Publications by authors named "Guochun Liao"

23 Publications

  • Page 1 of 1

High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

Proc Natl Acad Sci U S A 2015 Dec 23;112(49):15196-201. Epub 2015 Nov 23.

Novira Therapeutics, Doylestown, PA 18902;

The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.
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http://dx.doi.org/10.1073/pnas.1513803112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679053PMC
December 2015

H5N1 influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load.

mBio 2011 6;2(5). Epub 2011 Sep 6.

Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

Unlabelled: The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus.

Importance: Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.
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http://dx.doi.org/10.1128/mBio.00171-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171982PMC
January 2012

Genetic susceptibility to the delayed sequelae of neonatal respiratory syncytial virus infection is MHC dependent.

J Immunol 2010 Nov 4;185(9):5384-91. Epub 2010 Oct 4.

Department of Respiratory Medicine, Centre for Respiratory Infection, National Heart and Lung Institute, Imperial College London, London, United Kingdom.

Respiratory syncytial virus (RSV) is a major cause of respiratory morbidity, resulting in hospitalization for bronchiolitis in some infected infants that is associated with wheeze in later life. Genetic factors are known to affect the severity of the sequelae after RSV infection, but the complexity of the temporal and genetic effects makes it difficult to analyze this response in studies in man. Therefore, we developed a murine genetic model to analyze the sequelae occurring after RSV infection in early life. Haplotype-based genetic analysis of interstrain differences in severity identified the MHC as an important genetic determinant. This was confirmed by analysis of responses in congenic mice with different MHC haplotypes. We also found that susceptible strains had high CD8 levels during secondary infection. Analysis of first filial generation, second filial generation, and back-cross progeny produced by intercrossing resistant (H-2(k), C3H/HeN) and sensitive (H-2(b), BALB/c) strains indicated that susceptibility to sequelae after RSV infection was dominantly inherited but also segregated in a non-MHC-dependent manner. Thus, MHC haplotype and its effect on CD8 cell response is an important determinant of the outcome of neonatal RSV infection.
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http://dx.doi.org/10.4049/jimmunol.1001594DOI Listing
November 2010

Computational genetic mapping in mice: the ship has sailed.

Sci Transl Med 2009 Oct;1(3):3ps4

Department of Anesthesia, Stanford University, Stanford, CA, USA.

Computational haplotype-based genetic mapping can be used to discover new biological mechanisms, disease-related pathways, and unexpected uses for existing drugs. Here we discuss the benefits and limitations of this methodology, its impact on translational medicine, and its future course.
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http://dx.doi.org/10.1126/scitranslmed.3000377DOI Listing
October 2009

An integrative genomic analysis identifies Bhmt2 as a diet-dependent genetic factor protecting against acetaminophen-induced liver toxicity.

Genome Res 2010 Jan 18;20(1):28-35. Epub 2009 Nov 18.

Department of Genetics and Genomics, Roche Palo Alto, Palo Alto, California 94304, USA.

Acetaminophen-induced liver toxicity is the most frequent precipitating cause of acute liver failure and liver transplant, but contemporary medical practice has mainly focused on patient management after a liver injury has been induced. An integrative genetic, transcriptional, and two-dimensional NMR-based metabolomic analysis performed using multiple inbred mouse strains, along with knowledge-based filtering of these data, identified betaine-homocysteine methyltransferase 2 (Bhmt2) as a diet-dependent genetic factor that affected susceptibility to acetaminophen-induced liver toxicity in mice. Through an effect on methionine and glutathione biosynthesis, Bhmt2 could utilize its substrate (S-methylmethionine [SMM]) to confer protection against acetaminophen-induced injury in vivo. Since SMM is only synthesized in plants, Bhmt2 exerts its beneficial effect in a diet-dependent manner. Identification of Bhmt2 and the affected biosynthetic pathway demonstrates how a novel method of integrative genomic analysis in mice can provide a unique and clinically applicable approach to a major public health problem.
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http://dx.doi.org/10.1101/gr.097212.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798828PMC
January 2010

The beta3 subunit of the Na+,K+-ATPase mediates variable nociceptive sensitivity in the formalin test.

Pain 2009 Aug 22;144(3):294-302. Epub 2009 May 22.

Department of Psychology and Centre for Research on Pain, McGill University, 1205 Dr. Penfield Ave., Montreal, Que., Canada H3A 1B1 Department of Neurology and Neurosurgery, Montreal Neurological Institute, and Centre for Research on Pain, McGill University, Montreal, Que., Canada H3A 2B4 Department of Anesthesiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Department of Genetics and Genomics, Roche Palo Alto, Palo Alto, CA 94304, USA Integrated DNA Technologies Inc., Coralville, IA 52241, USA Department of Anesthesiology, Stanford University, Palo Alto, CA 94304, USA Department of Anesthesia, Stanford University, Stanford, CA 94305, USA.

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the beta3 subunit of the Na+,K+-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, beta3 protein expression, and biophysical properties of the Na+,K+ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the beta3 subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the beta3 subunit of the Na+,K+-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.
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http://dx.doi.org/10.1016/j.pain.2009.04.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744953PMC
August 2009

From mouse to man: the 5-HT3 receptor modulates physical dependence on opioid narcotics.

Pharmacogenet Genomics 2009 Mar;19(3):193-205

Department of Anesthesiology, Stanford University, Palo Alto, California, USA.

Objectives: Addiction to opioid narcotics represents a major public health challenge. Animal models of one component of addiction, physical dependence, show this trait to be highly heritable. The analysis of opioid dependence using contemporary in-silico techniques offers an approach to discover novel treatments for dependence and addiction.

Methods: In these experiments, opioid withdrawal behavior in 18 inbred strains of mice was assessed. Mice were treated for 4 days with escalating doses of morphine before the administration of naloxone allowing the quantification of opioid dependence. After haplotypic analysis, experiments were designed to evaluate the top gene candidate as a modulator of physical dependence. Behavioral studies as well as measurements of gene expression on the mRNA and protein levels were completed. Finally, a human model of opioid dependence was used to quantify the effects of the 5-HT3 antagonist ondansetron on signs and symptoms of withdrawal.

Results: The Htr3a gene corresponding to the 5-HT3 receptor emerged as the leading candidate. Pharmacological studies using the selective 5-HT3 antagonist ondansetron supported the link in mice. Morphine strongly regulated the expression of the Htr3a gene in various central nervous system regions including the amygdala, dorsal raphe, and periaqueductal gray nuclei, which have been linked to opioid dependence in previous studies. Using an acute morphine administration model, the role of 5-HT3 in controlling the objective signs of withdrawal in humans was confirmed.

Conclusion: These studies show the power of in-silico genetic mapping, and reveal a novel target for treating an important component of opioid addiction.
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http://dx.doi.org/10.1097/FPC.0b013e328322e73dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730361PMC
March 2009

Plasminogen alleles influence susceptibility to invasive aspergillosis.

PLoS Genet 2008 Jun 20;4(6):e1000101. Epub 2008 Jun 20.

Duke University Medical Center, Durham, North Carolina, United States of America.

Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.
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http://dx.doi.org/10.1371/journal.pgen.1000101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423485PMC
June 2008

A genomic "roadmap" to "better" drugs.

Drug Metab Rev 2008 ;40(2):225-39

Department of Genetics & Genomics, Roche Palo Alto, Palo Alto, California 94304-1397, USA.

Utilization of pharmacogenomic information has the potential to significantly improve treatment outcome and markedly reduce the rate of attrition of drugs in clinical development. A major gap that limits our ability to utilize pharmacogenomic information in drug discovery, drug development or clinical practice is that we often do not know the genetic variants responsible for inter-individual differences in drug metabolism or drug response. We examine emerging genomic methods that can fill this gap; these methods can be used to generate new information about drug metabolism or mechanism of action, or to identify predictors of drug response. Although they have not yet had their full impact, a wider application of these emerging genomic technologies has the potential to significantly improve the safety of drugs, the quality of patient care and the efficiency of clinical drug development.
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http://dx.doi.org/10.1080/03602530801952815DOI Listing
June 2008

Quantitative trait locus and computational mapping identifies Kcnj9 (GIRK3) as a candidate gene affecting analgesia from multiple drug classes.

Pharmacogenet Genomics 2008 Mar;18(3):231-41

Department of Psychology and Centre for Research on Pain, McGill University, Montreal, Canada.

Aims: Interindividual differences in analgesic drug response complicate the clinical management of pain. We aimed to identify genetic factors responsible for variable sensitivity to analgesic drugs of disparate neurochemical classes.

Methods And Results: Quantitative trait locus mapping in 872 (C57BL/6x129P3)F2 mice was used to identify genetic factors contributing to variability in the analgesic effect of opioid (morphine), alpha2-adrenergic (clonidine), and cannabinoid (WIN55,212-2) drugs against thermal nociception. A region on distal chromosome 1 showing significant linkage to analgesia from all three drugs was identified. Computational (in silico) genetic analysis of analgesic responses measured in a panel of inbred strains identified a haplotype block within this region containing the Kcnj9 and Kcnj10 genes, encoding the Kir3.3 (GIRK3) and Kir4.1 inwardly rectifying potassium channel subunits. The genes are differentially expressed in the midbrain periaqueductal gray of 129P3 versus C57BL/6 mice, owing to cis-acting genetic elements. The potential role of Kcnj9 was confirmed by the demonstration that knockout mice have attenuated analgesic responses.

Conclusion: A single locus is partially responsible for the genetic mediation of pain inhibition, and genetic variation associated with the potassium channel gene, Kcnj9, is a prime candidate for explaining the variable response to these analgesic drugs.
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http://dx.doi.org/10.1097/FPC.0b013e3282f55ab2DOI Listing
March 2008

In silico and in vitro pharmacogenetic analysis in mice.

Proc Natl Acad Sci U S A 2007 Nov 31;104(45):17735-40. Epub 2007 Oct 31.

Department of Genetics and Genomics, Roche Palo Alto, Palo Alto, CA 94304, USA.

Combining the experimental efficiency of a murine hepatic in vitro drug biotransformation system with in silico genetic analysis produces a model system that can rapidly analyze interindividual differences in drug metabolism. This model system was tested by using two clinically important drugs, testosterone and irinotecan, whose metabolism was previously well characterized. The metabolites produced after these drugs were incubated with hepatic in vitro biotransformation systems prepared from the 15 inbred mouse strains were measured. Strain-specific differences in the rate of 16 alpha-hydroxytestosterone generation and irinotecan glucuronidation correlated with the pattern of genetic variation within Cyp2b9 and Ugt1a loci, respectively. These computational predictions were experimentally confirmed using expressed recombinant enzymes. The genetic changes affecting irinotecan metabolism in mice mirrored those in humans that are known to affect the pharmacokinetics and incidence of adverse responses to this medication.
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http://dx.doi.org/10.1073/pnas.0700724104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077071PMC
November 2007

Bone microstructure and its associated genetic variability in 12 inbred mouse strains: microCT study and in silico genome scan.

Bone 2008 Feb 22;42(2):439-51. Epub 2007 Sep 22.

Physical Medicine and Rehabilitation Service, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, USA.

Unlabelled: MicroCT analysis of 12 inbred strains of mice identified 5 novel chromosomal regions influencing skeletal phenotype. Bone morphology varied in a compartment- and site-specific fashion across strains and genetic influences contributed to the morphometric similarities observed in femoral and vertebral bone within the trabecular bone compartment.

Introduction: Skeletal development is known to be regulated by both heritable and environmental factors, but whether genetic influence on peak bone mass is site- or compartment-specific is unknown. This study examined the genetic variation of cortical and trabecular bone microarchitecture across 12 strains of mice.

Materials And Methods: MicroCT scanning was used to measure trabecular and cortical bone morphometry in the femur and vertebra of 12 strains of 4-month-old inbred male mice. A computational genome mapping technique was used to identify chromosomal intervals associated with skeletal traits.

Results: Skeletal microarchitecture varied in a compartment- and site-specific fashion across strains. Genome mapping identified 13 chromosomal intervals associated with skeletal traits and 5 of these intervals were novel. Trabecular microarchitecture in different bone sites correlated across strains and most of the chromosomal intervals associated with these trabecular traits were shared between skeletal sites. Conversely, no chromosomal intervals were shared between the trabecular and cortical bone compartments in the femur, even though there was a strong correlation for these different bone compartments across strains, suggesting site-specific regulation by environmental or intrinsic factors.

Conclusion: In summary, these data confirm that there are distinct genetic determinants that define the skeletal phenotype at the time when peak bone mass is being acquired, and that genomic regulation of bone morphology is specific for skeletal compartment.
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http://dx.doi.org/10.1016/j.bone.2007.09.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704123PMC
February 2008

2D NMR metabonomic analysis: a novel method for automated peak alignment.

Bioinformatics 2007 Nov 10;23(21):2926-33. Epub 2007 Sep 10.

Department of Genetics & Genomics, Roche Palo Alto LLC, Palo Alto, CA 94304, USA.

Motivation: Comparative metabolic profiling by nuclear magnetic resonance (NMR) is showing increasing promise for identifying inter-individual differences to drug response. Two dimensional (2D) (1)H (13)C NMR can reduce spectral overlap, a common problem of 1D (1)H NMR. However, the peak alignment tools for 1D NMR spectra are not well suited for 2D NMR. An automated and statistically robust method for aligning 2D NMR peaks is required to enable comparative metabonomic analysis using 2D NMR.

Results: A novel statistical method was developed to align NMR peaks that represent the same chemical groups across multiple 2D NMR spectra. The degree of local pattern match among peaks in different spectra is assessed using a similarity measure, and a heuristic algorithm maximizes the similarity measure for peaks across the whole spectrum. This peak alignment method was used to align peaks in 2D NMR spectra of endogenous metabolites in liver extracts obtained from four inbred mouse strains in the study of acetaminophen-induced liver toxicity. This automated alignment method was validated by manual examination of the top 50 peaks as ranked by signal intensity. Manual inspection of 1872 peaks in 39 different spectra demonstrated that the automated algorithm correctly aligned 1810 (96.7%) peaks.

Availability: Algorithm is available upon request.
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http://dx.doi.org/10.1093/bioinformatics/btm427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174787PMC
November 2007

Genetic variants of the P-glycoprotein gene Abcb1b modulate opioid-induced hyperalgesia, tolerance and dependence.

Pharmacogenet Genomics 2006 Nov;16(11):825-35

Department of Anesthesiology, Stanford University, California, USA.

Objective: Opioid-induced hyperalgesia (OIH) is a state of paradoxically increased nociceptive sensitivity seen in both humans and rodents following the resolution of the acute opioid antinociceptive effects or during periods of chronic opioid administration. Using the power of genetic analysis, we hoped to discover novel mechanisms modulating this trait.

Basic Methods: The degree of opioid-induced hyperalgesia displayed in response to a thermal stimulus applied to the hind paw was measured in 16 strains of inbred mice after 4 days of morphine administration. The degree of thermal sensitization was then used in a recently developed in silico haplotypic mapping algorithm along with a haplotypic map constructed from a database containing 209,000 single nucleotide polymorphisms.

Main Results: Analysis of the data resulted in the identification of several haplotype blocks strongly associated with the thermal opioid-induced hyperalgesia trait. The most strongly associated block was located within the Abcb1b P-glycoprotein drug transporter gene. Experiments using the P-glycoprotein inhibitor cyclosporine A and P-glycoprotein null mutant mice supported the hypothesis that a functional association exists between P-glycoprotein transporters and opioid-induced hyperalgesia. The observation of a correlation between morphine brain concentrations and the development of opioid-induced hyperalgesia was consistent with this hypothesis as well. In addition, P-glycoprotein gene deletion and pharmacological inhibition altered morphine ED50, tolerance and physical dependence.

Conclusions: We conclude that the use of haplotypic mapping to identify novel mechanisms controlling complex traits is a viable approach. Variants of the Abcb1b gene may explain some portion of the interstrain differences in OIH and perhaps other consequences of chronic opioid administration.
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http://dx.doi.org/10.1097/01.fpc.0000236321.94271.f8DOI Listing
November 2006

Understanding our drugs and our diseases.

Proc Am Thorac Soc 2006 Jul;3(5):409-12

Departments of Genetics and Genomics, Roche Palo Alto, Palo Alto, CA 94304, USA.

Analysis of mouse genetic models of human disease-associated traits has provided important insight into the pathogenesis of human disease. As one example, analysis of a murine genetic model of osteoporosis demonstrated that genetic variation within the 15-lipoxygenase (Alox15) gene affected peak bone mass, and that treatment with inhibitors of this enzyme improved bone mass and quality in rodent models. However, the method that has been used to analyze mouse genetic models is very time consuming, inefficient, and costly. To overcome these limitations, a computational method for analysis of mouse genetic models was developed that markedly accelerates the pace of genetic discovery. It was used to identify a genetic factor affecting the rate of metabolism of warfarin, an anticoagulant that is commonly used to treat clotting disorders. Computational analysis of a murine genetic model of narcotic drug withdrawal suggested a potential new approach for treatment of narcotic drug addiction. Thus, the results derived from computational mouse genetic analysis can suggest new treatment strategies, and can provide new information about currently available medicines.
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http://dx.doi.org/10.1513/pats.200601-014AWDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2658704PMC
July 2006

In silico pharmacogenetics of warfarin metabolism.

Nat Biotechnol 2006 May;24(5):531-6

Department of Genetics and Genomics, Roche Palo Alto S3-1, 3431 Hillview Ave., Palo Alto, California 94304, USA.

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.
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http://dx.doi.org/10.1038/nbt1195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1459533PMC
May 2006

A genetic analysis of opioid-induced hyperalgesia in mice.

Anesthesiology 2006 May;104(5):1054-62

Department of Anesthesiology, Stanford University, Palo Alto, California, USA.

Background: Opioid-induced hyperalgesia (OIH) is a syndrome of increased sensitivity to noxious stimuli, seen after both the acute and chronic administration of opioids, that has been observed in humans and rodent models. This syndrome may reduce the clinical utility of opioids in treating acute and chronic pain.

Methods: In these studies, the authors measured the propensity of 15 strains of inbred mice to develop mechanical manifestations of OIH. These data were subjected to in silico genetic analysis, which resulted in the association of haplotypic blocks within or near several known genes. Both pharmacologic agents and transgenic mice were used to confirm the functional association of the most strongly linked gene with OIH.

Results: Both baseline mechanical nociceptive thresholds and the percentage changes in these thresholds after 4 days of morphine treatment were found to be highly strain dependent. The haplotypic blocks most strongly associated with the mechanical OIH data were located within the beta2 adrenergic receptor gene (beta2-AR). Using the selective beta2-AR antagonist butoxamine, the authors observed a dose-dependent reversal of OIH. Furthermore, deletion of the beta2-AR gene sharply reduced the mechanical allodynia present after morphine treatment in the wild-type mouse strain. Analysis of the associated beta2-AR haplotypic block identified single nucleotide polymorphisms potentially explaining in part the strain specific differences in OIH.

Conclusions: Genetic variants of the beta2-AR gene seem to explain some part of the differences between various strains of mice to develop OIH. The association of this gene with OIH suggests specific pharmacologic strategies for reducing the impact of OIH on patients consuming opioids.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1464476PMC
http://dx.doi.org/10.1097/00000542-200605000-00023DOI Listing
May 2006

Chronic pain and genetic background interact and influence opioid analgesia, tolerance, and physical dependence.

Pain 2006 Apr 3;121(3):232-240. Epub 2006 Mar 3.

Department of Anesthesia, Stanford University School of Medicine, Stanford, CA, USA Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA, USA Roche Palo Alto, Palo Alto, CA, USA.

Opioids are commonly used in the treatment of moderate to severe pain. However, their chronic use is limited by analgesic tolerance and physical dependence. Few studies have examined how chronic pain affects the development of tolerance or dependence, and essentially no studies have looked at the role of both genetics and pain together. For these studies we used 12 strains of inbred mice. Groups of mice from each strain were tested at baseline for morphine analgesic sensitivity, mechanical nociceptive threshold, and thermal nociceptive threshold. Mice were then given morphine in a 4-day escalating morphine administration paradigm followed by reassessment of the morphine dose-response relationship. Finally, physical dependence was measured by administering naloxone. Parallel groups of mice underwent hind paw injection of complete Freund's adjuvant (CFA) to induce chronic hind paw inflammation 7 days prior to the beginning of testing. The data showed that CFA treatment tended to lower baseline ED(50) values for morphine and enhanced the degree of analgesic tolerance observed after 4 days of morphine treatment. In addition, the degree of jumping behavior indicative of physical dependence was often altered if mice had been treated with CFA. The influence of background strain was substantial for all traits measured. In silico haplotypic mapping of the tolerance and physical dependence data demonstrated that CFA pretreatment altered the pattern of the predicted associations and greatly reduced their statistical significance. We conclude that chronic inflammatory pain and genetics interact to modulate the analgesic potency of morphine, tolerance, and physical dependence.
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http://dx.doi.org/10.1016/j.pain.2005.12.026DOI Listing
April 2006

Computational genetics: from mouse to human?

Trends Genet 2005 Sep;21(9):526-32

Department of Genetics and Genomics, Roche Palo Alto S3-1, Palo Alto, CA 94304, USA.

In this article, we describe a novel computational-analysis method that rapidly identified the genetic basis for several trait differences among inbred mouse strains. This approach enables researchers to identify a causative genetic factor by correlating a pattern of observable physiological or pathological differences among selected inbred strains with a pattern of genetic variation. Compared with conventional methods used for mouse genetic analysis, which require many years to produce results, this haplotype-based computational analysis can be rapidly performed. We discuss the factors affecting the performance and precision of this computational method. Although it currently can analyze traits of limited genetic complexity in mouse, the potential application of this genetic-analysis method to other experimental organisms, and possibly humans, is evaluated.
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http://dx.doi.org/10.1016/j.tig.2005.06.010DOI Listing
September 2005

In silico genetics: identification of a functional element regulating H2-Ealpha gene expression.

Science 2004 Oct;306(5696):690-5

Department of Genetics and Genomics, Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304-1397, USA.

Computational tools can markedly accelerate the rate at which murine genetic models can be analyzed. We developed a computational method for mapping phenotypic traits that vary among inbred strains onto haplotypic blocks. This method correctly predicted the genetic basis for strain-specific differences in several biologically important traits. It was also used to identify an allele-specific functional genomic element regulating H2-Ealpha gene expression. This functional element, which contained the binding sites for YY1 and a second transcription factor that is probably serum response factor, is located within the first intron of the H2-Ealpha gene. This computational method will greatly improve our ability to identify the genetic basis for a variety of phenotypic traits, ranging from qualitative trait information to quantitative gene expression data, which vary among inbred mouse strains.
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http://dx.doi.org/10.1126/science.1100636DOI Listing
October 2004

The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes.

Genetics 2004 Jun;167(2):761-81

Department of Molecular and Human Genetics, Howard Hughes Medical Institute, Program in Developmental Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The Berkeley Drosophila Genome Project (BDGP) strives to disrupt each Drosophila gene by the insertion of a single transposable element. As part of this effort, transposons in >30,000 fly strains were localized and analyzed relative to predicted Drosophila gene structures. Approximately 6300 lines that maximize genomic coverage were selected to be sent to the Bloomington Stock Center for public distribution, bringing the size of the BDGP gene disruption collection to 7140 lines. It now includes individual lines predicted to disrupt 5362 of the 13,666 currently annotated Drosophila genes (39%). Other lines contain an insertion at least 2 kb from others in the collection and likely mutate additional incompletely annotated or uncharacterized genes and chromosomal regulatory elements. The remaining strains contain insertions likely to disrupt alternative gene promoters or to allow gene misexpression. The expanded BDGP gene disruption collection provides a public resource that will facilitate the application of Drosophila genetics to diverse biological problems. Finally, the project reveals new insight into how transposons interact with a eukaryotic genome and helps define optimal strategies for using insertional mutagenesis as a genomic tool.
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http://dx.doi.org/10.1534/genetics.104.026427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470905PMC
June 2004

Ozone-induced acute pulmonary injury in inbred mouse strains.

Am J Respir Cell Mol Biol 2004 Jul 19;31(1):69-77. Epub 2004 Feb 19.

Department of Medicine, Duke University Medical Center and VA Medical Center, Durham, NC 27710, USA.

To determine if host factors influence the time course and extent of lung injury after acute inhalation of ozone (O3), we evaluated the physiologic and biologic response of nine genetically diverse inbred strains of mice (C57BL/6J, 129/SvIm, BTBR, BALB/cJ, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ) exposed to O3 (2.0 ppm x 3 h). Whole lung lavage determined that 129/Svlm, BTBR, DBA/2J, and FVB/NJ had a peak increase in polymorphonuclear cells (PMNs) at 6 h, whereas C57BL/6J and CAST/Ei had a peak increase at 24 h after exposure; airway PMNs were minimally elevated in A/J and C3H/HeJ; BALB/cJ had a predominant lymphocytic influx. Interleukin-6 concentration in the lavage fluid was associated with the influx of PMNs, whereas the total protein in the lavage fluid did not always correlate with lavage cellularity. Respiratory responses were monitored using whole body plethysmography and enhanced pause index. C57BL/6J, BALB/cJ, 129/SvIm, and BTBR were highly sensitive to O3 and exhibited significant increases in enhanced pause to methacholine aerosol stimulation at 6 and 24 h after exposure to O3. In contrast, DBA/2J, A/J, FVB/NJ, CAST/Ei, and C3H/HeJ strains had demonstrated increases in sensitivity to MCh at 6 h after exposure, but responses had returned to near baseline by 24 h after exposure to O3. Epithelial cell proliferation as assessed by proliferating cell nuclear antigen staining was evident at 24 h after exposure to O3. C57BL/6J and A/J showed 4% proliferating cell nuclear antigen-positive cells; 129/SvIm, DBA/2J, and FVB/NJ had 1-3%; and BTBR, BALB/cJ, CAST/Ei, and C3H/HeJ had < 1%. Phenotypic measurements in six inbred strains were used for an in silico genome analysis based on the Roche mouse database. Consistent loci on chromosomes 1, 7, and 15 were among those identified to have a significant association with the phenotypes studied. In aggregate, our approach has identified O3-resistant (C3H/HeJ and A/J) and -vulnerable (C57BL/6J and 129/SvIm) strains of mice, and determined novel genomic loci, suggesting a clear genetic basis for the lung response to inhaled O3.
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http://dx.doi.org/10.1165/rcmb.2003-0001OCDOI Listing
July 2004

The Drosophila gene collection: identification of putative full-length cDNAs for 70% of D. melanogaster genes.

Genome Res 2002 Aug;12(8):1294-300

Berkeley Drosophila Genome Project, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5' expressed sequence tags (5' expressed sequence tags [ESTs]from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to ~40% of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remaining genes, we have generated an additional 157,835 5' ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22-h embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70% of the predicted genes in Drosophila.
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http://dx.doi.org/10.1101/gr.269102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC186637PMC
August 2002