FOUR NOVEL CAUSATIVE MUTATIONS IDENTIFIED IN THE PROS1 GENE OF JAPANESE DEEP VEIN THROMBOSIS PATIENTS WITH PROTEIN S DEFICIENCY
Abstract number: P-T-074
Sanda1 N., Fujimori1 Y., Kashiwagi1 T., Yamada1 T., Okumura1 K., Takagi1 A., Murate1 T., Iwasaki2 T., Katsumi2 A., Yamamoto3 K., Matsushita2 T., Naoe2 T., Saito4 H., Kojima1 T.
11Department of Pathophysiological Laboratory Sciences 22Department of Hematology-Oncology, Nagoya University Graduate School of Medicine 33Division of Transfusion Medicine, Nagoya University Hospital 44Director, Nagoya Central Hospital, Nagoya, Japan
How-to-cite Sanda N, Fujimori Y, Kashiwagi T, Yamada T, Okumura K, Takagi A, Murate T, Iwasaki T, Katsumi A, Yamamoto K, Matsushita T, Naoe T, Saito H, Kojima T. FOUR NOVEL CAUSATIVE MUTATIONS IDENTIFIED IN THE PROS1 GENE OF JAPANESE DEEP VEIN THROMBOSIS PATIENTS WITH PROTEIN S DEFICIENCY. J Thromb Haemost 2007; 5 Supplement 2: P-T-074
Abstract
Introduction: Protein S (PS) is a vitamin K-dependent plasma glycoprotein that functions as a natural anticoagulant, and its deficiency is known to be one of the major risk factors for deep vein thrombosis. In this study, we performed molecular analyses of protein S deficiency in 4 unrelated Japanese patients.
Methods: We amplified each of the 15 exons including the exon/intron boundaries of the PROS1 gene by polymerase chain reaction (PCR) using allele-specific primers, and analyzed DNA sequences of the patients with PS deficiency. To investigate whether the 5'-flanking mutation influenced the PROS1 promoter activity, we analyzed the mutant promoter activity by luciferase reporter assay in the transiently transfected HepG2 cells. We also evaluated the behavior of an oligonucleotide containing the 5'-flanking mutation in the interaction with nuclear extracts from HepG2 cells by electrophoretic mobility shift assay.
Results: The entire analysis of the coding regions and intron/exon boundaries of the PROS1 gene revealed the presence of 4 distinct novel mutations respectively. Three were premature termination mutations, 2 frameshift mutations (152-153delAG (FS11X), 1355-1356insG (FS432X)) and a nonsense mutation (1680T>A (Y519X)), which would cause to be a type I protein S deficiency. The other was a C to T substitution at position -168bp upstream from the translational start site (-168C>T), which located within a putative Sp1 binding site. In the luciferase assay, the relative activity of the -168C>T mutant reporter plasmid was 23.3 12.9% of that of the wild type plasmid. We also observed that the -168C>T mutation decreased the Sp1 binding to the human PROS1 promoter sequence. These data suggested that a -168C>T mutation would reduce PS mRNA transcription, due to an impairment of Sp1 binding to PROS1 promoter sequence, leading to PS deficiency.
Conclusions: We identified 4 novel causative mutations in the PROS1 gene of 4 Japanese patients with protein S deficiency respectively.
