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Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high‐resolution melting curve analysis

Boonyuen, Usa, Songdej, Duantida, Tanyaratsrisakul, Sasipa, Phuanukoonnon, Suparat, Chamchoy, Kamonwan, Praoparotai, Aun, Pakparnich, Phonchanan, Sudsumrit, Sirapapha, Edwards, Thomas, Williams, Christopher, Byrne, Rachel, Adams, Emily ORCID: https://orcid.org/0000-0002-0816-2835 and Imwong, Mallika (2021) 'Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high‐resolution melting curve analysis'. Malaria Journal, Vol 20, p. 194.

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Abstract

Background
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.

Methods
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.

Results
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.

Conclusions
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.

Item Type: Article
Subjects: QU Biochemistry > Enzymes > QU 135 Enzymes
QU Biochemistry > Genetics > QU 500 Genetic phenomena
WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
WH Hemic and Lymphatic Systems > Hematologic Diseases. Immunologic Factors. Blood Banks > WH 170 Hemolytic anemia (e.g., Sickle cell anemia)
Faculty: Department: Biological Sciences > Department of Tropical Disease Biology
Digital Object Identifer (DOI): https://doi.org/10.1186/s12936-021-03731-0
Depositing User: Marie Hatton
Date Deposited: 14 May 2021 14:57
Last Modified: 14 May 2021 14:57
URI: https://archive.lstmed.ac.uk/id/eprint/17837

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