Indiana University-Purdue University Indianapolis (IUPUI) / Cytochrome P450 2D6 (CYP2D6) is an important drug metabolizing enzyme that is involved in the metabolism of 20-25% of commonly prescribed drugs. There is interindividual variability in CYP2D6 enzyme activity and this leads to compromised metabolism of many drugs. Genetic and environmental factors explain only a part of the interindividual variability; the other factors that contribute to this variability are largely unknown. Hence, it becomes important to study CYP2D6 to understand the endogenous and exogenous factors that control its activity. The specific objective of this research was to determine the contribution of genetic and epigenetic factors in the regulation of CYP2D6 expression and activity. The specific aims were: (1) to identify the common CYP2D6 polymorphisms in Vietnamese and Filipino women with breast cancer and evaluate its association with plasma concentrations of endoxifen (an active metabolite of the breast cancer therapeutic drug, tamoxifen); (2) to identify the CYP2D6 copy number variations (CNVs) in these women and evaluate their association with endoxifen concentration; and (3) to identify microRNAs (miRNAs) that regulate the expression of CYP2D6 directly or indirectly. The results of this study indicated that: (1) in Vietnamese and Filipino women, the reduced function allele CYP2D6*10 was frequent (~55%) and it was significantly associated with reduced endoxifen concentration; (2) in these women, only 39% carried two copies of the CYP2D6 gene, the rest had a genomic imbalance for CYP2D6, primarily involving the CYP2D6(*36)n-*10 allele. However, carrying multiple copies of CYP2D6*36 allele did not significantly affect CYP2D6 activity, suggesting that multiple copies of a gene does not always translate to additive effects; and (3) microRNAs were identified to target HNF4A, a transcriptional factor that regulates CYP2D6 expression. These miRNAs are likely to play an important role in the indirect regulation of CYP2D6. Taken together, these results emphasize on the role of polymorphisms, CNVs and possibly miRNAs in the regulation of CYP2D6. These clinically important biomarkers will help to improve the efficacy and reduce the side effects of many CYP2D6 substrate drugs and thus contribute to personalization of drug therapy.

Mitochondria are essential cellular organelles and the location of the electron transport chain, the site of the majority of energy production in the cell. Mitochondria contain their own circular genome approximately 16,000 base pairs in length. The mitochondrial genome (mtDNA) encodes 11 protein-coding genes essential for the electron transport chain, 22 tRNA genes, and two rRNA genes. Mitochondrial malfunction occurs in many diseases, and changes in the mitochondrial genome lead to numerous disorders. Multiple mitochondrial haplotypes and sequence features are associated with Alzheimer's disease. In this dissertation we utilized TreeScanning, an evolutionary-based haplotype approach to identify haplotypes and sequence variation associated with specific phenotypes: Alzheimer's disease case-control status, mitochondrial copy number, and 16 neuroimaging phenotypes related to Alzheimer's disease neurodegeneration. In the first two studies we utilized 1007 complete mitochondrial genomes from participants in the Cache County Study on Memory Health and Aging. First, individuals with mitochondrial haplotypes H6A1A and H6A1B showed a reduced risk of AD. Our study is the largest to date and the only study with complete mtDNA genome sequence data. Next, each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. Each of these three variants was associated with higher mitochondrial copy number and we suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that lead to changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes. Lastly, we used an endophenotype-based approach to further characterize mitochondrial genetic variation and its relationship to risk markers for Alzheimer's disease. We analyzed longitudinal data from non-demented, mild cognitive impairment, and late onset Alzheimer's disease participants in the Alzheimer's Disease Neuroimaging Initiative with genetic, brain imaging, and behavioral data. Four clades were associated with three different endophenotypes: whole brain volume, percent change in temporal pole thickness, and left hippocampal atrophy over two years. This was the first study of its kind to identify mitochondrial variation associated with brain imaging endophenotypes of Alzheimer's disease. Together, these projects provide evidence of mtDNA involvement in the risk and physiological changes of Alzheimer's disease.

Frequently evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Here, various methods of post PCR purification were evaluated for their effects on the sensitivity of fluophore-based allelic detection. A method of post PCR purification is described which increases the sensitivity of standard 28 cycle PCR such that low copy number DNA templates (<100 pg DNA) can be analyzed. Full profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele drop-out, increased stutter, and contamination (allele drop-in) typical of LCN analysis were observed. A single incident of contamination was observed in a reagent blank (not duplicated upon re-amplification) however, no contamination was observed in negative amplification controls.

Unlike the autosomes, the Y chromosome in humans and other primates has few protein coding genes, with only a few dozen single-copy genes and several tandem duplicated gene families, called the "ampliconic" genes. The interaction of many biological and evolutionary factors is responsible for this structural heterogeneity among different parts of the genome. We sequenced and assayed the copy numbers of Y-linked, single-copy genes and ampliconic genes in a group of closely related macaque monkeys, then fit models of gene family evolution to this data along with whole genome data from human, chimpanzee, and rhesus macaque. Our results (i) recovered evidence for several novel examples of gene conversion in papionin monkeys, (ii) indicate that ampliconic gene families evolve faster than autosomal gene families and than single-copy genes on the Y chromosome, and that (iii) Y-linked singleton and autosomal gene families evolved faster in great apes than they do in other Old World higher Primates. These findings highlight the evolutionary eccentricity of duplicated genes on the Y chromosome and suggest an important role for natural selection and gene conversion in the evolution of Y-linked gene duplicates. / Thesis / Master of Science (MSc)

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