Transcript Service Meeting Department of Medicine 內科部 部務會議

Clinical and Genetic Features
of Inherited Antithrombin
Deficiency
Yeu-Chin Chen MD
Division of Hematology/Oncology, Internal
Medicine
Tri-Service General Hospital, National
Defense Medical Center
January 12, 2016
Outline
A case illustration
 Pathogenesis of venous thromboembolism
 Inherited thrombophilia
 Antithrombin (AT) deficiency

 Clinical
and genetic features
 Diagnosis
TSGH experience
 Take home message

A Case Report


General data: A 37 y/o man
Chief complaint:
– Shortness of breath and anterior chest pain for one day.

Present illness:




Family Hx: His niece had been found to have antithrombin deficiency
assoicated DVTs.
Physical examination:



He suffered from these symptoms after long distance bicycle riding ( 100 KM ).
He has visited a local clinic without definite diagnosis. He visited our ER
because of worse symptoms.
Chest and lungs, bil: clear BS, no rales and rhonchi.
Heart: rapid and regular heart beats, normal S1 and S2, no murmur.
Laboratory data:

B/R: Hb: 15.0 g/dL; MCV: 89.4 fl WBC: 5700 N/L/M/E/B: 63/20/14/2
Platelet: 141K/cumm
Answer
D-Dimer ( near 10,000 ng/mL )
 Image diagnostic tool

 Ventilation/perfusion
lung scan
 Spiral ( helical ) chest CT scan with contrast.
Antithrombin gene screening-沈XX, the patient’s niece
Clinical data: 11/12/09 AT: 36.3%
E4: c.1012delG ; heterozygous (no reported data)
沈X君家族 AT 基因檢測結果:
This patient
AT:39.1%
張XX
AT:45.9%
沈XX
AT:97.2%
c.981A>G; 295V (P)
c.1011A>G; 305Q (P)
c.981A>G; 295V (P)
c.1011A>G; 305Q (P)
c.981A>G; 295V (P)
c.1011A>G; 305Q (P)
c.1012delG
c.1012delG
沈XX
AT:36.3%
c.981A>G; 295V (P)
c.1011A>G; 305Q (P)
c.1012delG
295V & 305Q : reported: polymorphism
c.1012delG : no reported data
Pathogenesis for Venous
Thromboembolism
Vessel Wall Trauma
Virchow’s
Triad
1856
Hypercoagulable state in
blood
( increased procoagulant or
decreased anticoagulant )
Blood flow stasis
One or more
Inherited
Prothrombotic
Mutation(s)
Factor V Leiden
Prothrombin 20210A
MTFHR mutation
Protein C deficiency
Protein S deficiency
Antithrombin deficiency
Thrombosis
Acquired
Prothrombotic
Stimulus
After Schafer
Antiphospholipid antibodies
Malignancy
Immobilization
Surgery
Pregnancy
Estrogen
Hyperhomocysteinemia
Heparin-induced thrombocytopenia
Coagulation pathway
Rogders GM Thromb Haemost 2009;101:806-812.
Clinical features of patients with inherited deficiencies of
AT, PC, PS, and APC-resistance
• Venous thrombosis (>90% of cases)
–
–
–
–
–
•
•
•
•
Deep vein thrombosis of the lower limbs (common)
Pulmonary embolism (common)
Superficial thrombophlebitis
Mesenteric vein thrombosis (rare but characteristic)
Cerebral vein thrombosis (rare but characteristic)
Frequent family history of thrombosis
First thrombosis usually at young age (<40yr*)
Frequent recurrences*
Neonatal purpura fulminans (homozygous PC or PS
deficiency)
*- all these features are less evident in patients with APC-resistance,
who appear to be less severely affected clinically
Factor V-Leiden:
• First reported from Bertina et al from the Leiden city,
Dutch in 1994.
• One of the most common causes for thrombophilia
– A single point mutation cause Arg506Gln of APC cleavage
site.
– 20% in first thrombotic event
– 50% in recurrent thormbosis
• Risk for VTE (DVT +/- PE)
– Heterozygous: 5-10 times .
– Homozygous: 50-100 times.
• 5-8% of general population affected (Caucasians).
Activation and degradation of normal
FV and FV Leiden
Dahlback B. Blood 2008; 112: 19-27
Frequency of inherited thrombophilia among unselected patients with VTE
Frequency of inherited thrombophilia among healthy subjects
Angchaisuksiri P. Thromb Haemost 2011;106:585-590.
Inherited AT deficiency

Inherited antithrombin (AT) deficiency: an uncommon
autosomal dominant disorder accounts for 0.5-4.9% in
patients with venous thromboembolism (VTE),
associated with a 5 to 50 fold increased risk for VTE.
AJH (1991) l36:249, Blood Coagul Fibrinolysis (1992) 3:749

The prevalence of AT deficiency among general population is
around 1/2000 to 1/5000.
Vosen et al. J of Tromb Hemostasis 2005.

No gender, racial or ethnic predilection
BJH (1994) 87: 106–12, AJH (1994) 45: 321–4.
Shen et al. Thromb Research 1997;87:377-385.
Protein C and protein S deficiencies are
the most important risk factors in
Chinese VTE in Taiwan
•116 Chinese VTE patients without acquired causes ( 58 male
and 58 female) and 125 healthy control subjects. (mean age:
47.5 vs 45.5 y/o)
•None of the 116 patients had prothrombin G20210A and factor
V Leiden mutation or an APC sensitivity ratio < 2.0
Shen et al. Thrombosis Research 2000; 99: 447-52.
Rogders GM Thromb Haemost 2009;101:806-812.
Rogders GM Thromb Haemost 2009;101:806-812.
Ornaghi et al. Thromb Res 2014;113:972-984.
Antithrombin

AT is a single chain plasma glycoprotein ( 58 KDa ) and
contains 464 amino acids, which belongs to the superfamily of
the Serine Protease Inhibitor ( SERPINS ).

The protein includes two functional domains:
 the heparin binding-domain at the N-terminus of the
mature protein
 the reactive site domain at the C-terminus.
AT is a globular protein
composed of three β-sheets (A,
B and C),nine α-helices and a
reactive center loop (RCL).
The RCL protrudes
above the core of the serpin
molecule and has a sequence
of amino acids at the reactive
center that is complementary
to binding pockets in the active
sites of target proteases
Type of AT deficiency
Several AT deficiency phenotypes are defined according to the plasma
level of functional and antigenic AT
Type I AT deficiency is defined by equally low functional and antigenic
antithrombin (about 50%) and type II deficiency by the synthesis of a
variant protein with altered function.
According to which function of the protein is affected,
three type II subtypes were defined :
• reactive site defect (RS),
• heparin binding site defect (HBS)
• pleiotropic defect, caused by mutations clustered in the s1C-s4B region.
Haemophilia (2008), 14, 1229–1239
Antithrombin gene
Official Symbol: SERPINC1
Official Full Name: serpin peptidase inhibitor, clade C (antithrombin),
member 1
chromosome: 1; Location: 1q23.1-q23.9
Exons: 7
Transcript length: 1,559 bps
Translation length: 464 residues
Structure of the antithrombin gene
More than 300 different mutations in the AT gene have been
reported.
Luxembourg et al. Thromb Haemost 2011;105:635-646.
Ding et al. Thromb Research 2013;132:367-373.
Treatment of VTE
• Initial treatment:
– Heparin: unfractioned vs LMWH
• Heparin resistance
– AT concentrate
• Long-term treatment: VKA for 3-6 months
• Extended treatment: VKA
– Lifelong: recurrent episodes, VTE in life-threatening site
such as cerebral vein and pulmonary embolism.
spontaneous episode with proven cause (DVT vs PE).
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Monitor of treatment
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Heparin – PTT.
LMW – anti FXa activity; LMWH does not affect PTT.
Warfarin: PT / INR 2-3.
New oral anticoagulants
Niagara falls
Clinical and Genetic Features of
Antithrombin Deficiency in 17
Taiwanese Patients
Yeu-Chin Chen* 1, Jyh-Pyng Gau2, Tsu-Yi Chao3
1Division
of Hematology/Oncology, Tri-Service General
Hospital, National Defense Medical Center
2Division of Hematology/Oncology, Department of Medicine,
Taipei Veteran General Hospital
3Division of Hematology/Oncology,
Department of Medicine, Shuang Ho Hospital, Taipei Medical
University, Taipei, Taiwan
June 24, 2015 at ISTH Congress
Introduction

Inherited anticoagulant deficiency associated VTE is
more important and encountered in Taiwan because no
FV Leiden and prothrombin gene mutations were
reported.
The
prevalence of AT deficiency in patients with VTE
in Taiwan was around 5.2~7.0%.
•Ho CH. Thromb Res 1997;88:409-412 Lin et al. Thromb Haemost 1998;80:343.
•Shen, et al. Thromb Res. 1997;87(4):377-85,
•Thromb Res. 2000;99(5):447-52,
• Chen TY, et al. Ann Hematol. 2003 Feb;82(2):114-7.
The aim of this study
The
aim of this study was to analyze
clinical features
 genetic defects
of patients with inherited AT deficiency associated VTE.

Patient and Methods
• 17 patients from 14 unrelated families were diagnosed to
have inherited AT deficiency associated VTE from
August 2009 to April 2014 from the 3 institutions located
in Taipei city territory.
• There were 10 male and 7 female patients.
• Their clinical presentations were recorded
★ demographic
★age at first VTE
★ number and sites of VTE
★ provoked events for VTE
Patient and Methods
• AT activity was assayed by chromogenic assay based
on FXa inactivation ( Liquid Antithrombin, HemosIL).
• Peripheral blood DNA was extracted after each patient’s
informed consent was obtained
• The 7 encoded exons including the boundary of
intron/exon of SERPINC1 gene were amplified by
polymerase chain reaction, followed by direct
sequencing.
Result
• The median age of our cohort:
– 40 y/o ( 16-60 y/o )
• The median age of first VTE attack
– 24 y/o ( 5-46 y/o )
• The median AT activity
– 43.7%, range from 32~64% ( normal range: 70120%).
Age of first VTE
7
6
Patient's number
5
4
3
2
1
0
1-10
11-20
21-30
Age
31-40
40-50
Sites and percentage of different VTE events in the 17 patients
Venous
thromoembolism
(VTE)
Deep vein
thrombosis
Patient Number
Percentage
11
64.7%
Plumonary embolism
7
41.2%
CNS thrombosis
2
11.8%
4
23.5%
4
23.5%
Mesenteric
thrombosis
Portal venous
thrombosis
Number of various VTE events in the 17 patients
Episodes of thrombosis
(Site)
Episodes
Patients
Patient number(%)
once
8
47.1%
twice
3
17.6%
Plumonary embolism
once
7
41.2%
CNS thrombosis
once
2
11.8%
Mesenteric thrombosis
once
4
23.5%
Portal venous thrombosis
once
4
23.5%
Deep vein thrombosis
Provocative events or associated risk for VTE in in the
17 patients
Provocative events or risk
Patient Number
Smoking
4
Excessive exercise
2
Post operation
2
Postpartum
1
Pregnancy
1
Muscle strain
1
Chemotherapy
1
Genetic defect of the 17 patients
Patient
Number
Genetic defect
Reference
1
E6: c.1301T>C; TTC>TCC; Phe(F) > Ser(S); F434S heterozygous Thrombosis and Haemostasis 1991; 65: 670
2
E3a: c.539delG ; heterozygous
3
E3a: c.539delG ; heterozygous
4
E6: c.1301T>C; TTC>TCC; Phe(F) > Ser(S); F434S heterozygous Thrombosis and Haemostasis 1991; 65: 670
5
E3b: c.663G>T; TGG>TGT; Trp(W) > Cys(C); W221C
heterozygous
6
E4: c.1012delG ; heterozygous
7
E4: c.1012delG ; heterozygous
8
E4: c.1148insC; heterozygous
9
E3a: c.442T>C; TCT>CCT; Ser(S) >Pro(P); S148P heterozygous
10
11
E4:c.851T>G ; ATG>AGG; Met(M)>Arg(R); M284R; heterozygous
E4 c.1148insC; heterozygous
12
E4 c.992delTCAC; heterozygous
13
E2: c.374G>A; GGT>GAT; Gly(G)>Asp(D); G125D; heterozygous
14
E5:c.1171C>T,CGA>TGA; Arg(R)>Ter(X); R391X
15
E4, c953C>T, CCC>CTC; Pro(P)>Leu(L); P318L
16
E3a:c456-464 del CTT delF
17
Intron 4, IVS4-1G>A, splice site mutation
Blood 1993; 81: 1300-1305
British Journal of Haematology 1993; 84:
656-661.
British Journal of Haematology 1993; 84:
656-661.
Genetic mutation type
8
7
Patient's number
6
5
4
3
2
1
0
Series1
missense
7
nonsense
1
deletion
6
insertion
2
splicing site
1
Genetic mutation type
Patient's number %
60%
50%
40%
30%
20%
10%
0%
Our Lab.
HGMD
missense/nonsen
se
47%
56%
deletion
insertion
splicing site
other type
35%
18%
12%
8%
6%
7%
0%
11%
HGMD: The Human Gene Mutation Database, total patients: 311
http://www.hgmd.cf.ac.uk/ac/all.php
Nine novel mutations were identified in this study
Genetic Defects
Number
E3a: c.539delG ; heterozygous; frameshift
Deletions
E4: c.1012delG ; heterozygous; frameshift
3
E4: c.992delTCAC ; heterozygous; frameshift
Insertions
Missense
E4: c.1148insC; heterozygous ; frameshift
E3b: c.663G>T; TGG>TGT; Trp(W) > Cys(C);
W221C; heterozygous
E4: c.851T>G ; ATG>AGG; Met(M)>Arg(R); M284R;
heterozygous
E2: c.374G>A; GGT>GAT; Gly(G)>Asp(D); G125D;
heterozygous
1
4
E4: c.953C>T; CCC>CTC; Pro(P)>Leu(L); P318L
Splicing site
Mut.
Intron 4, IVS4-1G>A
According to the report from Human Genetic Mutation Data
base ( www.hgmd.org )
1
Table. Clinical characteristics in 12 patients with VTE caused by inherited AT
deficiency in TSGH
AT antigen : 250-360 ug/mL
Discussion
• This is the first collaborative study to describe the
clinical features and genotype for AT deficiency
patients in Taiwan.
• We demonstrated that the majority of patients
( 65%) experienced DVT of lower limbs, followed by
pulmonary embolism (40%). Each a quarter of
patients had mesenteric and portal venous
thrombosis, respectively.
• 10% of patients had CNS thrombosis.
Discussion
• Most patients experienced first VTE attack at 20-30 years
of age. All patients developed VTE by age of 50. One
patient had portal venous thrombosis in childhood.
• Excessive exercise, post-operation, pregnancy and
postpartum were found to be the provoked events.
• Missense mutation ( 41% ) and deletion ( 35% ) were the
more common defect in our cohort.
• There was no repetitive mutation in our cohort and more
deletion and insertion mutations were noted as compared
to that reported from mainland China ( missense
mutation).
Ding et al. Thromb Res 2013;132:367-373.
Discussion and Conclusion

Nine novel AT gene defects were found and in vitro
protein expression and functional studies are indicated to
confirm the pathologically causative effect.
 The weakness of this study
 No AT antigen assay for type classification.
 Small patients number
 Patients lived in Northern Taiwan and urban area.
 In conclusion



AT deficiency patients developed first VTE in young age, and all
patients had VTE attack by 50 of age.
Majority of patients had experienced DVT of lower limbs.
Genetic study is very helpful for disease diagnosis.
Acknowledgments
Taipei Vetran General Hospital
Dr. Jyh-Pyng Gau
Shuang Ho Hospital
Dr. Tsu-Yi Chao
Tri-service General Hospital
Ms. Ya-Fang Yang
Ms. Shu-Hsia Hu
Take home message

AT deficiency carry the highest risk for VTE formation,
although its prevalence among VTE patients is the lowest.
 Type 1 AT deficiency is more common in inherited
thrombophilia.
 The majority of patients with AT deficiency experience first
VTE attack at 3rd decade.
 DVT and PE are the most commonly developed in patients
with inherited AT deficiency.
 Acquired AT deficiency should be ruled out by repetitive AT
testings.
 Genetic testing is very helpful for inherited AT diagnosis and
almost all patients can be identified to have genetic mutation.
 Missense/nonsense mutation are the most common type of
AT mutations.
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