COEXISTENCE OF A COMMON PROTHROMBOTIC RISK FACTOR AND HEMOPHILIA IN THE BULGARIAN HEMOPHILIC POPULATION: GENOTYPE/PHENOTYPE CORRELATIONS
Petkova R1, Chakarov S2, Horvath A3, Ganev V3, Kremensky I1
*Corresponding Author: Dr. Rumena Petkova, Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynaecology “Maichin dom’, No 2 Zdrave Str, 1432 Sofia, Bulgaria; Tel: +359 2 952 049 0; Fax: +359 2 952 012 4; E-mail: RumenaPetkova@yahoo.com
page: 37

MATERIALS AND METHODS

In the present study were included 30 hemophilia A-affected patients and one hemophilia B-affected patient, suffering from severe hemophilia as determined by deficient factor activity levels below 1%. DNA was extracted either by the salting-out method [10] or by phenol-extraction and subsequent ethanol precipitation [11]. A factor V fragment, containing the Leiden mutation site, was amplified using flanking primers FVF (5'-TGCCCAGTGCTT AACAAGACCA-3') and FVR (5'-TGTTATCACACTGG TGCTAA-3') [1,12]. The amplification mix contained 1X STS polymerase reaction buffer; 5 nmoles each deoxynu­cleotidetriphoshate; 0.5 pM of each primer per mL of reac­tion mix; 0.2 U STS DNA polymerase and 50-100 ng genomic DNA. An initial denaturation step at 940C for 4 minutes was followed by 30 cycles of amplification, each consisting of denaturation step at 940C for 45 seconds, annealing step at 560C for 45 seconds, and elongation step at 720C for 45 seconds, that yielded a 267 bp fragment. Amplification products were chloroform extracted, ethanol precipitated and dissolved in TE buffer. An aliquot of the purified DNA was digested with 0.5-1 U MnlI for 2-6 hours. The DNA digestion profile was analyzed by electrophoresis in 8-10% polyacrylamide gels (29:1 acrylamide/ bisacrylamide). The presence of the G1691A mutation abolishes a restriction site in the amplified fragment. MnlI digestion of the wild-type allele results in 163, 67 and 37 bp fragments, whereas the mutant allele gives fragments of 200 and 67 bp. Screening for common defects causing severe hemophilia A was carried out by Southern hybridization [13].




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