Nanosphere, Inc.
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Northbrook, IL, 60062
Website: http://www.nanosphere-inc.com
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Finding Factor II and V Leiden Polymorphisms with Gold Nanoparticles
By Paul J. Jannetto, Laura Tooke, Charles Schur, Run Zhang Shi, Gregory J. Tsongalis
Abstract
Venous thrombosis is a multi-factor disease that is associated with genetic and
acquired risk factors. Genetic variations in Factor V Leiden and Factor II (Prothrombin)
represent two of the most common causes of familial thrombophilia, a disease that
constitutes major health-related problems and, in some cases, even death.
Schematic representation of the imaging
spots and capture strand
configuration for the Factor V Leiden and Factor II assay.
Click
here to enlarge
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Venous thrombosis is often difficult to spot in patients during routine clinical examinations and, for those with a family history of the disease and including people in other high-risk categories, prevention of the disease by way of screening for pre-disposition is appropriate.
Laboratory investigation of thrombophilic disorders has been enabled by molecular assays that primarily rely on DNA amplification using PCR. In this study, we evaluated the feasibility and accuracy of using an in-development chip-based nanoparticle technology to detect specific nucleotide sequences that are important for familial thrombophilia without using PCR.
Nanoparticle probes for disease markers
There is potential for the detection of protein and nucleic acid biomarkers with
ultra-sensitive molecular diagnostic tools that
Representative patient imaging spots for the Factor V Leiden and Factor
II assay.
A. Factor II Wild-type, Factor V Wild-type;
B. Factor II Wild-type, Factor V Heterozygous;
C. Factor II Homozygous variant, Factor V Wild-type;
D. Factor II Wild-type, Factor V Homozygous variant.
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identify disease-related changes in biomarker levels, before they manifest with
advanced clinical signs secondary to disease progression.
Nanoparticle probes that will help enable the detection of very low levels of proteins found in specific diseases through signal amplification are at the core of a new diagnostic tool capable of detecting disease at the molecular level in blood, cerebrospinal fluid and other body specimens. The Verigene System combines gold nanoparticle probes and ultimately Biobarcode technology to enable multiplexed detection of protein and nucleic acid biomarkers using enhanced signal amplification techniques without the need for PCR.
Nanoparticle probes for Factor II and V
For this study, the Verigene System was used to detect Factor V Leiden, and Prothrombin mutations in unamplified human genomic DNA. The method is based on hybridization of the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes, followed by a proprietary signal amplification process for target detection.
Factor II (prothrombin) and Factor V are involved in blood clotting and coagulation. Mutations can lead to upwards of a five-fold increased risk for prothrombin and up to 80-fold for Factor V. Factor V Leiden is an arginine to glutamine substitution at position 506 of coagulation factor V that interferes with the cleavage and inactivation of Factor V. Factor V Leiden is the cause of resistance against activated protein. Prothrombin 20210 is a G to A substitution that occurs in the 3'-untranslated region of the prothrombin gene. Prothrombin 20210 has also been associated with myocardial infarction and cerebral vein thrombosis. Together, Factor V Leiden and Prothrombin polymorphisms are the two most common causes of familial thrombophilia.
Method
A combined total of 166 random, de-identified DNA samples were genotyped at two separate institutions (90 at the Medical College of Wisconsin and 76 at Dartmouth-Hitchcock Medical Center) after IRB approval was obtained. Steps follow:
—Isolation of genomic DNA from whole blood (45 minutes): DNA was extracted from whole blood (400 μl) using a standard manual extraction kit.
—Chemical fragmentation of DNA (45 minutes): DNA samples underwent Nanosphere's chemical fragmentation process to achieve approximately 500 bp fragment lengths.
—DNA samples loaded into the hybridization chamber (10 minutes).
—Hybridization and integrated fluid processing (90 minutes).
—Detection and Factor II/V results (3 minutes): processed hybridization units were rinsed with MilliQ water (Millipore Corp.), spun dry and inserted into the Verigene ID System (Nanosphere Inc.). Hybridization units were scanned and genotype results were displayed.
Results
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A,
B, C. Principle of the Verigene ID detection system. A. After isolation
from a clinical sample, DNA is hybridized to both nanoparticle probes and
capture strands. B. Only the perfect DNA match is bound to the test site.
C. Silver is catalyzed on the gold resulting in six orders of magnitude
amplification of signal.
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The Verigene ID results showed 100 percent concordance with the commercial based
assay or PCR for both Factor V Leiden and Prothrombin polymorphisms. The average
first pass call rate for Factor V Leiden and Prothrombin was 81.5 percent and
84.6 percent, respectively, this with a non-commercial, pre-manufactured product
using genomic DNA. In the samples where genotype calls were made, 92.7 percent
were wild-type for Factor V Leiden, 6.6 percent were wild-type, 0.7 percent were
heterozygous, and 0.7 percent were homozygous variants.
Conclusions
The benefits to society from this semi-automated assay could be substantial, since it allows simple, rapid and accurate detection of polymorphisms in two genes implicated in the diagnosis of hyper-coagulation disorders. The Verigene system's ClearRead technology also offers sensitivity sufficient to allow the direct analysis of samples without PCR amplification. Furthermore, the chip-based format makes it readily adaptable to multiplexing, and provides the capability to detect DNA, RNA and protein-based targets.
The Verigene ID optical detection
instrument and Naptune Auto-Processing System. |
About the authors
The authors are (need short author bio…). More information about the Verigene
System and the methods discussed in this article is available from: Nanosphere,
Inc.847-400-9000www.nanosphere-inc.com/
References
1. Zivelin, A., Rosenberg, N., Faier, S., et al. A single genetic origin for the common prothrombotic G20210A polymorphism in the prothrombin gene. Blood 92:1119-1124 (1998).
2. Lee, R. Am J Med Sci. 322:88-102 (2001).
3. Bertina R.M., Koeleman B.P., Koster T., et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 369:64-7 (1994).
4. Poort S.R., Rosendaal F.R., Reitsma P.H., Bertina R.M. 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 88:3698-703 (1996).
5. Rosendaal F.R., Siscovick D.S., Schwartz S.M., Psaty B.M., Raghunathan T.E., Vos H.L. A common prothrombin variant (20210 G to A) increases the risk of myocardial infarction in young women. Blood 90:1747-50 (1997).
6. Nguyen A. Prothrombin G20210A polymorphism and thrombophilia. Mayo Clin Proc 75:595-604 (2000).
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