Polymorphism analysis of multidrug resistance protein 1 gene in imported Plasmodium vivax in Yunnan Province

doi:10.12140/j.issn.1000-7423.2023.04.002

Abstract

Abstract:

Objective To analyze the polymorphism of Plasmodium vivax multidrug resistance protein 1 gene (Pvmdr1) from imported vivax malaria patients in Yunnan Province. Methods The whole Pvmdr1 gene was amplified and sequenced in the blood samples of patients who diagnosed with vivax malaria in 2020 and 2021 in Yunnan Province. The sequence of P. vivax SalⅠ isolate (GenBank accession number: NC_009915.1) was taken as the reference sequence for designing primers. MEGA 5.04 software was used to align the spliced sequences with the coding region reference sequence (GenBank accession number: XM_001613678.1). Haplotype and single nucleotide polymorphism (SNP) sites and their mutation types, nucleotide diversity π, expected heterozygosity (He), etc were analyzed by DnaSP 6.11.01. Haplotype medium network diagram was constructed using Network 10.0 Software. Results A total of 276 blood samples were collected from vivax malaria patients and were imported from overseas. The full gene sequences with 4 392 bp of Pvmdr1 were obtained from 259 blood samples, and the accession numbers were OP559204-OP559462 assigned by GenBank. The π of 259 DNA coding sequences was 0.000 76. A total of 22 SNPs were detected from 259 coding sequences, including 13 non-synonymous mutant and 9 synonymous mutant loci. Only the detection rate difference of c.4074C>T mutation between 2020 and 2021 [15.0% (21/140), 5.9% (7/119)] was statistically significant (χ² = 5.546, P < 0.05). The minor allele frequency was c.1587A>G (97.9%, 253/259), and the newly discovered SNPs included c.2499G>T (2.3%, 6/259), c.3358C>T (0.4%, 1/259), and c.3832C>T (0.4%, 1/259). All of c.3064C>T, c.4065A>G, c.3358C>T and c.3832C>T mutant sites were detected from the blood samples in 2021. The 259 complete coding region sequences of Pvmdr1 were defined as 26 haplotypes (Hap_2-Hap_27) by comparison with the reference sequence (haplotype Hap_1, all of which were mutant type haplotypes, and He was 0.890 7. Among them, the detection rate of Hap_8 was the highest (18.5%, 48/259). There were 15 and 22 haplotypes found from blood samples in 2020 and 2021, respectively. Among them, 4 haplotypes were only detected in blood samples from 2020, and 10 haplotype were only detected in blood samples from 2021. The difference of Hap_10 detection rate between 2020 and 2021 [15.0% (21/140), 3.4% (4/119)] was statistically significant (χ² = 22.264, P < 0.05). The proportions of quadruple, quintuple, six-fold, seven-fold, nine-fold and ten-fold multiple mutations in 26 haplotypes were 0.4% (1/26), 19.2% (5/26), 38.5% (10/26), 30.8% (8/26), 0.4% (1/26), and 0.4% (1/26), respectively. The haplotype medium network diagram displayed that the gradual evolution of 26 haplotypes started with Hap_1, and then passed through quadruple mutation (Hap_24), moved towards quintuple, six-fold, seven-fold, nine-fold, and ten-fold multiple mutations away from. Conclusion There is high polymorphism in the Pvmdr1 gene in the P. vivax from the imported vivax malaria cases in Yunnan Province.

Key words: Plasmodium vivax, Multidrug resistance protein 1, Mutation, Yunnan Province

CLC Number:

R382.311

DING Hongyun, DONG Ying, XU Yanchun, DENG Yan, LIU Yan, WU Jing, CHEN Mengni, ZHANG Canglin. Polymorphism analysis of multidrug resistance protein 1 gene in imported Plasmodium vivax in Yunnan Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(4): 404-411.

Figures/Tables 5

Table 1

Fig. 1

Table 2

Table 3

Different haplotypes mutations in Pvmdr1 gene in imported P. vivax in Yunnan Province

序号 Order	单倍型 Haplotype	不同程度的多重突变型 Multiple mutants of different degrees		单倍型数（检出率/%） No. haplotype (Detection rate/%)
序号 Order	单倍型 Haplotype	类型 Type	重性 Multiplicity	2020 （n = 140）	2021 （n = 119）	合计 Total
1	Hap_24	G698S/M908L/T958M/F1076L	4	1(0.7)	-	1(0.4)
2	Hap_8	T529T/G698S/M908L/T958M/F1076L	5	29(20.7)	19(16.0)	48(18.5)
3	Hap_9	T529T/M908L/T958M/F1076L/K1393N	5	-	4(3.4)	4(1.5)
4	Hap_21	G698S/L845F/M908L/T958M/F1076L	5	2(1.4)	1(0.8)	3(1.2)
5	Hap_26	K44K/G698S/M908L/T958M/F1076L	5	1(0.7)	-	1(0.4)
6	Hap_27	K44K/T529T/M908L/T958M/F1076L	5	1(0.7)	-	1(0.4)
7	Hap_7	S513R/T529T/G698S/M908L/T958M/K1393N	6	6(4.3)	4(3.4)	10(3.9)
8	Hap_4	P8L/L310L/T529T/M908L/T958M/F1076L	6	19(13.6)	24(20.2)	43(16.6)
9	Hap_10	S513R/T529T/G698S/M908L/T958M/S1358S	6	21(15.0)	4(3.4)^a	25(9.7)
10	Hap_12	T529T/G698S/M908L/T958M/F1076L/K1393N	6	-	1(0.8)	1(0.4)
11	Hap_13	S513R/T529T/G698S/M908L/T958M/F1076L	6	-	1(0.8)	1(0.4)
12	Hap_16	S513R/T529T/M908L/T958M/F1076L/S1358S	6	-	1(0.8)	1(0.4)
13	Hap_18	T529T/G698S/M908L/T958M/F1076L/S1358S	6	-	1(0.8)	1(0.4)
14	Hap_20	T409M/T529T/M908L/T958M/F1076L/K1393N	6	-	1(0.8)	1(0.4)
15	Hap_23	K44K/T529T/G698S/M908L/T958M/F1076L	6	-	1(0.8)	1(0.4)
16	Hap_25	T529T/G698S/A861E/M908L/T958M/F1076L	6	1(0.7)	-	1(0.4)
17	Hap_2	K44K/T529T/G698S/L845F/M908L/T958M/F1076L	7	10(7.1)	14(11.8)	24(9.3)
18	Hap_3	P8L/L310L/T529T/G698S/M908L/T958M/F1076L	7	-	1(0.8)	1(0.4)
19	Hap_5	K44K/T529T/G698S/A861E/M908L/T958M/F1076L	7	15(10.7)	17(14.3)	32(12.4)
20	Hap_6	K44K/T529T/G698S/M908L/T958M/F1076L/S1450L	7	16(11.4)	13(11.0)	29(11.2)
21	Hap_11	K44K/T529T/G698S/M833I/M908L/T958M/F1076L	7	3(2.1)	3(2.5)	6(2.3)
22	Hap_14	T409M/T529T/G698S/M908L/T958M/F1076L/K1393N	7	8(5.7)	4(3.4)	12(4.6)
23	Hap_17	K44K/S513R/T529T/G698S/M908L/T958M/S1358S	7	-	1(0.8)	1(0.4)
24	Hap_19	S513R/T529T/M908L/T958M/L1022L/F1076L/K1355K	7	-	1(0.8)	1(0.4)
25	Hap_22	K44K/G698S/L845F/M908L/T958M/F1076L/L1120L/L1278L/S1450L	9	-	1(0.8)	1(0.4)
26	Hap_15	K44K/G520D/T529T/G698S/A861E/M908L/T958M/F1076L/I1348I/S1450L	10	7(5.0)	2(1.7)	9(3.5)

Table 3

Fig. 2

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片段 Fragment	引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')	PCR轮次 PCR round	产物长度/bp Product length/bp
F1	MD-1F	TAACTCCTCACCGTTTGGGAAT	第一轮	1 245
F1	MD-1R	TCATTGTTTGGTTGCTGGTTGC	First round	1 245
F2	MD-2F	TTTATTACCATATTTACGTACGCAAG	第一轮	1 385
F2	MD-2R	ATGATGATCGTAATTCTGTTTTCG	First round	1 385
F3	MD-3F1	CAACATCAAGTATAGTTTGTACAGC	第一轮	1 368
	MD-3R1	TGAACATCTCTGTTAATATGTGCTG	First round	1 368
	MD-3F2	TTAGTGTTTCGAAGAAGGTGCA	第二轮	959
	MD-3R2	GTAGAGGGAGTACTTATTCGAGT	Second round	959
F4	MD-4F	GCAGCATTTATAAGGACTCCG	第一轮	1 387
F4	MD-4R	CTCATCACGGTAGATTTGCC	First round	1 387
F5	MD-5F1	GAGAAGGCTATTGATTATTCGAAT	第一轮	1 571
	MD-5R1	TTAACTATGTTTACTACGGTTAAGGG	First round
	MD-5F2	TCCTTCGAAAGGTACTACCCACT	第二轮
	MD-5R2	CATATGATCTGTGCACGTGCAT	Second round