This study describes a method for simultaneous assessment of G6PD-deficiency markers and HBB polymorphisms. These were successfully genotyped from small volumes (5 µl) of DNA extracted from human blood. Gene segments were amplified in a single, multiplex PCR reaction and genotyped by magnetic bead-based allele specific multiplex assay. The assay is fast, relatively inexpensive and performed as well as established genotyping methods.
Assessing G6PD deficiency status is relevant to predict the susceptibility of individuals for the disadvantageous haemolytic consequences of treatment with 8-aminoquinolines like primaquine and tafenoquine [
29‐
33]. Although G6PD phenotyping assays [
21,
34,
35] may be most informative in this respect and allow meaningful assessments of varying levels of G6PD due to random X-inactivation or lyonization [
24‐
26,
36,
37], genotyping approaches are frequently used in population research to plan public health interventions [
11,
38]. Here, a multiplex assay that concurrently detects common SNPs for the A−
G6PD variant and
HBB mutations was successfully developed using a mix of previously published and novel oligonucleotides [
23] and optimized to maximize the SNP specific fluorescence signal. Using this microsphere multiplex assay, samples from G6PD deficient and G6PD normal individuals were assessed for mutations with good agreement with conventional genotyping methods. To assess the correlation in between
G6PD genotype and G6PD enzyme activity, the % G6PD-positive cells was assessed for different
G6PD genotypes. In line with published data, the A− genotype (376G and 202A) had the lowest percentage G6PD positive cells compared to wild type and A+ individuals [
39]. A small proportion of genetically deficient individuals did not have a lower percentage G6PD positive cells and, conversely, a fraction of genetically
G6PD normal individuals displayed unexpected low percentage G6PD positive cells. Including additional SNPs that have been associated with lower G6PD activity in The Gambia [
8], improved the correlation between percentage G6PD positive cells and genotypes. This suggests that genotyping approaches that focus only on 376G and 202A may misclassify individuals with low G6PD enzyme activity. The inability to explain all variation in G6PD activity by the examined SNPs suggests that other (currently unidentified) genetic variants that affect G6PD status may exist.
The current genotyping results were based on populations enriched for G6PD deficient individuals and findings thus do not represent G6PD deficiency prevalence in the two countries. Whilst the current selective sample set does not allow unbiased population prevalence estimates, the HbS gene was more common in The Gambia than Burkina Faso (13.8% vs. 2.7%) and the HbC trait was more common in Burkina Faso (14.7% vs. 10.3%) [
16,
40].
The assay presented here has an advantage that it can be adapted to multiple SNPs of interest and is increasingly used for detecting SNPs associated with drug resistance in parasites [
41,
42]. The assay does not detect large deletions such as either of the thalassaemias (α-thalassaemia and β-thalassaemia), which is one of its limitations. At present the assay contains two common
G6PD SNPs and two
HBB gene polymorphisms, but the flexible multiplex nature of the assay allows users to include additional markers. The assay is less time consuming and labour intensive than traditional end-point PCR/agarose gel genotyping methods and requires less operator time. Up to 50 markers can be tested in a single reaction and markers can be mixed and matched according to population of interest and parasite epidemiology.