[Show abstract][Hide abstract] ABSTRACT: 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.
Genome Research 11/2009; 20(1):28-35. DOI:10.1101/gr.097212.109 · 13.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 12/2007; 104(45):17735-40. DOI:10.1073/pnas.0700724104 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The pathophysiology of pelvic floor dysfunction resulting in stress urinary incontinence (SUI) in women is complex. Evidence suggests that there is also a genetic predisposition towards SUI. We sought to identify differentially expressed genes involved in extracellular matrix (ECM) metabolism in vaginal tissues from women with SUI in the secretory phase of menses compared with asymptomatic women.
Tissue samples were taken from the periurethral vaginal wall of five pairs of premenopausal, age-matched SUI and continent women and subjected to microarray analysis using the GeneChip Human Genome U133 oligonucleotide chip set.
Extensive statistical analyses generated a list of 79 differentially expressed genes. Elafin, keratin 16, collagen type XVII and plakophilin 1 were consistently identified as up-regulated ECM genes. Elafin, a serine protease inhibitor involved in the elastin degradation pathway and wound healing, was expressed in pelvic fibroblasts and confirmed by Western blot, quantitative competitive PCR and immunofluorescence cell staining.
Genes involved in elastin metabolism were differentially expressed in vaginal tissue from women with SUI, suggesting that elastin remodelling may be important in the molecular aetiology of SUI.
Human Reproduction 02/2006; 21(1):22-9. DOI:10.1093/humrep/dei276 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the variation in gene expression profiles of prostate cancer caused by zone specific genes.
Ten normal central zone, 10 transition zone (benign prostatic hyperplasia) and 6 normal peripheral zone tissues from radical retropubic prostatectomies were compared to each other and to 12 peripheral zone Gleason grade 4/5 cancers. Test chips and HuGeneFL6800 (Affymetrix, Inc., Santa Clara, California) chips were used to assay the transcribed genes. Data were obtained with the Microarray Suite Version 4.0.1 (Affymetrix, Inc.) and analyzed statistically.
Substantially different gene expression profiles were found depending upon which of the 3 zonal tissues were used as a control. All 3 profiles were compared for efficiency (ability to locate genes) and for robustness (the magnitude of difference between the control and the Gleason grade 4/5 tissue). Microscopically normal appearing peripheral zone tissue at the gene level shows many characteristics of Gleason grade 4/5 cancer.
Gene expression profiles of prostate cancer are affected by the zonal location of the control tissue and the cancer.
The Journal of Urology 01/2004; 170(6 Pt 1):2263-8. DOI:10.1097/01.ju.0000096414.25583.0d · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanical stability of the genitourinary tract is dependent on intact collagen fibers that support the bladder neck, urethra, and pelvic organs. We hypothesize that genetic differences in collagen metabolism may contribute to stress urinary incontinence. Because sex hormones have substantial influence on the female lower urinary tract throughout adult life, we investigated the gene expression of vaginal tissue of women with stress incontinence compared with women with no stress incontinence in the proliferative phase of the menstrual cycle.
Quantitative competitive polymerase chain reaction was used to verify that the gene expressions were similar between periurethral vaginal tissue and pelvic ligamentous tissue. Labeled complementary RNA was obtained from periurethral vaginal tissue in five pairs of age- and menstrual phase-matched, premenopausal women with and without stress urinary incontinence. The vaginal tissues were then hybridized on HuGeneFL arrays that contained probes representing 6800 full-length human genes. The Student t test and Mann-Whitney ranking were used independently to select candidates with probability values <.05. Hierarchical clustering analysis was performed on the selected candidates to assess the ability of these genes to discriminate between normal and affected individuals.
Tissue inhibitor of metalloproteinases-1 and estrogen receptor-alpha messenger RNA expressions were found to be similar between uterosacral ligament and periurethral vaginal tissue in six participants. Of the 90 candidate genes that were identified, 62 genes were up-regulated and 28 were down-regulated in the stress urinary incontinence group. Genes that were involved in extracellular matrix activity in the up-regulated group include transforming growth factor-beta3, laminin, and collagen type VI. Down-regulated genes that may participate in collagen metabolism include laminin-related protein, collagen XVII, serine/threonine protein kinase, type II interleukin-1 receptor, and platelet-derived growth factor-associated protein.
In this preliminary study, we identified differential gene expressions that may contribute to extracellular matrix remodeling in pelvic tissue from women with stress urinary incontinence in the proliferative phase versus continent control subjects. The alteration in expression of these candidate genes suggests that they should be targets for further investigation.
American Journal of Obstetrics and Gynecology 07/2003; 189(1):89-97. DOI:10.1067/mob.2003.373 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that hepsin, a serine protease, is over expressed in prostate cancers, implicating hepsin activity in tumor invasion. Using microarray technology we have previously identified 22 genes that were up-regulated in high grade prostate cancers compared with benign prostatic hyperplasia. Of them hepsin was the most differentially over expressed. In the current report we compare hepsin to maspin (BD Transduction Laboratories, San Diego, California), a serine protease inhibitor (serpin), to measure the balance between levels of serine proteases and serpins, which are considered to be a critical determinant of net proteolytic activity.
We combined the technique of laser capture microdissection with gene expression monitoring by micro-array analysis to investigate the gene expression profiles of prostate cells of different histological types. We also studied maspin immunohistochemically.
We observed that hepsin as well as 7 of 22 previously reported up-regulated genes demonstrated a pattern of increasing expression with increasing malignant phenotype. In contrast, the expression of maspin (a serpin) decreased with increasing malignancy of prostate cancers. Using immunohistochemistry we observed that maspin protein is expressed strongly in benign prostatic tissues and slightly in grade 3 prostate cancers, and is absent in grade 4/5 cancers.
We conclude that the increased ratio of hepsin-to-maspin may have an important role in prostate cancer progression and invasion.
The Journal of Urology 05/2003; 169(4):1316-9. DOI:10.1097/01.ju.0000050648.40164.0d · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a high-throughput method for resequencing for single nucleotide polymorphism (SNP) discovery using high-density microarrays. Over the two-year course of this study a number of improvements in sample preparation methods, hybridization assay, array handling, and analysis method were developed and implemented. DNA from 40 unrelated individuals of three different ethnic origins was amplified, labeled, and hybridized to arrays designed with probes representing genomic, coding, and regulatory regions. Protocol improvements including the use of long PCR and semi-automation reduced labeling and fragmentation costs by 33%. Automation improvements include the development of a scanner autoloader for arrays, a faster array wash station, and a linked laboratory tracking and data management system. Validation of a smaller feature size, 20 x 24 microns, allowed the simultaneous screening of 30-kb sense and 30-kb antisense DNA on each microarray, increasing throughput to 1.4 Mb per day per two laboratory personnel. More than 15,000 SNPs were identified in 8.3 Mb of the human genome using high-density resequencing and variation detection arrays (microarrays).
Human Mutation 04/2002; 19(4):402-9. DOI:10.1002/humu.10075 · 5.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PurposeBecause Gleason grade 4/5 cancer is the primary cause of failure to cure prostate cancer, we examined the molecular profiles of this high grade cancer in search of potentially new therapeutic interventions as well as better serum markers than prostate specific antigen.Materials and MethodsWe compared the gene expressions in fresh frozen tissues from 9 men with Gleason grade 4/5 cancer to 8 men with benign prostatic hyperplasia (BPH) treated with radical retropubic prostatectomy. Labeled complementary RNA from each of the 17 tissues was applied to HuGeneFL probe arrays representing approximately 6,800 genes (Affymetrix, Inc., Santa Clara, California). After removing all genes undetectable in BPH and grade 4/5 cancers, and transforming the data into a parametric distribution, we chose only those up and down-regulated genes with a p difference in fluorescence between grade 4/5 cancer and BPH of p <0.0005. This value reduced the data set to 40 up-regulated and 111 down-regulated genes. We then eliminated all genes that were not expressed in all 8 BPH and 9 grade 4/5 tissues, which produced a final set of 86 genes, of which 22 were up-regulated and 64 were down-regulated.ResultsCluster analysis cleanly separated men with grade 4/5 cancers from those with BPH. Only 17 of the 86 candidate genes (20%) were known to be prostate cancer related and 42 (49%) were related to other cancers. The most up-regulated gene is Hepsin, a trypsin-like serine protease with its enzyme catalytic domain oriented extracellularly. Prostate specific membrane antigen is the second most up-regulated gene (all other reports on prostate specific membrane antigen have been at the protein level). The genes for prostate specific antigen (hK3) and human glandular kallikrein2 (hK2) showed equivalent expression levels 10 times the average of other genes. Complete lists of all 22 up-regulated genes and 64 down-regulated genes, together with their locus on the chromosome, are presented in rank order.ConclusionsWe characterize for the first time 64 down-regulated and 22 up-regulated genes in Gleason grade 4/5 cancer, using the gene profile from BPH as control tissue. A number of interesting new genes, previously undescribed in prostate cancer, are presented as possibilities for further study.
The Journal of Urology 12/2001; 166(6-166):2171-2177. DOI:10.1016/S0022-5347(05)65528-0 · 3.75 Impact Factor