This is the fourth blog post in the series of posts focused on the G6PD gene and G6PD deficiency. In this blog post, I wrote about how you can test for G6PD deficiency and what are the most common methodologies for G6PD deficiency testing. For other blog posts on the G6PD gene, please check the following list:
What are G6PD mutations tested to detect G6PD deficiency?
The measurement of enzyme activity or level is sufficient to determine a patient’s expression of the G6PD gene and deficiency if expressed [citation].
We already mentioned that over 60 mutations are described as Class I mutations, which comprise the most severe form of G6PD deficiency and lead to chronic non-spherocytic hemolytic anemia (CNSHA) [citation].
Specific and frequent mutations:
What type of genetic testing is used for detecting G6PD mutations?
The diagnosis of G6PD deficiency can be made through several methods.
PCR tests are used for the detection of G6PD mutations. They are usually used for prenatal diagnosis, population screening, and family studies [citation].
The level of enzyme activity can be measured to determine a patient’s expression of G6PD. Additionally, molecular analysis can detect mutations of the gene encoding G6PD.
PCR-single-strand conformational polymorphism analysis, DNA sequencing, amplification refractory mutation system, and denaturing high-performance liquid chromatography are involved in molecular testing for G6PD mutations.
High sensitivity and specificity are some of the properties of High-resolution melting (HRM) analysis (simultaneous work of PCR and mutation scanning) [citation]. HRM is used for rapid and reliable analysis [citation].
Are there any known certified commercial tests for the G6PD mutations?
RT-PCR sequencing has a high detection rate of G6PD mutations [citation]. Multiplex allele-specific PCR-based systems like the DiaPlexC G6PD genotyping kit (simple and rapid alternative to PCR) [citation].
How do you test for G6PD in adults?
There are only two male genotypes: hemizygous normal and hemizygous G6PD deficient. In females, there are three genotypes: homozygous normal, homozygous deficient, and heterozygous [citation].
The G6PD test for deficiency in males and homozygous females is simple and related to the measurement of enzymatic activity.
Sometimes, detection in females can be challenging because X-linked disorders affect males and females differently. Development of G6PD deficiency in females depends on X chromosome inactivation. In every female cell one X chromosome is active and one is turned off so results may vary from normal to deficient. For all ambiguities in this case, molecular testing methods are recommended.
It is recommended to use different test for male and female when testing for G6PD deficiency. Inexpensive fluorescent spot test for males, and cytochemical assay for females in order to discriminate between healthy, heterozygous-deficiency and homozygously-deficient females [citation].
There are: Spectrophotometric tests (gold standard for testing, NADPH detection)
Molecular tests (PCR, used for detection of known mutations)
Fluorescent spot test (FST)
Chromophore test (formazan based test),
Point-of-care Rapid diagnostics tests [citation].
How do you test for G6PD in newborns?
G6PD test can be done using one of a variety of qualitative or quantitative tests. Many are commercially available or may be set up in an individual hospital laboratory. Both qualitative and quantitative enzyme activity tests are based on the reduction of NADP to NADPH that, further reflects G6PD activity. World Health Organization recommends the fluorescent spot test for the G6PD screening [citation]. This is a type of blood test. FST for G6PD can be done from umbilical cord blood. The G6PD fluorescent spot test (FST) is an inexpensive and reliable qualitative phenotypic test with sensitivity and specificity above 95%. However, quantitative tests are better but are expensive and require skilled technicians and well-equipped laboratories [citation]. Confirmatory tests involve quantitative tests. G6PD screening is routine in Pennsylvania and Washington, D.C. (United States) [citation].
What does G6PD positive mean? What must G6PD mutations be detected to be considered as G6PD deficiency positive?
Low levels of G6PD in patients' blood indicate an inherited deficiency, marking positive G6PD test results. Molecular tests are further used for specific mutations, and this diagnostic is performed only in specialized laboratories [citation].
Over 60 mutations are described as Class I variants, which comprise the most severe form of G6PD deficiency and lead to chronic non-spherocytic hemolytic anemia (CNSHA) [citation].
The biggest number of mutations are related to exon ten but other exons do not drop behind (37 mutations are related to exon 10, from which 32 are of Class I type and related to more severe phenotype) [citation].
What does G6PD negative mean? What G6PD mutations must be detected to be considered as G6PD deficiency negative?
Diagnosis for G6PD deficiency is based on an assay for G6PD [citation]. Regular G6PD activity is considered: 7–10 IU/g hemoglobin; however, some laboratories use wider reference ranges (i.e., 4–12 IU/g) [citation].
What happens if G6PD is high? What is a normal G6PD level? What is an average G6PD level?
Normal G6PD activity is 10.15–14.71 U/g hemoglobin (Hb) for neonates and 6.75–11.95 U/g Hb for adults [citation].
What does a high G6PD level mean?
High G6PD level usually reflects the presence of a young red blood cell population and reticulocytosis. Levels of G6PD are higher in newborns and do not bias the measurement of enzyme activity [citation]. In one study, leukemia cells showed higher levels of G6PD compared to normal peripheral blood mononuclear cells. Also, higher concentrations were observed in leukemic cells with G6PD deficiency compared to cells that have G6PD deficiency without leukemia. High G6PD activity occurs in proliferating untransformed cells and tumor cells, suggesting an important role of G6PD in cell division [citation].
Park et al. proposed that high levels of G6PD in adipocytes may mediate metabolic disorders in obesity by increasing oxidative stress and inflammatory signals. G6PD expression levels in adipose tissue may be a promising indicator of obesity and insulin resistance, and regulation of G6PD may be a novel therapeutic target in treating metabolic disorders [citation].