La PCR-temps réel dans le diagnostic des alpha-thalassémies: une alternative d’avenir ?

Résumé

Résumé

Les lésions génétiques associées aux alpha thalassémies sont souvent dues à des délétions touchant un/deux loci de la globine. Le but de cette étude est d’évaluer l’intérêt des températures et courbes de fusions obtenues en GAP-PCR temps réel dans le diagnostic des alpha-thalassémies. Cinq patients thalassémiques ainsi que cinq contrôles ont été évalués. Les températures de fusion ainsi que l’allure des courbes de fusion sont différentes dans les deux groupes. Cette stratégie prometteuse mérite d’être évaluée sur d’autres types de délétions.

Abstract

Abstract

The commonest genetic defects leading to α-thalassemia are genomic deletions encompassing one or both α-globin loci. The aim of this work was to evaluate the interest of melting curves characteristics obtained in gap real-time polymerase chain reaction for the diagnosis of α-thalassemia deletions. Five patients with known deletional α-thalassemia, and five control subjects were tested. The melting temperature and melting curve shapes of the PCR products allowed us to discriminate controls from α-thalassemic patients. This promising strategy should be further tested with samples carrying other α-thalassemic deletions.

Background

The commonest genetic defects leading to α-thalassemia are genomic deletions encompassing one or both α-globin loci. The aim of this work was to evaluate the interest of melting curves characteristics obtained in gap real-time polymerase chain reaction for the diagnosis of α-thalassemia deletions.

Methods

Five patients with known deletional α thalassemia, —(MED)(2), —(SEA)(2), —(FIL)(1), and five control subjects were tested. Specific primers were used for each deletion. Real-time-gap PCRs, using SYBER-Green mix, were performed on a « light-cycler 480 from Roche® ». At the melting temperature, the released of the SYBER-Green dye bonded to double strained DNA is associated with a dramatic decrease in fluorescence intensity. The rate of this variation was determined by plotting the negative derivative of fluorescence vs temperature (lightcycler 480 software, Roche®).

Results

The melting temperature (Tm) and melting curve shapes of the PCR products derived from α-thalassemic patients or control subjects, allowed us to discriminate controls from α-thalassemic patients (eg —Phil Tm = 91°C whereas control Tm = 81 and 85,6°C). Some non specific amplified products are still present, probably because of the high GC content and high homology of sequences in the α-globin cluster.

Conclusion

This promising strategy should be further tested with samples carrying other α-thalassemic deletions. Nevertheless it appears reliable, cost-effective and safe offering additional benefits including minimal labor, rapid turnaround time and decreased risk of PCR carryover contamination. It may be one alternative technology available for routine diagnosis of all types of deletional α-thalassemia.