The association of CYP2D6 gene polymorphisms in the full-length coding region with higher recurrence rate of vivax malaria in Yunnan Province, China

Huang

et al. 2021

Malar J

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Background In recent years, the incidence rate of vivax malaria recurrence still had 3.1% in Yunnan Province population after eradication therapy using primaquine (PQ). In order to understand the specific failure reasons for preventing vivax malaria relapses, a preliminary exploration on the CYP2D6 enzyme activity was carried out in the vivax malaria patients in Yunnan Province population by analysing mutational polymorphism in the coding region of CYP2D6 gene. Methods Blood samples were collected from vivax malaria patients with suspected relapse (SR) and non-relapsed (NR) malaria in Yunnan Province. The DNA fragments containing 9 exons regions of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were aligned by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The alleles of CYP2D6 gene were identified according to the criteria prescribed by Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database and the CYP2D6 enzyme activity was predicted based on diploid genotype. Results A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1,457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). The high-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group ($${x}^{2}$$ x 2 = 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. Allele *10 was the most frequent among these genotypes (0.423). Among the allele *10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group ($${x}^{2}$$ x 2 = 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group ($${x}^{2}$$ x 2 = 10.050, P < 0.05), and genotype *4/*4was detected only in the SR group. Conclusion In the patients receiving PQ dosage in Yunnan Province population, both rs1135840 single nucleotide polymorphism and *10 allele form was common in the CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse.

Section: Methodsmentioning

confidence: 99%

Prediction of the CYP2D6 enzymatic activity based on investigating of the CYP2D6 genotypes around the vivax malaria patients in Yunnan Province, China

Huang

et al. 2021

Malar J

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“…Reference sequence (ID: NC_000022.11) was used as the template to design the PCR primers and to determine the reaction conditions for CYP2D6 gene, according to the methods described in previous studies [17][18][19]. The coding regions covering exons 1-4 and exons 5-9 were ampli ed by segmentation.…”

Section: Extraction Of Human Genomic Dna and Pcr Ampli Cation Of Cyp2d6 Gene Fragmentsmentioning

confidence: 99%

“…The sequencing results were collated by using DNAStar 11.0 and BioEdit 7.2.5, and the rst CDS chain of CYP2D6 gene of each sample, known as maternal CDS chain, was obtained by splicing method, according to the protocol described in the literature [19]. The maternal CDS chain was used to derive the paternal CDS chain by replacing the bases of the unread double allelic heterozygous sites [20], which were identi ed by searching the DNA sequencing peak chromatograms.…”

Section: Analysis Of Cyp2d6 Gene Coding Region Polymorphism and Prediction Of Enzyme Activitymentioning

confidence: 99%

“…The haplotypes (allelic forms) of the CDS chain of CYP2D6 gene were determined in accordance with the criteria of the "Human Cytochrome P450 Allele Nomenclature Committee Database" (NM_000106.5) [21] and published literatures [22,19]. The haplotypes at the sub-allelic level.…”

Section: Analysis Of Cyp2d6 Gene Coding Region Polymorphism and Prediction Of Enzyme Activitymentioning

confidence: 99%

“…For the haplotypes devoid of the criteria for the allelic forms, they were customized as "* + a single lowercase letter" [19].…”

Section: Analysis Of Cyp2d6 Gene Coding Region Polymorphism and Prediction Of Enzyme Activitymentioning

confidence: 99%

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Prediction of The CYP2D6 Enzymatic Activity Based on Investigating of The CYP2D6 Genotypes Around The Vivax Malaria Patients in Yunnan Province, China

Huang

et al. 2021

Preprint

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BackgroundTo preliminary explore CYP2D6 enzyme activity in patients with recurrent vivax malaria in Yunnan Province by analyzing mutational polymorphism in the coding region of CYP2D6 gene.Methods Blood samples were collected from vivax malaria patients with suspected relapse (SR) malaria and non-relapsed (NR) in Yunnan Province. The DNA fragments containing 9 exons region of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were compiled by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The allelic forms of CYP2D6 gene were identified according to the criteria prescribed by "Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database" and the CYP2D6 enzyme activity was predicted based on diploid genotype. Results A total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). High-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group (x2 = 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. *10 allele was the most frequent among these genotypes (0.423). Among the 10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group (x2 = 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group (x2 = 10.050, P < 0.05), and genotype *4/*4 was detected only in the SR group. Conclusion In the patients receiving primaquine dosage in Yunnan Province, both rs1135840 single nucleotide polymorphism and *10 allele form was the common in the coding region CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse patients.

Molecular identification of vivax malaria relapse patients in the Yunnan Province based on homology analysis of the Plasmodium vivax circumsporozoite protein gene

et al. 2022

Parasitol Res

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More than 85% of the malaria burden in the Yunnan Province is caused by imported vivax malaria, and Yunnan is also where the majority of vivax malaria patients are diagnosed in China. Timely removal of the infection sources of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To that end, blood samples were collected from cases diagnosed and revalidated as single species infection with P. vivax in the Yunnan Province from 2013 to 2020. Specifically, samples from vivax malaria patients with suspected relapses episodes were subjected to PCR amplification, product sequencing, and analysis of the P. vivax circumsporozoite protein (pvcsp) gene. In total, 77 suspected relapse patients were identified out of 2484 cases infected with P. vivax, with a total of 81 recurrent episodes. A total of 156 CDS (coding DNA sequence) chains were obtained through PCR amplification and sequencing of the pvcsp gene from 159 blood samples, 121 of which can be matched to the paired sequences of 59 vivax malaria patients with both primary attack and recurrent experience. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites (VS), meaning every two paired sequence was completely homologous. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, indicating that the paired sequences was “weakly heterologous” with no fragment insertions (or deletions). All 59 vivax malaria patients with recurrences were caused by the activation of P. vivax hypnozoites originated from the same population as the primary infection. The paired analysis of the similarity between high variant genes allowed the identification of relapse episodes caused by P. vivax homologous hypnozoites and also demonstrated pvcsp gene as one of the candidate molecular markers for tracing infection origin.