The Hb A₂ value of all heterozygous carriers was normal or decreased (to 0.55%), while the Hb F value was slightly increased (1.6-2.4%). Hb analysis by electrophoresis on cellulose acetate of the 18 subjects (both patients and their relatives) revealed the presence of an abnormal Hb fraction. Its electrophoretic mobility pointed to four possible Hb variants: Hb Lepore, Hb D, Hb G and Hb S (Fig. 1). PCR analysis of the patients’ DNA confirmed the presence of the Hb Lepore gene in all of them (Fig. 2). Detection of two fragments (356 and 751 bp) in each gap-PCR analysis showed that all patients were heterozygous carriers of Hb LBW or Hb Lepore-Baltimore. The presence of Hb Lepore-Hollandia was excluded due to deletion of the sequence complementary to primer A in this type of Hb Lepore.

In order to determine the type of Hb Lepore, we performed a sequencing analysis of the 356 bp fragment (both strands). All the genes showed the same DNA sequence, indicating that the mutation was of the Hb LBW type (Fig. 3). Moreover, within the second intervening sequence of all analyzed hybrid genes, a single base substitution was detected [IVS-II-74 (G®T)]. This single base substitution defines it as FW2 [10].

Table 1. Hematological data of heterozygous Hb Lepore carriers (average values - SD)

Fig. 1. Hb electrophoresis on cellulose acetate at pH 8.4. Lanes 1: normal adult; lane 2: heterozygous Hb Lepore carrier.

Fig. 2. Detection of the Hb Lepore gene by gap-PCR analysis. Lanes 1: heterozygous Hb Lepore carrier; lane 2: normal adult; lane 3: control PCR amplification done in the absence of the template (water control); lane 4: l/ BstEII DNA marker.

CATTCTAAAC  TGTACCCTGT    TACTTATCCC 

 CTTCCTATGA    CATGAACTTA     ACCATAGAA

 AGAAGGGGAA  AGAAAACATC  AAGGGTCCCA 

TAGACTCACC  CTGAAGTTCT   CAGGATCCAC

 GTGCAGCTTG  TCACAGTGCA  GCTCACTCAG 

CTGAGAAAAA  GTGCCCTTGA  GGTTGTCCAG 

 GTGAGCCAGG  CCATCACTAA  AGGCACCTG 

CACCTTCTTG  CCATGAGCCT  TCACCTTAGG 

GTTGCCCATA  ACAGCATCAG  GAGAGGACAG 

 ATCCCCAAAG  GACTCAAAGA  ACCTCTGGGT 

 CCAAGGGTAG  ACCACCAGTA      ATCTGAGGGT