A RAPID AND INNOVATIVE METHOD FOR THE IDENTIFICATION OF THE
COMMONEST G6PD ITALIAN MUTATIONS
A. Minucci, L. Gentile, S. Rocchetti, C. Zuppi, B. Giardina and E. Capoluongo
Laboratory of Clinical Molecular Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy
BACKGROUND
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most prevalent enzyme
deficiency in the world (Fig.1). To date, more than 190 different G6PD mutations, mainly
represented by single nucleotide substitutions (SNSs), have been identified among
different ethnic populations and geographical areas, and each ethnic population presents a
characteristic mutation profile. For this reason, in this study we set-up an economic
molecular approach for rapid and simple detection of the commonest G6PD
Italian
mutations.
Figure 1. Global prevalence of the G6PD deficiency.
MATERIALS
AND
METHODS
In the last 5 years, our Clinical Molecular Diagnostics Laboratory collected clinical and
laboratory data regarding numerous G6PD deficient patients and healthy controls. Based on our
diagnostic results, we established that the most common G6PD mutations were: G6PD
Mediterranean, G6PD Seattle, G6PD A-(c.202+c.376) and G6PD Cassano. For the identification
of these four mutations we used a rapid DNA extraction, directly performed in a 0.2 ml PCR
tube, followed by both PCR and restriction digestion, performed in the same DNA mix tube
ready to use (STAT-NATTM DNA-Mix, Sentinel, Diagnostic) (Fig.2).
Figure 2. Flow chart of the diagnostic approach reported in this study.
RESULTS
Using 3-4 µL of DNA extracted from only 2 µL of whole blood or 5 µL of saliva, the correct
identification of four G6PD mutations was carried out. The genotypes obtained by PCR-RFLP
were 100% concordant with the same DNA previously sequenced. PCR amplification showed
efficiency similar to classic PCR amplifications where DNA template was obtained by the
classical phenol–chloroform method, by commercial DNA kits or by automated DNA
A
B
Figure 3. A: PCR from DNA extracted by 2uL of fresh blood sample and B: PCR from
DNA extracted by 5uL of saliva
extraction (Fig.3). Furthermore, the rational primer design allowed to obtain amplicons of
approximately 300bp which is an optimal size to easily interpret the restriction patterns.
DISCUSSION
Several tests can be used for the detection of G6PD deficiency, but only very few tests are able to reliably diagnose G6PD deficiency
heterozygous women reliably. DNA based tests present absolute diagnostic value for the heterozygous women identification. However, they are
expensive and required often sophisticated equipment. The simplicity and rapidity of the molecular approach presented in this study is that DNA
extraction is directly performed in 200µL tube by a thermal cycler using a simple cycle consisting of two steps. The subsequent PCR and
restriction reactions are performed in the same tube.
CONCLUSIONS
We believe that this assay could be directed to those Countries and laboratories which don’t have large financial resources and facilities. In fact,
as compared to other genotyping techniques, this molecular approach doesn’t require large equipment, while saves time and dedicated personnel.