*2/*41
|
1
|
3
|
C/C
|
T/T
|
G/A
|
C/C
|
|
1.5
|
NM
|
*1/*41
|
1
|
1
|
C/C
|
C/T
|
G/A
|
C/C
|
|
1.5
|
NM
|
B. Hap_4
|
Genotypes
|
Relapse times
|
No. of samples
|
Scored loci and their diploid
|
|
Caudle's method
|
c.100
|
c.408
|
--
|
c.1457
|
|
Score of diploid
|
Degree
|
NC_000022.11
|
--
|
--
|
C/C
|
G/G
|
--
|
G/G
|
|
2
|
NM
|
*10/*10
|
0
|
2
|
T/T
|
C/C
|
--
|
C/C
|
|
0.5
|
IM
|
*1/*10-1
|
0
|
3
|
C/T
|
C/C
|
--
|
G/C
|
|
1.25
|
NM
|
*1/*10-2
|
0
|
6
|
C/T
|
C/C
|
--
|
C/C
|
|
1.25
|
NM
|
*1/*10-2
|
1
|
2
|
C/T
|
C/C
|
--
|
C/C
|
|
1.25
|
NM
|
a Intronic position c.1023+36(Intron 2988)has been considered as defining allele *41 because the predictability of this mutation for impaired enzyme function has been demonstrated recently
|
The 13 CDS chains belonging to Hap_4 was found in 2 relapsed and 11 non-relapsed patients (Table 4). In the diploid of relapsed cases, only one genotype was found, which is *1/*10-2. Its diploid expression at the three scoring loci c.100, c.408 and c.1457 showed mutant heterozygosity (C/T), mutant homozygosity (C/C) and mutant homozygosity (C/C), respectively. Such pattern resembled the genotype of 54.6% (6/11) of non-relapsed cases, and reached the genotype score of 1.25. The CYP2D6 enzyme activity based on this score was predicted at "NM" level (Table 4). The remaining 5 non-relapsed cases were classified as two genotypes of *10/*10 and *1/*10-1. The diploids of genotypes *10/*10 at the three scoring loci of c.100, c.408 and c.1457 were all mutant homozygous (T/T), (C/C) and (C/C), with the lowest genotype score being 0.5. In 3 cases of *1/*10-1 genotype, mutation heterozygosity (C/T) was found at the c.100 scoring locus, the genotype score was 1.25, and its CYP2D6 enzyme activity was predicted as "NM" level (Table 4).