SCANNING METHOD TO IDENTIFY THE MOLECULAR HETEROGENEITY OF DELTA-GLOBIN GENE ESPECIALLY IN DELTA-THALASSEMIAS - DETECTION OF 3 NOVEL SUBSTITUTIONS IN THE PROMOTOR REGION OF THE GENE

A scanning strategy for the detection of delta-globin gene mutations a nd polymorphisms is presented. This procedure is based on the denaturi ng gradient gel electrophoresis (DGGE) of four different artificially amplified DNA fragments which cover the promoter, the exons, as well a s IVS I of the reported gene. To estimate the efficiency and sensitivi ty of the proposed procedure, we analysed the appropriate controls of delta-thalassemic carriers, uncharacterised delta-thalassemias and cas es with normal hematological phenotype, but slightly increased (up to 3.5%) HbA(2). DGGE results permitted the identification of delta-globi n gene mutations and the polymorphism -199 (T-->C). Three novel base s ubstitutions inside the promoter region of the gene [-65(A->G), -55(T- ->C), -36(C-->A)], were also revealed. These changes are either linked in cis with other mutations or are responsible for thalassemias or fo r positive regulatory effect in delta-globin gene expression. The prop osed experimental strategy consists of an accurate, curate, rapid, saf e and inexpensive screening procedure for establishing the molecular b asis of delta-globin gene defects, suitable for the application for bo th research and diagnostics. (C) 1997 Wiley-Liss, Inc.