School of Basic Medical Sciences, Dali University, Dali, 667000, China.
Yunnan Institute of Parasitic Diseases Control, Yunnan Provincial Key Laboratory of Vector-Borne Diseases Control and Research, Yunnan Centre of Malaria Research, Pu'er, 665000, China.
Malar J. 2021 Mar 20;20(1):160. doi: 10.1186/s12936-021-03685-3.
Accumulating evidence suggest that compromised CYP2D6 enzyme activity caused by gene mutation could contribute to primaquine failure for the radical cure of vivax malaria. The current study aims to preliminarily reveal the association between the recurrence of vivax malaria in Yunnan Province and CYP2D6 gene mutation by analysing polymorphisms in the entire coding region of human CYP2D6 gene.
Blood samples were collected from patients with vivax malaria, who received "chloroquine and 8-day course of primaquine therapy" in Yunnan Province. The suspected relapsed cases were determined by epidemiological approaches and gene sequence alignment. PCR was conducted to amplify the CYP2D6 gene in the human genome, and the amplified products were then sequenced to compare with the non-mutation "reference" sequence, so as to ensure correct sequencing results and to determine 9 exon regions. Subsequently, the DNA sequences of 9 exons were spliced into the coding DNA sequence (CDS), which, by default, is known as maternal CDS. The paternal CDS was obtained by adjusting the bases according to the sequencing peaks. The mutation loci, haplotypes (star alleles), genotypes and odds ratios (OR) of all the CDSs were analysed.
Of the119 maternal CDS chains in total with 1491 bp in length, 12 mutation sites in the 238 maternal and paternal CDS chains were detected. The c.408G > C mutation was most frequently detected in both suspected relapsed group (SR) and non-relapsed group (NR), reaching 85.2% (75/88) and 76.0% (114/150), respectively. The c.886C > T mutation was most closely related to the recurrence of vivax malaria (OR = 2.167, 95% CI 1.104-4.252, P < 0.05). Among the 23 haplotypes (Hap_1 ~ Hap_23), Hap_3 was non-mutant, and the sequence structure of Hap_9 was the most complicated one. Five star alleles, including *1, *2, *4, 10 and 39, were confirmed by comparison, and CYP2D610 allele accounted for the largest percentage (45.4%, 108/238). The frequency of CYP2D62 allele in the SR group was significantly higher than that in the NR group (Χ = 16.177, P < 0.05). Of the defined 24 genotypes, 8 genotypes, including *4/*4, *4/*o, *2/*39, *39/*m, *39/*x, *1/*r, *1/*n, and *v/*10, were detected only in the SR group.
Mutation of CYP2D610 allele accounts for the highest proportion of vivax malaria cases in Yunnan Province. The mutations of c. 886C > T and CYP2D62 allele, which correspond to impaired PQ metabolizer phenotype, are most closely related to the relapse of vivax malaria. In addition, the genotype *4/*4 with null CYP2D6 enzyme function was only detected in the SR group. These results reveal the risk of defected CYP2D6 enzyme activity that diminishes the therapeutic effect of primaquine on vivax malaria.
越来越多的证据表明，CYP2D6 酶活性的基因突变为引起伯氨喹根治性治疗失败的原因。本研究旨在通过分析人 CYP2D6 基因全长编码区的多态性，初步揭示云南省间日疟复发与 CYP2D6 基因突变之间的关联。
采集云南省接受“氯喹和 8 天伯氨喹疗程”治疗的间日疟患者的血样。通过流行病学方法和基因序列比对来确定疑似复发病例。采用 PCR 扩增人基因组中的 CYP2D6 基因，然后对扩增产物进行测序，与非突变“参考”序列进行比较，以确保正确的测序结果，并确定 9 个外显子区域。随后，将 9 个外显子的 DNA 序列拼接成编码 DNA 序列（CDS），默认情况下，这被称为母本 CDS。通过根据测序峰调整碱基获得父本 CDS。分析所有 CDS 的突变位点、单倍型（星等位基因）、基因型和比值比（OR）。
总共 119 条母本 CDS 链，长度为 1491bp，在 238 条母本和父本 CDS 链中检测到 12 个突变位点。c.408G>C 突变在疑似复发组（SR）和非复发组（NR）中均最为常见，分别达到 85.2%（75/88）和 76.0%（114/150）。c.886C>T 突变与间日疟复发最为密切相关（OR=2.167，95%CI 1.104-4.252，P<0.05）。在 23 种单倍型（Hap_1~Hap_23）中，Hap_3 是非突变的，Hap_9 的序列结构最为复杂。通过比较，确定了 5 个星等位基因，包括1、2、4、10 和39，CYP2D610 等位基因占比最大（45.4%，108/238）。SR 组中 CYP2D62 等位基因的频率明显高于 NR 组（Χ=16.177，P<0.05）。在所定义的 24 种基因型中，仅在 SR 组中检测到 8 种基因型，包括4/*4、*4/*o、*2/*39、*39/*m、*39/*x、*1/*r、*1/n 和v/*10。
CYP2D610 等位基因的突变在云南省间日疟病例中占比最高。与间日疟复发最密切相关的是 c.886C>T 和 CYP2D62 等位基因的突变，这对应于 PQ 代谢不良表型。此外，仅在 SR 组中检测到无 CYP2D6 酶功能的缺失基因型*4/*4。这些结果揭示了 CYP2D6 酶活性缺陷的风险，降低了伯氨喹治疗间日疟的疗效。
