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].
|
|
|
|
|
Number 26 Number 26 VOL. 26(2), 2023 All in one |
Number 26 VOL. 26(2), 2023 |
Number 26 VOL. 26, 2023 Supplement |
Number 26 VOL. 26(1), 2023 |
Number 25 VOL. 25(2), 2022 |
Number 25 VOL. 25 (1), 2022 |
Number 24 VOL. 24(2), 2021 |
Number 24 VOL. 24(1), 2021 |
Number 23 VOL. 23(2), 2020 |
Number 22 VOL. 22(2), 2019 |
Number 22 VOL. 22(1), 2019 |
Number 22 VOL. 22, 2019 Supplement |
Number 21 VOL. 21(2), 2018 |
Number 21 VOL. 21 (1), 2018 |
Number 21 VOL. 21, 2018 Supplement |
Number 20 VOL. 20 (2), 2017 |
Number 20 VOL. 20 (1), 2017 |
Number 19 VOL. 19 (2), 2016 |
Number 19 VOL. 19 (1), 2016 |
Number 18 VOL. 18 (2), 2015 |
Number 18 VOL. 18 (1), 2015 |
Number 17 VOL. 17 (2), 2014 |
Number 17 VOL. 17 (1), 2014 |
Number 16 VOL. 16 (2), 2013 |
Number 16 VOL. 16 (1), 2013 |
Number 15 VOL. 15 (2), 2012 |
Number 15 VOL. 15, 2012 Supplement |
Number 15 Vol. 15 (1), 2012 |
Number 14 14 - Vol. 14 (2), 2011 |
Number 14 The 9th Balkan Congress of Medical Genetics |
Number 14 14 - Vol. 14 (1), 2011 |
Number 13 Vol. 13 (2), 2010 |
Number 13 Vol.13 (1), 2010 |
Number 12 Vol.12 (2), 2009 |
Number 12 Vol.12 (1), 2009 |
Number 11 Vol.11 (2),2008 |
Number 11 Vol.11 (1),2008 |
Number 10 Vol.10 (2), 2007 |
Number 10 10 (1),2007 |
Number 9 1&2, 2006 |
Number 9 3&4, 2006 |
Number 8 1&2, 2005 |
Number 8 3&4, 2004 |
Number 7 1&2, 2004 |
Number 6 3&4, 2003 |
Number 6 1&2, 2003 |
Number 5 3&4, 2002 |
Number 5 1&2, 2002 |
Number 4 Vol.3 (4), 2000 |
Number 4 Vol.2 (4), 1999 |
Number 4 Vol.1 (4), 1998 |
Number 4 3&4, 2001 |
Number 4 1&2, 2001 |
Number 3 Vol.3 (3), 2000 |
Number 3 Vol.2 (3), 1999 |
Number 3 Vol.1 (3), 1998 |
Number 2 Vol.3(2), 2000 |
Number 2 Vol.1 (2), 1998 |
Number 2 Vol.2 (2), 1999 |
Number 1 Vol.3 (1), 2000 |
Number 1 Vol.2 (1), 1999 |
Number 1 Vol.1 (1), 1998 |
|
|