Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
Malar J. 2021 Apr 20;20(1):194. doi: 10.1186/s12936-021-03731-0.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test.
A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed.
The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females.
The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.
葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是人类最常见的酶病，在疟疾流行的热带和亚热带地区普遍存在。抗疟药物，如伯氨喹和泰法喹，可导致 G6PD 缺乏症患者发生溶血。因此，建议在对间日疟原虫进行根治性治疗之前进行 G6PD 检测。表型检测已广泛用于 G6PD 缺乏症的筛查，但在杂合子女性中，随机 lyonization 导致结果解读困难。已经鉴定出超过 200 种 G6PD 变异体，它们形成与 G6PD 缺乏症程度和溶血易感性相关的基因型。本研究旨在使用新开发的分子基因分型检测方法评估 G6PD 突变的频率。
开发了一种多重高分辨率熔解（HRM）检测方法来检测 8 种 G6PD 突变，其中 4 种突变可以同时检测。使用 70 个 G6PD 缺乏样本验证该方法的有效性。然后，该方法用于筛查来自泰缅边境地区的 725 个人的血液样本。这些样本的酶活性也使用水溶性四唑盐（WST-8）测定法测定。然后，分析了基因型与酶活性之间的相关性。
多重 HRM 检测方法检测 G6PD 突变的灵敏度为 100%（95%置信区间[CI]：94.87-100%），特异性为 100%（95%CI：87.66-100%）。表型 WST-8 检测显示，研究人群中 G6PD 缺乏症的总体患病率为 20.55%（149/725）。相比之下，通过多重 HRM 检测，27.17%（197/725）的受试者存在 G6PD 突变。本研究中检测到的突变包括四个单变体，G6PD Mahidol（187/197）、G6PD Canton（4/197）、G6PD Viangchan（3/197）和 G6PD Chinese-5（1/197），以及两个双突变，G6PD Mahidol+Canton（1/197）和 G6PD Chinese-4+Viangchan（1/197）。携带 G6PD Mahidol 的个体中观察到广泛的 G6PD 酶活性，尤其是女性。
基于多重 HRM 的检测方法对 G6PD 突变的检测既敏感又可靠。这种基因分型检测方法可以有助于检测杂合子，这可能是在疟疾流行地区使用伯氨喹和泰法喹之前进行 G6PD 缺乏症高通量筛查的有用方法。
