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1.  Polymorphisms affecting gene transcription and mRNA processing in pharmacogenetic candidate genes: detection through allelic expression imbalance in human target tissues 
Pharmacogenetics and genomics  2008;18(9):781-791.
Genetic variation in mRNA expression plays a critical role in human phenotypic diversity, but it has proven difficult to detect regulatory polymorphisms - mostly single nucleotide polymorphisms (rSNPs). Additionally, variants in the transcribed region, termed here ‘structural RNA SNPs’ (srSNPs), can affect mRNA processing and turnover. Both rSNPs and srSNPs cause allelic mRNA expression imbalance (AEI) in heterozygous individuals. We have applied a rapid and accurate AEI methodology for testing 42 genes implicated in human diseases and drug response, specifically cardiovascular and CNS diseases, and affecting drug metabolism and transport. Each gene was analyzed in physiologically relevant human autopsy tissues, including brain, heart, liver, intestines, and lymphocytes. Substantial AEI was observed in ∼55% of the surveyed genes. Focusing on cardiovascular candidate genes in human hearts, AEI analysis revealed frequent cis-acting regulatory factors in SOD2 and ACE mRNA expression, having potential clinical significance. SNP scanning to locate regulatory polymorphisms in a number of genes failed to support several previously proposed promoter SNPs discovered with use of reporter gene assays in heterologous tissues, while srSNPs appear more frequent than expected. Computational analysis of mRNA folding indicates that ∼90% of srSNPs affects mRNA folding, and hence potentially function. Our results indicate that both rSNPs and srSNPs represent a still largely untapped reservoir of variants that contribute to human phenotypic diversity.
doi:10.1097/FPC.0b013e3283050107
PMCID: PMC2779843  PMID: 18698231
2.  Highly variable mRNA expression and splicing of L-type voltage-dependent calcium channel alpha subunit 1C in human heart tissues 
Pharmacogenetics and genomics  2006;16(10):735-745.
Objectives
The voltage-dependent L-type calcium channel α-subunit 1c (Cav1.2, CACNA1C) undergoes extensive mRNA splicing, leading to numerous isoforms with different functions. L-type calcium channel blockers are used in the treatment of hypertension and arrhythmias, but response varies between individuals. We have studied the interindividual variability in mRNA expression and splicing of CACNA1C, in 65 heart tissue samples, taken from heart transplant recipients.
Methods
Splice variants were measured quantitatively by polymerase chain reaction in 12 splicing loci of CACNA1C mRNA. To search for functional cis-acting polymorphisms, we determined allelic expression ratios for total CACNA1C mRNA and several splice variants using marker single nucleotide polymorphisms in exon 4 and exon 30.
Results
Total CACNA1C mRNA levels varied ∼50-fold. Substantial splicing occurred in six loci generating two or more splice variants, some with known functional differences. Splice patterns varied broadly between individuals. Two heart tissues expressed predominantly the dihydropyridine-sensitive smooth muscle isoform of CACNA1C (containing exon 8), rather than the cardiac isoform (containing exon 8a). Lack of significant allelic expression imbalance, observed with total mRNA and several splice variants, argued against CACNA1C polymorphisms as a cause of variability. Taken together, highly variable splicing can cause profound phenotypic variations of CACNA1C function, potentially associated with disease susceptibility and response to L-type calcium channel blockers.
doi:10.1097/01.fpc.0000230119.34205.8a
PMCID: PMC2688811  PMID: 17001293
cis-acting polymorphism; L-type calcium channel α-subunit 1c; mRNA splicing
3.  Searching for polymorphisms that affect gene expression and mRNA processing: Example ABCB1 (MDR1) 
The AAPS Journal  2006;8(3):E515-E520.
Cis-acting genetic variations can affect the amount and structure of mRNA/protein. Genomic surveys indicate that polymorphisms affecting transcription and mRNA processing, including splicing and turnover, may account for main share of genetic factors in human phenotypic variability; however, most of these polymorphisms remain yet to be discovered. We use allelic expression imbalance (AEI) as a quantitative phenotype in the search for functionalcis-acting polymorphisms in many genes includingABCB1 (multidrug resistance 1 gene, MDR1, Pgp). Previous studies have shown that ABCB1 activity correlates with a synonymous polymorphism. C3435T; however, the functional polymorphism and molecular mechanisms underlying this clinical association remained unknown. Analysis of allele-specific expression in liver autopsy samples and in vitro expression experiments showed that C3435T represents a main functional polymorphism, accounting for 1.5-to 2-fold changes in mRNA levels. The mechanism appears to involve increased mRNA turnover, probably as a result of different folding structures calculated for mRNA with the Mfold program. Other examples of the successful application of AEI analysis for studying functional polymorphism include5-HTT (serotonin transporter, SLC6A4) andOPRM1 (μ opioid receptor). AEI is therefore a powerful approach for detectingcis-acting polymorphisms affecting gene expression and mRNA processing.
doi:10.1208/aapsj080361
PMCID: PMC2761059  PMID: 17025270
ABCB1; allele-specific expression; mRNA stability; cis-acting polymorphism
5.  Pharmacogenomics of multigenic diseases: Sex-specific differences in disease and treatment outcome 
AAPS PharmSci  2003;5(4):49-61.
Numerous genetic variations have been shown to affect disease susceptibility and drug response. Pharmacogenomics aims at improving therapy on the basis of genetic information for each individual patient. Furthermore, sex chromosomes broadly determine biological differences between males and females. Consequently, substantial sex differences exist in phenotypic manifestation of disease and treatment response. This review discusses the role of sex in coronary artery disease, schizophrenia, and depression—complex multigenic disorders with considerable sex differences in frequency and presentation. Moreover, genetic factors underlying disease and drug response appear to differ between male and female patients. This appears to result at least in part from different physiological effects exerted by sex hormones such that polymorphisms in susceptibility genes may have physiological relevance only in males or females. However, few examples have been discovered to play a role in complex multigenic diseases, and the mechanistic basis of genetic variants as sex-dependent susceptibility factors has yet to be explored. Therefore, pharmacogenomic studies must consider sex differences in an effort to optimize individual drug therapy.
doi:10.1208/ps050429
PMCID: PMC2750991  PMID: 15198517
pharmacogenomics; sex differences; multigenic disease; candidate genes; coronary artery disease; depression; schizophrenia
6.  Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein 
AAPS PharmSci  2002;4(4):21-28.
Intracellular free H+ concentration (pHi) responds to numerous extracellular stimuli. The use of fluorescent indicator dyes to measure pHi is strongly influenced by the ability of target cells to retain activated dye within the cytoplasmic compartment. Here, 3 pH-sensitive indicator dyes—acetoxymethyl (AM) esters of SNARF-1 and BCECF, and the thiol-reactive 5-chloromethyfluorescein (CMFDA)—were examined for monitoring pHi. The stability of pH measurements was strongly affected by temperature, cell type, indicator dye, and use of transport inhibitors to prevent dye export. Cellular retention of CMFDA, which forms covalent complexes, was sufficient to permit monitoring of transient pHi changes over extended time periods in a multi-well plate assay format. In human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells, increasing osmotic pressure caused a significant rise in pHi. In contrast, activation of native or transfected β-adrenergic, cholinergic, and d and m opioid receptors did not measurably affect pHi in HEK293 cells. Decreases in pHi were observed in CHO cells expressing the human H+/peptide transporter PEPT1 upon addition of dipeptide substrates. The use of CMFDA in multi-well formats should facilitate study of osmotic and transport activity and screening for drugs that affect pHi.
doi:10.1208/ps040421
PMCID: PMC2751310  PMID: 12645993
intracellular pH; fluorescent pH-sensitive; dyes; Chinese hamster ovary cells; human embryonic; kidney cells; human colon adrenocarcinoma cells; hypertonic stress
7.  Evolutionary relationships among G protein-coupled receptors using a clustered database approach 
AAPS PharmSci  2001;3(2):25-42.
Guanine nucleotide-binding proteincoupled receptors (GPCRs) comprise large and diverse gene families in fungi, plants, and the animal kingdom. GPCRs appear to share a common structure with 7 transmembrane segments, but sequence similarity is minimal among the most distant GPCRs. To reevaluate the question of evolutionary relationships among the disparate GPCR families, this study takes advantage of the dramatically increased number of cloned GPCRs. Sequences were selected from the National Center for Biotechnology Information (NCBI) nonredundant peptide database using iterative BLAST (Basic Local Alignment Search Tool) searches to yield a database of ∼1700 GPCRs and unrelated membrane proteins as controls, divided into 34 distinet clusters. For each cluster, separate position-specific matrices were established to optimize sequence comparisons among GPCRs. This approach resulted in significant alignments between distant GPCR families, including receptors for the biogenic amine/peptide, VIP/secretin, cAMP, STE3/MAP3 fungal pheromones, latrophilin, developmental receptors frizzled and smoothened, as well as the more distant metabotrobic glutamate receptors, the STE2/MAM2 fungal pheromone receptors, and GPR1, a fungal glucose receptor. On the other hand, alignment scores between these recognized GPCR clades with p40 (putative GPCR) and pml (putative GPCR), as well as bacteriorhodopsins, failed to support a finding of homology. This study provides a refined view of GPCR ancestry and serves as a reference database with hyperlinks to other sources. Moreover, it may facilitate database annotation and the assignment of orphan receptors to GPCR families.
doi:10.1208/ps030212
PMCID: PMC2779559  PMID: 11741263
8.  Human membrane transporter database: A web-accessible relational database for drug transport studies and pharmacogenomics 
AAPS PharmSci  2000;2(3):11-17.
The human genome contains numerous genes that encode membrane transporters and related proteins. For drug discovery, development, and targeting, one needs to know which transporters play a role in drug disposition and effects. Moreover, genetic polymorphisms in human membrane transporters may contribute to interindividual differences in the response to drugs. Pharmacogenetics, and, on a genome-wide basis, pharmacogenomics, address the effect of genetic variants on an individual's response to drugs and xenobiotics. However, our knowledge of the relevant transporters is limited at present. To facilitate the study of drug transporters on a broad scale, including the use of microarray technology, we have constructed a human membrane transporter database (HMTD). Even though it is still largely incomplete, the database contains information on more than 250 human membrane transporters, such as sequence, gene family, structure, function, substrate, tissue distribution, and genetic disorders associated with transporter polymorphisms. Readers are invited to submit additional data. Implemented as a relational database, HMTD supports complex biological queries. Accessible through a Web browser user interface via Common Gateway Interface (CGI) and Java Database Connection (JDBC) (http://128.218.208.23/transporter/trans.html),
HMTD also provides useful links and references, allowing interactive searching and downloading of data. Taking advantage of the features of an electronic journal, this paper serves as an interactive tutorial for using the database, which we expect to develop into a research tool.
doi:10.1208/ps020320
PMCID: PMC2761131  PMID: 11741236
9.  Human proton/oligopeptide transporter (POT) genes: Identification of putative human genes using bioinformatics 
AAPS PharmSci  2000;2(2):76-97.
Purpose: The proton-dependent oligopeptide transporters (POT) gene family currently consists of ∼70 cloned cDNAs derived from diverse organisms. In mammals, two genes encoding peptide transporters, PepT1 and PepT2 have been cloned in several species including humans, in addition to a rat histidine/peptide transporter (rPHT1). Because the Candida elegans genome contains five putative POT genes, we searched the available protein and nucleic acid databases for additional mammalian/human POT genes, using iterative BLAST runs and the human expressed sequence tags (EST) database. The apparent human orthologue of rPHT1 (expression largely confined to rat brain and retina) was represented by numerous ESTs originating from many tissues. Assembly of these ESTs resulted in a contiguous sequence covering ∼95% of the suspected coding region. The contig sequences and analyses revealed the presence of several possible splice variants of hPHT1. A second closely related human EST-contig displayed high identity to a recently cloned mouse cDNA encoding cyclic adenosine monophosphate (cAMP)-inducible 1 protein (gi:4580995). This contig served to identify a PAC clone containing deduced exons and introns of the likely human orthologue (termed hPHT2). Northern analyses with EST clones indicated that hPHT1 is primarily expressed in skeletal muscle and spleen, whereas hPHT2 is found in spleen, placenta, lung, leukocytes, and heart. These results suggest considerable complexity of the human POT gene family, with relevance to the absorption and distribution of cephalosporins and other peptoid drugs.
doi:10.1208/ps020216
PMCID: PMC2751030  PMID: 11741232
10.  Pharmacogenomics: The promise of personalized medicine 
AAPS PharmSci  2000;2(1):29-41.
Pharmacogenetics and pharmacogenomics deal with the genetic basis underlying variable drug response in individual patients. The traditional pharmacogenetic approach relies on studying sequence variations in candidate genes suspected of affecting drug response. On the other hand, pharmacogenomic studies encompass the sum of all genes, i.e., the genome. Numerous genes may play a role in drug response and toxicity, introducing a daunting level of complexity into the search for candidate genes. The high speed and specificity associated with newly emerging genomic technologies enable the search for relevant genes and their variants to include the entire genome. These new technologies have essentially spawned a new discipline, termed pharmacogenomics, which seeks to identify the variant genes affecting the response to drugs in individual patients. Moreover, pharmacogenomic analysis can identify disease susceptibility genes representing potential new drug targets. All of this will lead to novel approaches in drug discovery, an individualized application of drug therapy, and new insights into disease prevention. Current concepts in drug therapy often attempt treatment of large patient populations as groups, irrespective of the potential for individual, genetically-based differences in drug response. In contrast, pharmacogenomics may help focus effective therapy on smaller patient subpopulations which although demonstrating the same disease phenotype are characterized by distinct genetic profiles. Whether and to what extent this individual, genetics-based approach to medicine results in improved, economically feasible therapy remain to be seen.
To exploit these opportunities in genetic medicine, novel technologies will be needed, legal and ethical questions must be clarified, health care professionals must be educated, and the public must be informed about the implications of genetic testing in drug therapy and disease management.
doi:10.1208/ps020104
PMCID: PMC2750999  PMID: 11741220
Pharmacogenomics
11.  Molecular modeling of G-protein coupled receptor kinase 2: Docking and biochemical evaluation of inhibitors 
AAPS PharmSci  2000;2(1):9-21.
G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 μM (pA2 6.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 μM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.
doi:10.1208/ps020102
PMCID: PMC2750997  PMID: 11741218
12.  Genomic structure of proton-coupled oligopeptide transporter hPEPT1 and pH-sensing regulatory splice variant 
AAPS PharmSci  2000;3(1):66-79.
Proton-coupled oligopeptide transporter PEPT1 facilitates the transport of dipeptides and peptoid drugs (including antibiotics) across the cell membranes of endothelial and epithelial cells. Substrate transport by the proton symport is driven by pH gradients, while the profile of pH sensitivity is regulated by a closely related protein. hPEPT1-RF. We investigated the genomic structure of hPEPT1 and hPEPT1-RF. Analysis of the high-throughput genomic sequence (HTGS) database revealed that hPEPT1 and hPEPT1-RF are splice variants encoded by the same gene located in chromosome 13, consisting of 24 exons. hPEPT1 is encoded by 23 exons and hPEPT1-RF by 6 exons. Coding sequences of hPEPT1-RF share 3 exons completely and 2 exons partially with hPEPT1. The genomic organization of hPEPT1 shows high similarity with its mouse orthologue. Exon-intron boundaries occur mostly in the loops connecting transmembrane segments (TMSs), suggesting a modular gene structure reflecting the TMS-loop repeat units in hPEPT1. The putative promoter region of hPEPT1 contains TATA boxes and GC-rich regions and a potential insulin responsive element.
doi:10.1208/ps030106
PMCID: PMC2751238  PMID: 11741257
Peptide transporter; Genomic structure; Splice variant
13.  Pharmacogenomics 
Western Journal of Medicine  1999;171(5-6):328-332.
Images
PMCID: PMC1308751  PMID: 18751198

Results 1-13 (13)