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Electronic Letters to:
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Electronic letters published:
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Real time PCR using hybridization probes for rapid prenatal diagnosis of HBS-beta thalassemia
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Joaquín Martínez-López, Ph D Haematologist S. de Hematologia . Hospital Universitario 12 de Octubre. Madrid. Spain, Rosa Ayala, Queta Albizua, Alberto Galindo and Florinda Gilsanz
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jmartinezlo{at}yahoo.es Joaquín Martínez-López, et al.
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Compound heterozygosity for S hemoglobin and thalassemia is observed in countries with coexistence of these two pathologies. These inherited diseases were infrequent in Spain, but their incidence has increased here due to the population flow. As Vrettou et al assessed in a recent paper (1) real time PCR methods are extraordinarily useful for both population studies and prenatal diagnosis of hemogobinopathies and thalassemias.
We have employed real time PCR for prenatal diagnosis in sickle cell-beta thalassemia case with a different set of primers and probes but with the same accuracy and speed as Vrettou et al (1). Our case underscores the genotyping identification problems that can be found with HBS and these very close thalassemic mutations. In a first step, we studied a 3-year-old boy with sickle cell anemia-beta thalassemia, the mother, an 11-week-pregnant woman from the Dominican Republic with sickle cell trait and her husband, born in Apulia (Italy), with Beta thalassemia minor phenotype. Real time PCR was developed, employing a specific primer for beta-globin gene and hybridization probes. PCR amplifications were carried out in a Light CyclerTM (Roche), using Fast Start Light Cycler TM DNAMaster, which contains Taq-polymerase, reaction buffer and dNTPs (Roche Biochemical Mannheim Ge). Real-time PCR to identify sickle cell anemia: 0.5 microM of each primer and 0.2 microM of the corresponding hybridation probes were used, Mg concentration was 3 mM and cycling conditions were 35 cycles of denaturation (94ºC / 0 sec), annealing and fluorescence acquisition (55ºC /10 sec) and elongation (72ºC /10sec). The sequences of primers and hybridization probes for sickle cell anemia PCR were: HBS Fw 5´-GGGCTGGGCATAAAAGTCA-3; HBS Re 5´-AATAGACC AATAGGCAGAG-3´; HBS Anchor 5´-CATCCACGTTCACCTTGCCCCACA-3´-flu; HBS sensor 5´red640-CAGTAACGGCAGACTTCTCCCACAGGA-3´. Real time PCR in beta thalassemia to identify IVS I-110 (G62388A) as well as the most frequent beta thalassemia mutations in Spain was performed as previously published (2). The mother was heterozygous for HbS and the father, heterozygous for mutation IVS I-110 A, the most frequent beta-thalassemia mutation in the Italian population. Their son was heterozygous for mutation IVS I-110 A and, in a first approach, apparently homozygous for HbS mutation, as only one peak of fluorescence at 68.5ºC was detected. Analysis of the primer and the beta-globin gene sequence showed that the HbS PCR reverse primer we were using was unable to hybridize correctly with the allele that included IVS I-110 A mutation, and thus failed to amplify it. The reverse primer of HbS PCR presented a mismatch that corresponded to the IVS-I 110 A mutation. For this reason, a new primer, which hybridized exactly to the mutated allele IVS I-110, was designed and real-time PCR was performed, with similar conditions as previously described. With this new primer for HbS PCR, two melting peaks, for heterozygous HbS and a normal HbS allele (at 65ºC and 68.5ºC), were found in the son while only the normal peak (at 65ºC) was found in the father. Amniocentesis was performed on 11th week of gestation. DNA from 100 microL amniotic fluid was obtained by an automatic nucleic acid extractor, which employs magnetic bead technology for DNA extraction (Magnapure from Roche), without further manipulation. The DNA extraction technique was previously standardized using five amniotic fluid samples. In every amniocentesis sample, the concentrations of DNA ranged between 10-2 and 10-3 times that of DNA obtained from 100 microL of peripheral blood. DNA amount obtained from amniocentesis was enough to identify the mutations.The advantages of this DNA extraction method are its speed, minimal sample required and low manipulation. Immediately after, we developed real time PCR to detected both: beta-globin gene, IVS I-110 A heterozigous and HbS heterozigous. The results were confirmed repeating both PCR and the fetus was diagnosed with sickle cell anemia Beta-thalassemia only 24 hours after amniocentesis. However the parents decided to continue with pregnancy and the diagnosis was confirmed later in the newborn. Some of the specific advantages of Real time PCR should be emphasized: less contamination, minimal errors and speed in results but, although accurate, RT-PCR may show failures in technique as we described. They can be avoided by a careful previous family study and a detailed design of primers or probes; this will minimise errors. Thus, as Vrettou et al assessed, real-time PCR probably constitutes one of the best methods in prenatal diagnosis for point mutation diseases and must be the goal for prenatal diagnosis of well-characterised molecular targets for a quick and accurate diagnosis(1). Bibliography 1. Vrettou C, Traeger-Synodinos J, Tzetis M, Malamis G, Kanavakis E. Rapid screening of multiple beta-globin gene mutations by real-time PCR on the LightCycler: application to carrier screening and prenatal diagnosis of thalassemia syndromes. Clin Chem 2003;49:769-76. 2. Moreno I, Bolufer P, Perez M, Barragan E, Sanz M. Rapid detection of the major Mediterranean beta-thalassaemia mutations by real-time polymerase chain reaction using fluorophore-labelled hybridization probes. Br J Haematol 2002;119:554-7. |
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