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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.
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