Genetic regulation of epigenetic processes in mouse: DNA methylation and X chromosome inactivation

Epigenetics is the study of inheritance not encoded by primary DNA sequence. In mammals, epigenetic processes are required for proper development, gene regulation, chromosome function (e.g., X-chromosome inactivation (XCI)), and genome stability. Misregulation of epigenetic processes is typically a hallmark of disease. Epigenetic marks vary depending on genomic position, cell type, environment, time, sex, and even between individuals within a population. Genetic variation is one source of epigenetic variability that has only recently been appreciated. It is unknown how prevalent and to what extent underlying genetic variation influences epigenetic variability, and furthermore, how this epigenetic variability contributes to phenotypic variation within a population. It has been postulated that epigenetic variation between individuals may help solve the `missing heritability' problem. In an attempt to address these questions and further characterize the influence of genetics on epigenetics, I demonstrate that DNA sequence variation in cis affects two epigenetic processes, DNA methylation and XCI. In the first section, I performed a genome-wide allele-specific methylation survey in the mouse brain to show widespread loci that influence nearby DNA methylation at CpGs. These differentially methylated CpGs tend to reside near transcription start sites and may serve a functional role. We estimate that there are roughly 13,000 of these loci genome-wide. Additionally, I show that these strain-specific cis-acting loci also influence a parent-of-origin differentially methylated region in the 3'UTR of the Actn1 gene, which suggests that genetic variation might also influence highly conserved imprinted regions as well. In the second section, I mapped a cis-acting locus called the X-chromosome controlling element (Xce) that influences XCI choice in mouse. I reduced the Xce candidate interval to a 176 kb region located approximately 500 kb proximal to Xist. I extensively characterized the genetic architecture of the new candidate interval in over 300 inbred and wild-caught mice. I conclude that each mouse taxa examined has a different functional Xce allele and there is no sharing. I identified two new Xce alleles (Xcee and (Xcef) that bring the number to six functional alleles in Mus. I propose that structural variation of segmental duplications within this interval explains the presence of multiple functional Xce alleles. Overall these results provide new insights into the genetic regulation of epigenetic processes in mouse. Furthermore, this work creates a foundation for future work to untangle the molecular mechanisms behind differential DNA methylation and X- chromosome inactivation choice.Doctor of Philosoph

Genetic Architecture of Skewed X Inactivation in the Laboratory Mouse

X chromosome inactivation (XCI) is the mammalian mechanism of dosage compensation that balances X-linked gene expression between the sexes. Early during female development, each cell of the embryo proper independently inactivates one of its two parental X-chromosomes. In mice, the choice of which X chromosome is inactivated is affected by the genotype of a cis-acting locus, the X-chromosome controlling element (Xce). Xce has been localized to a 1.9 Mb interval within the X-inactivation center (Xic), yet its molecular identity and mechanism of action remain unknown. We combined genotype and sequence data for mouse stocks with detailed phenotyping of ten inbred strains and with the development of a statistical model that incorporates phenotyping data from multiple sources to disentangle sources of XCI phenotypic variance in natural female populations on X inactivation. We have reduced the Xce candidate 10-fold to a 176 kb region located approximately 500 kb proximal to Xist. We propose that structural variation in this interval explains the presence of multiple functional Xce alleles in the genus Mus. We have identified a new allele, Xcee present in Mus musculus and a possible sixth functional allele in Mus spicilegus. We have also confirmed a parent-of-origin effect on X inactivation choice and provide evidence that maternal inheritance magnifies the skewing associated with strong Xce alleles. Based on the phylogenetic analysis of 155 laboratory strains and wild mice we conclude that Xcea is either a derived allele that arose concurrently with the domestication of fancy mice but prior the derivation of most classical inbred strains or a rare allele in the wild. Furthermore, we have found that despite the presence of multiple haplotypes in the wild Mus musculus domesticus has only one functional Xce allele, Xceb. Lastly, we conclude that each mouse taxa examined has a different functional Xce allele

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes. MATERIALS AND METHODS: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material. RESULTS: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA. CONCLUSIONS: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine

Development of type-specific assays for detecting human papillomavirus in preserved pap smear samples

Specific high-risk types of human papillomavirus (HPV) have been shown to cause cervical carcinomas. The purpose of this MQP was to develop type-specific assays for detecting specific HPV types in preserved pap smear samples. Three kinds of assays were tested: 1) immunofluorescence using type-specific antibodies, 2) RT-PCR to assay episomal (low risk) versus integrated (high-risk) HPV genomes, and 3) genomic PCR using high-risk type-specific primers while simultaneously inhibiting low-risk HPV amplification using PNA probes. Our data indicates that 1) immunofluorescence staining of cancerous cell lines was more effective than specimen cells, 2) RT-PCR of HPV mRNA was effective on HeLa cell lines, and more optimization is needed, 3) PCR amplification of high-risk HPV types could be induced with the devised primer cocktail while minimizing the amplification of low risk HPV types. 4) PNA probes specific for optimal binding to optimal HPV type 11 can inhibit amplification in PCR

AMPHIBROMUS SCABRIVALVIS (GRAMINEAE) IN LOUISIANA

Volume: 11Start Page: 207End Page: 21

The stock market and the worldwide web -- mathematics or gambling?

This project deals with the use of mathematics to model the modern day stock market, and also the influence of online trading on the financial community. With the hopes of better understanding option investing, a computer program was created to calculate the prices of options. The program was developed in Java so that it would be placed online to better educate people in their investing. Lastly, an analysis of how the Internet impacts the market was conducted