Journal of clinical laboratory analysis · 2026
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.
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Chen Hongjian, Yao Qi, Fu Mianai
Аннотация
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.
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.