Comparisons between RT-PCR, real-time PCR, and in vitro globin chain synthesis by α/ß ratio calculation for diagnosis of α- from ß-thalassemia carriers.

BACKGROUND: Thalassemia, which may be due to point mutations, translocations, and deletions involving the α or ßglobin gene, is the most prevalent single gene disorder in Iran.This study aims to calculate the α/ß ratio in normal cases, α- and ß-thalassemia carriers by RT-PCR, real-time PCR, and in vitro globin chain synthesis (GCS) in order to establish the most accurate technique to distinguish between α- and ß-thalassemia carriers in suspicious cases. METHODS: The α/ß ratios were calculated in all samples by RT-PCR, real-time RT-PCR, and in vitro GCS. RESULTS: Using RT-PCR, the ratios were 1.09 ± 0.07 in normal samples, 1.2 ± 0.17 in ß-thalassemia, 1.08 ± 0.19 in mild α-thalassemia, and 0.96 ± 0.19 in severe α-thalassemia carriers. In real-time RT-PCR, the ratios were 2.21 ± 1.36 in normal samples, 5.12 ± 1.83 in ß-thalassemia, 2.88 ± 0.81 in mild α-thalassemia, and 1.18 ± 0.52 in severe α-thalassemia carriers. With GCS, the ratios were 1.03 ± 0.1 in normal samples, 1.9 ± 0.37 in ß-thalassemia, 0.8 ± 0.13 in mild α-thalassemia, and 0.59 ± 0.12 in severe α-thalassemia carriers. CONCLUSION: To determine the most accurate technique, we statistically analyzed the α/ß ratios obtained from the three standard methods. The ratio obtained by GCS and real-time PCR were helpful in distinguishing between α and ß carriers in suspicious patients in whom the mutation detection was limited and the risk for offspring was not clear. The use of this technique is more obvious when time is restricted (i.e. during the pregnancy period).

Validation and comparison of two quantitative real-time PCR assays for direct detection of DMD/BMD carriers.

OBJECTIVES: To develop a robust and reliable assay for direct identification of female carriers of deletions in the dystrophin gene. DESIGN AND METHODS: We compared two quantitative real-time PCR approaches for the detection of the deletions of exons 4, 17, 47, and 50 in DMD/BMD carriers. One hundred and ten individuals from 26 unrelated families, including 8 large pedigrees characterized by having at least two DMD affected males, were studied. Carrier status of the subjects was also evaluated by MLPA. RESULTS: The results showed the gene dosage ratio of 0.99+/-0.14 and 1.09+/-0.19 for normal individuals and 0.48+/-0.06 and 0.50+/-0.10 for carriers in SYBR green and TaqMan probe assays, respectively. Carrier status was accurately attributed in 100% of cases and confirmed by MLPA. CONCLUSION: Quantitative real-time PCR can be used as a direct method for carrier detection in female relatives of DMD patients with known deletions. The results are comparable to the MLPA data.