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Personalized Warfarin Dosing Enhanced by MS-Based SNP Genotyping

The anticoagulant drug warfarin (also known under the brand name Coumadin) is commonly used to prevent blood clots and embolisms. However, the drug exhibits significant inter-individual variability in dosing requirements. This variability is partly due to single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. A group led by Dr. Haifeng Wu of Ohio State University has developed a new, rapid method to genotype SNPs that will help clinicians to choose appropriate doses of warfarin for individual patients. Using surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS), which can determine the elemental composition of a sample, the researchers were able to determine the genotype of three warfarin-related SNPs (CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A) in under five hours with high levels of accuracy. The researchers suggested that "on-site application of this method in hospital laboratories will greatly help clinicians to determine appropriate doses of warfarin to treat patients with thromboembolic disorders." In future studies, the Ohio State scientists plan to apply the SELDI-TOF platform to genotype other medically important SNPs that influence the efficacy and safety profiles of many drug therapies and to thus ultimately promote personalized health care. This work was reported in the March issue of the Journal of Molecular Diagnostics. [Press release] [Journal of Molecular Diagnostics abstract]

Animal Model Suggests Possible Cause for ADHD

Using a mouse model they created, scientists at Rockefeller University and collaborating institutions have identified a gene (CK1 delta) that they believe merits investigation as a possible cause of attention-deficit/hyperactivity disorder (ADHD). Currently, the cause of ADHD is unknown, but there is increasing evidence that dopamine, a neurotransmitter involved in the brain’s reward-motivation system, is involved. Scientists have previously found that the levels of dopamine, and of the D2 receptor it binds to, are involved in the progression of ADHD, as are four connected regions in the frontal region of the brain, two of which are directly linked to reward and motivation. In their work, the scientists focused on an enzyme called casein kinase I (CK1), which is involved in regulating the dopamine signaling pathway. They created a line of mice genetically modified to overexpress a form of CK1 called CK1 delta, specifically in the forebrain of the mouse. Under normal conditions and in response to stimulation by drugs such as the ones used today to treat ADHD, the mice that overexpressed CK1 delta showed behavioral symptoms and responses to drugs similar to those observed in people with ADHD. “The genetically modified mice that we generated present interesting features such as hyperactivity and altered nesting capacities that might be related to attention deficit, and possibly altered impulsivity,” said Dr. Marc Flajolet, senior author of the report. Biochemical studies showed that both classes of dopamine receptors, D1R and D2R, were significantly reduced in the CK1 delta-overexpressing mice, providing further evidence that the dopaminergic system is severely affected.

Genetic Locus Associated with Increased Need for Orthodontia

Researchers have reported that the teeth of babies with certain genetic variants tend to appear later and that these children have lower numbers of teeth by age one. Additionally, certain of these children whose teeth develop later are more likely to need orthodontic treatment later in life. In a a SNP-based genome-wide association study conducted in approximately 6,000 individuals, the scientists identified five genetic loci (the KCNJ2, EDA, MSRB3, IGF2BP1, and RAD51L1 gene regions) that were significantly associated with both time of first tooth eruption and number of teeth at age one. The researchers also identified five additional loci that were suggestively associated with these same variables. The international team further found that a SNP at one of these suggestive loci (a SNP within the HOXB gene cluster) was associated with a 35 percent increased risk of requiring orthodontia treatment by the age of 31 years. The discovery of genes influencing tooth growth may lead to innovations in the early treatment and prevention of congenital dental and occlusion problems, the authors noted. They also said their findings should provide a strong foundation for the study of the genetic architecture of tooth development, which in addition to its relevance to medicine and dentistry, may have implications in evolutionary biology because teeth represent important markers of evolution. The scientists emphasized that tooth development is not an isolated event. Teeth and several other organs have common growth and developmental pathways in early life. Some of the genes identified here have been linked in previous studies with the development of the skull, jaws, ears, fingers, toes, and heart. The article describing the current research was published online on February 26, 2010, in PLoS Genetics.

Normal Version of Fusion Gene Component Is Required by Leukemia

Researchers have found that a particularly aggressive type of blood cancer called mixed lineage leukemia (MLL) requires the normal version of a translocated gene (the MLL gene) to survive. The findings were featured as the cover article in the February 17, 2010 issue of Cancer Cell. MLL develops when a piece of chromosome 11 breaks at the normal MLL gene. The chromosome piece attaches to another broken chromosome, resulting in the joining of the MLL gene to a now-neighboring gene on the other chromosome. The joined genes code for a new “fusion protein” that eventually causes uncontrolled growth of blood cells. The researchers found that the runaway growth triggered by the fusion protein is blocked when the gene for the normal MLL protein is deleted from leukemia cells. The results showed that the normal protein is required for fusion-protein generation of the leukemia and for the maintenance of transformed cells. This suggested that the normal protein cooperates with the fusion protein in the generation of the leukemia. "This research not only uncovers the crucial role of a normal protein key to the development of MLL, but also how the cancer cells stay alive in the first place," said Dr. Xianxin Hua, from the University of Pennsylvania, senior author of the article. The results point to the normal MLL gene as a potential target for new therapies, possibly through repression of the gene in leukemia stem cells. [Press release] [Cancer Cell abstract]

Gene-Based Stem Cell Therapy Knocks Down HIV Receptor Expression

UCLA researchers have reported successfully removing CCR5 from human cells in a humanized mouse model. CCR5 is a cell receptor to which HIV-1 binds for infection, but which the human body apparently does not need. Individuals who naturally lack the CCR5 receptor have been found to be essentially resistant to HIV. In the humanized mouse model, the researchers transplanted a specific short hairpin RNA (shRNA), targeted against the CCR5 gene, into human blood stem cells, in order to inhibit the expression of CCR5 in the human immune cells arising from the stem cells. According to the authors, the positive results provide evidence that this strategy may be an effective way to treat HIV-infected individuals, i.e., by prompting the potent long-term and stable reduction of CCR5 in systemic lymphoid organs. The results of the UCLA work were published in the February 25 issue of Blood. [Press release] [Blood abstract]

Prion Protein Functions in Maintenance of Peripheral Myelin

The neuronal expression and regulated proteolysis of the normal, cellular prion protein (PrPc) are essential for the maintenance of peripheral myelin, according to an international team of researchers from institutions including Cal Tech and the University Hospital of Zurich. The integrity of peripheral nerves depends on communication between axons and the Schwann cells that produce myelin. Myelin insulates peripheral nerve axons and speeds electrical transmission. The axon signals that ensure myelin maintenance are distinct from those that direct myelination and are largely unknown, the authors wrote. The normal function of PrPc--that when misfolded into the scarpie-associated form (PrPsc) causes various transmissible fatal neurodegenerative diseases, including scrapie in sheep, mad cow disease in cattle, and Creutzfeldt-Jakob disease in humans--is also unknown. The current results appear to represent progress toward solving, at least in part, both of these puzzles. The authors showed that ablation of PrPc triggers a chronic demyelinating polyneuropathy (CDP) in four independently targeted mouse strains. CDP was triggered by depletion of PrPc specifically in neurons, but not in Schwann cells, and was suppressed by PrPc expression restricted to neurons but not to Schwann cells. CDP was prevented by PrPc variants that undergo proteolytic amino-proximal cleavage, but not by variants that are nonpermissive for cleavage, including secreted PrPc lacking its glycolipid membrane anchor. This work was published in the March issue of Nature Neuroscience. The seminal research identifying and characterizing prion proteins was recognized by the awarding of the Nobel Prize in Physiology or Medicine to Dr. Stanley Prusiner in 1997.

Second DISC-1 Study Suggests Reason for Adolescent Onset of Schizophrenia

In a second study, researchers at Johns Hopkins and collaborating institutions have identified a mechanism whereby alterations in the DISC-1 (disrupted in schizophrenia-1) gene may underlie the adolescent onset of schizophrenia. In this second study, published in the March issue of Nature Neuroscience, the research team examined DISC-1's role in forming connections between nerve cells. The first study looked at the long-term effects of transient DISC-1 gene expression changes near the time of birth in a mouse model. This first study was published in the February 25, 2010 issue of Neuron and has previously been reported on in BioQuick News. Taken together, the results of both studies suggest that anatomical differences that seem to be influenced by the DISC-1 gene cause problems that start before birth, but surface only in young adulthood. "If we can learn more about the cascade of events that lead to these anatomical differences, we may eventually be able to alter the course of schizophrenia. During adolescence, we may be able to intervene to prevent or lessen symptoms," said second study senior author and first study co-author Dr. Akira Sawa, professor of psychiatry and director of the program in molecular psychiatry at the Johns Hopkins University School of Medicine. Numerous studies have previously suggested that schizophrenia results from abnormal connectivity. The fact that symptoms typically arise soon after adolescence, a time of massive reorganization of connections between nerve cells, supports this idea. The scientists began their second study by surveying rat nerve cells to see where DISC1 was most active. Unsurprisingly, they found the highest DISC-1 activity in connections between nerve cells.

Female Sex Hormone Found in Plant

In a finding that overturns conventional wisdom, scientists have reported the first discovery of the female sex hormone progesterone in a plant. Until now, scientists thought that only animals could make progesterone. A steroid hormone secreted by the ovaries, progesterone prepares the uterus for pregnancy and maintains pregnancy. A synthetic version, progestin, is used in birth control pills and other medications. "The significance of the unequivocal identification of progesterone cannot be overstated," the article, by Dr. Guido F. Pauli and colleagues, claimed. "While the biological role of progesterone has been extensively studied in mammals, the reason for its presence in plants is less apparent." The authors speculate that the hormone, like other steroid hormones, might be an ancient bioregulator that evolved billions of years ago, before the appearance of modern plants and animals. The new discovery may change scientific understanding of the evolution and function of progesterone in living things. Scientists had previously identified progesterone-like substances in plants and speculated that the hormone itself could exist in plants. But researchers had not found the actual hormone in plants until now. Dr. Pauli and colleagues used two powerful laboratory techniques, nuclear magnetic resonance and mass spectroscopy, to detect progesterone in leaves of the Common Walnut (or English Walnut) tree (Juglans regia). They also identified five new progesterone-related steroids in a plant belonging to the buttercup family. The discoveries were reported online on January 28, 2010 in the American Chemical Society's Journal of Natural Products. [Press release] [Journal of Natural Products abstract]

New Strategy Rapidly Identifies Two Prototype Drugs

A new and comprehensive drug development strategy that starts with extensive screening of potential targeting peptides to rapidly identify prototype small-molecule drugs has produced two that target the EGFR and VEGFR pathways in novel ways, according to a research team led by scientists at The University of Texas M.D. Anderson Cancer Center "The conceptual advance here is to demonstrate how to go rapidly from screening to structural-functional analysis to drug prototype in a few years," said co-senior author Dr. Wadih Arap, of the David H. Koch Center at M.D. Anderson. "The practical outcome is a pair of new drug candidates, one that acts as a decoy to inhibit a cancer-promoting pathway and another that blocks angiogenesis (the development of new blood vessels), which has the potential to treat both cancer and retinopathies that cause blindness," said co-senior author Dr. Renata Pasqualini, also of the David H. Koch Center. The group's approach begins by screening the target receptors with a phage display library used by Drs. Arap and Pasqualini. This method screens billions of viral particles that each display a different peptide on its outer coat to find those that fit into the receptor as a key goes into a lock. Candidate peptides are next winnowed by using structural and functional analysis. Once a peptide is identified and tested, the researchers take an additional step to synthesize a new version of the peptide more suited for use as a drug.

Stem Cells Restore Sight in Retina-Damaged Mice

Researchers have successfully used mouse embryonic stem cells to replace diseased retinal cells and restore sight in a mouse model of retinitis pigmentosa. This strategy could potentially become a new treatment for retinitis pigmentosa, a leading cause of blindness that affects approximately one in 3,000 to 4,000 people, or a total of 1.5 million people worldwide. The approach also holds promise for the treatment of other retina-damaging diseases. "This research is promising because we successfully turned stem cells into retinal cells, and these retinal cells restored vision in a mouse model of retinitis pigmentosa," said Dr. Stephen Tsang, assistant professor of ophthalmology, pathology, and cell biology at Columbia University Medical Center, and lead author of the paper. "The transplanted cells not only looked like retinal cells, but they functioned like them too." In Dr. Tsang's study, sight was restored in one-fourth of the mice that received the stem cells. However, complications of benign tumors and retinal detachments were seen in some of the mice, so Dr. Tsang and colleagues will optimize techniques to decrease the incidence of these complications in human embryonic stem cells before testing in human patients can begin. "Once the complication issues are addressed, we believe this technique could become a new therapeutic approach for not only retinitis pigmentosa, but age-related macular degeneration, Stargardt disease, and other forms of retinal disease that also feature loss of retinal cells," said Dr. Tsang. Specialized retinal cells called the retinal pigment epithelium help maintain vision.

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