Balkan Journal of Medical Genetics

MOLECULAR GENETIC CHARACTERIZATION OF b-THALASSEMIA AND SICKLE CELL SYNDROME IN THE ALBANIAN POPULATION
Babameto-Laku A1,*, Mitre A2, Berisha S2, Mokini V1, Roko D1
*Corresponding Author: Anila Babameto-Laku, University Hospital Center “Mother Teresa”, Faculty of Medicine, Service of Medical Genetics, Rruga Dibres, 307, Tirana, Albania; Tel.: +68-20-94-170; Fax: +355-4-227-2782; E-mail: laku62@yahoo.com
page: 45

MATERIALS AND METHODS

DNA was obtained from the blood of 68 Albanian patients with b-thal, 26 with sickle cell anemia or sickle cell/b-thal, 54 parents of these patients and 14 heterozygotes related to these families. Most of the patients were from districts from southwest Albania. In 108 patients and heterozygotes, the sex ratio was 47 males to 61 females. Their age group ranged from 2 to 15 years old. We used the TTGE method (temporal temperature gradient electrophoresis) for the detection of mutations and polymorphisms in the sequences of the b-globin gene as described by Shaji et al. [5]. This method was developed at the Laboratory of Biochemistry and Molecular Genetics, Henri Mondor Hospital (APHP), Creteil, Paris, France. The DCode™ instrument (Bio-Rad Laboratories, Hercules, CA, USA) and TTGE technique are simple to use and have enormous potential for high throughput screening since gel casting is easy; the constant denaturing gel used eliminates the need to pour chemical gradient gels. DNA extraction was performed using the kit Nucleon BACC3, DNA (manufactured for Amersham BioSciences UK Ltd. by Tepnel Life Sciences, Manchester, UK). The b-globin gene was amplified as seven fragments (A, B, C, D, E, F, and G) [6]. Fragments A-F contain the coding and non coding sequences of the bglobin gene, and fragment G can detect three gene polymorphisms [IVS-II-16 (C>G), IVS-II-74 (G>T) and IVS-II-81 (C>T)], while fragments B, C, and D contain the polymorphic sites at codon 2 (C>T), IVS-II-16 and IVS-II-666 (T>C), respectively (Figure 1) [5]. The PCR products were analyzed in 16-20 cm 6% polyacrylamide gels prepared in 1.5X Tris acetate- EDTA buffer containing 6 mol/L of urea. We loaded 10 mL of PCR product with 5 mL of 2X gel loading dye onto the gel. Electrophoresis was carried out at 130 V with constant temperature increments of 2°C/hour on the DCode™ mutation detection system (Bio-Rad Laboratories) that has an automatic thermal regulator. The temperature range for TTGE for each PCR fragment was determined by reducing the upper and lower temperatures by 12°C because each mol of urea lowers the melting temperature by 2°C. The temperature ranges for the different PCR fragments were: 57°C to 69°C for fragment A, 56°C to 70°C for fragments B and C, 48°C to 59°C for fragments D, F and G and 57°C to 69°C for fragment E. Of the nine previously reported mutations common in the Albanian population, eight (96.5%) can be searched for on fragments B and C of the b-globin gene [4,7,8]. Thus, fragments G, B and C were analyzed first, followed by the remainder. Fragment G was used for the study of the frameworks and interpretation of the results of fragments B, C and D. Fragments B and C were used because they contain the more frequent mutations found in the Albanian population. The PCR products from healthy controls were also loaded onto every TTGE gel. Because fragments B, C and D each contain one polymorphic site, DNA samples heterozygous for these polymorphisms (frameworks 1/3) were used as controls, whereas for fragments A, E, and F, a control from a healthy individual was used. Confirmation of the b-thal alleles was obtained following restriction endonuclease digestion.

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