The alpha-thalassemias are common global genetic disorders that arise from reduced synthesis of the alpha globin chains of fetal and adult hemoglobins. At present, for a variety of technical, logistic and economic reasons, large-scale carrier screening and appropriate antenatal detection programs have not been established. We have successfully established a simple, rapid, sensitive PCR assay for detecting common alpha gene deletions using DNA from fresh blood cells from alpha-thalassemia patients. In alpha-thalassemia syndromes, alpha-globin synthesis is either diminished or absent due to either deletional or non-deletional abnormalities involving the alpha-globin genes. In our strategy, deletion of hemoglobin gene alpha 1 or alpha 2 could be determined using three sets of primers (two sets for alpha 1 and alpha 2 gene respectively and one set for beta-actin as internal control) from alpha-thalassemia syndrome patients by allele-specific amplification and differential PCR. The number of each alpha-globin gene present in the tested sample could be quantitatively determined by comparing the intensity of bands with that of beta-actin gene. We have tested hemoglobin alpha gene status in 21 normal subjects and 74 alpha-thalassemia patients in the majority of whom the diagnosis was confirmed by standard Southern Blotting techniques. We applied regression analysis to the densitometric data and demonstrated that the number and specific alpha gene deletion in these patients could be differentiated by measuring the band intensity of target genes (alpha 1, alpha 2) and reference gene (beta-actin) after PCR amplification and gel electrophoresis. We also plan to examine the feasibility of a color complementary assay with the new technique of real time quantitative PCR which may permit a more sensitive, accurate and quicker method to detect alpha gene deletion.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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