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 male-female ratio was 2.66:1, and the proportions of these cases imported from Myanmar infection, imported from Africa countries infection, Laos infection and Yunnan indigenous infection 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. The results showed that only the age groups of 5-20 years old and 21-60 years old had statistical significance (P ˂ 0.05), which were 0.380 (95% CI: 0.986~0.146) and 2.471 (95% CI: 5.417~1.127) respectively, that is, the risk of relapsed of vivax malaria in patients aged 5-20 years old was reduced. The risk of relapsed between the patients ages of 21-60 was 2.471 times higher than that of other age groups. There was no significant correlation between other age groups or gender and the relapsed of vivax malaria.
Table 1 Information of 150 vivax malaria cases whose blood samples successful amplified CYP2D6 gene exon1-9 fragments
Characteristic
|
Years
|
Total
|
2014
|
2015
|
2016
|
2017
|
2018
|
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
|
|
|
|
|
|
|
0-4
|
0
|
0
|
1
|
2
|
1
|
4
|
5-20
|
0
|
2
|
4
|
1
|
16
|
23
|
21-60
|
17
|
25
|
15
|
6
|
51
|
114
|
above 60
|
1
|
0
|
0
|
1
|
7
|
9
|
3.Genderb
|
|
|
|
|
|
|
Male
|
16
|
22
|
14
|
6
|
51
|
109
|
Female
|
2
|
5
|
6
|
4
|
24
|
41
|
4.Infection sourcec
|
|
|
|
|
|
|
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
|
One hundred and fifty-six blood samples of relapsed cases 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 bands were all observed at >2000bp, which were considered to be the target bands (Fig.2).
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(Genbank accession number: MT339044-MT339193) 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 substitutions at 12 loci such as c.31 and c.100 were found (Table 2). The proportion of the third and the first base substitution of the codon triplet was 41.7% (5/12), and the proportion of the second base substitution was 16.6% (2/12). There were 7 missense mutation loci and 5 synonymous mutation loci. The mutation loci of relapsed cases accounted for 91.7% (11/12), and the mutation loci of non-relapsed cases accounted for 66.7% (8/12) (Table 2)
Table 2 Polymorphism Comparison of Relapse cases and Non-relapse cases in the coding domain of CYP2D6 Genes from 1st aa to 497th aa
|
Relapse cases
|
Non-relapse cases
|
Loci
|
Codon changea
|
Amino acid change
|
|
Loci
|
Codon changea
|
Amino acid change
|
c.31
|
GTG>ATG
|
V11M
|
|
--
|
--
|
--
|
c.100
|
CCA>TCA
|
P34S
|
|
c.100
|
CCA>TCA
|
P34S
|
c.271
|
CTG>ATG
|
L91M
|
|
c.271
|
CTG>TTG
|
L91L
|
c.281
|
CAC>CGC
|
H94R
|
|
--
|
--
|
--
|
c.294
|
ACC>ACG
|
T98T
|
|
c.294
|
ACC>ACG
|
T98T
|
c.297
|
GCC>GCT
|
A99A
|
|
--
|
--
|
--
|
c.336
|
TTC>TTT
|
F112F
|
|
c.336
|
TTC>TTT
|
F112F
|
c.408
|
GTC>GTG
|
V136V
|
|
c.408
|
GTC>GTG
|
V136V
|
c.505
|
GGT>AGT
|
G169S
|
|
--
|
--
|
--
|
--
|
--
|
--
|
|
c.801
|
CCC>CCA
|
P267P
|
c.886
|
CGC>TGC
|
R296C
|
|
c.886
|
CGC>TGC
|
R296C
|
c.1457
|
AGC>ACC
|
S486T
|
|
c.1457
|
AGC>ACC
|
S486T
|
a DNA base highlighted in bold are where the SNP occur
Polymorphism of haplotypes
A total of 24 CYP2D6 haplotypes (Hap_1~Hap_24) were clustered amongst the 150 vivax malaria samples examined: 17 haplotypes were observed in the sequences of relapsed patients, π and He equal to 0.0015 and 0.8191, respectively. 15 haplotypes were observed in the sequences of non-relapsed patients (π = 0.0014 and He = 0.8065). There are 8 haplotypes both in the sequences of relapsed and non-relapsed patients, including Hap_2, Hap_3, Hap_4, Hap_5, Hap_6, Hap_7, Hap_14 and Hap_17. Haplotype and frequency were shown in Fig. 3. Among them, Hap_3 was wild-type sequence, and all 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).
Haplotypes of CYP2D6 gene coding region and their association with relapse
Six haplotype sequences with sequence number > 1 that simultaneously existed in patients with and without recurrence was selected to calculate the odds ratio (Table 3). Among them, only two haplotypes, Hap_4 and Hap_6, showed statistical significance (P ˂0.05). The OR values of Hap_4 was 0.159 (95% CI: 0.746 ~ 0.034) and Hap_6 was 5.615 (95% CI: 26.577 ~ 1.186). So, it demonstrated that the occur of Hap_4 indicates reduced the risk of relapsing vivax malaria. By contrast, the risk of relapsing vivax malaria in Hap_6 was about 5.615-fold higher than in 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 relationship 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)
|
n: number of cases; NS: not significant; S: significant; a: Chi-square test
|
Enzyme activity prediction of significant haplotypes and their diploids
The enzyme activity of Hap_6 and Hap_4 genotypes was predicted for they were proved to play an important role in the recurrence. 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 mutation loci of c.408, c.886, c.1023+36 and c.1457, only the mutations at c.886 and c.1023+36 were diploid alleles. 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 couldn’t reach the full score of 2.0. In this case, the score was 1.5 and the enzyme activity of CYP2D6 was “NM” (Table 4). The genotypes of *1/*2 and *1/*41 were found in the non-relapsed cases. However, *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 the full genotype score (Table 4).
Table 4 Prediction of enzyme activities of Hap-6 and Hap-4 genotypes
|
A. Hap_6
|
Genotypes
|
Relapse times
|
No. of samples
|
Mutation 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
|
Mutation 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 predictivity 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 relapsed cases, only one diploid genotype was found, the *1/*10-2. The mutations of *1/*10-2 at loci c.100, c.408 and c.1457 were heterozygosity (C/T), homozygosity (C/C) and 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 the genotypes of *10/*10 and *1/*10-1. The diploids of genotypes *10/*10 at the mutation 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. It was predicted that the activity of CYP2D6 enzyme is "IM" (Table 4). In 3 cases of *1/*10-1 genotype, mutation heterozygosity (C/T) was found at the c.100, and the genotype score was 1.25, suggesting that its CYP2D6 enzyme activity was predicted as "NM" level (Table 4).