Molecular assays for DNA diagnosis and single cell analysis of alpha- and beta-thalassemia mutations

Abstract

<P>THALASSEMIA IS THE MOST COMMON MONOGENIC HEMOGLOBIN DISORDER GLOBALLY. DELETIONS INVOLVING THE A-GLOBIN GENE ACCOUNTS FOR MORE THAN 95% OF A-THALASSEMIA MUTATIONS WHILE POINT MUTATIONS OR THE LOST OF ONE OR TWO BASES IN THE B-GLOBIN GENE ACCOUNTS FOR MAJORITY OF B-THALASSEMIA MUTATIONS. A SINGLE-TUBE MULTIPLEX-PCR ASSAY WAS DEVELOPED TO DETECT HETEROZYGOSITY, HOMOZYGOSITY AND COMPOUND HETEROZYGOSITY FOR THE SEVEN MOST COMMON DELETIONAL DETERMINANTS OF A-THALASSEMIA (-A3.7, -A4.2, --SEA, --FIL, --THAI, --MED AND -(A)20.5). A NON-DELETION CONTROL AND A SEPARATE CONTROL FOR AMPLIFICATION SUCCESS WERE ALSO INCLUDED. IN ADDITION, TWO SINGLE CELL ASSAYS WERE DEVELOPED FOR APPLICATION TO PREIMPLANTATION GENETIC DIAGNOSIS OF THE MOST COMMON --SEA DOUBLE A-GLOBIN GENE DELETION, WHICH IN HOMOZYGOSITY LEADS TO HEMOGLOBIN BARTA??S HYDROPS FETALIS SYNDROME; AND THE IVS II, 654 (C-T) B-GLOBIN GENE MUTATION, WHICH IN HOMOZYGOSITY LEADS TO B-THALASSEMIA MAJOR. SIMULTANEOUS AMPLIFICATION OF D1S80