Regular ArticleRapid detection of G6PD mutations by multicolor melting curve analysis
Introduction
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked hereditary defect caused by mutations in the G6PD gene. G6PD deficiency is one of the most prevalent human enzymopathies affecting over 400 million individuals worldwide, and particularly in those undeveloped and resource-limited countries. The clinical phenotype of G6PD deficiency varies significantly from asymptomatic to neonatal jaundice, kernicterus, or acute hemolytic anemia following the ingestion of certain drugs during some infections, and notably through eating fava beans (favism). This variability in clinical phenotypes has been attributed to diverse mutant types in the G6PD gene [1], [2], [3]. To date, > 180 mutations have been reported worldwide, and each ethnic population presents a characteristic mutation spectrum [4]. For example, in the Chinese population, at least 21 different mutations have been associated with G6PD deficiency [4], [5], [6], [7]. These mutations cause class II (severe) or class III (mild) deficiencies, in which anemia is not present in daily life, but hemolytic attack can occur upon ingestion of certain oxidative medicines or foods [8]. Therefore, screening for affected individuals is critical for prevention of the disease [9].
Biochemical assays based on G6PD-catalyzed production of nicotinamide adenine dinucleotide phosphate (NADPH) are widely used for newborn screening. Despite the success in identifying male patients, measurement of G6PD activity appears to be inadequate for the detection of heterozygous females due to lyonization (inactivation of one X chromosome) [5], [10]. To overcome this limitation, many alternative molecular assays have been developed, including denaturing high-performance liquid chromatography (DHPLC) [11], amplification refractory mutation system (ARMS) [12], microarray-based assay [13] and reverse dot blot assay (RDB) [7], [14]. Although each assay has unique advantages in terms of specificity and sensitivity, a common shortcoming of these methods is that they often involving multiple steps of post-PCR manipulations, which increase the technical complexity and the risk of amplicon contamination. High-resolution melting (HRM) [15], [16] is a good choice to obviate the post-PCR complexity; nevertheless, the performance of the dye-based methods is compromised by an inability to precisely identify the mutation.
The MeltPro G6PD (Zeesan, Xiamen, China) assay is a qualitative diagnostic assay developed based on multicolor melting curve analysis (MMCA) using dual-labeled, self-quenched probes [17], [18], [19]. This assay was designed to detect the genotypes of 16 mutations in the G6PD gene, which covers > 95% of the Chinese G6PD mutations. The MeltPro assay is a closed-tube format performed on a real-time PCR platform, from which the mutation information is retrieved based on differences in melting temperature (ΔTm) compared to the wild-type. One distinct feature of this assay is its ease-of-use due to the omission of complex post-PCR manipulations. Moreover, the exact mutation type can be identified based on the predefined Tm values and the detection channels.
In this study, we systematically evaluated the analytical and clinical performances of the MeltPro G6PD assay. For the analytical study, the accuracy of mutation detection, the limit of detection, the reproducibility, and the cross-platform compatibility were evaluated. For the clinical study, a multicenter validation study was performed using 763 clinical samples collected from three different hospitals in China. We examined both G6PD enzyme activity results and DNA sequencing results.
Section snippets
Clinical samples
A total of 763 clinical, unrelated, peripheral blood samples (428 males and 335 females) were collected from Zhuhai Municipal Maternity and Child Healthcare Hospital (Zhuhai, Guangdong province), Liuzhou Municipal Maternity and Child Healthcare Hospital (Liuzhou, Guangxi Zhuang Autonomous Region), and the First Affiliated Hospital of Guangxi Medical University (Nanning, Guangxi Zhuang Autonomous Region). The Research Ethics Committee of each hospital approved this study, and informed consent
Design of the MeltPro G6PD assay
The MeltPro G6PD assay is a real-time PCR-based four-color melting curve analysis that detects 16 mutations in two reactions (Fig. 1). Reaction 1 detects eight mutations using two primer pairs (F1, R1, F2, and R2) and four differently labeled self-quenched probes (P1, P2, P3, and P4). Reaction 2 detects another eight mutations using three primer pairs (F3, R3, F4, R4, F5, and R5) and five differently labeled self-quenched probes (P5, P6, P7, P8, and P9). According to the MeltPro G6PD assay, Tm
Discussion
G6PD deficiency is a serious but often neglected disease in developing countries. Due to the lyonization effect, biochemical screening often miss heterozygous female patients who might have either normal or deficient G6PD activity. In addition, because severity of this disease is mutant type dependent, accurate identification of the mutant type is a preliminary requirement for clinical use. Other factors, such as wide coverage of mutation types and time- and cost-effectiveness are also
Author contributions
Zhongmin Xia, Qiuying Huang and Qingge Li wrote the manuscript. Zhongmin Xia and Qiuying Huang designed the research, performed experiments, and analyzed data. Ping Chen, Ning Tang, Tizhen Yan, Yuqiu Zhou, and Qizhi Xiao collected the clinical samples and analyzed data. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
This work has been financially supported by the Natural Science Foundation of Fujian Province [Grant No. 2013J01355], National High Technology Research and Development Program [863 Programme] of China [Grant No. 2013AA020205], and Guangxi Key Laboratory Project [Grant No. 15-140-11]. This work has been supported by 2011 Collaborative Innovation Center of Guangxi Biological Medicine. The funding organizations played no role in the study design; in the collection, analysis, and interpretation of
References (23)
G6PD: population genetics and clinical manifestations
Blood Rev.
(1996)- et al.
Glucose-6-phosphate dehydrogenase deficiency Baillieres
Best Pract. Res. Clin. Haematol.
(2000) - et al.
Glucose-6-phosphate dehydrogenase (G6PD) mutations database: review of the “old” and update of the new mutations
Blood Cells Mol. Dis.
(2012) - et al.
Development and evaluation of a reverse dot blot assay for the simultaneous detection of six common Chinese G6PD mutations and one polymorphism
Blood Cells Mol. Dis.
(2008) - et al.
The first case of a class I glucose-6-phosphate dehydrogenase deficiency, G6PD Santiago de Cuba (1339G > A), in a Chinese population as found in a survey for G6PD deficiency in northeastern and central China
Acta Med. Okayama
(2010) - et al.
Identification of G6PD Mediterranean mutation by amplification refractory mutation system
Clin. Chim. Acta
(2002) - et al.
A reverse dot blot assay for the expanded screening of eleven Chinese G6PD mutations
Clin. Chim. Acta
(2013) - et al.
Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis
J. Mol. Diagn.
(2010) - et al.
A melting curve analysis-based PCR assay for one-step genotyping of beta-thalassemia mutations a multicenter validation
J. Mol. Diagn.
(2011) Severe neonatal jaundice associated with glucose-6-phosphate dehydrogenase deficiency: pathogenesis and global epidemiology
Acta Paediatr. Suppl.
(1994)
Structure and function of glucose-6-phosphate dehydrogenase-deficient variants in Chinese population
Hum. Genet.
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