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s12185-017-2352-8

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Int J Hematol
DOI 10.1007/s12185-017-2352-8
CASE REPORT
Gene analysis of inherited antithrombin deficiency and functional
analysis of abnormal antithrombin protein (N87D)
Sayaka Kamijima1 · Akiko Sekiya1 · Mao Takata2 · Haruka Nakano1 ·
Morika Murakami1 · Tomonori Nakazato3 · Hidesaku Asakura4 · Eriko Morishita1,4 Received: 22 May 2017 / Revised: 27 September 2017 / Accepted: 9 October 2017
© The Japanese Society of Hematology 2017
Abstract Inherited antithrombin (AT) deficiency is one of
the most clinically significant forms of congenital thrombophilia and follows an autosomal dominant mode of inheritance. We analyzed SERPINC1 in a patient who developed
deep-vein thrombosis and low AT activity during pregnancy, and identified a novel missense mutation c.259A>G
(p.Asn87Asp; N87D). Surprisingly, analysis of the parents’ DNA showed that they did not possess this mutant,
and thus, it may have been due to a de novo mutation. We
also expressed this mutant AT protein in COS-1 cells and
compared its intracellular localization and intracellular and
extracellular antigen levels with that of wild-type AT. The
expression experiment did not reveal a significant difference
in the antigen levels of the mutant and wild-type AT in the
cell lysate, but the mutant AT antigen level was markedly
lower than that of its wild-type counterpart in the COS-1
cell supernatant. Immunofluorescence did not indicate any
difference between the mutant and wild-type AT in terms of
cytoplasmic localization of fluorescence signals. Our findings suggest that the patient’s AT deficiency may have been
* Eriko Morishita
eriko86@staff.kanazawa‑u.ac.jp
1
Division of Health Sciences, Kanazawa University Graduate
School of Medical Science, 5‑11‑80 Kodatsuno, Kanazawa,
Ishikawa 920‑0942, Japan
2
Department of Central Clinical Laboratory, Kanazawa
Medical University Hospital, 1‑1 Daigaku, Uchinada,
Kahoku, Ishikawa 920‑0293, Japan
3
Department of Hematology, Yokohama Municipal
Citizen’s Hospital, 56 Okasawa, Hotogaya, Yokohama,
Kanagawa 240‑8555, Japan
4
Department of Internal Medicine (III), Kanazawa University
School of Medicine, 13‑1 Takara‑machi, Kanazawa,
Ishikawa 920‑8641, Japan
caused by impaired extracellular secretion of mutant AT
protein p.Asn87Asp.
Keywords Antithrombin deficiency · Venous
thrombosis · Missense mutation · Functional analysis · De
novo mutation
Introduction
Antithrombin (AT) is a plasma serine protease inhibitor that
inactivates coagulation factors such as thrombin and factors
Xa, IXa, XIa, and XIIa. AT is a 432-amino acid single-chain
glycoprotein that is synthesized by hepatocytes as a 464amino acid precursor, from which a 32-amino acid signal peptide is cleaved off before secretion. The molecular weight of
AT is approximately 58 kDa, and it circulates at a concentration of 15–27 mg/dL. The human antithrombin gene is located
on chromosome 1 at q23.1–23.9 and spans 13.5 kb in the
genome. The gene comprises seven exons and six introns [1].
AT inactivates its target proteases through stable binding
to the active sites of these proteases. AT activity is accelerated approximately 1000-fold by the binding of heparin to
arginine residues in the D-helix of the AT protein [2, 3].
Inherited AT deficiency is an autosomal dominant thrombotic disorder caused by various mutations in SERPINC1.
The prevalence of inherited AT deficiency in the general
population is estimated to be 0.02–0.25% among different
populations, and 2–5% in patients with venous thromboembolism [4]. According to the plasma levels of functional
and antigenic AT, AT deficiency is divided into two phenotypes: type I deficiency in which both functional and antigenic AT levels are low, and type II deficiency in which
the functional AT level is low but the antigenic AT level is
normal. Depending on the location of the mutation, type II
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deficiency is further subdivided into three groups: type II
reactive site defects, type II heparin binding site defects,
and type II defects associated with pleiotropic defects [5, 6].
In the present study, we identified a novel missense mutation in a Japanese patient with type I AT deficiency. Surprisingly, the analysis of the parents’ DNA showed that they did
not possess this mutant, and thus, it may have been due to a
de novo mutation. We also expressed this mutant AT protein
in African green monkey kidney (COS-1) cells and compared its intracellular antigen production and extracellular
secretion levels with those of the wild-type AT. Moreover,
we analyzed the protein’s cytoplasmic localization using
fluorescence immunostaining.
Ethical approval for this study was obtained from the Ethics Committee of Kanazawa University School of Medicine,
Japan.
Case report
The proband was a 30-year-old woman. She developed deep
vein thrombosis (DVT) in her leg while pregnant with her first
child at the age of 23 years, and underwent an induced abortion to treat the DVT. While pregnant with her second child,
the patient was diagnosed with type I AT deficiency because
her AT activity using the FXa inhibition activity assay (Sekisui Medical, Tokyo, Japan) and AT antigen level using an
enzyme-linked immunosorbent assay (ELISA) (Affinity Biologicals, Inc., Ontario, Canada) decreased to 42% and 8.4 mg/
dL (normal range 23.6–33.5 mg/dL), respectively. Following
heparin anticoagulant therapy and AT replacement therapy,
the patient gave birth without incident. Although her paternal cousin had a previous history of cerebral infarction when
24 years old, both of the proband’s parents had normal AT
activity: 94% in the father and 115% in the mother.
After obtaining their informed consent, we extracted
DNA from peripheral blood leukocyte samples of the
proband and her parents. We then used the polymerase
chain reaction (PCR) method to amplify all seven exons and
exon–intron boundaries of the SERPINC1, and used direct
sequencing to determine the base sequence [7, 8]. Next, we
performed a PCR-restriction fragment length polymorphism
(PCR–RFLP) assay using the restriction enzyme MluC1 to
determine whether the mutation was present in the proband
and her parents, as well as in 50 healthy volunteers.
The mutation detected in the proband was analyzed in
silico to investigate whether these mutations could be pathogenic. For in silico analysis, three bioinformatics tools,
Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org),
Polymorphism Phenotyping (PolyPhen-2; http://genetics.
bwh.harvard.edu/pph2), and Mutation Taster (http://www.
mutationtaster.org/), were used.
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S. Kamijima et al.
We constructed an AT expression vector for the
mutation identified in the proband by performing sitedirected mutagenesis using a mutation primer (forward:
5′-GCCGATTC
​ CCG
​ CTT
​ TGC
​ TAC-3′ underlined nucleotide
indicates the introduced mutation, reverse: 5′-CTTG
​ GAC
​ AG​
TTC​CCA​GAC​AC-3′) with full-length, wild-type AT cDNA
in the PCR3.0(+) vector (kindly provided by Dr. Tsuneo
Imanaka of Toyama University, Japan). We then transfected
COS-1 cells with the wild-type and mutant AT cDNA using
HilyMax Transfection Reagent (Dojindo, Kumamoto, Japan)
and cultured the cells for 48 h. Using the culture cell lysate
and supernatant, we identified the AT protein in accordance
with the attached manual using the Matched-Pair Antibody
set for ELISA of the human AT antigen (Affinity Biologicals, Inc.). We then performed immunofluorescent staining
using mouse anti-human AT monoclonal antibody (Sigma)
as the primary antibody and tetramethylrhodamine isothiocyanate-labeled goat anti-mouse IgG antibody (Sigma) as
the secondary antibody, and observed the intracellular localization of the AT protein with a fluorescence microscope.
The data obtained from our experiment are expressed as
the mean ± standard error of the mean (SEM). Statistical
analysis was performed using the Student’s t test with a significance level of P < 0.05.
After analyzing the SERPINC1 in the proband’s DNA
sample, we identified a missense mutation caused by a single base substitution from A to G at position c.259 in exon
2. We presume that this base substitution caused the 87th
amino acid of the AT protein—namely aspartic acid (Asn,
N)—to mutate into aspartic acid (Asp, D) (Fig. 1). We used
PCR–RFLP to determine whether this mutation was present
Fig. 1 A missense mutation identified in SERPINC1 in the patient.
Using direct sequencing to analyze the SERPINC1 in the proband’s
DNA sample, we identified a single base substitution from A to G at
position c.259 in exon 2. We presume that this substitution caused the
87th amino acid of the AT protein—namely asparagine (Asn, N)—to
mutate into aspartic acid (Asp, D) (wherein A in the initiation codon
ATG is represented as base number + 1, and methionine is amino
acid number + 1)
Gene analysis of inherited antithrombin deficiency and functional analysis of abnormal…
Fig. 2 PCR-RFLP using restriction endonuclease MluC1 in the
patient and patient’s parents. When the N87D mutation is not present,
cleaving the DNA with MluC1 and performing electrophoresis causes
the 232- and 273-bp bands to appear. Conversely, when the N87D
mutation is present, the DNA is not cleaved and the 505-bp band
appears. This is why both bands were seen in the proband, whereas
only one band was seen in her parents
in the proband and in 50 healthy volunteers. The proband
had the 273-bp/232-bp bands, and all of the 505-bp bands,
indicating the presence of the mutation. Conversely, none
of the 50 volunteers had the 505-bp band, demonstrating
that this single base substitution is not a polymorphism.
Moreover, neither of the proband’s parents were deemed to
possess the mutation because they did not have any of the
corresponding bands (Fig. 2).
Fig. 3 The levels of recombinant AT antigen in cell lysates and
culture medium of COS-1 cells transfected with either wild-type or
N87D mutant AT expression vectors. The levels of the AT antigen
in cell lysates and culture medium measured by ELISA are shown in
a relative manner in which antigen levels of wild-type AT are set at
All three methods of in silico analysis revealed that
this mutation is pathogenic, because the results of SIFT,
PolyPhen-2, and MutationTaster were “probably damaging
(score = 1.000)”, “damaging (score = 0.040)”, and “disease
causing (score = 0.999)”, respectively.
Assuming an antigen level of 100% for the wild-type AT,
the relative levels of the N87D mutant AT antigen detected
in the cell culture and in the supernatant using ELISA were
67 ± 16 and 11 ± 5%, respectively. The antigen level in the
cell culture was therefore slightly lower, but not significantly
different. On the other hand, the antigen level in the supernatant was significantly lower (P = 0.003) (Fig. 3).
The results of fluorescence immunostaining showed
that both the wild-type and mutant AT had a reticular
distribution of fluorescence inside the cytoplasm, and that
there was no discernible difference in the localization of
their respective fluorescence signals (Fig. 4).
Discussion
In this study, we identified a novel missense mutation
(p.N87D) in the patient with type I AT deficiency. In the
expression experiment, a newly identified mutation showed
a slight decrease in levels of the AT antigen in the cell lysate
and a significant decrease in the medium, indicating diminished secretion of mutant AT. The three methods of in silico
analysis indicated that this mutation is pathogenic. Asn87
and its surrounding region are highly conserved among
multiple mammalian species. Multiple causal missense
100%. MOCK indicates the expression vector, which does not contain
AT cDNA. Data obtained by three independent experiments are presented as the mean ± standard error of the mean. P values obtained
by Student’s t test are shown
13
S. Kamijima et al.
Fig. 4 Fluorescence immunostaining. Both the wild-type
and mutant AT exhibited a
reticular distribution of fluorescence inside the cytoplasm.
No fluorescent substance was
visible inside the cells using
MOCK
mutations of AT deficiency, such as Ala86Val, Ser88Pro,
and Arg89Cys, have been identified at the adjacent locus
[4, 9]. Taken together, we speculate that Asn87 and its surrounding region play a significant role in determining the
appropriate conformation of AT. Asn87 is located in the
middle of helix A on the N-terminus of mature AT. Helix A
(Arg78–Ser101) is mainly composed of uncharged amino
Fig.  5 a Modeling of
antithrombin. The area marked
in red is the reactive site to serine protease on the C-terminal
end, and the area marked in
yellow on the N-terminal end
is the heparin binding site. This
mutation exists within the helical structure. b Electric charge
in helix A. The amino acids in
helix A have been color-coded
according to their electrical
charge. Specifically, the negatively charged amino acids are
colored blue, and the positively
charged amino acids are colored
red. The spheres represent the
87th amino acid that switched
from polar asparagine to negatively charged aspartic acid
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acid residues and a few positively charged residues (Fig. 5a).
The conformation of helix A, and possibly the entire conformation of AT, may be affected by the substitution of an
uncharged asparagine with a negatively charged aspartic acid
in the middle of helix A (Fig. 5b).
There are some reports of type I AT deficiency in which
more detailed functional analysis by pulse-chase analysis
Gene analysis of inherited antithrombin deficiency and functional analysis of abnormal…
were performed [4, 10]. Zhou et al. showed, in their cases
of Thr98Ile, that a substitution of Thr by Ile alters the conformation of helix C, affecting AT protein folding, resulting
in a secretion defect and intracellular degradation [10]. In
our case, substitution of Asn 87 by Asp may have caused
intracellular degradation of AT in a similar manner.
We did not attempt to quantify mRNA expression or
investigate its intracellular transport and degradation mechanisms in the present study. As such, we cannot deny the
possibility that the decrease in the mutant AT antigen level
was due to mRNA degradation or defective intracellular
synthesis of the AT protein. Further research is needed to
determine the actual mechanism by which this mutation triggers a reduction in the AT antigen.
The fact that the mutation was not found in our analysis
of the parents’ DNA suggests that it could be a de novo
mutation. In fact, there have been several studies of AT
deficiency caused by de novo mutations [11, 12]. While a
detailed patient and family history and paternity/maternity
test results based on several DNA samples are typically
required to determine whether a genetic defect is a de novo
mutation, we could not perform such analyses in the present
study due to ethical constraints.
In conclusion, our data showed that the N87D mutation in
AT caused AT deficiency with decreased level of AT antigen
and activity. To clarify mechanisms in which genetic mutations affect protein expression and function, and relationship between genetic abnormalities and clinical phenotypes,
may contribute further understanding of pathogenesis of AT
deficiencies.
Acknowledgements The authors would like to thank Dr. Tsuneo
Imanaka for providing the pcDNA3.1/AT expression plasmid. This
study was partly supported by a grant from the Ministry of Health,
Labor and Welfare to E.M. (Grant number 26070201), and grants of the
Ministry Education, Culture, Sports, Science and Technology of Japan
to E.M. (Grant number 15K08643), A.S. (Grant number 25462818) and
H.A. (Grant number 15K19176).
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Compliance with ethical standards Conflict of interest The authors declare that they have no conflict
of interest.
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