Materials: Blood samples from the proband and 18 members of his family were collected in vacutainers with EDTA as an anticoagulant.

Methods: Routine hematological and Hb analyses included complete blood counts obtained by an automated cell counter, peripheral blood smears, n-butanol stability test, starch and cellulose acetate gel electrophoresis and reversed phase high performance liquid chromatography (HPLC) on Vydac C₄ (The Separations Group, Hesperia, CA, USA) columns [8].

The abnormal Hbs were characterized by DNA analyses. DNA was isolated from the leukocytes using the Proteinase K digestion – phenol / chloroform extraction - ethanol precipitation method [9]. The Hb C mutation was determined by polymerase chain reaction (PCR) amplification of the b-globin gene, followed by sequencing on an automated DNA sequencer (ABI PRISM 310 Genetic Analyzer; Applied BioSystems, Palo Alto, CA, USA) using Big Dye terminator cycle sequencing.  The  presence  of  the  Hb LBW  gene  was determined using  5' d - and 3' b- specific primers designed in our laboratory [5' d (5'-GGAGCAGGGAGGACAGGACCAGCA-3'); 3' b (5'-GTATTTTCCCAAGGTTTGAACTAGCTCT-3')]. The type of Lepore Hb (LBW, Lepore-Baltimore and Lepore-Hollandia)  was   determined  by PvuII   restriction enzyme digestion of the PCR amplified fragment from the Lepore gene using the method of Lindeman et al. [10]. Haplotype analysis, including seven standard polymorphic sites (HincII/e, HindIII/^Gg, HindIII/^Ag, HincII/3'yb, AvaII/b, and BamHI/3'b) was performed by restriction enzyme digestion of PCR-amplified fragments. Two additional polymorphic sites (TaqI/3'^Gg and HpaI/b) were analyzed by Southern blot analysis. The b-globin gene framework and the (AT)_X(T)_Y motif at –530 5' to the b-globin gene were identified by direct sequencing of PCR-amplified fragments.