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| Dharmacon in the Real World |
| Dharmacon SMARTpool® siRNA - identification of a gene
critical to the blood clotting pathway |
In 2004, Darrel Stafford's research group at the University of North Carolina, in
collaboration with Dharmacon researchers, published work highlighting a novel
application of Dharmacon's SMARTselection™ and SMARTpool®
technologies. In this work, SMARTpool siRNA reagents were used to complement
traditional positional cloning methods to identify the gene for vitamin K epoxide
reductase (VKOR), a major enzyme in the blood-clotting pathway.[1]
The coagulation, or clotting, of blood is a deadly problem for people afflicted with
bleeding disorders such as hemophilia. Hemophilia affects <0.01% of men and is an
inherited bleeding disorder that results from the body's inability to produce proteins
that cause blood to clot. There are many other causes of bleeding disorders including
vitamin K deficiency and severe liver disease. Individuals with these types of
disorders lack an important factor in the blood clotting process and may be treated
with regular injections of the deficient clotting factor. In contrast, over 600,000
Americans (~0.2% of the US population) die each year from the opposite problem,
abnormally active coagulation. Leading causes of blood clots include irregular or rapid
heartbeat, heart-valve replacement, and heart attacks. These patients actually require
blood thinners to prevent clotting. Warfarin, a coumarin derivative, is commonly used
today to prevent clotting. However, care must be taken in the use of this drug as it is
difficult to control the dosage and there is a risk of inducing bleeding. Thus, there
has been extensive ongoing research into the blood-clotting pathway and the role of the
various factors involved.
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In 1943, with the Nobel Prize in Medicine awarded for the characterization of vitamin
K's role in the clotting pathway, research began in earnest to define other critical
components working in concert with vitamin K. Concurrent with the time-frame of the Nobel
award, warfarin was identified as a vitamin K antagonist interfering with the vitamin K
regeneration (oxidation-reduction) cycle. Over the course of several decades, vitamin K
was found to be an essential factor in the carboxylation of blood-clotting factors
(Figure 1) and in 1991, the gene coding for vitamin-K-dependent carboxylase (VKDC) was
finally cloned and characterized. However the other participant in the vitamin K cycle,
vitamin K epoxide reductase (VKOR), proved difficult to purify and the gene remained
unidentified. The difficulty in administering warfarin and the motivation to develop
better treatments, fueled interest in characterizing the gene that codes for VKOR to
better understand and exploit the interactions of warfarin in the vitamin-K cycle. |
Figure 1. The Vitamin K oxidation-reduction cycle |
The Stafford and Dharmacon research teams, met the challenge of identifying the gene
encoding VKOR using SMARTpool reagents comprised of four individual
SMARTselection siRNA reagents.[1] They narrowed down the possible
gene candidates to 13 open reading frames that encode proteins containing putative
transmembrane proteins (VKOR is a predicted membrane protein). Dharmacon synthesized
SMARTpool reagents to silence each of the 13 candidates identified by Li, et
al.[1] It was found that the knockdown of only one gene, MGC11276 mRNA,
caused a marked reduction in VKOR activity. Furthermore, when this gene was expressed
in insect cells, the gene produced active VKOR that was inhibited by warfarin. This
discovery not only further extends the understanding of the coagulation pathway, but
also will assist in the development of better anticoagulant drugs. Specifically, there
appears to be a genetic dependence on the necessary warfarin dosage due to polymorphisms
in the VKOR gene. Thus, the discovery of the VKOR gene will facilitate the association
between specific VKOR polymorphisms and the warfarin requirement.
In summary, this study demonstrates the utility of siRNA-mediated silencing as a novel
tool to complement positional cloning where a functional assay is available. The utility
of RNA interference as a critical molecular tool is further emphasized by the lengthy
parallel study published by Clemens Müeller's lab at the University of Würzburg, where
the VKOR gene was confirmed through an extensive genetic screening study involving patients
with VKOR mutations.[2]
1. Li, T., C.-Y. Chang, et al. Nature, 2004. 427(6974): p. 541-544.
2. Rost, S., A. Fregin, et al. Nature, 2004. 427(6974): p. 537-41.
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