David P. Yarnall

A single-nucleotide polymorphism tagging set for human drug metabolism and transport

Interindividual variability in drug response, ranging from no therapeutic benefit to life-threatening adverse reactions, is influenced by variation in genes that control the absorption, distribution, metabolism and excretion of drugs. We genotyped 904 single-nucleotide polymorphisms (SNPs) from 55 such genes in two population samples (European and Japanese) and identified a set of tagging SNPs that represents the common variation in these genes, both known and unknown. Extensive empirical evaluations, including a direct assessment of association with candidate functional SNPs in a new, larger population sample, validated the performance of these tagging SNPs and confirmed their utility for linkage-disequilibrium mapping in pharmacogenetics. The analyses also suggest that rare variation is not amenable to tagging strategies.

Elevated levels of TNF in the joints of adjuvant arthritic rats

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians.

In order to characterize the endogenous gene product for rad (ras‐related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109–121, 178–195, 254–271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP‐binding activity was inhibited by the addition of N‐ethylmaliemide, GTP, GTPγS, and GDPβS but not ATP or dithiothreitol. Moreover, cytosol‐dependent rad‐GTPase activity was stimulated by the peptide corresponding to amino acids 109–121. Antibodies corresponding to this epitope inhibited cytosol‐dependent rad‐GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP‐binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109–121) may be involved in regulating rad‐GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis. J. Cell. Biochem. 65:527–541.

Linkage Analysis Using Single Nucleotide Polymorphisms

We performed multipoint linkage analysis using 83 markers from the SNP Consortium (TSC) SNP linkage map in 3 regions covering 190 cM previously scanned with microsatellite markers and found to be linked to type 2 diabetes. Since the average linkage disequilibrium present in the TSC SNP marker clusters is relatively low, we assumed the intracluster genetic distances were a reasonable small nonzero distance (0.03 cM) and performed linkage analysis using GENEHUNTER PLUS and ASM linkage analysis software. We found that for the pedigree structures and missing data patterns in our samples the average information content in all three regions and the LOD score curves in two regions obtained from the TSC SNP markers were similar to results obtained from microsatellite marker maps with 10 cM average spacing. We also give an algorithm which extends the Lander-Green algorithm to permit multipoint linkage analysis of clusters of tightly linked markers with arbitrarily high levels of intracluster linkage disequilibrium.

Single Nucleotide Polymorphism Analysis by ZipCode-Tagged Microspheres

Publisher Summary Microsphere-based platforms offer a high level of multiplexing capability and provide an “open” system in which reactions can be modified easily by combining different sets of reagent-bearing microspheres. With the completion of a representative human genomic sequence, emphasis is increasingly shifting toward rapid, cost-effective, high-throughput analysis of individual sequence variation. Although a portion of this variation occurs as nucleotide insertions and deletions, substitutions in the form of single nucleotide polymorphisms (SNPs) are the most common. Occasionally, a capture probe will consistently produce reaction products that display low signal strength or high backgrounds, resulting in poor signal-to-noise ratios. It is sometimes possible to correct this problem by redesigning the capture probe from the opposite genomic strand. Effective removal of unincorporated biotinylated dideoxynucleotides during the wash step can be critical to maximize signal-to-noise ratios. Microspheres can be photobleached by ambient laboratory light, causing them to lose their spectral identities. Because this is a cumulative process, long-term microsphere stocks, such as bulk carboxylated microspheres and oligonucleotide-coupled stocks, should be rigorously protected by wrapping in aluminum foil.