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 B ACKGROUND 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. 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-NAT TM DNA-Mix, Sentinel, Diagnostic) (Fig.2). M ATERIALS AND M ETHODS 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 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. R ESULTS 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. D ISCUSSION 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. C ONCLUSIONS Figure 1. Global prevalence of the G6PD deficiency. Figure 3. A: PCR from DNA extracted by 2uL of fresh blood sample and B: PCR from DNA extracted by 5uL of saliva Figure 2. Flow chart of the diagnostic approach reported in this study. A B