Single nucleotide polymorphism (SNP) selection for genotype-phenotype association studies

With hundreds of single nucleotide polymorphisms (SNPs) in a candidate gene and millions of SNPs across the genome, selecting an informative subset of SNPs to maximize the ability to detect genotype-phenotype association is of great interest and importance. In addition, with a large number of SNPs, analytic methods are needed that allow investigators to control the false positive rate resulting from large numbers of SNP genotype-phenotype analyses. This dissertation uses simulated data to explore methods for selecting SNPs for genotype-phenotype association studies. I examined the pattern of linkage disequilibrium (LD) across a candidate gene region and used this pattern to aid in localizing a disease-influencing mutation. The results indicate that the r2 measure of linkage disequilibrium is preferred over the common D′ measure for use in genotype-phenotype association studies. Using step-wise linear regression, the best predictor of the quantitative trait was not usually the single functional mutation. Rather it was a SNP that was in high linkage disequilibrium with the functional mutation. Next, I compared three strategies for selecting SNPs for application to phenotype association studies: based on measures of linkage disequilibrium, based on a measure of haplotype diversity, and random selection. The results demonstrate that SNPs selected based on maximum haplotype diversity are more informative and yield higher power than randomly selected SNPs or SNPs selected based on low pair-wise LD. The data also indicate that for genes with small contribution to the phenotype, it is more prudent for investigators to increase their sample size than to continuously increase the number of SNPs in order to improve statistical power. When typing large numbers of SNPs, researchers are faced with the challenge of utilizing an appropriate statistical method that controls the type I error rate while maintaining adequate power. We show that an empirical genotype based multi-locus global test that uses permutation testing to investigate the null distribution of the maximum test statistic maintains a desired overall type I error rate while not overly sacrificing statistical power. The results also show that when the penetrance model is simple the multi-locus global test does as well or better than the haplotype analysis. However, for more complex models, haplotype analyses offer advantages. The results of this dissertation will be of utility to human geneticists designing large-scale multi-locus genotype-phenotype association studies. ^

Hazardous air pollutants and childhood lymphohematopoietic cancer in Southeast Texas, 1995--2004

Southeast Texas, including Houston, has a large presence of industrial facilities and has been documented to have poorer air quality and significantly higher cancer rates than the remainder of Texas. Given citizens’ concerns in this 4th largest city in the U.S., Mayor Bill White recently partnered with the UT School of Public Health to determine methods to evaluate the health risks of hazardous air pollutants (HAPs). Sexton et al. (2007) published a report that strongly encouraged analytic studies linking these pollutants with health outcomes. In response, we set out to complete the following aims: 1. determine the optimal exposure assessment strategy to assess the association between childhood cancer rates and increased ambient levels of benzene and 1,3-butadiene (in an ecologic setting) and 2. evaluate whether census tracts with the highest levels of benzene or 1,3-butadiene have higher incidence of childhood lymphohematopoietic cancer compared with census tracts with the lowest levels of benzene or 1,3-butadiene, using Poisson regression. The first aim was achieved by evaluating the usefulness of four data sources: geographic information systems (GIS) to identify proximity to point sources of industrial air pollution, industrial emission data from the U.S. EPA’s Toxic Release Inventory (TRI), routine monitoring data from the U.S. EPA Air Quality System (AQS) from 1999-2000 and modeled ambient air levels from the U.S. EPA’s 1999 National Air Toxic Assessment Project (NATA) ASPEN model. Further, once these four data sources were evaluated, we narrowed them down to two: the routine monitoring data from the AQS for the years 1998-2000 and the 1999 U.S. EPA NATA ASPEN modeled data. We applied kriging (spatial interpolation) methodology to the monitoring data and compared the kriged values to the ASPEN modeled data. Our results indicated poor agreement between the two methods. Relative to the U.S. EPA ASPEN modeled estimates, relying on kriging to classify census tracts into exposure groups would have caused a great deal of misclassification. To address the second aim, we additionally obtained childhood lymphohematopoietic cancer data for 1995-2004 from the Texas Cancer Registry. The U.S. EPA ASPEN modeled data were used to estimate ambient levels of benzene and 1,3-butadiene in separate Poisson regression analyses. All data were analyzed at the census tract level. We found that census tracts with the highest benzene levels had elevated rates of all leukemia (rate ratio (RR) = 1.37; 95% confidence interval (CI), 1.05-1.78). Among census tracts with the highest 1,3-butadiene levels, we observed RRs of 1.40 (95% CI, 1.07-1.81) for all leukemia. We detected no associations between benzene or 1,3-butadiene levels and childhood lymphoma incidence. This study is the first to examine this association in Harris and surrounding counties in Texas and is among the first to correlate monitored levels of HAPs with childhood lymphohematopoietic cancer incidence, evaluating several analytic methods in an effort to determine the most appropriate approach to test this association. Despite recognized weakness of ecologic analyses, our analysis suggests an association between childhood leukemia and hazardous air pollution.^

Role played by serum, a biological cue, in the adherence of Enterococcus faecalis to extracellular matrix proteins, collagen, fibrinogen, and fibronectin.

BACKGROUND: Most previous studies have found that Enterococcus faecalis isolates do not show significant adherence to fibronectin and fibrinogen. METHODS: The influence of various conditions on E. faecalis adherence to extracellular matrix (ECM) proteins was evaluated using a radiolabeled-cell adherence assay. RESULTS: Among the conditions studied, growth in 40% horse serum (a biological cue with potential clinical relevance) elicited adherence of all 46 E. faecalis strains tested to fibronectin and fibrinogen but not to elastin; adherence levels were independent of strain source, and adherence was eliminated by treating cells with trypsin. As previously reported, serum also elicited adherence to collagen. Although prolonged exposure to serum during growth was needed for enhancement of adherence to fibrinogen, brief exposure (<5 >min) to serum had an immediate, although partial, enhancing effect on adherence to fibronectin and, to a lesser extent, collagen; pretreatment of bacteria with chloramphenicol did not decrease this enhanced adherence to fibronectin and collagen, indicating that protein synthesis is not required for the latter effect. CONCLUSION: Taken together, these data suggest that serum components may serve (1) as host environmental stimuli to induce the production of ECM protein-binding adhesin(s), as previously seen with collagen adherence, and also (2) as activators of adherence, perhaps by forming bridges between ECM proteins and adhesins.

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^