Download A novel deletion mutation is recurrent in von Willebrand disease

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THROMBOSIS AND HEMOSTASIS
A novel deletion mutation is recurrent in von Willebrand disease types 1 and 3
Megan S. Sutherland,1 Anthony M. Cumming,1 Mackenzie Bowman,2 Paula H. B. Bolton-Maggs,1 Derrick J. Bowen,3
Peter W. Collins,3 Charles R. M. Hay,1 Andrew M. Will,4 and Stephen Keeney1
1University
Department of Haematology, Manchester Royal Infirmary, Manchester, United Kingdom; 2Pathology and Molecular Medicine, Queen’s University,
Kingston, ON; 3Department of Haematology, School of Medicine, Cardiff University, Cardiff, United Kingdom; and 4Department of Haematology, Royal
Manchester Children’s Hospital, Manchester, United Kingdom
Direct sequencing of VWF genomic DNA
in 21 patients with type 3 von Willebrand
disease (VWD) failed to reveal a causative
homozygous or compound heterozygous
VWF genotype in 5 cases. Subsequent
analysis of VWF mRNA led to the discovery of a deletion (c.221-977_532 ⴙ 7059del
[p.Asp75_Gly178del]) of VWF in 7 of
12 white type 3 VWD patients from 6
unrelated families. This deletion of VWF
exons 4 and 5 was absent in 9 patients of
Asian origin. We developed a genomic
DNA-based assay for the deletion, which
also revealed its presence in 2 of 34 type
1 VWD families, segregating with VWD in
an autosomal dominant fashion. The deletion was associated with a specific VWF
haplotype, indicating a possible founder
origin. Expression studies indicated markedly decreased secretion and defective
multimerization of the mutant VWF protein. Further studies have found the muta-
tion in additional type 1 VWD patients and
in a family expressing both type 3 and
type 1 VWD. The c.221-977_532 ⴙ 7059del
mutation represents a previously unreported cause of both types 1 and 3 VWD.
Screening for this mutation in other type 1
and type 3 VWD patient populations is
required to elucidate further its overall
contribution to VWD arising from quantitative deficiencies of VWF. (Blood. 2009;
114:1091-1098)
Introduction
von Willebrand disease (VWD) is a hereditary bleeding disorder
resulting from quantitative (type 1 and type 3 VWD) or qualitative (type
2 VWD) abnormalities of the multimeric plasma glycoprotein von
Willebrand factor (VWF).1-3 VWF has essential roles in primary
hemostasis where it functions in an adhesive matrix between platelets
and subendothelial components at sites of vascular injury in vessels
subject to high shear stress.4,5 VWF also acts as a carrier for procoagulant factor VIII (FVIII) in the circulation.
Type 3 VWD, the result of markedly decreased or absent
VWF, is associated with moderate to severe bleeding symptoms,
including epistaxis, menorrhagia, arthropathy, and postoperative
bleeding.6 It is an autosomal recessive inherited bleeding
disorder with a prevalence of approximately 0.5 to 1 person per
million in the general population,7 although prevalence may be
as high as 6 per million in populations where consanguinity is
common.8 Originally, it was assumed that type 3 VWD was
caused by large deletions of the gene encoding VWF (VWF). Of
107 mutations reported to cause type 3 VWD (International
Society on Thrombosis and Hemostasis [ISTH] SSC VWF
database9), there are only 11 (10%) reports of large or partial
gene deletions. These deletions range in size from a single
exon10 through several exons11-14 to the entire gene.15-18 Heterozygous carriers of deletions have so far all been reported to be
asymptomatic and do not have significant bleeding symptoms.
Approximately 83% of VWF mutations in type 3 VWD are made
up of mutations associated with a VWF-null allele (ie, nonsense
mutations, deletions, splice site mutations, and small insertions). Missense mutations account for 17% of sequence variants responsible for type 3 VWD.
Type 1 VWD, reportedly the most common form of the disorder,19 is
caused by a partial quantitative deficiency of VWF and is considered
classically to show an autosomal dominant inheritance pattern.1,2 The
main symptom of type 1 VWD is a significant mucocutaneous bleeding
history; however, a definitive diagnosis is often complicated by variable
expressivity and incomplete penetrance.20 There are several environmental factors, such as age and stress as well as other genetic modifiers at
different loci, particularly ABO blood group, which may influence the
observed phenotype.2,21-28 Recent studies have investigated the molecular pathogenesis of type 1 VWD and have found inconclusive results
regarding the underlying genetic defects associated with the disease in a
large proportion of patients.29-31 These findings support the hypothesis
that the type 1 VWD phenotype is not always associated with
pathogenic mechanisms involving VWF and indicate limited utility
for conventional genetic diagnosis in type 1 VWD.32
The ISTH VWF mutation database9 reports 144 candidate VWF
mutations associated with type 1 VWD. These include 93 missense
mutations, 9 small deletions, 13 splice site mutations, 19 promoter
region mutations, 2 insertions, one duplication, and 7 nonsense
mutations. It may be unclear how a mutation on a single VWF allele
gives rise to a clinical bleeding disorder when the second allele
may be predicted to maintain VWF at levels sufficient for normal
hemostasis, although a dominant-negative mechanism has been
demonstrated in some cases.33,34 In this respect, heterozygous
carriers of type 3 VWD are generally clinically asymptomatic.
The primary aim of our study was to identify the underlying
molecular pathogenesis of type 3 VWD in 21 patients (20 index
cases and 1 family member) with this disorder who attend the adult
and pediatric hemophilia centers at Central Manchester National
Submitted August 7, 2008; accepted April 8, 2009. Prepublished online as
Blood First Edition paper, April 16, 2009; DOI 10.1182/blood-2008-08-173278.
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 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
© 2009 by The American Society of Hematology
BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
1091
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The phenotypic data, bleeding history, and genotypic data are shown for the type 3 and type 1 VWD patient cohorts. All patients for whom data are reported here are white and of British origin. Laboratory reference ranges: VWF:Ag, 50 to
200 IU/dL; VWF:RCo, 50 to 200 IU/dL; and FVIII, 50 to 150 IU/dL.
*Bleeding symptoms: E, epistaxis; O, oral bleeding; S, bleeding from cuts; D, dental bleeding; Sur, surgical bleed; B, bruising; M, menorrhagia; P, postpartum bleeding; J, joint bleeding; I, intracerebral bleed; and G, gastrointestinal bleed.
†Zygosity: Hom, homozygous; Cpd het, compound heterozygous; and Het, heterozygous.
‡Individual is unaffected.
Het
Ex4-5del
90
1
10:1
18
O
15
11
Normal
E, B
Cpd het
Het
Ex4-5del ⫹ Splice site
Ex4-5del
No clinical history available
Normal
I, Sur
2
145
37
Not detected
9:2
1
3
Possible 1
9:1
32
A
Not detected
39
Dimers only detected
Het
Het
Ex4-5del
B, E, surgery with DDAVP cover
47
A
1
8:2
8
⬍ 20
⬍ 20
Reduced HMW
Het
Ex4-5del
E, M, B, Sur
89
O
1
8:1
32
20
28
Normal
Het
None, all surgery with DDAVP cover
Ex4-5del
Ex4-5del
None, all surgery with DDAVP cover
Normal
89
107
21
O
25
A
1
7:4
15
1
7:3
18
41
43
Normal
Het
Ex4-5del
O, D, S, B, M
37
O
1
7:2
45
16
11
Normal
Het
Hom
Ex4-5del
None
99
B
1‡
7:1
81
81
105
Normal
Hom
Ex4-5del
Ex4-5del
D, J, G, O
E, D, J, I, G
2
None detected
2
Not detected
O
Not detected
A
3
6:2
42
3
6:1
46
Not detected
Not detected
None detected
Cpd het
Hom
Ex4-5del
E, O, J, G
1
AB
3
5:1
19
Not detected
Not detected
None detected
Cpd het
Ex4-5del ⫹ missense
E, O, J
13
A
3
4:1
46
3
Not detected
Trace
Het
Ex4-5del
Ex4-5del ⫹ frameshift
M, E
E, S, J, G
Not done
None detected
19
⬍1
Not detected
Not detected
4
⬍1
A
B
48
55
3
3
2:1
3:1
Cpd het
Het
Ex4-5del
B, D
34
O
1
1:2
18
22
13
Normal
Zygosity†
Ex4-5del ⫹ Indel
Mutation present
Bleeding symptoms*
E, D, S, O, B, M, J, G
Not detected
Multimer pattern
FVIII, IU/dL
Not detected
VWF:RCo, IU/dL
Not detected
AB
55
VWF:Ag, IU/dL
ABO blood group
Age, y
3
VWD type
1:1
Family:patient
Table 1. Phenotypic data for all patients
BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
SUTHERLAND et al
None detected
1092
Health Service Trust. We initially used genomic DNA sequence
analysis of the essential regions of VWF (52 exons and flanking
intronic regions, 5⬘ promoter region, and the 3⬘ untranslated
region)29 to identify homozygosity or compound heterozygosity in
16 type 3 VWD patients for a variety of VWF mutations (M.S.S.,
A.M.C., P.H.B.B.-M., C.R.M.H., A.M.W., S.K., manuscript submitted). Homozygosity for a previously unreported in-frame deletion
mutation removing exons 4 and 5 of VWF was found in 2 index
cases and one family member. Subsequent RNA analysis revealed
this deletion to be present in the heterozygous state in a further
4 unrelated type 3 VWD index cases. This information was used to
design an assay for the deletion that could be applied at the
genomic DNA level and was used to investigate the presence of the
deletion in 34 index cases previously recruited into a United
Kingdom national study of type 1 VWD.29
Methods
Ethical approval
The study of type 3 VWD patients in Manchester was approved by the Central
Manchester Local Research Ethics Committee (Manchester, United Kingdom).
The study of VWF genotype in United Kingdom patients with type 1 VWD was
approved by the Multi-Center Research Ethics Committee for Wales.
Patients
Twenty index cases and one family member with an existing diagnosis of
type 3 VWD (VWF:antigen [VWF:Ag] ⬍ 5 IU/dL, VWF:ristocetin cofactor activity [VWF:RCo] undetectable) were recruited (Table 1 phenotypic
data). Thirty-four patients with type 1 VWD previously recruited into a
United Kingdom national study29 were also investigated. Written informed
consent was obtained from all patients in accordance with the Declaration
of Helsinki. A total of 20 mL of blood was collected from each patient in
four 5 mL tubes containing 0.105 M sodium citrate. Samples were
processed as soon as possible after collection to ensure the integrity of the
platelets for RNA extraction.
Nucleic acid extraction
Genomic DNA was extracted from leukocytes using an in-house ammonium acetate method. RNA was isolated from platelets using TRIzol reagent
(Invitrogen).
PCR primer design
Fifty-one primer sets were used to amplify the essential regions of VWF (52
exons and flanking intronic regions, 5⬘ promoter region, and the 3⬘
untranslated region). Primers had previously been designed in-house
(sequences available on request) to amplify the sequence of interest at an
annealing temperature of either 57°C or 59°C. Thirteen primer sets were
designed using Primer 3 software35 to amplify the VWF cDNA. A further
18 primer sets, designated A to R, were designed to amplify the 26-kb
region of genomic DNA from exon 3 to exon 6 of VWF.
Mutation analysis
Polymerase chain reaction (PCR) was performed to amplify the essential
regions of VWF. The PCR mix contained 2.5 ␮L 10⫻ PCR buffer, 0.75 ␮L
50 mM MgCl2, 0.5 units Taq polymerase, 2 ␮L 10 mM deoxynucleotide
triphosphates (all supplied by Invitrogen), 6 ␮L appropriate forward and
reverse primer (2 ␮M each), 12.65 ␮L sterile H2O, and 1 ␮L (approximately 250 ng) genomic DNA in a final volume of 25 ␮L. PCR was
performed on a DNA thermal cycler (MWG Biotech) as follows: 95°C for 2
minutes, followed by 32 cycles of 94°C for 30 seconds, 59°C or 57°C for
45 seconds, 72°C for 1 minute 30 seconds, and a final extension at 72°C for
5 minutes. PCR products were visualized on a 1.5% (wt/vol) agarose gel.
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BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
A NOVEL DELETION MUTATION IN VWD TYPES 1 AND 3
1093
Table 2. The haplotype panel studied in the patients with the ex4-5del mutation
Haplotype panel
Polymorphic frequency
SNP reference number
Common haplotype 1
Haplotype 2
c.-11254A⬎G
0.37/0.63
rs10774398
G
G
c.-6231G⬎A
0.35/0.65
rs10774394
A
A
c.-6117G⬎A
0.33/0.67
rs10849387
A
A
c.-2709C⬎T
0.71/0.29
rs7964777
T
T
c.-2661A⬎G
0.71/0.29
rs7954855
G
G
c.-2527G⬎A
0.71/0.29
rs7965413
A
A
c.-2522C⬎T
0.96/0.04
—
C
C
c.-64C⬎T
0.35/0.65
rs2286608
T
T
C
c.-20C⬎T
0.99/0.01
rs41276742
C
c.220⫹2421C⬎T
0.35/0.65
rs10849385
T
T
c.220⫹3364G⬎A
0.35/0.65
rs7961844
A
A
c.220⫹3793A⬎T
0.34/0.66
rs12307072
T
T
c.221-1953G⬎T
0.34/0.66
rs3782716
T
T
c.954T⬎A
0.96/0.04
rs1800387
T*
T*
c.1411G⬎A
0.45/0.55
rs1800377
G*
G*
G*
c.1451G⬎A
0.62/0.38
rs1800378
G*
c.2282-42C⬎A
0.46/0.54
rs216293
A*
C*
c.2365A⬎G
0.67/0.33
rs1063856
A*
G*
c.2385T⬎C
Unknown
rs1063857
T*
C*
c.3414C⬎T
0.98/0.02
rs4008538
T*
C*
c.4141A⬎G
0.46/0.54
rs216311
A*
G*
c.4641C⬎T
0.42/0.58
rs216310
T*
C*
A*
c.4665A⬎C
0.64/0.36
rs1800384
A*
c.5844C⬎T
0.71/0.29
rs216902
C*
T*
c.7682T⬎A
0.94/0.06
rs35335161
T*
T*
Common haplotype 1 was observed in all patients heterozygous or homozygous for the deletion. Haplotype 2 was seen in 2 related type 3 VWD cases in the compound
heterozygous state with haplotype 1. Common haplotype 1 and haplotype 2 are identical 5⬘ of the deletion and differ 3⬘ of the deletion.
— indicates not applicable.
* 3⬘ haplotype panel.
PCR product purification was performed using microCLEAN (Web Scientific) before cycle sequencing using Big-Dye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems). Sequencing reaction products were
purified using an in-house ethanol precipitation method and resuspended in
formamide (Applied Biosystems) before direct sequencing on an Applied
Biosystems AB3130xl DNA Sequencer. Mutation detection was performed
using the Staden software package.36 Each candidate mutation was
confirmed by repeat PCR and sequencing.
cDNA analysis
cDNA synthesis of platelet-derived RNA was achieved using random
hexameric primers according to the manufacturer’s instructions (M-MLV
Reverse Transcription System; Invitrogen). VWF cDNA was then analyzed
by standard PCR methods (see “Mutation analysis”).
Mapping of the VWF exons 4 and 5 deletion breakpoint
The exact locations of the deletion breakpoints in 3 type 3 VWD patients in
whom the mutation was initially identified at the genomic level were
determined by primer walking. The genomic region from exon 3 to exon 6
(26 kb) was amplified in approximately 1.5 kb overlapping fragments using
18 primer sets designated A to R. Direct sequencing of a truncated product
obtained using combinations of these primers was performed, and further
primers were then designed to amplify a smaller product (1084 bp) so that
sequencing could be used to determine the precise deletion breakpoint. The
Delmap primer sequences used in the assay specific for the detection of the
deletion were as follows: forward primer: 5⬘-gtagcgcgacggccagtCCTATCTTTCCTATTTCCATTAATTTCT-3⬘; reverse primer: 5⬘-cagggcgcagcgatgacATGCTAATGGATCAGAATTCATATTGT-3⬘.
These primers were tailed with universal “N13” tails (lower case letters
in primer sequences), which are modifications of the universal M13
sequencing primer sequences, for use in subsequent cycle sequencing
reactions.
The base and amino acid numbering of the novel VWF exons 4 and 5
deletion mutation was determined by reference to the published VWF
sequence9 and the published VWF cDNA sequence. The mutation was
described both at the cDNA level (c.) (RefSeq NCBI accession number
NM_000552.3) and at the amino acid level (p.) (RefSeq NCBI accession
number NP_000543.2) according to guidance issued by the Human
Genome Variation Society.37
Confirmation of zygosity
A multiplex PCR was developed to confirm the homozygous/heterozygous
status of the deletion. The reaction was performed based on the conditions
described for genomic DNA using 35 cycles of denaturation, annealing, and
extension. Two primer sets were used: (1) Delmap primers to amplify
across the breakpoint (1084-bp product; 10 ␮L appropriate forward and
reverse primer mix, 2 ␮M each); and (2) primers to amplify across exons 4
and 5 (1694-bp product) as an internal control (6 ␮L appropriate forward
and reverse primer mix, 2 ␮M each). The volume of H2O added to the
reaction was altered accordingly to achieve a total reaction volume of
25 ␮L. “N13” tailed primer sequences for the internal control were as follows:
forward primer: 5⬘-gtagcgcgacggccagtCAACAGAGCACAACCCTGTC-3⬘;
reverse primer: 5⬘-cagggcgcagcgatgacCTGCTCACTGCAAGTTCTCC-3⬘.
Mutation mechanism
The mutation mechanism was investigated by screening the DNA sequence
flanking the deletion for common repetitive elements, interspersed repeats,
and low complexity sequences.38 These are found throughout the genome
and are implicated in the illegitimate recombination of DNA sequences.
MarWiz39 was used to identify Matrix Association Regions within DNA
sequences.40
Haplotype analysis
To investigate the possibility of a founder effect for the exons 4 and 5
deletion mutation, a haplotype panel of 25 VWF polymorphisms was
constructed by reference to the ISTH SSC VWF database9 and the
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1094
SUTHERLAND et al
HapMap project.41 Polymorphisms were chosen to represent the length
of the gene (Table 2). These were analyzed by DNA sequence analysis.
Expression and characterization of recombinant VWF
To create the construct lacking exons 4 and 5, site-directed mutagenesis was
used using the QuikChange II XL site-directed mutagenesis kit (Stratagene)
performed on the VWF expression vector pClneoVWFES (kindly provided
by Dr P. Kroner, Medical College of Wisconsin, Milwaukee, WI) according
to the manufacturer’s instructions (primers available on request). The
fragment was excised with AfeI and NheI (located in VWF cDNA at
1067 bp and 14 042 bp, respectively) and ligated back into the expression
vector pClneoVWFES to produce the cDNA expression vector pClneoVWFdel4-5. Plasmids were digested with HaeII to identify those with the
deletion, and the mutant product was sequenced to confirm the presence of
the deletion. Plasmid DNA was purified for transfection.
To evaluate the expression of the exon 4 and 5 deletion, HEK-293T cells
were cultured in Dulbecco modified Eagle medium containing 2 mM
L-glutamine, 100 U/mL penicillin, 100 ␮g/mL streptomycin, and 10%
(vol/vol) fetal bovine serum at 37°C and 5% CO2. Cells in the log phase of
growth were seeded at a density such that cells were approximately 50%
confluent the following day. Cells were transfected with 20 ␮g of DNA
(3.2 ␮g ␤-galactosidase reporter construct, 6.8 ␮g calf thymus DNA, and
10 ␮g of the mutant or wild-type plasmids or 5 ␮g of each of these
plasmids) using the calcium phosphate method. VWF was secreted into
serum-free OptiMEM containing 100 U/mL penicillin, 100 ␮g/mL streptomycin, 1⫻ insulin/selenium/transferrin G (Invitrogen). Forty-eight hours
after transfection, media was collected and cells were lysed. Transfection
efficiency was determined by measuring ␤-galactosidase reporter transcript
using Berthold Lumat LB 9501 luminometer (Fisher Scientific) and the
Galacto Light Plus reporter gene assay (Tropix). Quantification of the
VWF:Ag present in the media and cell lysates was determined by
enzyme-linked immunosorbent assay using polyclonal rabbit antihuman
antibody (Dako North America) against a standard normal human reference
plasma (CRYOcheck, lot no. 7128; PrecisionBioLogic). Recombinant
VWF (rVWF) from the media was concentrated and multimers analyzed by
sodium dodecyl sulfate-agarose gel electrophoresis. VWF:RCo was measured in duplicate by platelet aggregometry.
Results
Patient information and geographic origin
Of 21 patients (20 index cases and one family member) with type 3
VWD who were recruited into the study, 12 were of white origin
and 9 were of Asian origin. Of the 12 white patients, 7 were found
to carry a previously unreported deletion mutation spanning VWF
exons 4 and 5. This deletion was not present in any of the Asian
patients. All of the 7 white patients with the deletion, consisting of
6 index cases and 1 family member, were of British origin, so far as
could be ascertained (Table 1).
Two of 34 index cases from type 1 VWD families also had the
VWF exon 4 and 5 deletion. Both of these were of white British
origin (Table 1).
Characterization of the deletion of exons 4 and 5
On initial genomic DNA amplification of VWF in type 3 VWD
patients 5:1, 6:1, and 6:2 (Table 1), exons 4 and 5 failed to amplify,
suggesting that these exons had undergone a homozygous deletion.
Through the method of “primer walking,” using primer sets A to R,
the deletion mutation was confirmed and the approximate breakpoints identified by failure to generate amplified products from G to
M. Because primer sets A to R overlapped, the G forward and M
reverse primers were used to amplify across the deleted region.
This produced a PCR product of 1788 bp, rather than the expected
BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
product size of 10.4 kb. Further primers (Delmap) were designed
and DNA sequencing used to map the deletion breakpoint.
A wild-type PCR product using the Delmap primers would be
expected to be 9715 bp in size. In the type 3 VWD patients
homozygous for the exons 4 and 5 deletion, a product of 1084 bp
was observed. Sequencing of this product identified a total in-frame
deletion of 8631 bp from within intron 3 to a point within intron 5;
c.221-977_532 ⫹ 7059del (p.Asp75_Gly178del), hereafter referred to as ex4-5del.
Identification of heterozygosity for ex4-5del
PCR analysis of platelet-derived VWF cDNA was performed on type 3
VWD patients 1:1, 2:1, 3:1, and 4:1 (Table 1) for whom the phenotype
was unexplained by sequencing of genomic DNA (either no mutation or
heterozygosity for only one mutation in VWF). This revealed a truncated
PCR product in the region comprising VWF exon 2 to exon 7; an
expected wild-type PCR product of 995 bp was replaced by one of
683 bp. At the VWF cDNA level, 2 patients (2:1 and 4:1) were
compound heterozygous for the wild-type and the 683-bp products and
2 patients (1:1 and 3:1) were apparently homozygous for the mutant
683-bp product. The control was homozygous for the wild-type 995-bp
product. Patients 1:1 and 3:1 had previously been found to be heterozygous for VWF nonsense mutations; associated RNA transcripts would
therefore probably undergo nonsense-mediated decay, thereby explaining the apparent homozygosity for the deletion at the cDNA level in
these 2 persons. Direct sequencing of the 683-bp truncated PCR product
from all 4 patients revealed a deletion of the entire coding sequence for
exon 4 and exon 5. The 995-bp product in patients 2:1 and 4:1 was
sequenced and shown to be the expected wild-type sequence.
Given that the VWF exon 4 and exon 5 sequences were present
at the genomic DNA level in type 3 VWD patients 1:1, 2:1, 3:1, and
4:1, it was considered probable that their absence at the RNA level
was the result of the presence of a heterozygous deletion, masked
during PCR amplification of genomic DNA by the presence of the
normal sequence on the other VWF allele. To confirm this
hypothesis, the Delmap primers were used at the genomic DNA
level in these patients. This revealed the presence of an identical
1084-bp truncated PCR product, confirmed by DNA sequencing, as
observed in the 3 patients homozygous for the deletion (5:1, 6:1,
and 6:2). Examination of the DNA sequence surrounding the
breakpoints in these PCR products showed complete homology
between the homozygotes and heterozygotes for the deletion. The
heterozygous nature of this deletion was confirmed in patients 1:1,
2:1, 3:1, and 4:1 by designing an assay containing 2 primer sets.
One set of primers amplified across the breakpoint (1084-bp
product); the second set amplified across exons 4 and 5 (1694-bp
product) as a control (Figure 1). Both the 1084-bp and 1694-bp
products were amplified in all 4 patients.
Identification of the deletion in type 1 VWD patients
Because of the high overall frequency of the ex4-5del mutation in
the type 3 VWD cohort and the fact that heterozygosity for this
mutation may not be identified by conventional analysis at the
genomic DNA level, the possibility that this deletion may occur in
patients with type 1 VWD was investigated. Thirty-four index
cases from type 1 VWD families recruited into a United Kingdom
national study29 were analyzed for the ex4-5del mutation using the
Delmap primers at the genomic DNA level, with confirmation by
the multiplex assay. Heterozygosity for the deletion was identified
in 2 index cases (7:2 and 8:2, Table 1) in whom no candidate VWF
mutation had previously been identified. The families of these
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BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
1
2
3
2 kb
A NOVEL DELETION MUTATION IN VWD TYPES 1 AND 3
1095
deletion. RepeatMasker38 indicated that the 5⬘ and 3⬘ breakpoints of the
deleted sequence lie within inverted, imperfect AluY elements, which
show high homology in the vicinity of the breakpoints (Figure 2). The
breakpoints are also situated in the vicinity of topoisomerase II binding
and cleavage sites, which are concentrated at sites of nuclear attachment.
Further investigation (Mar-Wiz39) showed that the 5⬘ and 3⬘ breakpoints
lie within regions of high Matrix Associated Region potential. These
AT-rich regions contain numerous origin of replication patterns and are
implicated in the interaction between chromatin and the nuclear
matrix.14,42
4
Upper band 1694 bp
1.5 kb
Lower band 1084 bp
1 kb
Haplotype analysis
Figure 1. Gel image to illustrate the deletion of exons 4 and 5 at the DNA level.
The top band (1694 bp) represents the wild-type PCR product, which amplifies
across exons 4 and 5; bottom band (1084 bp), the PCR product designed to amplify
across the deletion breakpoint. Lane 1 indicates a patient heterozygous for the
deletion; lane 2, a patient homozygous for the deletion; lane 3, a wild-type control;
and lane 4, a negative control.
index cases (families 7 and 8, Table 1) were screened. The mutation
was shown to segregate with the type 1 VWD phenotype in family
8. In family 7, heterozygosity for ex4-5del segregated with VWD,
with the exception of unaffected person 7:1, suggesting incomplete
penetrance of the mutation in this family.
Investigation of the deletion mechanism
In silico analysis of the DNA sequences adjacent to the deletion
breakpoint was performed to investigate the mechanism underlying the
I
A
Haplotype analysis was performed to investigate the possibility of
a founder effect in the patients with the ex4-5del mutation. In the
7 type 3 VWD patients as well as the 2 type 1 VWD index cases and
affected family members with this mutation, the deletion segregated with a common VWF haplotype. In the 2 related type 3 VWD
patients who were homozygous for the deletion, a second VWF
haplotype was observed, in the compound heterozygous state with
the common haplotype (Table 2). The second haplotype and
common haplotype were identical 5⬘ to the deletion breakpoint but
were different 3⬘ to this location.
Expression and characterization of recombinant VWF
To determine whether or not the mutant recombinant protein was
retained within the cell or was inefficiently secreted, VWF:Ag
levels were assayed in cell lysates and in the conditioned media
using enzyme-linked immunosorbent assay. Secretion of the rVWF
protein from the heterozygous and homozygous ex4-5del transfections was decreased by 86% (P ⬍ .001) and 98% (P ⬍ .001),
5’
3’
X
II
5’
3’
VWF ex4-5del
Deleted DNA
8631 bp
Figure 2. A possible model for the pathogenesis of the
deletion. (Ai) AluY repeats, highlighted in dark gray (5⬘) and
light gray (3⬘) at the 5⬘ and 3⬘ deletion breakpoints line up while
attached to the nuclear matrix, followed by a recombination
event. This results (Aii) in the deletion of 8631 bp, including
exons 4 and 5 from VWF. (B) An 85.2% (52 of 61) homology
between the 5⬘ and 3⬘ AluY sequences 30 bp on either side of
the deletion breakpoints (highlighted in dark gray).
B
c.221-977
TTATTTTTTATTTTTTTTGAGACAGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGC
||||| | | ||||||||||| |||||| ||||||| |||||||||||||||||||||
CCATTTTATTTATTTTTTGAGACGGAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGGC
c.532+7059
5’ sequence
3’ sequence
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1096
SUTHERLAND et al
respectively, relative to the secretion of the wild-type rVWF
protein (VWF:Ag levels for the wild-type, heterozygous, and
homozygous mutant were 1.53 ⫾ 0.04 U/mL, 0.21 ⫾ 0.01 U/mL,
and 0.03 ⫾ 0.00 U/mL, respectively; n ⫽ 5). VWF:RCo levels for
the wild-type, heterozygous, and homozygous mutant concentrated
media were 1.58 U/mL, 0.52 U/mL, and 0.00 U/mL, respectively.
The decrease in recombinant protein (heterozygous and homozygous mutant) in the media did not correspond to an increase in
intracellular retention. VWF:Ag levels for the wild-type, heterozygous, and homozygous mutant cell lysates were 0.09 U/mL,
0.08 U/mL, and 0.08 U/mL, respectively. These results demonstrate that the deletion of VWF exons 4 and 5 results in markedly
decreased secretion of VWF and indicate the dominant-negative
effect of the mutation in the heterozygous state.
To determine the effect of the ex4-5del mutation on VWF
multimers, conditioned media from the wild-type, homozygous
mutant, and 50:50 heterozygous transfections were concentrated
and analyzed by sodium dodecyl sulfate–agarose gel electrophoresis. A full range of multimers was observed in the media from the
heterozygous transfections. Only dimers were observed in the
media of the recombinant homozygous mutant protein, indicating
that this mutation interferes with the multimerization process.
Control screening
A total of 100 anonymized blood samples received by the
laboratory for hereditary hemochromatosis genotyping were
screened to confirm that the deletion event observed was a
pathogenic mutation and not a polymorphism present in the general
population. All of the 200 alleles screened were negative for the
ex4-5del mutation.
Discussion
The present study aimed to identify the molecular pathogenesis of
type 3 VWD in a cohort of patients from the North West of England
by direct sequencing of VWF at the genomic DNA level and of
VWF mRNA-derived cDNA. This approach identified homozygous or compound heterozygous mutations consistent with the type
3 VWD phenotype in 19 of 21 patients from 20 unrelated families
(M.S.S., A.M.C., P.H.B.B.-M., C.R.M.H., A.M.W., S.K., manuscript submitted). Seven patients were found to have a previously
unreported c.221-977_532 ⫹ 7059del mutation (ex4-5del), which
removed 8631 bp of VWF genomic sequence, including exons
4 and 5. Three of these 7 patients, including 2 from the same family,
were homozygous for this mutation, as detected and characterized
at the genomic DNA level. The remaining 4 persons were
heterozygous for the deletion, which was detected initially at the
RNA level only. Three of these 4 persons were compound
heterozygous for the deletion and a second VWF mutation, thus
explaining their type 3 VWD phenotype. A second VWF mutation
to explain the type 3 VWD phenotype has not been identified in the
fourth person (patient 2:1, Table 1). Overall, the ex4-5del mutation
occurred frequently in our type 3 VWD patients of white origin (7
of 12 persons from 6 unrelated families) but was absent in
9 patients of Asian origin. The prevalence of the deletion among the
apparently unrelated type 3 VWD families in our cohort was 8 of
40 alleles (20%) from 20 unrelated families.
The high prevalence of the ex4-5del mutation in our type 3
VWD study population, together with failure to detect heterozygosity for this mutation when genomic DNA was analyzed by standard
methods because of the presence of a normal allele, led us to
BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
investigate the occurrence of this mutation in 34 unrelated type 1
VWD index cases previously recruited into a United Kingdom
national study.29 Heterozygosity for ex4-5del was identified in
2 cases. In both, a type 1 VWD-causative candidate VWF mutation
had not been previously identified. Heterozygosity was subsequently identified in other VWD-affected family members of these
index cases, suggesting that heterozygosity for this mutation was
causative of type 1 VWD in these kindreds. Heterozygosity for
ex4-5del was identified in one unaffected person (person 7:1, Table
1), suggesting incomplete penetrance of the mutation. Data were
not available to us to indicate whether or not the normal VWF
laboratory parameters in this elderly subject may be the result of
VWF levels having increased with age.
Since the investigation of the original type 3 and type 1 VWD
patient cohorts, we have also identified the ex4-5del mutation in a
further 4 persons (Table 1), including compound heterozygosity in
a type 3 VWD index case (9:1) and heterozygosity in his possible
type 1 VWD affected mother (9:2), heterozygosity in a type 1
VWD affected relative (1:2) of a patient from the original type 3
VWD patient cohort (1:1), and heterozygosity in a newly investigated patient with type 1 VWD (10:1). All 4 of these persons were
of white British origin.
Haplotype analysis was performed using a panel of 25 previously described polymorphisms associated with VWF (Table 2).
This identified a common haplotype associated with ex4-5del in
5 type 3 VWD patients who were heterozygous for this mutation,
3 homozygous type 3 VWD patients (2 related), 2 heterozygous
type 1 VWD-affected family members of type 3 VWD index cases,
3 type 1 VWD index cases heterozygous for the deletion, and a
further 4 heterozygous family members from the families of the
type 1 VWD index cases. This suggested a common origin of the
deletion among these apparently unrelated kindreds. The presence
of a second VWF haplotype in 2 related persons (6:1 and 6:2) may
indicate that a recombination event has occurred in this family,
resulting in the deletion being associated with a different haplotype. To support this suggestion, in these 2 persons, the haplotype
5⬘ of the deletion was identical to the “common” deletionassociated haplotype, with variance only 3⬘ of the deletion.
Further analysis is required to examine the prevalence of the
ex4-5del mutation in other VWD patient populations, for example,
in the Canadian and European type 1 VWD patient cohorts.
Candidate mechanism for the VWF ex4-5del deletion
The 5⬘ and 3⬘ breakpoints of the deletion were shown to lie within
regions of AluY repetitive elements and in the proximity of
topoisomerase II binding and cleavage sites, which are responsible
for the splicing of DNA. Alu repeats are the most abundant class of
short interspersed repeat elements in the human genome, and they
are known to promote unequal homologous recombination. The
breakpoints also lie within regions of high matrix association
potential, suggesting that the regions may be in close proximity
when attached to the nuclear matrix during DNA replication. Taken
together, it is probable that the AluY regions line up when attached
to the nuclear matrix, causing the formation of a hairpin loop.
A further mechanism, eg, an incorrect cleavage and rejoining event
mediated by topoisomerase II, may be responsible for the incorrect
recombination of the 2 DNA strands either side of the breakpoints
and the splicing of the 8631 bp encompassing exons 4 and 5 as well
as flanking intronic sequences (Figure 2). There are previous
reports of large deletions causing type 3 VWD with a similar
Alu-mediated deletion mechanism to that proposed here.13,14
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BLOOD, 30 JULY 2009 䡠 VOLUME 114, NUMBER 5
A NOVEL DELETION MUTATION IN VWD TYPES 1 AND 3
Significance of the deletion in type 1 and type 3 VWD
Type 3 VWD is a severe bleeding disorder inherited in an
autosomal recessive manner with reported VWF mutations mostly
resulting in a null allele, ie, nonsense, frameshift, and deletion
mutations. Despite some previous reports,15,17,43,44 the different
inheritance patterns and genetics of types 1 and 3 VWD mean that
it is unusual to find a type 3 VWD linked mutation that, in the
heterozygous state, is clearly associated with dominant type 1
VWD. The ex4-5del mutation may be expected to have an
autosomal recessive inheritance pattern, requiring the presence of
the deletion in either the homozygous or compound heterozygous
state. However, in association with type 1 VWD, ex4-5del appears
to operate in an autosomal dominant fashion with normal VWF
multimers, as observed in the type 1 VWD index cases and their
affected family members (Table 1). Expression studies have
demonstrated an 86% decrease in secretion of VWF from heterozygous transfections compared with secretion of VWF from wildtype cells, providing further evidence of a dominant-negative effect
of ex4-5del and the association of this mutation with type 1 VWD.
In conclusion, we have identified and described the investigation of a previously unreported deletion of VWF exons 4 and 5.
Expression studies have indicated that this mutation causes markedly decreased secretion and defective multimerization of the
resultant mutant VWF protein. The absence of increased intracellular retention of the mutant VWF may be the result of proteasomal
degradation of the protein in the cytoplasm, mooted as a possible
general mechanism for mutations associated with dominant type 1
VWD.33 The ex4-5del mutation is recurrent among type 3 VWD
patients of British white origin and, in the heterozygous form,
appears to underlie a proportion of cases of dominant type 1 VWD.
Our data indicate that, in the majority of cases, the deletion
originates from a single founder event; however, there is evidence
for a possible recombination event associating the mutation with a
second VWF haplotype. Together with the theoretical mechanism
for the deletion, this raises the possibility that ex4-5del may be
present in persons of other ethnicities. The deletion described may
go undetected using current VWF screening strategies; however, it
is readily detected using the PCR-based analysis described herein.
Screening for the ex4-5del mutation in other type 1 and type 3
1097
VWD patient populations is required to provide further information
on the overall contribution of this mutation to quantitative deficiencies of VWF.
Acknowledgments
The authors thank the members of the United Kingdom Hemophilia Center Doctors’ Organisation von Willebrand Disease Working Party and other scientists and clinicians who participated in the
recent United Kingdom study of the molecular pathogenesis of
type 1 VWD,29 index case patient samples from which were among
those screened for the VWF exons 4 and 5 deletion described in the
current manuscript: S. Brown (London), E. Chalmers (Glasgow),
S. Enayat (Birmingham), G. Evans (Canterbury), P. Grundy
(Manchester), A. Guilliatt (Birmingham), J. Hanley (Newcastle),
F. Hill (Birmingham), D. Keeling (Oxford), K. Khair (London),
R. Leisner (London), W. Lester (Birmingham), C. Millar (London),
J. Pasi (London), C. Tait (Glasgow), L. Tillyer (Lewisham), and
J. Wilde (Birmingham).
Authorship
Contribution: M.S.S. and D.J.B. designed and performed the
research, analyzed and interpreted data, and contributed to
writing the manuscript; A.M.C. and S.K. designed the research,
analyzed and interpreted data, and contributed to writing the
manuscript; M.B. designed and performed the expression studies, analyzed and interpreted data, and contributed to writing the
manuscript; P.H.B.B.-M., C.R.M.H., and A.M.W. recruited
patients and provided clinical information; and P.W.C. recruited
patients, provided clinical information, and contributed to
writing the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Stephen Keeney, Molecular Diagnostics Centre, Top-Floor Multipurpose Bldg, Manchester Royal Infirmary,
Oxford Rd, Manchester, M13 9WL, United Kingdom; e-mail:
[email protected].
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2009 114: 1091-1098
doi:10.1182/blood-2008-08-173278 originally published
online April 16, 2009
A novel deletion mutation is recurrent in von Willebrand disease types 1
and 3
Megan S. Sutherland, Anthony M. Cumming, Mackenzie Bowman, Paula H. B. Bolton-Maggs, Derrick
J. Bowen, Peter W. Collins, Charles R. M. Hay, Andrew M. Will and Stephen Keeney
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