Use of pairwise marker combination and recursive partitioning in a pharmacogenetic genome-wide scan.

The objective of pharmacogenetic research is to identify a genetic marker, or a set of genetic markers, that can predict how a given person will respond to a given medicine. To search for such marker combinations that are predictive of adverse drug events, we have developed and applied two complementary methods to a pharmacogenetic study of the hypersensitivity reaction (HSR) associated with treatment with abacavir, a medicine that is used to treat HIV-infected patients. Our results show that both of these methods can be used to uncover potentially useful predictive marker combinations. The pairwise marker combination method yielded a collection of marker pairs that featured a spectrum of sensitivities and specificities. Recursive partitioning results led to the genetic delineation of multiple risk categories, including those with extremely high and extremely low risk of HSR. These methods can be readily applied in pharmacogenetic candidate gene studies as well as in genome-wide scans.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

A cyclic adenosine 3',5'-monophosphate signal is required for the induction of IL-1 beta by TNF-alpha in human monocytes.

IL-1 beta is a cytokine generally considered to be a major component involved in the pathogenesis of rheumatoid arthritis and other inflammatory diseases. Of the agents found in high concentrations in inflamed rheumatoid arthritis joints, TNF-alpha is among the most strongly implicated as an in vivo inducer of IL-1 beta. Here we report that in human PBMC and in a stable transfectant of the promonocytic cell line, THP-1, TNF-alpha indeed appears to be an inducer of IL-1 beta production, but only in the presence of dibutyryl cAMP or agents such as the PG that elevate intracellular cAMP levels. This TNF-alpha/cAMP pathway regulates IL-1 beta production at the level of transcription and requires a cAMP response element located between -2762 and -2755 bp in the upstream regulatory sequence of IL-1 beta. Because PG, which are known to elevate cAMP levels in vivo, and TNF-alpha are both found in significant quantities in the synovial fluid of rheumatoid arthritis joints, the observed synergistic up-regulation in IL-1 beta synthesis by TNF-alpha/cAMP (PG) may provide valuable insight into the potential pathways involved in the continuous production of IL-1 beta in the chronically inflamed joint.

A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.

Transfection of U937 and THP-1 cells with a recombinant plasmid, pIL1(4.0kb)-CAT, containing 4 kb of the interleukin 1 beta (IL-1 beta) gene upstream regulatory sequence resulted in inducer-dependent expression of chloramphenicol acetyltransferase activity. Treatment of the transfected cells with various combinations of the inducers lipopolysaccharide, phorbol myristate acetate, and dibutyryl cyclic AMP upregulated the IL-1 beta promoter. In U937 and THP-1 cells, maximum stimulation of both the endogenous IL-1 beta gene and pIL1(4.0kb)-CAT transfectants was observed following treatment with the combination of inducing agents lipopolysaccharide-phorbol myristate acetate-dibutyryl cyclic AMP. This combination of inducing agents was used to identify and study, at the molecular level, some of the regulatory elements necessary for induction of the IL-1 beta gene. A series of 5' deletion derivatives of the upstream regulatory sequence were used in transient transfection assays to identify an 80-bp fragment located between -2720 and -2800 bp upstream of the mRNA start site that was required for induction. Exonuclease III mapping, electrophoretic mobility shift assays (EMSA), and DNA sequence analysis of this region were used to identify a transcription factor binding sequence which contained a potential cyclic AMP response element (CRE/ATF)- and NF-kappa B-like binding site. Site-directed mutagenesis of the CRE/ATF-like site resulted in the loss of binding of a specific factor or factors as determined by EMSA. The loss of binding activity directly correlated with a loss of approximately 75% of promoter activity as determined in transient transfection assays. As determined by EMSA, the factor binding to the CRE/ATF-like site was present in nuclear extracts prepared from both uninduced and induced THP-1 and U937 cells. However, the intensity of the band appeared to be increased when nuclear extracts from induced cells were used. In contrast to the CRE/ATF mutation, which resulted in the loss of promoter activity, mutation of the NF-kappa B-like site resulted in a moderate increase in activity in U937 cells. A similar increase in promoter activity was not observed in THP-1 cells. From these studies, we conclude that a CRE/ATF-like site and a factor or factors interacting with this site are essential for the maximum induction of the IL-1 beta gene in stimulated U937 and THP-1 cells.