Analysis on co-mutation of chloroquine-resistant gene and artemisinin-resistant gene in Plasmodium falciparum in Yunnan Province

Abstract

Abstract: Objective To analyze the co-mutation property of chloroquine resistant transporter gene(Pfcrt) and artemisinin-resistant gene K13 in Plasmodium falciparum based on reported malaria cases in Yunnan Province, in order to help unveil the mechanisms of multidrug resistance of P. falciparum. Methods The dry blood samples on filter paper were collected from falciparum malaria patients reported through the Reporting System of Chinese Center for Disease Control and Prevention from August 2012 to December 2016. The corresponding epidemiological data were collected as well. DNA was extracted from P. falciparum, and the exon2 region in Pfcrt gene and K13 kelch domain were amplified with nest-PCR. PCR products were sequenced, and the sequences were aligned with the reference sequences 2655199 and PF3D7_1343700. The haplotype number, expected heterozygosity(He), and genetic differentiation(fixation index, Fst) of the exon 2 region of Pfcrt and K13 kelch domain were analyzed by MEGA 5.04 and Arlequin 3.01 softwares. The haplotype media evolution tree was drawn with the Network 4.6.0 software. Data were analyzed with SPSS 21.0 software. Results A total of 234 blood samples were collected. The PCR results showed that the targets were amplified and 218 products of exon2 region of Pfcrt and 192 products of kelch domain of K13 were sequenced. Among them, 192 blood samples were simultaneously analyzed for the genotypes of Pfcrt and K13, including 12 samples of Yunnan local origin, 30 of Africa origin, and 150 of Myanmar origin. There were seven haplotypes for the exon2 region of Pfcrt in 218 samples, with an He of 0.238 5 and 83.0%(181/218) missense mutations. The proportions of single-locus mutation(CVMNT genotype), double-loci mutation (SVMNT genotype) and three-loci mutation(CVIET genotype) in the 72-76 nucleotide locus were 1.8%(4/218), 6.4%(14/218) and 74.8%(163/218), respectively. The media network diagram from the seven haplotypes showed that the haplotype first occurred as 72-76 locus wild-type CVMNK, then evolved a pattern from single mutation to double mutation and to triple mutation. There were 21 haplotypes for the kelch domain of K13 in 192 samples, with an He of 0.044 9 and 35.9%(69/192) missense mutation. Single mutation was found at 9 loci including 16, 446 and 676, with a highest rate for F446I (25.0%, 48/192). The media network diagram from the 21 haplotypes showed a“Satellite” layout. The Fst of Pfcrt gene was smallest between Yunnan local population and Myanmar imported population of P. falciparum(0.020 3). Among blood samples of chloroquine-sensitive CVMNK wild-type, 20.6%(7/34) were found with singe locus mutation in kelch domain, and chloroquine-resistant mutation-types CVMNT and CVIET had 1/4 and 31.7%(51/161), respectively. Conclusion The co-mutation rate of chloroquine-resistant gene and artemisinin-resistant gene of P. falciparum isolates from reported malaria patients in Yunnan Province is 27.1%. The artemisinin-resistant mutation genotypes show a similar pattern as population expansion.

Key words: Yunnan Province, Plasmodium falciparum, Chloroquine resistance, Atemisinin resistance, Co-mutation, Analysis

CLC Number:

R382.312

Ying DONG, Ai-ming SUN, Yan DENG, Meng-ni CHEN, Yan-chun XU, Xiang-hua MAO. Analysis on co-mutation of chloroquine-resistant gene and artemisinin-resistant gene in Plasmodium falciparum in Yunnan Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(3): 202-208.

Figures/Tables 4

Fig. 1

Table 1

Alleles diversity of Pfcrt and K13 genes of falciparum malaria cases from different infection sources

突变位点及基因型Site of mutation and/or genotype	合计Total		云南本地感染病例 Yunnan indigenous cases		自缅甸输入性病例 Myanmar imported cases		自非洲输入性病例 Africa imported cases		Fst(P值) (P Value)
	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	Fst(P值) (P Value)
	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	突变型数^a No. mutations^a / %	He/H	Pfcrt基因序列数 Sequence number	218	-	15	-	170	-	33	-	0.020 3^b
单倍型 Haplotype	7	0.238 5/0.424 2	4	0.374 6/0.542 9	7	0.219 0/0.387 2	3	0.488 6/0.515 2	（0.207 2）
错义突变 Missense mutations	181（83.0）	0.121 2/0.204 8	14（93.3）	0.408 8/0.472 5	151（88.8）	0.116 9/0.198 0	21（63.6）	0.095 2/0.095 2	-
C72S	14（6.4）	0.263 7/0.263 7	3（2.0）	-	10（5.9）	-	0	-	-
M74I	163（74.8）	0.012 3/0.024 5	10（66.7）	-	155（91.2）	0.127 7/0.226 9	3（9.1）	-	-
N75E	160（73.4）	0.012 5/0.012 5	10（66.7）	-	138（81.2）	0.065 6/0.084 6	3（9.1）	-	0.058 0^c
K76T	179（82.1）	0.138 0/0.197 0	1（6.7）	-	151（88.8）	0.118 4/0.197 9	3（9.1）	-	（0.162 3）
同义突变 Synonymous mutations	3（1.4）	-	0	-	3（1.8）	-	0	-	-
野生型 Wild genotype
C₇₂V₇₃M₇₄ N₇₅K₇₆	37（17.0）	-	1（6.7）	-	23（13.5）	-	13（39.4）	-	-
单突变型 Single mutation genotype
C₇₂ V₇₃M₇₄ N₇₅T₇₆	4（1.8）	-	1（6.7）	-	2（1.2）	-	1（3.0）	-	0.112 4^de
双突变型 Double mutation genotype									（0）
S₇₂ V₇₃M₇₄N₇₅T₇₆	14（6.4）	0.263 7/0.263 7	3（2.0）	-	11（6.5）	-	0	-	-
三突变型Triple mutation genotype
C₇₂ V₇₃I₇₄E₇₅T₇₆	163（73.9）	0.012 3/0.024 5	10（66.7）	-	133（78.2）	0.015 0/0.030 0	20（60.6）	-	-
K13基因序列数 Sequence number	192	-	12	-	150	-	30	-	-
单倍型 Haplotype	21	0.044 9/0.521 6	4	0.545 5/0.545 5	17	0.063 1/0.554 3	8	0.087 8/0.330 0	0.043 5^b
错义突变 Missense mutations	69（35.9）	0.076 5/0.511 5	6（50.0）	-	59（39.3）	0.102 8/0.531 8	5（16.7）	0.400 0/0.600 0	（0.153 2）
同义突变 Synonymous mutations	10（5.2）	-	1（8.3）	-	8（5.3）	-	1（3.3）	-	-
野生型 Wild genotype
S₁₆F₄₄₆C₄₄₉N₄₅₈V₄₉₄E₅₅₆ P₅₇₄A₅₇₈A₆₇₆	128（66.6）	-	6（50.0）	-	97（64.7）	-	25（83.3）	-	-
单突变型 Single mutation genotype									0.256 0^c
F446I	48（25.0）	-	6（50.0）	-	39（26.0）	-	3（10.0）	-	（0.018 0）
A676D	6（3.1）	-	0	-	6（4.0）	-	0	-	-
S16L	2（1.0）	-	0	-	2（1.3）	-	0	-	-
G449A	1（0.5）	-	0	-	0	-	1（3.3）	-	0.036 2^de
N458Y	2（1.0）	-	0	-	2（1.3）	-	0	-	（0.045 1）
V494I	1（0.5）	-	0	-	0	-	1（3.3）	-	-
E556D	1（0.5）	-	0	-	1（0.7）	-	0	-	-
P574L	2（1.0）	-	0	-	2（1.3）	-	0	-	-
A578S	1（0.5）	-	0	-	1（0.7）	-	0	-	-

Table 1

Fig. 2

Fig. 3

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