An Integrated Genotyping Strategy for α/β-Thalassemia: Based on the Analysis of the Coding Sequences and Expression Levels of HBA2, HBA1, and HBB in Peripheral Blood mRNA.

OBJECTIVE

To analyze the clinical diagnostic value for thalassemia genotyping using peripheral blood mRNA.

METHODS

Total RNA was extracted from 103 pre-genotyped peripheral blood samples, followed by cDNA synthesis. These samples comprised 21 wild-type cases, 49 α-globin gene mutant cases and 33 HBB mutant cases. An Integrated strategy was adopted, combining single-reaction Sanger sequencing with the multiple quantitative fluorescence PCR (MQF-PCR), to analyze the full-length coding sequences and expression level of HBA2/HBA1/HBB respectively.

RESULTS

(1) The substitution mutations located in the coding region (n = 14) were successfully detected, and the mutations located in the HBB non-coding region displayed negative results (n = 13). The samples with β41-42M/βN (n = 10) and β71-72M/βN (n = 4) were not observed to have their small deletion or insertion mutation initially, but they were eventually identified after using allele-specific MQF-PCR. Moreover, MQF-PCR results indicated that the level ranges of Peak area ratio 1 (PAR1, PAHBA2/PAHBA1) and Peak area ratio 2 (PAR2, PAHBB/PAHBA) can be used to identify the common α-globin gross-deletion types and to predict mutations in the HBB non-coding region.

CONCLUSION

This mRNA-based strategy enabled detection of coding-region mutations in the HBA2/HBA1/HBB and prediction of non-coding-region mutations as well as gross deletions with high coverage, low cost and simple operation.