Rapid detection of G6PD mutations by multicolor melting curve analysis

Highlights

• The clinical and analytical performances of the MeltPro G6PD assay were investigated.

• Comparison studies with biochemical assays and DNA sequencing were performed.

• A novel mutation in the G6PD gene has been found in clinical evaluation.

Abstract

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured T_m was < 0.3 °C. The limit of detection was 1.0 ng/μL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150 min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings.

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 (ΔT_m) 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 T_m 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.