Molecular Diagnosis Kits for Beta Thalassemia

We will develop a simple PCR method to diagnosis alpha-thalassemias. Phase I of the project willconduct feasibility study for the development of a non-isotopic sensitive assay to detect the deletionsof five frequently occurring genotypes of alpha-thalassemia using differential PCR. The Investigator willuse newly developed DNA/PCR mediated color complimentary assay which they developed to test driedblood samples on filter papers. These will be obtained from patients with alpha thalassemic disorders.The specimens will be made available to them by Dr. Griffin Rodgers, the Chief of the MolecularHematology Unit at the National Institutes of Health. We will develop a diagnostic kit for the detectionand quantification of hemoglobin alpha-genes in patients with alpha-thalassemia. The kit will be usedfor screening potential carriers with alpha-thalassemia in the United States, Southeast Asia andSouthern China. This should identify patients at risk of having offspring with symptomatic alpha-thalass-emia disorders. The Investigators note correctly that current screening methods are not practical forwidespread use and do not differentiate different types of alpha-thalassemia syndromes. In pursuit ofthe above, they plan to conduct studies on a micro-DNA-PCR assay to detect and differentiate deletionsof alpha-hemoglobin genes from dried blood samples. A method for using dried blood samples will beuseful in performing field studies. They then plan to establish the feasibility of a DNA-PCR mediatedcolor complementary assay to detect hemoglobin alpha gene deletions from dried blood samples and tocombine these two methodologies in to a simple and sensitive diagnostic kit for the alpha-thalassemias.Preliminary work is provided to show the feasibility of the methodology. In these experiments, theydemonstrated that deletion of hemoglobin gene alpha1 or alpha2 can be determined using three sets ofprimers from alpha thalassemia syndrome patients by differential PCR. In preliminary studies, they usedthis method to examine the hemoglobin alpha gene status from nine normal subjects and sevenalpha-thalassemia patients. These studies show that they can clearly differentiate alpha gene deletionsin these patients. They can be differentiated by measuring the band intensity of the target chains(alpha1, alpha2) and reference chain (beta-actin) after PCR amplification and gel electrophoresis. Theyalso have applied PCR mediated color complimentary assays on several normal subjects and found thisPCR mediated CCA can be used as a rapid sensitive method to detect alpha gene deletions by omittinggel electrophoresis. They postulate that after the assay is improved and stabilized, it would be auseful method of establishing clinical diagnoses in this disorder.