Aims: The p53 regulatory network is crucial in directing the suppression of cancer formation and mediating the response to commonly used cancer therapies. Functional genetic variants in the genes comprising this network could help identify individuals at greater risk for cancer and patients with poorer responses to therapies, but few such variants have been identified as yet. Methods: We first develop and apply three different screens that utilize known characteristics of functional single nucleotide polymorphisms (SNPs) in the p53 network to search for variants that associate with allelic differences in (i) recent natural selection, (ii) chemosensitivity profiles, and (iii) the gender- and age- dependent incidence of soft-tissue sarcoma. Secondly, we study and explore the functional mechanisms associated with the identified variants. Results: We identify SNPs in the PPP2R5E, CD44, YWHAQ and ESR1 genes that associate with allelic differences in the age of tumour diagnosis (up to 32.5 years, p=0.031), cancer risk (up to 8.1 odds ratio, p=0.004) and overall survival (up to 2.85 relative risk, p=0.011) in sarcomas, ovarian and pancreatic cancers, and exhibit allelic differences in the cellular responses to cytotoxic chemotherapeutic agents (up to 5.4-fold, p=5.6x10^-47). Lastly, we identify candidate causal SNPs in those genes and describe the regulatory mechanisms by which they might affect human cancer. Conclusions: Together, our work suggests that the inherited genetics of the p53 pathway have a great potential to further define populations in their abilities to react to stress, suppress tumor formation and respond to therapies.

Abstract FUNCTIONAL AND BIOCHEMICAL CONSEQUENCES OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE HUMAN VESICULAR MONOAMINE TRANSPORTER 1 GENE (SLC18A1) By Sally Gamal Shukry, B.S. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology at Virginia Commonwealth University. Virginia Commonwealth University, 2012 Major Advisor: Jennifer K. Stewart Associate Professor and Graduate Director, Department of Biology Single nucleotide polymorphisms (SNP) in the human VMAT1 gene (SLC18A1) have been associated with schizophrenia in three different populations: Han Chinese, Western European and Japanese. Effects of these mutations on transport function of the hVMAT1 protein have not been reported. The goal of this study was to investigate functional and biochemical differences in human VMAT1 proteins with a threonine or proline at amino acid position 4 (Thr4Pro) and a serine or threonine at position 98 (Ser98Thr). COS1 cells were transfected with variant SNPs coding for 4Thr/98Ser, 4Pro/98Ser, or 4Thr98Thr. Western blotting demonstrated robust over expression of the genes and no differences in electrophoretic mobility of the proteins. Maximal transport of serotonin by the VMAT1 protein with 4Pro/98Ser was less than that of the 4Thr/98Ser or the 4Thr/98Thr. Response of the 4Pro/Ser98 to the VMAT inhibitor reserpine was lower than that of the 4Thr/98Thr. These findings suggest mechanisms for human VMAT1 links to schizophrenia.

No description available.

The relatively low cost of Next-Generation Sequencing (NGS) has enabled researchers to generate large amounts of sequencing data in order to identify disease-causing mutations and to assemble simple genomes. However, NGS has inherent limitations due to the short DNA read lengths and high error rate associated with the technique. The short read lengths of NGS prevent the assembly of genomes with long stretches of repetitive DNA, and the high error rate prevents the accurate detection of rare mutations in heterogeneous populations such as tumors and microbiomes. I have co-developed new NGS methods to overcome these challenges. In order to increase the effective read length of NGS reads, local de novo assembly of short reads into long contigs can be achieved through the use of Paired-End Restriction-site Associated DNA Sequencing (RAD-PE-Seq). With the RAD-PE method, I sequenced a stickleback fosmid and generated contigs with an N50 length of 480 nucleotides. In order to eliminate false-positive mutations caused by the high error rate of NGS, the Paired-End Low Error Sequencing (PELE-Seq) method was developed, which uses numerous quality control measures during the sequencing library preparation and data analysis steps in order to effectively eliminate sequencing errors. Control testing of the PELE-Seq demonstrates that the method completely eliminates false-positive mutations at sequencing read depths below 20,000X coverage, compared to a ~20% false-positive rate obtained with previous methods. The high accuracy of the PELE-Seq method allows for the detection of ultra-rare mutations in a genome, which was previously impossible with NGS. This dissertation includes previously published and unpublished co-authored material.

Em um cenário em que a qualidade da informação é essencial para a sustentação do processo de gestão do sistema ou do processo de seleção animal, corretas atribuições de paternidade se tornam importante para a manutenção das informações de pedigree e, consequentemente, para a estimativa dos valores genéticos dos animais em programas de seleção. Com o advento do uso de marcadores moleculares do tipo SNP na seleção genômica e em diferentes painéis de teste de parentesco, maiores estudos se fazem cada vez mais necessários. Dessa maneira esse estudo buscou analisar os ganhos em acurácia dos valores genéticos para características produtivas em um cenário de seleção real entre avaliações genéticas que continham ou não animais com atribuições de paternidade por teste de DNA e além disso, verificar os possíveis conflitos de seleção entre elas. Como resultados foram encontrados ganhos em acurácia principalmente em animais mais jovens, mas de maneira geral todos os animais foram beneficiados e apresentaram ganhos acima de 8% os animais diretamente submetidos ao teste. Conflitos de seleção variaram entre 14 a 52% quando considerada somatória das diferenças de seleção nas duas avaliações genéticas, mostrando também, que existiu uma alteração na ordem de classificação dos animais nas características analisadas. Como o fator custo é um ponto importante quando se fala em avaliação genética e tudo que envolve a nova fase dos marcadores moleculares, um painel de paternidade eficiente é aquele que apresenta o melhor desempenho na determinação da paternidade sob um menor custo. Dessa forma, foram comparados alguns grupos de marcadores e o painel de 195 SNP proposto pelo grupo ISAG se mostrou eficiente, assim como outros, na determinação de paternidade em um grupo especifico de animais. Os resultados deste trabalho indicam também que o teste de parentesco por DNA é uma ferramenta que pode aumentar a precisão do arquivo pedigree por aumentar a conexão dos animais, ou seja, por criar maiores ou mais laços genéticos entre os indivíduos. Assim, poder-se-ia melhorar o desempenho da avaliação genética em um programa de melhoramento genético bovino através de pequenos grupos de marcadores moleculares com um custo-benefício atrativo. / In a scenario where information quality is essential to support a process management system or an animal selection process, correct paternity assignments become important for maintaining pedigree information and hence to estimate animals genetic values in breeding programs. With the advent of the use of SNP molecular markers in genomic selection and different kinship test panels, larger studies are increasingly necessary. Thus, this study investigated the gains in accuracy of breeding values for production traits (in a real selection scenario) between the genetic evaluations of animals with or without parenting assignment by DNA testing. It also verified possible selection conflicts between them. The following results showed gains in accuracy, especially in younger animals. However, in general, all animals benefited having gains greater than 8% where animals were directly impacted. Checking conflicts ranged from 14-52% when the sum of the differences of selection in both genetic evaluations was considered. This also showed that there was a change in the ranking of animals in the analyzed characteristics. As cost is also an important factor in genetic evaluations using new molecular markers, an effective parenting panel would be one that had the best performance in the paternity determination under a lower cost. Therefore, we compared some small marker groups and the 195 SNP panel proposed by ISAG group and determined this was efficient in estimating paternity on a specific group of animals. These results also suggest that kinship testing by DNA is a tool that can increase the accuracy of the pedigree file to increase the animals\' connection, or to create larger or more genetic links between individuals. This study shows that testing with small groups of molecular markers improves the performance of genetic evaluation in a bovine genetic selection program with an attractive cost-benefit.

Thesis advisor: Gabor T. Marth / Thesis advisor: Eric F. Tsung / My work is part of an effort in Dr. Gabor Marth's population genetics lab to extend the work of Marth's 2004 Genetics paper "The allele frequency spectrum in genome-wide human variation data reveals signals of differential demographic history in three large world populations" by applying its methods to new datasets. My contribution toward this end has been to create computer code (in Perl and Bash) to quantify ascertainment bias and determine proxy ancestral alleles in human genome-wide polymorphic data for post-doctoral fellow Dr. Eric Tsung's use in the determination of human demographic history. The final results of my efforts will be part of a poster by Dr. Tsung (with myself as a second author) displayed at the 2007 Biology of Genomes Symposium at Cold Spring Harbor Laboratory in Cold Spring Harbor, New York. Our goal is to turn that poster into a paper (on which I will be an author) for submission for publication in a major scientific research periodical and which will also be available in the future at http://bioinformatics.bc.edu/marthlab/ascertainmentancestral/. / Thesis (BS) — Boston College, 2007. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Orientador: Flávia Lombardi Lopes / Resumo: O melhoramento genético em bovinos visa a seleção de características para facilitar o manejo, a qualidade da carne, a resistência a doenças e a adaptação ao meio ambiente. Polimorfismos de nucleotídeo único (SNPs) podem gerar grandes efeitos sobre essas características fenotípicas. Os microRNAs são pequenos RNAs não-codificadores que atuam como reguladores da expressão pós-transcricional através de sua ligação a mRNAs alvo. No presente estudo, realizamos o cruzamento de dados entre ~56 milhões de SNPs contra todas as seqüências conhecidas de miRNA bovino e analisamos in silico, seus possíveis efeitos. Seguindo a predição dos alvos, mostramos que 82% dos alvos foram alterados como consequência dos SNPs que ocorrem na região de seed de miRNAs maduros. Em seguida, identificamos variações na Energia Livre Mínima (MFE) que representam a capacidade de alterar a estabilidade das moléculas e, consequentemente, a maturação dos miRNAs. Também encontramos 129 SNPs em miRNAs, que alteraram sua predição com alvos, ocorrendo em regiões de QTL e, por último, a análise dos escores de conservação evolutiva para cada locus de SNP sugeriu que eles têm uma função biológica conservada através do processo evolutivo. Nossos resultados sugerem que os SNPs em microRNAs têm o potencial de alterar os fenótipos bovinos e são de grande valor para a pesquisa de melhoramento genético, bem como para a produção. / Abstract: Genetic improvement of cattle is aimed at selection of characteristics to facilitate the handling, quality of the meat, resistance to diseases and adaptation to the environment. Single nucleotide polymorphisms (SNPs) can generate large effects on these phenotypic characteristics. MicroRNAs are small non-coding RNAs that act as regulators of posttranscriptional expression through their binding to target mRNAs. In the present study, we scanned ~56 million SNPs against all known bovine miRNA sequences and analyzed in silico, their possible effects. Following target prediction, we show that 82% of targets were altered as a consequence of SNPs that occur in the seed region of mature miRNAs. Next, we identified variations in the Minimum Free Energy (MFE) which represent the capacity to alter molecule stability and, consequently, the maturation of the miRNAs. We have also found 129 SNPs in miRNAs, with altered target prediction, occurring in QTL regions and, lastly, analysis of evolutionary conservation scores for each SNP locus suggested that they have a conserved biological function through the evolutionary process. Our results suggest that SNPs in microRNAs have the potential to alter bovine phenotypes and are of great value for genetic improvement research, as well as production. / Mestre

Elevated concentrations of arsenic in groundwater pose a public health threat to millions of people worldwide. While arsenic is an established human carcinogen, a mode of action has yet to be determined for arsenic carcinogenesis. However, the oxidative stress and DNA repair pathways have been implicated in arsenic toxicity and have been hypothesized to underlie arsenic carcinogenesis. To date, few epidemiologic studies have evaluated genetic susceptibility to arsenical skin lesions based on single nucleotide polymorphisms (SNPs) in antioxidant enzyme or DNA repair genes. Utilizing cross-sectional data from the 2000-2002 survey of the Health Effects of Arsenic Longitudinal Study (HEALS) for 610 prevalent arsenical skin lesion cases and 1,079 randomly selected controls, I evaluated the associations of SNPs in genes encoding antioxidant enzymes and DNA repair enzymes on skin lesion prevalence. I also evaluated potential interactions between the SNPS as well as SNP-environment interactions in determining skin lesion prevalence. In the first study of this dissertation (Chapter 2), I assessed the relationship between SNPs in antioxidant enzyme genes and skin lesion prevalence, as well as possible interactions of these associations on the additive scale by various environmental factors. There were no statistically significant associations between these SNPs (SOD2, rs4880; CAT, rs1001179; GPX1, rs1050450; and MPO, rs2333227) and skin lesion prevalence. Additionally, there was no evidence of additive interaction by arsenic exposure levels, body mass index, smoking status, or fruit and vegetable intake with the SNPs in relation to skin lesion prevalence. However, there was marginal evidence that skin lesion prevalence was increased among individuals who carried 4 or more risk alleles compared to individuals carrying 0-3 risk alleles in these SNPs. Additionally, I observed a significant departure from additivity for the risk allele score and primary methylation index on skin lesion prevalence. In the second study of this dissertation (Chapter 3), I assessed the relationship between SNPs in DNA repair genes (OGG1, rs1052133; XRCC1, rs25487 and rs1799782; XRCC3, rs861539; ERCC2, rs1052559; ERCC5, rs17655; and LIG4, rs1805388) and skin lesion prevalence, as well as possible interactions of these associations on the additive scale by various environmental factors. In logistic regression models controlling for sex, age, and well water arsenic concentration, no associations were observed between measured SNPs and skin lesion prevalence. The results did not vary by arsenic exposure levels, body mass index, or smoking status. However, I did observe a significant inverse association of total fruit and vegetable consumption with skin lesion prevalence, and its additive interaction with the polymorphism in ERCC5. In the third study of this dissertation (Chapter 4), I utilized a multi-analytic approach to explore gene-gene, gene-environment, and higher-order interactions among 10 SNPs related to the oxidative stress and DNA repair pathways by MDR, CART, and logistic regression models. As shown in Chapters 2 and 3, none of these SNPs were associated with skin lesion prevalence, however, were evaluated for potential SNP-SNP interactions. MDR and CART modeling approaches were utilized for the selection of potential gene-gene and gene-environment interactions. Considerable overlap of the interactions detected by both these methods was observed, which were further evaluated by logistic regression. Results from logistic regression modeling, provided some evidence of these statistical interactions; however, their biological interpretation was limited. In summary, there was marginal evidence that skin lesion prevalence was increased among individuals who carried 4 or more risk alleles in genotyped SNPs related to the oxidative stress pathway compared to individuals carrying 0-3 risk alleles in these SNPs and, a significant departure from additivity was observed for the risk allele score and primary methylation index on skin lesion prevalence. Additionally, a significant inverse association of total fruit and vegetable consumption with skin lesion prevalence was observed and, a significant interaction between the polymorphism in ERCC5 and total fruit and vegetable intake was observed in relation to skin lesion prevalence on the additive scale. However, these finding require replication in other studies.

Tracking seed dispersal using traditional, direct measurement approaches is difficult and generally underestimates dispersal distances. Variation in chloroplast haplotypes (cpDNA) offers a way to trace past seed dispersal and to make inferences about factors contributing to present patterns of dispersal. Although cpDNA generally has low levels of intraspecific variation, this can be overcome by assaying the whole chloroplast genome. Whole-genome sequencing is more expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among SNPs are lost in pooled samples and treating SNP frequencies as independent estimates of variation provides biased estimates of genetic distance. I have developed an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP frequencies from pooled samples based on network topology, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially-constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from Whetstone Prairie, in Southern Oregon, USA. In test networks, CallHap reliably recovered network topologies and haplotype frequencies. Overall, the CallHap pipeline allows for the efficient use of resources for estimation of genetic distance for studies using non-recombining, whole-genome haplotypes, such as intra-specific variation in chloroplast, mitochondrial, bacterial, or viral DNA.

<p>This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer.</p>