Genetic Studies on Protein C Deficiency

• Main Authors: Tsay, Woei, 蔡偉
• Other Authors: Shen Ming-Ching, Chen Pei-Jer
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/15032830085342256321

博士 === 國立臺灣大學 === 臨床醫學研究所 === 85 === Protein C (PC) is an essential plasma anticoagulant produced from the liver. The activated PC can inactivate two important coagulation cofactors, Va and VIIIa. PC deficiency is known as important risk factor of hereditary thrombophilia. PC gene defects of eighteen symptomatic Chinese families with PC deficiency were analysed by polymerase chain reaction and direct sequencing. Nine unique point mutations and one in-frame deletionin 15 propositi were identified. Among them, six mutations were novel, that is, T66C substitution, G3134A (C64Y), T6128G (F139V), deletion of nucleotides (nt) 6161-6163 (des K-150), G8404A (R230H) and G8478C (D255H). Compound heterozygotic PC deficiency, combined protein S deficiency, and antiphospholipid antibodies were not rare in our study. Most interestingly, one common mutation (accounted for 44%), C6152T at exon VII, tightly linked to a silent T66C substitution at exon II suggesting a founder effect. A further study searched for the R147W mutation among normal population by using allele- specific oligonucleotides and dot hybridization. The crude prevalence rate of R147W of Chinese in Taiwan is about 0.85% (95%CI: 0.35%~1.35%). This mutation seems a common genetic defect causing venous thrombosis in Chinese population. I also present a novel gene polymorphism, C/T at nt 8480, only found in Asians. The allele frequency is 0.87/0.13 in Asians. The allele frequencies of other gene polymorphisms were quite different between Asians and Caucasians. Four heterozygous point mutations located in the promoter region have been identified in families with type I PC deficiency and recurrent venous thrombosis. However, detailed analysis of regulatory elements and their interacting factors remains to be undertaken. This study presents results of biochemical and functional characterizations of several cis-elements located in the 5''-upstream regulatory region, and the trans-acting factors that interact with them. A cloned DNA fragment from nt -88 to +45 was sufficient for basal promoter activity of PC gene. Five cis- elements corresponding to HNF-1, HNF-3 and NF-I/CTF binding sites have been identified. Four heterozygous mutations have been shown to disrupt HNF-3 [mutants of A(-32)G and T(-27)A] and HNF-1 [T(-14)C and C(-10)T] binding. Mutation in the NF-I- binding site also significantly impairs the promoter activity. Furthermore, the electrophoretic mobility shift assay and competitive footprinting analysis provided evidences that either HNF-1 ?or HNF-3 can cooperate with each other in binding to their cis-elements. The results from the cotransfection assays in HeLa cells showed a novel synergistic transactivation between HNF-1?and HNF-3. Our data further indicated that the unique overlapping of the HNF-3 sites, the specific spatial relationship of the sites, and the coactivator C/EBP, all contributed to the synergistic interaction. I also found that NF-I could synergistically enhance the transactivation of HNF-1 ?or HNF-3. Viewed as a whole, the combinatorial interplay of HNF-1? HNF-3 and NF-I made a significant contribution to the activation of the liver-specific PC gene. Since several type II deficient mutations defined the locations of multiple essential amino-acid residues. The recombinant expression system will provide further insight into the pathophysiology of PC deficiency and the structure/function relationship of PC molecule. A well-designed prospective molecular epidemiologic studies shall be performed to confirm the importanceof R147W allelotype in Chinese.