alpha/beta-Globin mRNA ratio determination by multiplex quantitative real-time reverse transcription-polymerase chain reaction as an indicator of globin gene function.

OBJECTIVES

Imbalance in alpha/beta-globin chains is an important determinant of thalassemia disease severity. This study examined the relationship between alpha/beta-globin mRNA ratio and disease severity in various thalassemia genotypes.

DESIGN AND METHODS

alpha- and beta-globin mRNA contents of red blood cells of 75 alpha- and 32 beta-thalassemia subjects (5 with beta(0)-thalassemia/Hb E) and 14 normal controls were measured using multiplex quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The alpha/beta-globin mRNA ratio of each sample was calculated based on the 2(-DeltaDeltaC)(T) method.

RESULTS

A decrease of alpha/beta-globin mRNA ratios in alpha-thalassemia subjects compared to normal controls correlated with the numbers of defective alpha-globin genes, whereas an increase of the ratios was observed in beta-thalassemia. Subjects with beta(0)-thalassemia/Hb E disease had the highest alpha/beta-globin mRNA ratio, followed by beta(0)-thalassemia trait and then beta(+)-thalassemia trait, which correlated with decrease in severity of anemia. Coinheritance of alpha-thalassemia in beta(0)-thalassemia/Hb E resulted in a more balanced alpha/beta-globin mRNA ratio and an amelioration of the anemia.

CONCLUSIONS

This study indicates that imbalance in globin gene expression, the major factor affecting clinical severity of thalassemia, could be demonstrated by measuring alpha/beta-globin mRNA ratio, which was conveniently and accurately determined by qRT-PCR. In alpha-thalassemia, alpha/beta-globin mRNA ratio correlated with the number of functional alpha-globin genes present, whereas in beta-thalassemia, the ratio provided a good indicator of disease severity.