JordanA. Shavit,1Ani Manichaikul,2Heidi L. Lemmerhirt,3Karl W. Broman,2and David Ginsburg1,3,4
1Department of Pediatrics, University of Michigan,AnnArbor;2Department of Biostatistics, Johns Hopkins University, Baltimore, MD;3Department of Human
Genetics, and4Howard Hughes Medical Institute and Department of Internal Medicine, University of Michigan,AnnArbor
Type 1 von Willebrand disease (VWD) is
the most common inherited human bleed-
ing disorder. However, diagnosis is com-
plicated by incomplete penetrance and
variable expressivity, as well as wide
variation in von Willebrand factor (VWF)
levels among the normal population. Pre-
vious work has exploited the highly vari-
able plasma VWF levels among inbred
strains of mice to identify 2 major regula-
tors, Mvwf1 and Mvwf2 (modifier of VWF).
is a natural variant in Vwf that alters
biosynthesis. We report the identification
of an additional alteration at the Vwf lo-
cus (Mvwf5), as well as 2 loci unlinked to
Vwf (Mvwf6-7) using a backcross ap-
proach with the inbred mouse strains
WSB/EiJ and C57BL/6J. Through posi-
tional cloning, we show that Mvwf5 is a
cis-regulatory variant that alters Vwf
mRNA expression. A similar mechanism
centage of human VWD cases, especially
those with no detectable mutation in the
VWF coding sequence. Mvwf6 displays
conservation of synteny with potential
VWF modifier loci identified in human
pedigrees, suggesting that its ortholog
may modify VWF in human populations.
von Willebrand factor (VWF) is a central component of hemosta-
sis, serving as the adhesive link between platelets and the injured
blood vessel wall, as well as the carrier for factor VIII. Deficiencies
in VWF result in von Willebrand disease (VWD), the most
common inherited bleeding disorder in humans. Elevated VWF
levels may also be an important risk factor for thrombosis, both
through a direct role in platelet adhesion,1as well as indirectly by
causing elevated levels of factor VIII.2-4Diagnosis of VWD is
elusive in many cases because of its variable expressivity and
incomplete penetrance5and the nonspecific nature of bleeding
symptoms.6VWF plasma protein levels also display a broad
distribution in the normal human population. Thus, it is often
difficult to determine whether a person has VWD and is at risk for
pathologic hemorrhage or simply has VWF levels in the low range
Levels of plasma VWF have been shown to be largely
determined by genetic factors, with estimates of heritability in
humans ranging from 25% to 32% by pedigree analysis,7,8to 66%
to 75% in twin studies.9,10ABO blood group is responsible for
one-third of the genetic variability in VWF plasma levels.11
However, the loci responsible for the remaining two-thirds of this
genetic component are unknown. Recent evidence from European
and Canadian cooperative studies on type 1 VWD have found that
disease diagnosis does not segregate with VWF genotype in
approximately 50% of families, supporting the existence of addi-
tional genetic factors.12-16
Laboratory mice display wide variation in VWF levels with
strikingly similar to the estimates for humans derived from twin
studies.9,10This variability among inbred mouse strains has been
used to identify genetic loci modifying VWF levels, including
Mvwf1 (modifier of Vwf), a mouse glycosyltransferase (B4galnt2)
that alters clearance of VWF.18A similar mechanism probably
explains the modification of human VWF levels by ABO blood
group and some cases of type 1 VWD.19,20A natural variant of the
murine Vwf gene has also been identified (Mvwf2).17
We now report a backcross between 2 additional inbred mouse
strains, WSB/EiJ (WSB) and C57BL/6J (B6), with relatively high
and low levels of plasma VWF, respectively. Genetic analysis
identified 3 significant loci regulating VWF levels. The first is a
novel cis-regulatory allele of Vwf, whereas the others map to novel
loci on chromosomes 5 and 10.
Mouse strains and bleeding
Mice were purchased from The Jackson Laboratory. Each individual mouse
was bled on 3 separate occasions, at least 1 week apart. Bleeds were
performed after isoflurane-induced anesthesia by retro-orbital technique on
alternating eyes from week to week, removing approximately 75 ?L whole
blood with each bleed into heparinized capillary tubes (Thermo Fisher
Scientific). For the strain survey, 3 females of each strain were bled between
4 and 8 weeks of age. Further analysis of the C57BL/6J (B6) and WSB/EiJ
(WSB) strains were as follows: 2 each of WSB males and females, 3 males
and 4 females of B6, and 4 males and 5 females of (B6 ? WSB) F1 mice
were bled between 3 and 8 weeks of age. For the backcross study, WSB
males were crossed to B6 females to generate F1 progeny. Both male and
female F1s were backcrossed to B6 to produce the N2 generation.
Two hundred seven N2 mice were also bled in the same manner described
above, with the first bleed at weaning, the second between 3.5 and
Submitted July 15, 2009; accepted August 15, 2009. Prepublished online as
Blood First Edition paper, September 29, 2009; DOI 10.1182/ blood-2009-07-
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge
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marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2009 by TheAmerican Society of Hematology
5368 BLOOD, 17 DECEMBER 2009?VOLUME 114, NUMBER 26
Contribution: J.A.S. participated in research design, performed
the research, and wrote the paper; A.M. aided in data analysis
and wrote the paper; H.L.L. participated in research design and
performed the research; K.W.B. aided in data analysis and wrote
the paper; and D.G. participated in research design and wrote
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
The current affiliation for A.M. is Department of Biomedical
Engineering, University of Virginia, Charlottesville, VA. The
current affiliation for K.W.B. is Department of Biostatistics and
Medical Informatics, University of Wisconsin, Madison, WI.
Correspondence: David Ginsburg, Howard Hughes Medical Insti-
tute, University of Michigan, 210Washtenaw Ave, Life Sciences
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