Key Points

  • We report a partial F8 duplication associated with markedly elevated FVIII levels and venous thrombosis in 2 Italian families.

  • This duplication contains transcriptional activators that may be involved in the upregulation of F8 messenger RNA and protein expression.

Abstract

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

REFERENCES

1.
Egeberg
O
.
Thrombophilia caused by inheritable deficiency of blood antithrombin
.
Scand J Clin Lab Invest
.
1965
;
17
:
92
.
2.
Griffin
JH
,
Evatt
B
,
Zimmerman
TS
,
Kleiss
AJ
,
Wideman
C
.
Deficiency of protein C in congenital thrombotic disease
.
J Clin Invest
.
1981
;
68
(
5
):
1370
-
1373
.
3.
Schwarz
HP
,
Fischer
M
,
Hopmeier
P
,
Batard
MA
,
Griffin
JH
.
Plasma protein S deficiency in familial thrombotic disease
.
Blood
.
1984
;
64
(
6
):
1297
-
1300
.
4.
Bertina
RM
,
Koeleman
BP
,
Koster
T
, et al
.
Mutation in blood coagulation factor V associated with resistance to activated protein C
.
Nature
.
1994
;
369
(
6475
):
64
-
67
.
5.
Poort
SR
,
Rosendaal
FR
,
Reitsma
PH
,
Bertina
RM
.
A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis
.
Blood
.
1996
;
88
(
10
):
3698
-
3703
.
6.
Miyawaki
Y
,
Suzuki
A
,
Fujita
J
, et al
.
Thrombosis from a prothrombin mutation conveying antithrombin resistance
.
N Engl J Med
.
2012
;
366
(
25
):
2390
-
2396
.
7.
Djordjevic
V
,
Kovac
M
,
Miljic
P
, et al
.
A novel prothrombin mutation in two families with prominent thrombophilia—the first cases of antithrombin resistance in a Caucasian population
.
J Thromb Haemost
.
2013
;
11
(
10
):
1936
-
1939
.
8.
Bulato
C
,
Radu
CM
,
Campello
E
, et al
.
New prothrombin mutation (Arg596Trp, prothrombin Padua 2) associated with venous thromboembolism
.
Arterioscler Thromb Vasc Biol
.
2016
;
36
(
5
):
1022
-
1029
.
9.
Nogami
K
,
Shinozawa
K
,
Ogiwara
K
, et al
.
Novel FV mutation (W1920R, FVNara) associated with serious deep vein thrombosis and more potent APC resistance relative to FVLeiden
.
Blood
.
2014
;
123
(
15
):
2420
-
2428
.
10.
Pezeshkpoor
B
,
Castoldi
E
,
Mahler
A
, et al
.
Identification and functional characterization of a novel F5 mutation (Ala512Val, FVB onn) associated with activated protein C resistance
.
J Thromb Haemost
.
2016
;
14
(
7
):
1353
-
1363
.
11.
Simioni
P
,
Tormene
D
,
Tognin
G
, et al
.
X-linked thrombophilia with a mutant factor IX (factor IX Padua)
.
N Engl J Med
.
2009
;
361
(
17
):
1671
-
1675
.
12.
Koster
T
,
Blann
AD
,
Briët
E
,
Vandenbroucke
JP
,
Rosendaal
FR
.
Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis
.
Lancet
.
1995
;
345
(
8943
):
152
-
155
.
13.
van Hylckama Vlieg
A
,
van der Linden
IK
,
Bertina
RM
,
Rosendaal
FR
.
High levels of factor IX increase the risk of venous thrombosis
.
Blood
.
2000
;
95
(
12
):
3678
-
3682
.
14.
Meijers
JC
,
Tekelenburg
WL
,
Bouma
BN
,
Bertina
RM
,
Rosendaal
FR
.
High levels of coagulation factor XI as a risk factor for venous thrombosis
.
N Engl J Med
.
2000
;
342
(
10
):
696
-
701
.
15.
Hollestelle
MJ
,
Thinnes
T
,
Crain
K
, et al
.
Tissue distribution of factor VIII gene expression in vivo—a closer look
.
Thromb Haemost
.
2001
;
86
(
3
):
855
-
861
.
16.
Shahani
T
,
Covens
K
,
Lavend’homme
R
, et al
.
Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII
.
J Thromb Haemost
.
2014
;
12
(
1
):
36
-
42
.
17.
Pan
J
,
Dinh
TT
,
Rajaraman
A
, et al
.
Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo
.
Blood
.
2016
;
128
(
1
):
104
-
109
.
18.
Merlin
S
,
Famà
R
,
Borroni
E
, et al
.
FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice
.
Blood Adv
.
2019
;
3
(
5
):
825
-
838
.
19.
Fay
PJ
.
Activation of factor VIII and mechanisms of cofactor action
.
Blood Rev
.
2004
;
18
(
1
):
1
-
15
.
20.
Lakich
D
,
Kazazian
HH
Jr.
,
Antonarakis
SE
,
Gitschier
J
.
Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A
.
Nat Genet
.
1993
;
5
(
3
):
236
-
241
.
21.
Bagnall
RD
,
Waseem
N
,
Green
PM
,
Giannelli
F
.
Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A
.
Blood
.
2002
;
99
(
1
):
168
-
174
.
22.
Jenkins
PV
,
Rawley
O
,
Smith
OP
,
O’Donnell
JS
.
Elevated factor VIII levels and risk of venous thrombosis
.
Br J Haematol
.
2012
;
157
(
6
):
653
-
663
.
23.
Kamphuisen
PW
,
Houwing-Duistermaat
JJ
,
van Houwelingen
HC
,
Eikenboom
JC
,
Bertina
RM
,
Rosendaal
FR
.
Familial clustering of factor VIII and von Willebrand factor levels
.
Thromb Haemost
.
1998
;
79
(
2
):
323
-
327
.
24.
Bank
I
,
Libourel
EJ
,
Middeldorp
S
, et al
.
Elevated levels of FVIII:C within families are associated with an increased risk for venous and arterial thrombosis
.
J Thromb Haemost
.
2005
;
3
(
1
):
79
-
84
.
25.
Orstavik
KH
,
Magnus
P
,
Reisner
H
,
Berg
K
,
Graham
JB
,
Nance
W
.
Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level
.
Am J Hum Genet
.
1985
;
37
(
1
):
89
-
101
.
26.
Souto
JC
,
Almasy
L
,
Borrell
M
, et al
.
Genetic determinants of hemostasis phenotypes in Spanish families
.
Circulation
.
2000
;
101
(
13
):
1546
-
1551
.
27.
de Lange
M
,
Snieder
H
,
Ariëns
RA
,
Spector
TD
,
Grant
PJ
.
The genetics of haemostasis: a twin study
.
Lancet
.
2001
;
357
(
9250
):
101
-
105
.
28.
Vossen
CY
,
Hasstedt
SJ
,
Rosendaal
FR
, et al
.
Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C-deficient family
.
J Thromb Haemost
.
2004
;
2
(
2
):
242
-
247
.
29.
O’Donnell
J
,
Laffan
MA
.
The relationship between ABO histo-blood group, factor VIII and von Willebrand factor
.
Transfus Med
.
2001
;
11
(
4
):
343
-
351
.
30.
Smith
NL
,
Chen
MH
,
Dehghan
A
, et al;
Wellcome Trust Case Control Consortium
.
Novel associations of multiple genetic loci with plasma levels of factor VII, factor VIII, and von Willebrand factor: The CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) Consortium
.
Circulation
.
2010
;
121
(
12
):
1382
-
1392
.
31.
Antoni
G
,
Oudot-Mellakh
T
,
Dimitromanolakis
A
, et al
.
Combined analysis of three genome-wide association studies on vWF and FVIII plasma levels
.
BMC Med Genet
.
2011
;
12
:
102
.
32.
Tang
W
,
Cushman
M
,
Green
D
, et al
.
Gene-centric approach identifies new and known loci for FVIII activity and VWF antigen levels in European Americans and African Americans
.
Am J Hematol
.
2015
;
90
(
6
):
534
-
540
.
33.
Sabater-Lleal
M
,
Huffman
JE
,
de Vries
PS
, et al;
INVENT Consortium
;
MEGASTROKE Consortium of the International Stroke Genetics Consortium (ISGC)
.
Genome-wide association transethnic meta-analyses identifies novel associations regulating coagulation factor VIII and von Willebrand factor plasma levels
.
Circulation
.
2019
;
139
(
5
):
620
-
635
.
34.
Nossent
AY
,
Eikenboom
JC
,
Vos
HL
, et al
.
Haplotypes encoding the factor VIII 1241 Glu variation, factor VIII levels and the risk of venous thrombosis
.
Thromb Haemost
.
2006
;
95
(
6
):
942
-
948
.
35.
Hinds
DA
,
Buil
A
,
Ziemek
D
, et al;
METASTROKE Consortium, INVENT Consortium
.
Genome-wide association analysis of self-reported events in 6135 individuals and 252 827 controls identifies 8 loci associated with thrombosis
.
Hum Mol Genet
.
2016
;
25
(
9
):
1867
-
1874
.
36.
Shen
W
,
Gu
Y
,
Zhu
R
,
Zhang
L
,
Zhang
J
,
Ying
C
.
Copy number variations of the F8 gene are associated with venous thromboembolism
.
Blood Cells Mol Dis
.
2013
;
50
(
4
):
259
-
262
.
37.
Tizzano
E
,
Venceslá
A
,
Cornet
M
,
Baena
M
,
Baiget
M
.
Utility of a (GT) dinucleotide repeat in intron 1 of the factor 8 gene for haemophilia A carrier diagnosis
.
Haemophilia
.
2005
;
11
(
2
):
142
-
144
.
38.
Lalloz
MR
,
McVey
JH
,
Pattinson
JK
,
Tuddenham
EG
.
Haemophilia A diagnosis by analysis of a hypervariable dinucleotide repeat within the factor VIII gene
.
Lancet
.
1991
;
338
(
8761
):
207
-
211
.
39.
Lalloz
MR
,
Schwaab
R
,
McVey
JH
,
Michaelides
K
,
Tuddenham
EG
.
Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene
.
Br J Haematol
.
1994
;
86
(
4
):
804
-
809
.
40.
Machado
FB
,
Duarte
LP
,
Medina-Acosta
E
.
A novel informative dinucleotide microsatellite marker located on human factor VIII intron 25
.
Haemophilia
.
2009
;
15
(
2
):
613
-
614
.
41.
Schouten
JP
,
McElgunn
CJ
,
Waaijer
R
,
Zwijnenburg
D
,
Diepvens
F
,
Pals
G
.
Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification
.
Nucleic Acids Res
.
2002
;
30
(
12
):
e57
.
42.
Miotke
L
,
Lau
BT
,
Rumma
RT
,
Ji
HP
.
High sensitivity detection and quantitation of DNA copy number and single nucleotide variants with single color droplet digital PCR
.
Anal Chem
.
2014
;
86
(
5
):
2618
-
2624
.
43.
Li
H
,
Durbin
R
.
Fast and accurate short read alignment with Burrows-Wheeler transform
.
Bioinformatics
.
2009
;
25
(
14
):
1754
-
1760
.
44.
Abyzov
A
,
Urban
AE
,
Snyder
M
,
Gerstein
M
.
CNVnator: an approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing
.
Genome Res
.
2011
;
21
(
6
):
974
-
984
.
45.
Layer
RM
,
Chiang
C
,
Quinlan
AR
,
Hall
IM
.
LUMPY: a probabilistic framework for structural variant discovery
.
Genome Biol
.
2014
;
15
(
6
):
R84
.
46.
Livak
KJ
,
Schmittgen
TD
.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method
.
Methods
.
2001
;
25
(
4
):
402
-
408
.
47.
Gao
T
,
He
B
,
Liu
S
,
Zhu
H
,
Tan
K
,
Qian
J
.
EnhancerAtlas: a resource for enhancer annotation and analysis in 105 human cell/tissue types
.
Bioinformatics
.
2016
;
32
(
23
):
3543
-
3551
.
48.
Creyghton
MP
,
Cheng
AW
,
Welstead
GG
, et al
.
Histone H3K27ac separates active from poised enhancers and predicts developmental state
.
Proc Natl Acad Sci USA
.
2010
;
107
(
50
):
21931
-
21936
.
49.
Rose
AB
,
Carter
A
,
Korf
I
,
Kojima
N
.
Intron sequences that stimulate gene expression in Arabidopsis
.
Plant Mol Biol
.
2016
;
92
(
3
):
337
-
346
.
50.
Grant
CE
,
Bailey
TL
,
Noble
WS
.
FIMO: scanning for occurrences of a given motif
.
Bioinformatics
.
2011
;
27
(
7
):
1017
-
1018
.
51.
Figueiredo
MS
,
Brownlee
GG
.
cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene
.
J Biol Chem
.
1995
;
270
(
20
):
11828
-
11838
.
52.
Ottaviani
D
,
LeCain
M
,
Sheer
D
.
The role of microhomology in genomic structural variation
.
Trends Genet
.
2014
;
30
(
3
):
85
-
94
.
53.
Li
JN
,
Carrero
IG
,
Dong
JF
,
Yu
FL
.
Complexity and diversity of F8 genetic variations in the 1000 genomes
.
J Thromb Haemost
.
2015
;
13
(
11
):
2031
-
2040
.
54.
Famà
R
,
Borroni
E
,
Merlin
S
, et al
.
Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy [published online ahead of print 28 May 2020]
.
Haematologica
.
doi:10.3324/haematol.2019.239202
.
55.
Jin
Y
,
Messmer-Blust
AF
,
Li
J
.
The role of transcription enhancer factors in cardiovascular biology
.
Trends Cardiovasc Med
.
2011
;
21
(
1
):
1
-
5
.
56.
Santhekadur
PK
,
Rajasekaran
D
,
Siddiq
A
, et al
.
The transcription factor LSF: a novel oncogene for hepatocellular carcinoma
.
Am J Cancer Res
.
2012
;
2
(
3
):
269
-
285
.
57.
Jia
J
,
Ye
T
,
Cui
P
,
Hua
Q
,
Zeng
H
,
Zhao
D
.
AP-1 transcription factor mediates VEGF-induced endothelial cell migration and proliferation
.
Microvasc Res
.
2016
;
105
:
103
-
108
.
58.
Chiang
C
,
Scott
AJ
,
Davis
JR
, et al;
GTEx Consortium
.
The impact of structural variation on human gene expression
.
Nat Genet
.
2017
;
49
(
5
):
692
-
699
.
59.
Li
H
,
Chen
D
,
Zhang
J
.
Analysis of intron sequence features associated with transcriptional regulation in human genes
.
PLoS One
.
2012
;
7
(
10
):
e46784
.
60.
Park
SG
,
Hannenhalli
S
,
Choi
SS
.
Conservation in first introns is positively associated with the number of exons within genes and the presence of regulatory epigenetic signals
.
BMC Genomics
.
2014
;
15
(
1
):
526
.
61.
Jo
BS
,
Choi
SS
.
Introns: The Functional Benefits of Introns in Genomes
.
Genomics Inform
.
2015
;
13
(
4
):
112
-
118
.
62.
Weischenfeldt
J
,
Symmons
O
,
Spitz
F
,
Korbel
JO
.
Phenotypic impact of genomic structural variation: insights from and for human disease
.
Nat Rev Genet
.
2013
;
14
(
2
):
125
-
138
.
63.
Shovlin
CL
,
Angus
G
,
Manning
RA
, et al
.
Endothelial cell processing and alternatively spliced transcripts of factor VIII: potential implications for coagulation cascades and pulmonary hypertension
.
PLoS One
.
2010
;
5
(
2
):
e9154
.
64.
Brinke
A
,
Tagliavacca
L
,
Naylor
J
,
Green
P
,
Giangrande
P
,
Giannelli
F
.
Two chimaeric transcription units result from an inversion breaking intron 1 of the factor VIII gene and a region reportedly affected by reciprocal translocations in T-cell leukaemia
.
Hum Mol Genet
.
1996
;
5
(
12
):
1945
-
1951
.
65.
Rost
S
,
Löffler
S
,
Pavlova
A
,
Müller
CR
,
Oldenburg
J
.
Detection of large duplications within the factor VIII gene by MLPA
.
J Thromb Haemost
.
2008
;
6
(
11
):
1996
-
1999
.
66.
Sanna
V
,
Ceglia
C
,
Tarsitano
M
, et al
.
Aberrant F8 gene intron 1 inversion with concomitant duplication and deletion in a severe hemophilia A patient from Southern Italy
.
J Thromb Haemost
.
2013
;
11
(
1
):
195
-
197
.
67.
You
G
,
Chi
K
,
Lu
Y
, et al
.
Identification and characterisation of a novel aberrant pattern of intron 1 inversion with concomitant large insertion and deletion within the F8 gene
.
Thromb Haemost
.
2014
;
112
(
2
):
264
-
270
.
68.
Lannoy
N
,
Bandelier
C
,
Grisart
B
, et al
.
Tandem inversion duplication within F8 Intron 1 associated with mild haemophilia A
.
Haemophilia
.
2015
;
21
(
4
):
516
-
522
.
69.
Lannoy
N
,
Lambert
C
,
Van Damme
A
,
Hermans
C
.
Incidental finding of unreported large duplication in F8 gene during prenatal analysis: which management for genetic counselling?
Thromb Res
.
2019
;
182
:
39
-
42
.
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