We identified two large French-Canadian (FC) pedigrees with idiopathic partial epilepsy. Family studies of over 500 members from these two families revealed over 63 individuals reported to have seizures or seizure-like histories. Pedigree analysis confirmed an autosomal dominant inheritance with reduced penetrance. Most of the affected individuals shared a number of clinical characteristics compatible with the definition of a novel genetic form of epilepsy syndrome. We termed it as familial partial epilepsy with variable foci (FPEVF), which was recently described by Scheffer et al. (1998) in one Australian family with eight possibly affected individuals. The most prominent clinical feature of FPEVF is the presence of variable seizure foci in different affected individuals within the same family. Most affected individuals in the two FC families present infrequent, brief, non-clustering, nocturnal, partial seizures. Age of onset is between 5 and 25 years. Affected individuals are neurologically intact without any detectable pathological lesion(s). / These two families were then subjected to a full-scale molecular genetic study. No linkage to any known loci for idiopathic partial epilepsy was detected, including the suggested chromosome (Chr) 2q locus for FPEVF in the Australian FPEVF family, the Chr 20q13.2 and 15q24 loci for autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and the Chr 10q locus for autosomal dominant temporal lobe epilepsy with auditory symptoms (ADTLEAS). Significant linkage (LOD score = 8.60) was detected with markers on Chr 22 q12 during a genome scan. Collaborating with a group from Australia, we confirmed another two FC families and two non-FC families with compatible linkage to the same locus and a cumulative LOD score of 14.79 with marker D22S689. The minimum candidate region of FPEVF has been finally defined to an interval of 3.8 cM between markers D22S 1163 and D22S 1686 on Chr 22q12. We proceeded to search for the gene mutation by directly sequencing the coding regions of all candidate genes within the FPEVF region, an interval of 5.04 Mb harboring approximately 100 known or predicted genes. So far, 77 candidate genes have been sequenced, and no mutation has yet been found. Since there is no known ion channel gene mapped to this region, FPEVF is probably the first non-ion channel gene causing human idiopathic epilepsy*. / *After submission of this thesis for examination, Kalachikov et al. (2002) identified LGI1 (leucine-rich gene, glioma inactivated), a possible tumor suppressor, as the gene for ADTLEAS. This is the first evidence for a non-ion channel gene causing idiopathic epilepsy in humans.

Results indicate that U9-IIIB and PLB-IIIB cells stimulated with PMA, LPS or recombinant cytokines transcribed and translated significantly higher levels of TNF-$ alpha$, IL-1$ alpha$, IFN-$ alpha$, IL-1$ beta$, and IFN-$ beta$. Furthermore it appears that HIV infection of PLB-985 cells induces monocytic differentiation and alterations in the binding of NF-$ kappa$B related proteins. PLB-IIIB cells demonstrated characteristics reflecting differentiation including morphological alterations, changes in c-fms and c-myc proto-oncogene expression, and increased expression of CD14 cell surface antigen. Examination of the IL-1$ beta$ promoter revealed a DNA sequence capable of interacting with the NF-$ kappa$B family of proteins; oligonucleotides containing 1-3 copies of this IL-1$ beta$-$ kappa$B sequence were synthesized and shown to bind NF-$ kappa$B proteins from LPS and PMA treated U937 and U9-IIIB cells. Recombinant NF-$ kappa$B protein subunits specifically bound monomeric and dimeric copies of this IL-1$ beta$-$ kappa$B sequence and binding was eliminated by competition with unlabeled wild type but not mutant oligonucleotides. Results indicate that IL-1$ beta$ gene expression may be significantly elevated in HIV infected cells and that transcriptional regulation by NF-$ kappa rm B /{ it rel /}$ proteins may mediate its activation.

The avian reticuloendothelial virus strain T (REV-T) is among the most overtly transforming of all known retroviruses, and has been shown to transform a wide range of lymphocytes. The transforming ability of REV-T is attributed the v-rel oncogene, a member of the Rel/NFkappaB family of transcription factors. Although it is believed that v- rel mediated transformation involves the disregulation of normal NF-kappaB function, the intracellular requirements for transformation are still unknown. / We have used the RCAS vectors (R&barbelow;eplication C&barbelow;ompetent A&barbelow;vian Leukosis LTR with S&barbelow;plice Acceptor) for infection and dissemination of viral particles in chicken cells, permitting somatic transgenesis of the v-rel oncogene in vivo. In addition, the RCAS system has been combined with a tetracycline regulated gene expression system, in order to allow for conditional expression of the v- rel oncogene in transgenic birds. / Using this novel system, we have been able to study in situ transformation by the v-rel oncogene. By addressing the questions pertaining to the cells targeted for transformation by v-rel, we hoped to gain a better understanding of the mechanism for v-rel mediated transformation. Preliminary data suggests that B cells were targeted for transformation by v-rel in the transgenic birds. Phenotypic analysis revealed that transformed cells were not representative of all stages of B cell development, but rather had a mature B cell phenotype.

<p> This thesis focuses on methodologies and applications of module-based analysis (MBA) in omics studies to investigate the relationships of phenotypes and biomarkers, e.g., SNPs, genes, and metabolites. As an alternative to traditional single&ndash;biomarker approaches, MBA may increase the detectability and reproducibility of results because biomarkers tend to have moderate individual effects but significant aggregate effect; it may improve the interpretability of findings and facilitate the construction of follow-up biological hypotheses because MBA assesses biomarker effects in a functional context, e.g., pathways and biological processes. Finally, for exploratory &ldquo;omics&rdquo; studies, which usually begin with a full scan of a long list of candidate biomarkers, MBA provides a natural way to reduce the total number of tests, and hence relax the multiple-testing burdens and improve power.</p><p> The first MBA project focuses on genetic association analysis that assesses the main and interaction effects for sets of genetic (G) and environmental (E) factors rather than for individual factors. We develop a kernel machine regression approach to evaluate the complete effect profile (i.e., the G, E, and G-by-E interaction effects separately or in combination) and construct a kernel function for the Gene-Environmental (GE) interaction directly from the genetic kernel and the environmental kernel. We use simulation studies and real data applications to show improved performance of the Kernel Machine (KM) regression method over the commonly adapted PC regression methods across a wide range of scenarios. The largest gain in power occurs when the underlying effect structure is involved complex GE interactions, suggesting that the proposed method could be a useful and powerful tool for performing exploratory or confirmatory analyses in GxE-GWAS.</p><p> In the second MBA project, we extend the kernel machine framework developed in the first project to model biomarkers with network structure. Network summarizes the functional interplay among biological units; incorporating network information can more precisely model the biological effects, enhance the ability to detect true signals, and facilitate our understanding of the underlying biological mechanisms. In the work, we develop two kernel functions to capture different network structure information. Through simulations and metabolomics study, we show that the proposed network-based methods can have markedly improved power over the approaches ignoring network information.</p><p> Metabolites are the end products of cellular processes and reflect the ultimate responses of biology system to genetic variations or environment exposures. Because of the unique properties of metabolites, pharmcometabolomics aims to understand the underlying signatures that contribute to individual variations in drug responses and identify biomarkers that can be helpful to response predictions. To facilitate mining pharmcometabolomic data, we establish an MBA pipeline that has great practical value in detection and interpretation of signatures, which may potentially indicate a functional basis for the drug response. We illustrate the utilities of the pipeline by investigating two scientific questions in aspirin study: (1) which metabolites changes can be attributed to aspirin intake, and (2) what are the metabolic signatures that can be helpful in predicting aspirin resistance. Results show that the MBA pipeline enables us to identify metabolic signatures that are not found in preliminary single-metabolites analysis.</p>

<p>Dover sole (<i>Microstomus pacificus</i>) is a commercially important, slope dwelling flatfish of the northeast Pacific coast. Its genetic diversity at the mitochondrial DNA control region appears substantially lower than another commercially important flatfish, Pacific sanddab (<i>Citharichthys sordidus</i>). I designed a comparative study along depth and latitudinal gradients using five flatfishes and one brotula. In the control region's left domain, genetic diversity of six species trended lower with increasing habitat depth at Palos Verdes: shallow species had high genetic diversity and deep dwelling species (ex. Dover sole) had low genetic diversity. This diversity gradient may follow decreases in mass specific metabolic rates as Dover sole grow, invade the oxygen minimum zone and assume higher tissue water content. The left domain from 64 Dover sole specimens was compared across 4 latitudinal locations. Genetic diversity trended higher with increasing latitude, possibly due to cold water emergence as biomass shift shallower with increasing latitude. </p>

Tumors develop and progress as a result of alterations in oncogene and tumor suppressor genes. Knudson's two hit model predicts that cancer arises when both alleles of a tumor suppressor gene are inactivated. The insulin-like growth factor 2 receptor gene (IGF2R) is known to be a tumor suppressor for breast cancer, as mutations inactivating both alleles have been found in these tumors, consistent with its known functions in degrading insulin-like growth factor 2 (IGF2) and activating transforming growth factor beta1 (TGF-beta1), a growth inhibitor. Tumor suppressors can be inactivated by several means, such as genomic imprinting (a preferential silencing of a particular parental chromosome), large deletions resulting in loss of heterozygosity (LOH), and point mutations. In both humans and mice, IGF2R has gamete-of-origin dependent methylation which results in exclusive maternal expression in mice, while in humans imprinted expression is seen only in rare individuals and could be a predisposing factor for cancer. Since it is known that LOH plays a role in the genesis of breast cancer, we hypothesized that in some cases the event inactivating the other gene copy is parental imprinting. Our hypothesis predicted that this LOH is preferentially maternal and, therefore, the remaining allele is unmethylated. We tested this prediction in 20 breast cancer samples, thirteen of which (65%) were heterozygous and 9/13 (69%) had LOH. Two of 9 tumors (~22%) showed complete lack of methylation, consistent with LOH of the maternal allele. However, surprisingly and potentially significantly, normal tissue from the breast cancer patients was completely methylated in 4 samples and completely unmethylated in 2. This suggests that IGF2R methylation disruption may be a pre-existing, cancer-predisposing molecular lesion in the breast of these patients. More work is needed to exclude technical artifacts in reaching this conclusion.

Mitochondrial background. Recent studies on cancer have detected many mutations and much variability in the mitochondrial genome, particularly in the non-coding region (D-loop). The present study set out to sequence and examine the hypervariable region I (HVR-I) of the D-loop and transfer RNA Leucine (tRNA Leu) for mutations in breast cancer patients. Methods. Tumor and normal tissues from 17 patients that carry mutations in either BRCA1 (n = 11), BRCA2 (n = 3) or are non-carriers of mutations in either gene (n = 3) were examined by direct genomic sequencing of PCR products. Results. We found 44 variants in the HVR-I of 16 patients, twenty-six were polymorphisms, four somatic variants, and fourteen variations were undetermined because corresponding (unaffected) normal tissue was not available. One BRCA1 mutated tumor had four somatic tumor variants (1/14). All other BRCA1/BRCA2 mutated tumors had no somatic variants. Nine out of 14 (64%) of these patients had a total 22 germline variants. One out of three (33%) non-carriers had four germ-line variants. No variants were found in tRNA Leu. A five kilobase deletion was also found as a germ-line variant in two of seven (29%) patients. There were no obvious differences in the frequency of homoplasmic variants in the mitochondrial genome between the BRCA1/BRCA2 mutation carriers and non-carriers. Conclusion. Direct genomic sequencing of PCR products showed that there were no striking differences in homoplasmic variants between tumor and normal tissue, thus homoplasmic variants in mtDNA did not have a role in tumorigenesis in our samples. We speculate that the marked differences in mutation frequencies observed amongst various studies could be the result of differences in the techniques used to generate and analyze the data. / EGFR background. Recent studies have identified mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) in lung carcinomas. These mutations make the tumors sensitive to a molecular targeted drug called getifinib. Methods. We also sequenced the TK domain of EGFR in 16 breast cancer patients (BRCA1 = 9, BRCA2 = 4, non-carriers = 3) for mutations. Results. We did not find mutations reported previously in lung carcinomas but we identified other variants, in exons 18, 20 and 21 of the mutation carvers. We found one out of thirteen (eight percent) of the BRCA1/BRCA2 mutation carriers had somatic tumor variants, three out of thirteen (23%) patients had somatic variants found only in the adjacent normal tissue, and four out of thirteen (31%) patients had germ-line variants. The non-carriers did not have any variants. The variants found in the exons were two missense variants in exon 18 of two patients, three 'silent' substitutions in exon 20 of three patients, and two patients had exon 21 variants; a missense variant and a 'silent' substitution. Intronic variants were also found in three patients. Patient 5420 harbored more than one variant in the tumor tissue and patient 5483 harbored more than one somatic variant in the adjacent normal tissue. Although the sample size is small, these preliminary results seem to show a difference in EGFR variants between BRCA1/BRCA2 mutation carriers and non-carriers. Conclusion . EGFR variants found in this study were not the same ones found in lung cancer, but other variants could be significant in breast cancer progression and could possibly represent drug targets for future therapy.

To further our understanding of the complex pathophysiology of human sickle cell disease (SCD) and to develop better therapies, three transgenic mouse lines have been produced that survive on mostly human hemoglobin (Hb). These lines have been generated by crossing HbSAD (SAD) transgenic mice with other mice carrying a null deletion of the endogenous globin genes (alpha or beta), SAD/homozygous alpha-globin null (SADalpha -/-) mice display a SCD-like phenotype and have a dramatically reduced mean lifespan; therefore, this line provides a novel model for assessment of anti-sickling protocols or identifying epistatic modifiers genes. Intriguingly, SAD/heterozygous alpha-globin null (SADalpha+/-) mice have a mild phenotype compared to SAD mice. In stark contrast, the highest cellular HbSAD fraction and degree of in vivo sickling was obtained in SAD/compound heterozygous alpha- and beta-globin null (SADalpha +/-beta+/-) mice. These SADalpha +/-beta+/- mice display chronic anemia and delayed growth indicative of a severe phenotype, similar to the human HbSS phenotype.

<p> Human development and health involves the complex and coordinated regulation of gene expression across diverse tissues. Gene regulation is therefore an essential process in human biology. In the field of human genetics, this has only become more apparent as genomic technologies have made genome-wide surveys of genetic variation underlying human traits possible. In my thesis work, I studied the impact of variation in gene regulation on human traits from three distinct perspectives of human genetics. I first examined the contribution of gene regulation to human disease susceptibility by combining gene expression data with a genome-wide association study to identify novel asthma susceptibility candidate genes. I then studied the effects of depleting specific transcription factors from the cell on downstream gene expression by incorporating gene expression data (following cellular depletion of those factors) with genomic transcription factor binding data. Finally, I considered the role of gene regulation in human evolution by integrating RNA-seq data collected in human, chimpanzee, and rhesus macaque lymphoblastoid cell lines with promoter reporter assays conducted in the same lines. Throughout this work, I have synthesized multiple genomic data sets and multiple distinct sub-disciplines of human genetics in order to arrive at a unified view of the role of gene regulation in determining human traits.</p>

Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme in the metabolism of folate and methionine. MTHFR catalyzes the conversion of 5,10 methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methyltetrahydrofolate (5-methylTHF). 5-MethylTHF is a co-substrate for homocysteine remethylation to methionine catalyzed by the vitamin B12-dependent enzyme methionine synthase. A common MTHFR variant, 677C &rarr; T substitution resulting in the conversion of an alanine to a valine residue, has been shown to be a thermolabile form of MTHFR and to have reduced activity (Frosst et al., 1995). Since many diverse cancer cells have been documented to be methionine-dependent in culture, the effects of MTHFR downregulation on cell survival of transformed cells was examined. In addition, the influence of the MTHFR 677C &rarr; T polymorphism on the sensitivity of transformed lines to antifolate drug treatment was studied. / To test the effect of decreased MTHFR expression on cell viability, we transfected antisense oligonucleotides (ASOs) complementary to the MTHFR mRNA into the colon carcinoma cell line SW620. (Abstract shortened by UMI.)