Considering the electrophoretic and HPLC elution patterns, as well as the hematology of the proband and members of his family, the presence of a Hb Lepore was suspected. The electrophoretic and HPLC patterns of the other Hb were suggestive of Hb C. However, since the presence of Hb C is very unusual in this region, the entire coding region of the b-globin gene from the proband was sequenced. DNA sequencing analysis did indeed show the presence of the GAG®AAG mutation at codon 6 of the b-globin gene leading to Hb C. The presence of the Hb LBW gene was determined by PCR amplification using 5'd- and 3'b-globin gene specific primers, followed by PvuII digestion (Fig. 1).

Family studies showed that the Hb LBW was inherited from the patient's father who is of Croatian ancestry, while Hb C was inherited from his mother who originates from Hungary. Since Hb C has been found very rarely in this region, haplotype analyses were performed to trace its origin in this family. The data of the extended haplotype analysis of the b-globin gene cluster in this family are shown in Table 2. The Hb C variant in this family is associated with haplotype [– + + + – + + + –] (referring to HincII/e, HindIII/ ^Gg, TaqI/3'^Gg, HindIII/^Ag, HincII/yb, HincII/3'yb, AvaII/b, HpaI/b and BamHI/3'b polymorphisms), b-globin framework 2 and (AT)₉(T)₅ motif at –530 5' to the b-globin gene. The b^C chromosome in this family differs from the typical African b^C chromosome [11] at several polymorphic sites: TaqI/3'^Gg, HindIII/^Ag, HpaI/b and BamHI/3'b polymorphisms, b-globin frame­work and (AT)_X(T)_Y motif at –530 5' to the b-globin gene (Table 3). These data demonstrate that the Hb C mutation in this family has not been derived from the African b^C chromosome.