Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3976-3977
CORRESPONDENCE
A First Mutation in the Human Tissue Factor Pathway Inhibitor
Gene Encoding [P151L]TFPI
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LETTER |
To the Editor:
Tissue factor pathway inhibitor (TFPI) is an important regulator in the
extrinsic blood coagulation pathway.1-3 Although the
regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The
definition of a clinical TFPI deficiency seems to be more complex than
that of other coagulation inhibitors, because the activity and
concentration of circulating TFPI cannot be considered a true measure
of in vivo levels. Its determination in plasma samples by immunological
methods or functional assays has been shown to be inadequate in the
detection of a clinical deficiency.4
Therefore, we investigated whether genetic variations in the TFPI gene
contribute to the occurrence of hitherto unexplained cases of
thrombophilia by analyzing DNA samples of patients with thromboembolic
diseases and blood donors for mutations in the coding region of the
TFPI gene by polymerase chain reaction followed by single-strand
conformation polymorphism (PCR-SSCP).
We selected 50 unrelated individuals with a thrombotic history for the
determination of the genetic basis of their thrombosis (27 of them were
shown to be carriers of the factor V Leiden mutation). While scanning
all coding exons of the TFPI gene and the adjacent 5
and 3 intronic
regions by PCR-SSCP analysis, an abnormal pattern suggesting the
presence of a genetic variation was observed in a PCR fragment from
exon 7 in one patient. DNA sequencing of this fragment showed a single
heterozygous C to T mutation at nucleotide position 1 of exon 7, changing the codon CCG151 to CTG151, resulting
in a Pro151 to Leu151 exchange in the amino
acid sequence of the mature protein (Fig 1).
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| Fig 1.
Proposed secondary structure of the TFPI and the
position of the amino acid exchange. The mature protein
consists of 276 amino acids, forming three tandem Kunitz-type
proteinase inhibitory domains, two connecting chains, an acidic
N-terminus with negatively charged amino acids ( ) and a basic
carboxy-terminal end, containing a cluster of positively charged amino
acids (+).5,6 The Pro151 to
Leu151 change is located near the Kunitz-type
inhibitor domain  2 within the second connecting chain
(magnification).
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The C
T transition at nucleotide position 536 is associated with the
creation of a new recognition site for the restriction enzyme
BseNI, providing a rapid means of screening further individuals for this mutation by PCR and restriction analysis. TFPI-exon 7 and its
5 and 3 flanking intronic regions were specifically amplified from
genomic DNA by PCR using the primers TFPI-Ex7F, 5-TCTATTTTAATTGGCTGTAT-3, and TFPI-Ex7R, 5-GCATGATAATAGTTTCCTGG-3, for the amplification reaction. If the nucleotide C is present at
position 536, the resulting 170-bp DNA fragment is not digestible with
BseNI. However, if the nucleotide T is present at this
position, a 27-bp and a 143-bp restriction fragment is generated.
To estimate the prevalence of the 536CT exchange in the general
population, 2,480 randomly chosen unrelated blood donors (18 to 60 years of age) were investigated by PCR and restriction analysis. Four
persons heterozygous for this mutation were found. According to these
results, the allelic frequency of the 536CT mutation in the German
population of the Westphalian area is approximately 0.08%.
To detect a greater number of subjects with the trait, which would
allow a more precise estimation of the relative risk of those
individuals to develop venous thrombosis, we investigated family
members of the heterozygote blood donors and patients. In total, we
found 13 heterozygote individuals within 6 different families. Two of
them suffered from deep vein thrombosis. Homozygous carriers of this
mutation were not found.
To determine a possible effect of the mutation on circulating TFPI, we
measured the TFPI activity by a functional assay (Actichrome TFPI
Activity Assay; American Diagnostica Inc, Greenwich, CT) and the
protein concentration by an immunological assay (Imubind Total TFPI
ELISA Kit; American Diagnostica Inc) in plasma of all individuals
showing the TFPI mutation. Compared with the control group (blood
donors without TFPI mutation), no statistically significant differences
were detected. The mean values of the TFPI activity and TFPI
concentration were 101% (SD, 15.5%; n = 12) and 40 ng/mL (SD, 10.4 ng/mL; n = 12) in individuals with the mutation and 100% (SD, 7.9%;
n = 22) and 46 ng/mL (SD, 10.7 ng/mL; n = 22) in the control group.
However, existing small differences might not have been recognized due
to the low number of cases investigated so far.
Although the number of subjects with the TFPI mutation is somewhat too
small to evaluate the risk of thrombophilia, we compared the prevalence
of venous thromboembolism in this group with the prevalence in 466 blood donors who were thoroughly questioned about a possible history of
venous thrombosis. Ten cases were found in this group. Our present
results of the statistical analysis show a probability of 94.5% for
the hypothesis that the presence of the TFPI trait is linked to
thrombophilia (P = .055; odds ratio, 5.9; 95% confidence
interval, 1.0 to 36.5). It is not yet possible to prove the thrombotic
risk of subjects with this trait, because the mutation is found in both
thrombotic and nonthrombotic subjects and the number of probands who
are available for statistical analysis is relatively small. The low
prevalence of the trait in the population makes an investigation of a
large number of appropriate patients (n > 1,000) inevitable.
Therefore, multicentric epidemiologic studies are warranted to
elucidate the clinical significance of this mutation.
Knut Kleesiek
Michael Schmidt
Christian Götting
Thomas Brinkmann
Wolfgang Prohaska
Institut für Laboratoriums
und Transfusionsmedizin
Herz und Diabeteszentrum
Nordrhein-Westfalen
Universitätsklinik der
Ruhr-Universität Bochum
Bad Oeynhausen, Germany
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