Academisch proefschrift--Utrecht. / Bibliography: p. [257]-275.

Inaug.-Diss.--Bonn. / At head of title: Aus der Dr. Hertz'schen kuranstalt, Bonn ... Lebenslauf. "Literatur": p. 26.

Thése--Universit́e de Lausanne. / Bibliography: p. [243]-247.

Thesis (Ph.D.)--University of Iowa, 2006. / Supervisors: Todd E. Scheetz, Thomas L. Casavant. Includes bibliographical references (leaves 88-96).

A number of methodological considerations have been discussed in the area of economic evaluation of Clinical Genetic Services (CGSs) including the limited knowledge of psychosocial consequences of these services. This study aims to address this gap by identifying tangible and intangible benefits and disbenefits of CGSs and presenting these within a framework to assist in the design of a comprehensive welfarist economic evaluation. Mixed methods of data collection were adopted and a UK medical genetics service was used as a case study. Face-to-face interviews with genetic service providers were undertaken in Phase One to explore patient pathways and the perceived role of the service. Focus groups and face-to-face interviews with service users explored the perceived benefits and disbenefits of the CGS in Phase Two. Phase Three comprised a pilot study of using Audience Response Systems (ARSs) for exploring stakeholder preferences and tackled issues of respondent validation and transferability. Both process-related attributes and psychosocial outcomes emerged as utility-bearing for service users. Patient pathways i.e. patient experience, were found to be influenced by factors associated with the genetic condition and with individual patient/family characteristics and needs. The overall (dis)benefits of the service however were found to be common across conditions. The concept of Perceived Familial Control is proposed as a suitable outcome which encompasses the psychosocial dimension of CGSs. This study has demonstrated the use of qualitative methods in the context of health economics and economic evaluation. It has specifically demonstrated the use of various levels of qualitative analysis for obtaining attributes and outcomes of CGSs and has incorporated these within a framework directed towards the design of a welfarist economic evaluation. The use of ARSs was also tested for their usefulness as a method of establishing preferences and exploring the opinions of CGS stakeholders. Further research is required to establish whether the emerging (dis)benefits represent the experiences of users of other UK clinical genetic centres. Subsequently, user preferences for the identified (dis)benefits could be explored as a step towards the design of a welfarist economic evaluation. Further research is also required to develop the concept of Perceived Familial Control into an appropriate outcome measure for CGSs.

Genome editing based medicines are a new therapeutic modality being developed. They show great promise for a spectrum of genetic diseases lacking conventional treatment strategies. A major obstacle that prevents such therapies to reach the clinic is their safety. We focus on the development and establishment of newer approaches to capture and understand these safety risks during the drug development stage, to build safe drugs that would reach the clinic. To capture potential off-target double-stranded breaks generated by CRISPR, we worked on optimizing an next generation sequencing based method, INDUCE-seq. To capture larger scale on and off-target genetic changes induced by CRISPR editing, which are difficult to capture by next-generation short-read sequencing, we employed a novel imaging-based long-read genomic technology, Optical Genome Mapping (OGM). It enabled the detection of on-target integration with allelic frequency as low as 0.001%, uncovered previously unknown on-target allelic integration heterogeneity and found several off-target structural variants genome-wide, thereby proving that OGM is highly sensitive and can detect rare and complex DNA structural variants (SV) which would have been missed otherwise. Finally, to further characterize some of the identified structural variants in-depth, we employed two new approaches, Cas9 enrichment and X-drop enrichment, to selectively enrich the genomic region of interest, followed by reading it via long-read nanopore sequencing, to get a direct readout of the small genomic region of interest, without any interpretation errors that may happen by piecing together short reads. We demonstrate the successful performance of Cas9 enriched nanopore approach. And we show our progress in evaluating the X-drop approach.

No description available.

Tumor Necrosis Factor-Like Ligand 1a (TL1A) is a cytokine encoded by Tumor Necrosis Factor Super Family 15 gene (TNFSF15) gene mostly in endothelial cells which binds to T-cells and foments the production of pro-inflammatory cytokines including TNF-α, IL-6, IL-1b, IFN- γ and IL-13. TL1A level is elevated in inflammatory diseases including Crohn's Disease (CD). Although Single Nucleotide Polymorphisms (SNPs) in TNFSF15 have been reported in CD, no studies have investigated the effect of these SNPs on TL1A, inflammation, and susceptibility to Mycobacterium avium subspecies paratuberculosis (MAP) infection. MAP is a strong candidate in CD pathogenesis. This study is designed to elucidate the combined effect of MAP and SNPs in TNFSF15 (rs4263839, rs7848647, rs6478108, or rs6478109) on TL1A secretion and downstream effect on pro-inflammatory cytokines. Peripheral blood from CD and healthy subjects was analyzed for MAP DNA, TNFSF15 genotyping, circulating TL1A level, and IFN- γ and TNF-α gene expression. Our data is first to report that rs4263839, rs7848647, rs6478108, and rs6478109 in TNFSF15 resulted in increase in circulating TL1A level in healthy and CD samples. Specifically, in CD samples with rs7848647, the average TL1A level was 146.9 pg/mL ± 124.5 compared 62.4 pg/mL ± 82.8 in normal samples. Similarly, TL1A level in CD samples with rs6478109 was 141.9 pg/mL ± 127.7 compared to 71.5 pg/mL ± 88.4 in normal samples (p < 0.05). All 4 SNPs resulted in significant elevation in TL1A level in healthy samples (p < 0.05). Moreover, IFN-γ expression was significantly higher, by approximately 1.6-fold in CD patients with SNPs relative to CD patients with no SNPs (p < 0.05). Interestingly, SNPs in TNFS15 had no significant effect on TNF-α expression. MAP was detected in the blood of 63% of CD compared to 6% healthy subjects (p < .001). The data did not support a correlation between MAP presence and circulating TL1A levels, and no correlation between SNPs in TNSF15 and MAP susceptibility. This study strongly suggests, that SNPs in TNFSF15 increase TL1A levels and may be a contributory factor to the inflammation experienced by CD patients. Over all, the study emphasizes the need for a pharmacogenomic approach in treatment delivery for patients with CD by using TNFSF15 SNPs to identify patients that would benefit from biologics targeting TL1A rather than TNF-α for more efficacious treatment regiments for CD patients.

Until recently, complete understanding of the endogenous activity of pathologically relevant genes was out of reach and research was confined to in situ work, plasmid-based constructs and artificial model systems. The development and expansion of the CRISPR/Cas9 genome editing technique has enabled us to explore the molecular underpinnings of gene activation using the cell's own endogenous regulatory environment. In this work, we report on the development of a novel tool to monitor the endogenous activity of a causative gene in Parkinson's disease, a-synuclein. We use CRISPR/Cas9 to insert a highly sensitive engineered luciferase at the C-terminal of a-synuclein and assessed its responses to stimuli. Our system responds to epigenetic stimuli, which was unable to be recapitulated by previously available gene activity assays. After development of a sensitive detection tool for epigenetic stimuli, we focused on developed a modular suite of epigenetic writers and erasers by modification of the SunTag protein tagging system and used catalytically dead Cas9 (dCas9) to direct our modular epigenetic toolkit to individual genes. We show that our toolkit of epigenetic effectors successfully writes epigenetic information in a site-specific manner. Using the sensitive a-synuclein reporter we previously developed, we screen the promoter region of this pathologically relevant gene at high resolution and identify the most effective areas for epigenetic intervention in this cell line. These tools allow us to dissect and understand the endogenous regulatory mechanisms of almost any gene targetable by Cas9 in ways that were not previously available may prove to be an effective strategy for persistently altering pathologic transcriptional activity. This system offers a strong tool for to dissect and understand underlying epigenetic architecture and opens potential new avenues for therapeutic strategies for various disease conditions.

No description available.