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Brief report
A new mutation in the HNF4 binding region of the factor VII promoter in a
patient with severe factor VII deficiency
Josephine A. Carew, Eleanor S. Pollak, Stanislaw Lopaciuk, and Kenneth A. Bauer
Investigation of the molecular basis of a
severe factor VII (fVII) deficiency revealed
compound heterozygosity in the fVII gene.
On the paternal allele the patient had 3
structural gene abnormalities frequently
associated with fVII deficiency. A new
mutation, a C to T transition at position
ⴚ55 relative to the translational start site,
was found on the maternal allele. The
study demonstrates that this mutation
partially impeded binding of the transcriptional activator, hepatic nuclear factor 4,
to the fVII promoter while greatly reducing reporter gene expression in hepatic
cells. (Blood. 2000;96:4370-4372)
© 2000 by The American Society of Hematology
Introduction
We have recently characterized 2 independent point mutations
within the 5⬘ regulatory region of the factor VII (fVII) gene in
patients with severe fVII deficiency. One mutation, a T to G
substitution at position ⫺61, completely eliminates interaction
with the orphan nuclear receptor, hepatic nuclear factor 4 (HNF4).1,2
The second mutation, a C to G transversion at position ⫺94,
prevents binding and transactivation by Sp1 and other nuclear
proteins.3 These regulatory regions are particularly important for
expression of the fVII gene.4-6 In this report, we describe a new
mutation in the fVII promoter that reduced HNF4 binding but
nevertheless contributed to severe fVII deficiency.
Study design
Genetic analyses
Informed consent for this study, which was approved by the Human Studies
Committee of the Brockton-West Roxbury Department of Veterans Affairs
Medical Center, was obtained from both the patient and his mother. Plasma
levels of fVII coagulant activity (VII:C) and fVII antigen (VII:Ag) are
expressed as a percentage of normal levels.7 Leukocyte genomic DNA8 was
used for polymerase chain reaction (PCR) of the fVII 5⬘ flanking region and
exons with surrounding splice sites.1 Sequence analysis was done on a
373A DNA Sequencer (Applied Biosystems, Foster City, CA).9 PCR
fragments spanning nucleotides ⫺185 to ⫹1 were subcloned into a
promoterless reporter plasmid containing the human growth hormone
(hGH) structural gene (Nichols Diagnostics Institute, San Juan Capistrano, CA).1,6
Analysis of promoter mutation
HepG2 cells (ATCC HB 8065) were cultured and transfected with 3 ␮g of
reporter plasmid and 1.5 ␮g of pRSV-␤-galactosidase plasmid (Promega
Corp, Madison, WI). A total of 48 hours post-transfection, media were
assayed with the hGH assay kit (Nichols Diagnostics Institute) and lysates
with a colorimetric ␤-galactosidase assay kit (Promega).1 For cotransfec-
From the Hematology-Oncology Section, Boston VA Healthcare System, and
Department of Medicine, Harvard Medical School, Boston, MA; Department of
Pathology and Laboratory Medicine, University of Pennsylvania School of
Medicine, Philadelphia, PA; and the Laboratory of Blood Coagulation, Institute
of Hematology and Blood Transfusion, Warsaw, Poland.
tion, 6 ␮g of pCDNAI-HNF4, encoding rat ␣-HNF4, or inert pUC-19 were
also present. In vitro transcribed/translated HNF4 was prepared in TNTwheat germ system (Promega) and used in electrophoretic mobility shift
assays with [␥-32P]-labeled oligonucleotides.10 Factor VII oligonucleotides
spanned residues ⫺77 to ⫺47 and had either wild-type (WT) sequence, C to
T at position ⫺55 (MT55), or T to G at position ⫺61 (MT61). A control
oligonucleotide (residues ⫺92 to ⫺67 of human apolipoprotein [Apo]
CIIIB)11 was also used. In supershift assays, polyclonal anti-HNF4
antibody was added to binding reactions. Reactions were run on 5%
(wt/vol) polyacrylamide gels in Tris-borate buffer and analyzed by
autoradiography.
Results and discussion
The patient is a 25-year-old Polish male with a severe bleeding
disorder manifested by spontaneous deep muscle hematomas,
hemarthroses, ecchymoses, and epistaxis. He has required transfusions of fresh frozen plasma prophylactically and to control severe
bleeding episodes. His plasma levels of VII:Ag and VII:C were less
than 2% of normal. Sequence analysis of the coding regions and
intron/exon boundaries of his fVII gene identified several structural
gene mutations on the paternal allele: Ala294Val, Arg353Gln, and a
frameshift mutation in codon 404. These mutations are frequently
linked in Polish patients with fVII deficiency.7 The fVII protein
translated from the paternal allele is expected to be poorly secreted
and functionally abnormal.7,12-14 Heterozygosity for a neutral
dimorphism in codon His115 15 was also noted. Examination of
404 base pairs of the 5⬘ flanking region indicated heterozygosity for
base substitutions at positions ⫺55 (C to T) and ⫺402 (G to A).
Inheritance of these alterations was confirmed by genotyping the
patient’s mother, who exhibited modest reductions in plasma levels
of VII:Ag (38%) and VII:C (52%). The base substitution at position
⫺402, recently reported as a polymorphism associated with slight
increases in the plasma levels of VII:Ag and VII:C,16 seems
Reprints: Kenneth A. Bauer, VA Boston Healthcare System, 1400 VFW
Parkway, West Roxbury, MA 02132.
Submitted May 24, 2000; accepted August 17, 2000.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the Medical Research Service of the Department of Veterans
Affairs (K.A.B.).
© 2000 by The American Society of Hematology
4370
BLOOD, 15 DECEMBER 2000 䡠 VOLUME 96, NUMBER 13
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BLOOD, 15 DECEMBER 2000 䡠 VOLUME 96, NUMBER 13
A NEW MUTATION IN THE FACTOR VII PROMOTER
4371
were then used to direct expression of hGH in transiently transfected HepG2 cells, a human hepatoma cell line producing fVII and
other coagulation proteins.17 Experiments in which 12 dishes were
transfected with each reporter plasmid indicated that MT55 plasmid exhibited 9.7% ⫾ 5.4% (1 SD) of the activity observed with
WT plasmid.
The deleterious effect of the ⫺55 mutation on promoter activity
appeared to be due to decreased interaction with HNF4, as demonstrated
by electrophoretic mobility shift assays. Direct binding of fVII oligonucleotides to HNF4 protein made by in vitro transcription/translation was
strongest with WT oligonucleotide, intermediate with MT55 oligonucleotide and, as expected from prior analysis,1 undetectable with MT61
oligonucleotide (Figure 1A). The relative capacities of fVII oligonucleotides to compete with WT oligonucleotide for HNF4 mirrored their
abilities to bind HNF4 (Figure 1B). Thus, the WT oligonucleotide
served as the strongest competitor, with the MT55 oligonucleotide a
weaker competitor, and the MT61 oligonucleotide competed poorly, if
at all.
Binding and supershifted complexes of identical electrophoretic
mobilities were formed between HepG2 nuclear extract, or HNF4
protein made by in vitro transcription/translation, and the ApoCIIIB, WT, and MT55 oligonucleotides (data not shown). The
MT55 oligonucleotide interacted with HNF4 present in the nuclear
extracts more weakly than did the WT oligonucleotide.
The HNF4 binding ability of these oligonucleotides reflected
their relative capacity to be transactivated by this transcription
factor. Compared with the WT promoter fragment, reporter plasmids containing MT55 and MT61 caused similar reductions in
promoter activity as determined by transient transfection assays
(9.7% and 6.7% 1 of expression observed with WT reporter
Figure 1. Binding of fVII oligonucleotides. (A) The ⫺55 C to T mutation diminished
binding of HNF4 to fVII promoter sequence. A radiolabeled WT oligonucleotide
encompassing the HNF4 binding site (⫺77 to ⫺47 base pairs prior to the translation
start site) in the human fVII promoter region showed binding to HNF4 protein
prepared by in vitro transcription/translation (lane 3), and corresponding oligonucleotides having the ⫺55 mutation (MT55, lane 5) or the ⫺61 mutation (MT61, lane 7)
showed weaker and undetectable binding, respectively. All 3 oligonucleotides bound
nonspecifically to components of the mock transcription/translation reaction mixture
(lanes 2, 4, 6), which migrated to various different positions on the gel. The binding
reaction with a control oligonucleotide (the HNF4 binding site from the ApoCIIIB
promoter) is shown in lane 1. The amounts of WT, MT55, and MT61 oligonucleotides
used per lane were identical, but less control oligonucleotide was used to compensate for its comparatively strong binding to HNF4. (B) Competition assays show
specificity of binding to HNF4. The radiolabeled WT oligonucleotide bound to HNF4
prepared by in vitro transcription/translation (lane 2), and binding was subject to
competition by inclusion of increasing concentrations of unlabeled WT oligonucleotide (5 ⫻, 10 ⫻, 50 ⫻, and 100 ⫻ relative to the concentration of the labeled
oligonucleotide, lanes 3-6 successively). There was also competition, though less
effective, by unlabeled MT55 oligonucleotide (10 ⫻, 50 ⫻, 100 ⫻, and 200 ⫻, lanes
7-10 successively) but not at all by unlabeled MT61 oligonucleotide (100 ⫻ and
200 ⫻, lanes 11 and 12). Binding of the control ApoCIIIB-labeled oligonucleotide to
the HNF4 protein is shown in lane 13, and binding between the WT fVII probe and
components of the mock transcription/translation reaction mixture is shown in lane 1.
unlikely to influence fVII expression, given the severe phenotype
of the patient.
Position ⫺55 is in close proximity to the fVII transcriptional
start site and lies within a region essential for hepatic expression.4-6
Therefore, fragments of the 5⬘ flanking region extending from
position ⫺185 to position ⫹1 (the translational start site) were
prepared by PCR of the patient’s genomic DNA and inserted into
the promoterless reporter plasmid. The WT and MT55 plasmids
Figure 2. Cotransfection of an HNF4 expression vector alters expression of
reporter gene from vectors with WT and MT55 fVII promoter sequence. A total of
3 ␮g of WT or MT55 reporter vectors were transiently transfected into HepG2 cells,
with or without 6 ␮g of the pCDNAI-HNF4 expression vector or pUC-19 plasmid DNA,
as noted. The growth hormone values were corrected for expression from the
promoterless pOGH vector under parallel conditions, and for transfection efficiency,
then normalized to expression from the WT vector in the absence of coexpressed
HNF4. The results shown (3.97% ⫾ 2.87% [1 SD] for MT55 plasmid versus
100% ⫾ 13.1% [1 SD] for WT plasmid, in the absence of pCDNAI-HNF4; 187% ⫾ 99%
[1 SD] for MT55 plasmid versus 610% ⫾ 185% [1 SD] for WT plasmid, in the
presence of pCDNAI-HNF4) were the averages from 3 experiments. The total
number of replicates in each group is shown.
From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
4372
BLOOD, 15 DECEMBER 2000 䡠 VOLUME 96, NUMBER 13
CAREW et al
plasmid, respectively). Without cotransfected HNF4, MT55 plasmid exhibited negligible expression relative to WT plasmid.
However, inclusion of an HNF4 expression vector elevated hGH
expression from both reporter plasmids, although to a significantly
lesser extent with the MT55 plasmid than with the WT plasmid
(Figure 2). This is in contrast to the MT61 plasmid, for which we
previously observed no transactivation by an HNF4 expression vector.1
Even though cotransfected HNF4 interacted functionally with
the mutant promoter fragment in vitro, native HNF4 appears unable
to affect expression from the mutant allele in vivo. This may be due
to a limited amount of HNF4 or more avid binding between
available HNF4 and other promoter elements. In human liver, the
fVII promoter is rather weak compared with other promoters whose
expression is HNF4-dependent, and a poor interaction of this
transcription factor with the WT fVII promoter was suggested as
the cause.5 The C to T mutation at position ⫺55 would reduce
ability of HNF4 to bind the fVII promoter still further, thereby
resulting in a negligible in vivo interaction.
Acknowledgments
We thank Dr Margarita Hadzopoulou-Cladaras of Boston University School of Medicine, Boston, MA, both for generously
providing pCDNAI-HNF4 and the human ApoCIII HNF4-binding
oligonucleotide and for helpful advice during this study. We also
appreciate the gift of polyclonal antibody directed against an
epitope in the C-terminus of rodent and human HNF4, given by Dr
Frances Sladek of the University of California, Riverside, CA.
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From www.bloodjournal.org by guest on June 11, 2017. For personal use only.
2000 96: 4370-4372
A new mutation in the HNF4 binding region of the factor VII promoter in
a patient with severe factor VII deficiency
Josephine A. Carew, Eleanor S. Pollak, Stanislaw Lopaciuk and Kenneth A. Bauer
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