Abstract
Introduction. Coagulation factor IX plays an important intermediate role in the activation of blood coagulation. It is located within the blood plasma as a zymogen, in its inactivated state. Factor IX is dependent on the presence of Vitamin K. The structure of factor IX closely resembles the structures of many other Vitamin K dependent plasma proteins, such as prothrombin, factor X and protein C. After being activated, Factor IX forms a complex with calcium ions, membrane phospholipids and coagulation factor VIIIa to activate factor X.
The exact locus of the coagulation factor IX gene was found to exist in the Xq26-q27 region of the X chromosome. The FIX gene spans 34 kb and contains eight exons. Over 300 different mutations have been identified in the FIX gene, all of which result in the production of inactive FIX, causing hemophilia B.
Aim. In this study we searched for mutations in the FIX gene which result in an increased activity of FIX thus being the cause of thrombophilia syndromes.
Material: A total of 108 individuals from unrelated families were involved in this study, presenting thrombophilic syndromes. A control sample from a healthy non-thrombophilic individual was also used. Total DNA from the above individuals was supplied to us by the Haemostasis and Thrombosis Unit of AHEPA University Hospital, Thessaloniki, Greece. According to HAT (Heparin Antithrombin Test, Makris, Van Dreden 1998) method a mixture of human antithrombin and heparin is added in the plasma and partial thromboplastin time is estimated. 97% of normal individuals exhibit prolonged time values in this test, whereas in our patients the time was significantly reduced. However, after the addition of recombined human FIX (rhFIX) in the mixture, prolongation of PTT is noted.
Methods: The promoter region and the eight exons of the FIX gene were amplified by PCR using seven labelled primer pairs specific for these regions, that were described previously in literature. The amplification reactions were performed in a MJ Research P200 thermal cycler while the Tm of each primer pair was optimised as shown in the table. PCR products were analyzed using LI-COR DNA analyzer which is based on fragment separation by polyacrylamide gel electrophoresis. With this method PCR products presenting up to a 1 bp difference in their molecular weight create distinct bands on the gel and thus an insertion, or deletion of a base can be detected. However, no such differentiation was present among the samples examined.
Assuming that the potential mutations could involve point mutations and thus be undetectable by the above method, the samples were sequenced and compared with the control.
Sequencing the promoter and the 8 exons sites of the FIX gene of the most high risk cases.
A point mutation was detected in four of the samples. The mutation was a single base change (ACT →GCT) located at the 21975 bp of the FIX gene, in exon 6. This mutation causes a significant change, replacing the Thr194 residue with an Ala residue (T194A). The sequencing pattern of one of these patients and the control is shown in the figure.
Figure
