HOMOGENEITY OF THE Hb LEPORE GENE IN FR YUGOSLAVIA
Urosevic J1, Djureinovic T1, Poznanic J1, Cvorkov-Drazic M2, Bunjevacki G2, Janic D3, Krivokapic-Dokmanovic L3, Popovic Z1, Pavlovic S1
*Corresponding Author: Dr. Sonja Pavlovic, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, 11000 Belgrade, FR Yugoslavia; Tel: +381 11 3976 445; Fax: +381 11 3975 808; E-mail: sonya@sezampro.yu
page: 29

RESULTS

Screening analysis of Yugoslav patients with suspected thalassemia syndromes in last 4 years revealed six patients who were Hb Lepore carriers. Three were compound heterozygotes for Hb Lepore and b-thal, and they were affected with a thalassemia major syndrome. Family studies revealed 12 heterozygous relatives. All heterozygous carriers of Hb Lepore had a clinical phenotype of thalassemia trait. The hematological data for heterozygous Hb Lepore carriers (average values) are given in Table 1.

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

 

 

Fig. 3. The nt sequence of the 356 bp PCR fragment, comprising the d-b breakpoint region of the Hb Lepore carrier. Data were obtained using reverse primer (primer B). The underlined DNA sequence originates from the d-globin gene. The underlined bold sequence represents the 58 bp region common for both d- and b-globin genes. A single base substitution representing FW2 is indicated by an arrow

 

 




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