Comparative analysis of acute and chronic corticosteroid pharmacogenomic effects in rat liver: Transcriptional dynamics and regulatory structures

<p>Abstract</p> <p>Background</p> <p>Comprehensively understanding corticosteroid pharmacogenomic effects is an essential step towards an insight into the underlying molecular mechanisms for both beneficial and detrimental clinical effects. Nevertheless, even in a single tissue different methods of corticosteroid administration can induce different patterns of expression and regulatory control structures. Therefore, rich <it>in vivo </it>datasets of pharmacological time-series with two dosing regimens sampled from rat liver are examined for temporal patterns of changes in gene expression and their regulatory commonalities.</p> <p>Results</p> <p>The study addresses two issues, including (1) identifying significant transcriptional modules coupled with dynamic expression patterns and (2) predicting relevant common transcriptional controls to better understand the underlying mechanisms of corticosteroid adverse effects. Following the orientation of meta-analysis, an extended computational approach that explores the concept of agreement matrix from consensus clustering has been proposed with the aims of identifying gene clusters that share common expression patterns across multiple dosing regimens as well as handling challenges in the analysis of microarray data from heterogeneous sources, e.g. different platforms and time-grids in this study. Six significant transcriptional modules coupled with typical patterns of expression have been identified. Functional analysis reveals that virtually all enriched functions (gene ontologies, pathways) in these modules are shown to be related to metabolic processes, implying the importance of these modules in adverse effects under the administration of corticosteroids. Relevant putative transcriptional regulators (e.g. RXRF, FKHD, SP1F) are also predicted to provide another source of information towards better understanding the complexities of expression patterns and the underlying regulatory mechanisms of those modules.</p> <p>Conclusions</p> <p>We have proposed a framework to identify significant coexpressed clusters of genes across multiple conditions experimented from different microarray platforms, time-grids, and also tissues if applicable. Analysis on rich <it>in vivo </it>datasets of corticosteroid time-series yielded significant insights into the pharmacogenomic effects of corticosteroids, especially the relevance to metabolic side-effects. This has been illustrated through enriched metabolic functions in those transcriptional modules and the presence of GRE binding motifs in those enriched pathways, providing significant modules for further analysis on pharmacogenomic corticosteroid effects.</p

Glucocorticoid-induced osteoporosis: an update

Glucocorticoid-induced osteoporosis is the most common secondary cause of osteoporosis and the resulting fractures cause significant morbidity. Following initiation of oral glucocorticoids, rapid bone loss occurs and fracture risk increases within a few months in a dose-dependent manner. These adverse effects are due to inhibition of bone formation accompanied by an early but transient increase in bone resorption. Multiple mechanisms underlie these changes in bone remodeling; direct effects include up-regulation of PPARγR2, increased expression of sclerostin and increased RANKL/OPG ratio, whilst hypogonadism, altered renal and intestinal calcium handling, and reduced production of insulin-like growth factor 1 also contribute. Fracture risk assessment should be performed as soon as possible after glucocorticoids are initiated and bone protective therapy started promptly in individuals at high-risk, with calcium and vitamin D supplements where appropriate. Oral bisphosphonates are currently regarded as first line options on the grounds of their low cost. However, teriparatide has been shown to be superior in its effects on BMD and vertebral fracture risk in glucocorticoid-treated individuals with osteoporosis and should be considered as an alternative first line option in high-risk patients

Meta-analysis of Inter-species Liver Co-expression Networks Elucidates Traits Associated with Common Human Diseases

Co-expression networks are routinely used to study human diseases like obesity and diabetes. Systematic comparison of these networks between species has the potential to elucidate common mechanisms that are conserved between human and rodent species, as well as those that are species-specific characterizing evolutionary plasticity. We developed a semi-parametric meta-analysis approach for combining gene-gene co-expression relationships across expression profile datasets from multiple species. The simulation results showed that the semi-parametric method is robust against noise. When applied to human, mouse, and rat liver co-expression networks, our method out-performed existing methods in identifying gene pairs with coherent biological functions. We identified a network conserved across species that highlighted cell-cell signaling, cell-adhesion and sterol biosynthesis as main biological processes represented in genome-wide association study candidate gene sets for blood lipid levels. We further developed a heterogeneity statistic to test for network differences among multiple datasets, and demonstrated that genes with species-specific interactions tend to be under positive selection throughout evolution. Finally, we identified a human-specific sub-network regulated by RXRG, which has been validated to play a different role in hyperlipidemia and Type 2 diabetes between human and mouse. Taken together, our approach represents a novel step forward in integrating gene co-expression networks from multiple large scale datasets to leverage not only common information but also differences that are dataset-specific

New and alternative approaches to the assessment of pharmacokinetic and pharmacodynamic equivalence

La bioéquivalence, une mesure de substitution de l'innocuité et de l'efficacité à différents stades du processus de développement des médicaments, est tout particulièrement importante lors du développement d'un médicament générique. Entre autres critères, la bioéquivalence garantit que les médicaments génériques sont équivalents aux produits innovateurs ou de références approuvés en termes d’efficacité clinique et d’innocuité tout en contournant le long cours et le coût élevé des essais chez les animaux et des essais cliniques chez les patients exigés pour les médicaments innovants. Malgré les avancées dans le développement d'approches robustes au cours des dernières décennies, la pratique actuelle de la bioéquivalence fait toujours l'objet de controverses. Le but de cette thèse est d'explorer certaines de ces controverses et de les aborder en proposant des approches nouvelles et alternatives. L'une des questions les plus controversées dans la pratique actuelle de la bioéquivalence est l'extrapolation des résultats d'études de bioéquivalence d'une population à une autre. La majorité des études de bioéquivalence portant sur des formes pharmaceutiques orales efficaces par voie systémique reposent sur les critères de pharmacocinétique obtenus chez des sujets sains, alors que la population cible est constituée de patients. Ceci est basé sur l'hypothèse que si deux produits sont bioéquivalents dans une population, ils devraient l'être dans une autre. L'extrapolation des résultats des études de bioéquivalence ne se limite pas à celle des sujets sains aux patients. Depuis 2007, une proportion croissante d'études de bioéquivalence pharmacocinétique portant sur des soumissions génériques nord-américaines ou européennes a été réalisée auprès de populations géographiques/ethniques autres que celles visées, en raison du coût moins élevé de ces études en dehors de l'Amérique du Nord et de l'Europe. Dans le premier volet de cette thèse, nous avons examiné si les résultats de la bioéquivalence obtenus dans une population géographique ou ethnique pouvaient être extrapolés à une autre. À cette fin, nous avons extrait les résultats des études de bioéquivalence pharmacocinétique disponibles publiquement et provenant de soumissions génériques à Santé Canada et à la Food and Drug Administration des États-Unis. Pour dix médicaments différents, nous avons calculé l'effet d’un repas normalisé sur le produit de référence et comparé les résultats obtenus chez deux populations ethniques, les indiens et les nord-américains. Cette approche novatrice est basée sur le raisonnement suivant: si l'effet d’un repas sur le produit de référence est le même chez les populations indienne et nord-américaine, le produit générique et sa référence qui se sont révélés bioéquivalents dans la population indienne devraient également l'être dans la population nord-américaine. Pour 90% des médicaments à l'étude, une différence statistiquement significative a été détectée entre les deux populations après un repas. Pour 30% de ces médicaments, la différence s'est révélée d'une pertinence clinique possible. Les résultats de cette étude ont mis en évidence que l’extrapolation des résultats de bioéquivalence d’une population à l’autre devrait possiblement être reconsidérée pour certains médicaments. Les défis dans le contexte de la bioéquivalence ne se limitent pas toujours aux études pivots où la performance d’un produit générique est comparée à celle de la référence. En effet, une étude pilote peut être menée afin d’établir un protocole d’étude approprié pour cette étude pivot. Par conséquent, les résultats inexacts provenant d'une étude pilote, tels qu'une estimation imprécise du moment ou de la durée d’administration optimale de la dose lors de la comparaison du produit testé par rapport à la référence, pourront affecter négativement les résultats de l’étude de bioéquivalence. Ceci est particulièrement crucial pour les produits indiqués pour un usage topique dermatologique dont les corticostéroïdes constituent un cas d’espèce. En effet, leur bioéquivalence est démontrée par une mesure pharmacodynamique, le blanchiment cutané, à différents temps après application topique. L’intensité du blanchiment est comparée entre le produit générique et le produit de référence à une durée d’administration spécifique d’une dose donnée, la DD50, soit la durée associée à 50% de l’effet maximal observé. Par conséquent, cette durée d’administration de la dose doit d’abord être déterminée dans le cadre d’une étude pilote. L’agence réglementaire américaine recommande l’utilisation d’une approche populationnelle basée sur la modélisation non linéaire à effets mixtes pour l'estimation de la DD50 et ce, quelle que soit la méthode d'analyse. Étant donné qu’il existe différents types de méthodes d’analyse non linéaire à effets mixtes, chaque commanditaire peut en choisir une différente. Dans le deuxième volet de cette thèse, nous avons examiné si les mêmes estimations de DD50 pouvaient être obtenues en utilisant différentes méthodes non linéaires à effets mixtes. À cette fin, nous avons ajusté les données de blanchiment de la peau d’onze études avec deux méthodes non linéaires à effets mixtes différentes : le maximum de vraisemblance avec maximisation de l’espérance (MLEM) et l'estimation conditionnelle de premier ordre (FOCE). Les résultats ont favorisé MLEM, compte tenu d’une meilleure puissance discriminative pour l’estimation de la DD50 de population et d’une meilleure minimisation de la variabilité interindividuelle. Bien que l'approche de la bioéquivalence fondée sur la pharmacocinétique ait contribuée de manière significative au développement de versions génériques de haute qualité des formes pharmaceutiques orales indiquées pour un effet systémique, la disponibilité de versions génériques pour les produits dermatologiques topiques demeure limitée et ce, par manque de méthodes acceptées par les agences réglementaires pour l'évaluation de la bioéquivalence de ces produits. Dans le troisième volet de cette thèse, une nouvelle approche pour l’évaluation de la bioéquivalence de formulations de crème topique d’acyclovir a été développée en utilisant une analyse basée sur un modèle de données d’exposition locales récupérées à partir d’échantillons de peau abrasée prélevés à une seule durée d’administration de la dose, la DD50 à l’aide de bandes adhésives. Un seul échantillonnage de peau effectué à la DD50 a non seulement assuré que les données pharmacocinétiques étaient recueillies à la durée d’administration de la dose ayant le meilleur pouvoir discriminant pour détecter une différence au niveau des formulations, mais a également permis de diminuer considérablement le nombre d'échantillons à analyser. Et surtout, cette nouvelle approche a permis de générer un profil pharmacocinétique au niveau même de la peau. Ce faisant, nous avons pu utiliser l'analyse compartimentale populationnelle et contourner les nombreuses hypothèses et calculs sophistiqués requis par les méthodes précédentes. Notre approche a également permis de générer de nouveaux paramètres pharmacocinétiques permettant de décrire la vitesse et le degré d’exposition cutanée pour l'évaluation de la biodisponibilité et de la bioéquivalence topiques. Finalement, cette méthode a le potentiel de discerner une formulation bioéquivalente d’une autre qui ne l’est pas.Bioequivalence is a surrogate measure of safety and efficacy in different stages of drug development process with the most pronounced significance in the development of generic drugs. Bioequivalence, among other standards, ensures that generic drugs are equivalent to their approved innovator or reference products in terms of clinical efficacy and safety while circumventing the lengthy-time course and high cost of animal and clinical trials in patients required for innovator drugs. Despite the advancements in development of robust bioequivalence approaches over the past decades, there are still controversies in the current practice of bioequivalence. The aim of this thesis is to explore some of these controversies and address them by putting forward new and alternative approaches. One of the most controversial issues in the current practice of bioequivalence is the extrapolation of bioequivalence study results from one population to another. The majority of bioequivalence studies for systemic effective oral dosage forms are conducted based on pharmacokinetic endpoints in healthy volunteers whilst the targeted population is patients. This is based on the assumption that if two products are bioequivalent in one population, they should be bioequivalent in another one. The extrapolation of bioequivalence study results is not limited to that from healthy volunteers to patients. Since 2007, an ever-increasing proportion of pharmacokinetic bioequivalence studies for North American or European generic submissions have been performed in geographical/ethnic populations other than the intended ones, due to the lower cost of these studies outside North America and Europe. In the first part of this thesis, we investigated whether the bioequivalence results obtained in one geographical or ethnic population can be extrapolated to another one. To this purpose, we extracted pharmacokinetic bioequivalence studies results from generic submissions to Health Canada and the US Food and Drug Administration. We calculated food effect for ten different reference drug products and compared the results for each product between two ethnic populations, Indians and North Americans. This is based on the reasoning that if food effect is found to be the same between the Indian and North American populations, then the generic product and its reference that were found to be bioequivalent in the Indian population should also be bioequivalent in North American population. For 90% of the study drugs, statistically significant difference was detected in the food effect between two populations. For 30% of these drugs, the difference was found to be of possible clinical relevance. The results of this study raised a flag for extrapolating the bioequivalence results from one population to another. Challenges in the context of bioequivalence are not always limited to the pivotal studies where the performance of a generic product is compared to that of Reference. Prior to pivotal bioequivalence studies, a pilot study may be conducted to establish an appropriate study design for the pivotal bioequivalence study. Therefore, inaccurate results from a pilot study, such as inaccurate estimation of time point or dose duration for comparison of test versus reference, can affect the bioequivalence outcomes adversely. An example to this case is the comparison of the extent of skin blanching, the pharmacological effect of generic versus reference products of topical dermatological corticosteroids at specific dose duration, DD50, where the effect is half maximal. This dose duration should initially be determined in a pilot study. The US FDA 1995 Guidance document recommends the use of non-linear mixed effect population modeling for the estimation of DD50, irrespective of the method of analysis. Given the availability of different types of non-linear mixed effect modeling methods, each sponsor could choose a different one. In the second part of this thesis we investigated whether the same DD50 estimates can be obtained when different non-linear mixed effect modeling methods are used. To this purpose, we fitted the skin blanching data from eleven studies with two different non-linear mixed effect modeling methods, the Maximum Likelihood Expectation Maximization (MLEM) and the First Order Conditional Estimation (FOCE). The results favored MLEM given its lower population DD50 estimates that would locate in a more discriminative portion of the Emax curve and better minimization of inter-individual variability. Although the pharmacokinetic-based bioequivalence approach has contributed significantly to the development of high-quality generic versions of systemic effective oral dosage form, the availability of generic versions of topical dermatological products remains constrained due to the limited methods accepted for bioequivalence evaluation of these products. In the third part of this thesis, a novel approach for the bioequivalence assessment of topical acyclovir cream formulations was developed based on the model-based analysis of local exposure data recovered from tape stripping of the skin at a single dose duration, DD50. Conducting the stripping procedure only at DD50 not only ensured that the PK data was collected at the dose duration that is most discriminative of formulation differences, but it also decreased the number of samples to be analyzed significantly. More importantly, our novel approach in generating the local PK profile in the skin (dermatopharmacokinetic profile) and the implementation of population compartmental analysis circumvented the numerous assumptions and sophisticated calculations that were inherent to previous methods, while yielding the PK parameters relevant for topical bioavailability and bioequivalence assessment (rate and extent of exposure to the skin). This method successfully concluded bioequivalence and its absence

말초혈액 단핵구에서 유전자 발현을 통한 천식 병태생리의 이해

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 의학과, 2021. 2. 박흥우.코르티코스테로이드는 천식 치료의 중요한 약제이다. 하지만 코르티코스테로이드에 대한 치료 효과는 환자마다 상당한 차이가 있는 것으로 알려져 있다. 특히 코르티코스테로이드에 대한 낮은 반응성은 중증 천식 또는 잦은 급성 악화와 관련이 있을 수 있다. 비록 많은 유전체 연구들이 진행되었지만, 스테로이드 저반응성과 관련된 천식의 병태 생리에 대해서는 아직까지 충분히 연구되지 않았다. 따라서 생체외 덱사메타손 처리에 따른 유전자 발현 양상의 변화를 분석하는 것은 스테로이드 저반응성과 관련된 천식 급성악화의 기전을 연구하는데 도움이 될 수 있다. 본 연구는 천식 환자의 말초 혈액 단핵구 세포의 유전자 발현 양상 및 생체외 덱사메타손 처리에 따른 변화를 분석함으로써 스테로이드 저반응성과 관련된 병태생리 기전 및 생물학적 경로를 탐색해보고자 한다. 본 연구는 두 파트로 나누어 진행되었다. 첫번째 파트는 Weighted Gene Co-expression Network Analysis (WGCNA) 방법론을 통해, 소아천식 환자와 성인천식 환자에서 공통적으로 관찰되는 급성악화와 관련된 유전체 모듈이 존재하는지 그리고 해당 모듈에 생체외 덱사메타손 처리를 유전자 발현 양상의 변화를 탐색하였다. 소아 천식 환자 107명의 불멸화된 림프모세포 세포주와 성인천식 환자 29명의 말초혈액 단핵구에서 유전자 발현 양상을 분석하였다. 천식 급성악화는 전신스테로이드를 3일이상 복용하거나 천식으로 인해 응급 방문 또는 입원으로 정의하였다. 소아천식 환자군과 성인천식 환자군에서 공통적으로 관찰되는 총 77개의 유전자로 구성된 급성악화과 관련된 유전체 모듈을 찾았다. 해당 모듈의 EIF2AK2 유전체와 NOL11 유전체는 덱사메타손 처리시 소아 천식환자군과 성인천식 환자군 모두에서 유전체 발현양이 유의하게 감소하였다. 해당 모듈 중 64개의 유전체는 덱사메타손 처리시 유전자 발현양이 유의하게 변하지 않았는데, 이들 유전자들은 단백질 수리 경로 (protein repair pathway) 등과 관련성이 있었다. 단백질 수리 경로와 관련된 유전자 중에서 MSRA와 MSRB2의 중요한 역할은 산화 스트레스를 조절하는 것으로 알려져 있다. 본 연구의 두번째 파트는 유전자 조절 네트워크를 통해 성인 천식환자에서 흡입용 스테로이드에 대한 반응성에 영향을 미치는 요인들을 탐색하였다. 흡입용 스테로이드에 대한 치료 효과가 있었던 환자군과 치료 효과가 없었던 환자군에서 생체외 덱사메타손 처리시 전사인자 차별발현을 보였던 상위 5개의 전사인자효과는 GATA1, JUN, NFKB1, SPl1 그리고 RELA였다. 이들 전사인자는 흡입용 스테로이드에 반응이 있었던 환자군과 없었던 환자군에서 서로 다른 유전자들과 다양한 생물학적 경로에서 연결되어 있었다. TBX4 유전자는 흡입용 스테로이드에 좋은 치료효과를 보였던 환자군에서 염증반응과 관련된 NFKB1 전사인자와 연결되어 있었다. 본 연구를 통해 규명된 새로운 유전자 및 생물학적 경로 탐색을 통해 스테로이드 저반응성과 관련된 유전적 특질을 이해하는데 도움이 되었고, 이는 천식의 다양한 병태생리에 기반한 새로운 치료제 또는 생물지표를 개발하는데 이바지할 수 있을 것이다.Asthma is a chronic inflammatory airway disease characterized by bronchial hyperresponsiveness and reversible airway obstruction. Corticosteroids are known to the most effective treatment for asthma. However, there is substantial variability in response to corticosteroids in asthma patients. Ineffective response to corticosteroids may result in exacerbation of asthma. Although many genetic studies have been conducted, the mechanisms of asthma pathogenesis and steroid insensitivity in asthma have not been fully elucidated. Gene expression profile represents the complete set of RNA transcripts that are produced by the genome under specific circumstances or in a specific cell. High-throughput methods such as microarray, and recent advances in biostatistics based on network-based approaches provide a quick and effective way of identifying novel genes and pathways related to asthma. This study aimed to understand the pathogenesis and steroid insensitivity in asthma using gene expression profiles of blood cells from asthma patients. To obtain a comprehensive picture of the gene expression in these cells, we used network-based approaches. The study was divided into two separate parts. In the first part of the study, important genetic signatures of acute exacerbation (AE) in asthma were identified using weighted gene co-expression network analysis (WGCNA) in peripheral blood mononuclear cells (PBMCs) from 29 adult asthma patients and lymphoblastoid cell lines (LCLs) from 107 childhood asthma patients. An AE-associated gene module composed of 77 genes was identified from childhood asthma patients and the conservation of this gene module structure was validated in adult asthma patients. The identified module was found to be conserved in terms of the gene expression profile and associated with AE in both childhood and adult asthma patients, and thus it was defined as an AE-associated common gene module. Changes in the expression of genes in the AE-associated common gene module following in vitro dexamethasone (Dex) treatment were examined, to better understand the mechanisms associated with steroid insensitivity. The differential gene expression profiles were classified into two classes according to Dex-induced changes in childhood asthma patients. Thirteen genes showed significant Dex-induced differential expression and were categorized as the A gene set. Sixty-four genes were not significantly altered by Dex were categorized as the B gene set. In the A gene set, the expression of eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) showed significant Dex-induced differential expression in adult asthma patients as well. In addition, the basal expression of EIF2AK2 (pre-Dex) were significantly higher in asthma patients with AE compared to those without AE in both childhood and adult asthma. In the B gene set, based on a pathway-based approach, the protein repair pathway was found to be significantly enriched. Among the genes that belong to this pathway, the basal expression of methionine sulfoxide reductase A (MSRA) and methionine sulfoxide reductase B2 (MSRB2) were significantly lower in asthma patients with AE compared to those without AE in both childhood and adult asthma. These findings suggest that alternate treatment options, apart from corticosteroids, may be needed to prevent AE in asthma. Expression of EIF2AK2, MSRA, and MSRB2 in blood cells may help us to identify AE-susceptible asthma patients and adjust treatments to prevent AE events. In the second study, gene regulatory networks identified gene expression profiles of PBMCs from 23 adult asthma patients were assessed to elucidate the differences in responsiveness to inhaled corticosteroids (ICSs). Among these the top five (top-5) transcriptional factors (TFs; Top-5 TFs: GATA1, JUN, NFκB1, SPl1, and RELA) showing differential connections between good-responders (GRs) and poor-responders (PRs) were identified. Interestingly, GATA1 and JUN also showed differential connections in the gene regulatory networks identified gene expression profiles of LCLs from 107 childhood asthma patients in a previous study. The top-5 TFs and their connected genes were significantly enriched in distinct biological pathways associated with asthma. Among the genes connected to the top-5 TFs, the expression of TBX4, which is regulated by the TF, NFκB1, may be helpful in identifying GRs to ICS treatment. In conclusion, the novel genes and biological pathways identified in this study may deepen our understanding of asthma pathophysiology and steroid insensitivity in asthma.Table of Contents Chapter 1 Introduction 1 Chapter 2 Part I 10 2.1 Introduction 10 2.2 Methods 12 2.3 Results 23 2.4 Discussion 43 Chapter 3 Part II 48 3.1 Introduction 48 3.2 Methods 50 3.3 Results 53 3.4 Discussion 79 Chapter 4 Conclusions 84 Bibliography 86 Abstract in korean 101 List of Tables Table 1 26 Table 2 27 Table 3 29 Table 4 34 Table 5 55 Table 6 56 Table 7 57 Table 8 59 List of Figures Figure 1 14 Figure 2 35 Figure 3 36 Figure 4 37 Figure 5 38 Figure 6 39 Figure 7 40 Figure 8 41 Figure 9 42 Figure 10 77 Figure 11 78Docto

Individualized dosing algorithms for tacrolimus in kidney transplant recipients:current status and unmet needs

Introduction: Tacrolimus is a potent immunosuppressive drug with many side effects including nephrotoxicity and post-transplant diabetes mellitus. To limit its toxicity, therapeutic drug monitoring (TDM) is performed. However, tacrolimus’ pharmacokinetics are highly variable within and between individuals, which complicates their clinical management. Despite TDM, many kidney transplant recipients will experience under- or overexposure to tacrolimus. Therefore, dosing algorithms have been developed to limit the time a patient is exposed to off-target concentrations. Areas Covered: Tacrolimus starting dose algorithms and models for follow-up doses developed and/or tested since 2015, encompassing both adult and pediatric populations. Literature was searched in different databases, i.e. Embase, PubMed, Web of Science, Cochrane Register, and Google Scholar, from inception to February 2023 Expert Opinion: Many algorithms have been developed, but few have been prospectively evaluated. These performed better than bodyweight-based starting doses, regarding the time a patient is exposed to off-target tacrolimus concentrations. No benefit in reduced tacrolimus toxicity has yet been observed. Most algorithms were developed from small datasets, contained only a few tacrolimus concentrations per person, and were not externally validated. Moreover, other matrices should be considered which might better correlate with tacrolimus toxicity than the whole-blood concentration, e.g. unbound plasma or intra-lymphocytic tacrolimus concentrations.</p

Cortisol responsive gene networks in cardiovascular disease

Increased plasma cortisol levels are associated with Cardiovascular Disease (CVD) and CVD risk factors, however the tissue specific mechanisms underpinning this process are poorly understood. A genome wide meta-analysis by the CORtisol NETwork (CORNET) consortium identified genetic variants, spanning the SERPINA6/SERPINA1 locus on chromosome 14, associated with morning plasma cortisol and shown to be causal for ischaemic heart disease. SERPINA6 encodes Corticosteroid Binding Globulin (CBG), responsible for binding most cortisol in blood and putatively mediating delivery of cortisol to target tissues. This thesis addresses the hypothesis that genetic variants in SERPINA6 influence CBG expression in liver and cortisol delivery to extra-hepatic tissues, responsible for mediating cortisol-regulated gene expression. The Stockholm Tartu Atherosclerosis Reverse Networks Engineering Task study (STARNET) provides RNA sequencing data for 7 vascular and metabolic tissues from 600 genotyped individuals (mean age 65.8, 70.3% male) undergoing coronary artery bypass grafting. We have identified 21 Single Nucleotide Polymorphisms (SNPs) associated with both variation for plasma cortisol at genome wide significance in CORNET (p ≤ 5×10-8) and with SERPINA6 gene expression in STARNET-liver (q ≤ 0.05) as cis-expression Quantitative Trait Loci (eQTLs). We go on to describe the extra-hepatic consequences of genetic variation for plasma cortisol by linking SNPs associated with plasma cortisol to genes expressed in trans across STARNET tissues, finding the highest representation of trans-genes in liver, subcutaneous and visceral abdominal adipose tissue (FDR = 15%). Through the use of published evidence, we then identify a sub-set of cortisol associated trans-genes that are putatively regulated by the glucocorticoid receptor, the primary transcription factor activated by cortisol. Using causal methods, we have identified glucocorticoid regulated trans-genes that are responsible for the regulation of tissue specific gene networks. Cis-eQTLs were used as genetic instruments for the identification of pairwise causal relation- ships, from which cortisol associated gene networks could be reconstructed. Gene networks were identified in liver, subcutaneous fat and visceral abdominal fat, in- cluding a high confidence gene network specific to subcutaneous adipose (FDR < 10%) under the regulation of the interferon regulatory transcription factor, IRF2. Targets in this network include LDB2 and LIPA, both associated with coronary artery disease. Finally, we identify coordinated patterns of gene expression, representative of the STARNET gene networks, in gene expression data from the Metabolic Syndrome in Men (METSIM) and the Stockholm Atherosclerosis Gene Expression (STAGE) Study, both independent datasets from STARNET. This thesis describes genetic variation at the SERPINA6/ SERPINA1 locus that is associated with changes in both morning plasma cortisol and SERPINA6 expression in liver, the gene that encodes CBG. Altered CBG levels in turn impact gene expression in extra-hepatic tissues through modulation of cortisol delivery. This supports a dynamic role for CBG in modulating cortisol delivery to tissues. The cortisol-responsive gene networks identified here represent candidate pathways to mediate cardiovascular risk attributable to elevated cortisol

Predicting outcome in ulcerative colitis: clinical and genetic determinants of disease susceptibility and behaviour

Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as inflammatory bowel disease (IBD) are common, chronic inflammatory disorders of the intestines. The pathogenesis and subsequent clinical course of IBD remain unclear; although, inter¬ relating factors such as environment triggers, dysregulated immune response, defective gut barrier defence, variability of drug response and genetic susceptibility all contribute. The current work described in this thesis involves a series of clinical and genetic studies investigating their respective roles in determining susceptibility and course in UC. Firstly, the importance of corticosteroid resistance/dependence as a co-factor in disease progression was studied in a detailed 5-year inception cohort (1998-2003). This study demonstrated remarkable concordance with older series and paediatric cohorts suggesting a defined phenomenon within the innate response to corticosteroids. Secondly, a risk score to stratify the likelihood of response to standard medical therapy (high dose corticosteroids) identifying 3 distinct risk groups - low, intermediate and high risk; based on 167 consecutive patients (largest series studied) with acute severe UC was developed. A novel robust model was formulated with a sensitivity/specificity of 85% and 75% respectively to predict failure of medical therapy. These initial studies have critically provided the assimilation of highly accurate phenotypic and follow-up data, permitting very detailed statistical analyses to be performed in subsequent genetic studies. The multidrug resistance gene (MDR1 gene-encoding P-glycoprotein 170, an epithelial efflux transporter protein) was studied in detail due to its potential role in conferring susceptibility (based on knock-out animal models and genomic position within IBD locus of susceptibility) and its function in corticosteroid resistance. In this study, allelic variations of the MDR1 gene were found to be implicated in disease susceptibility in UC, in particular extensive and severe disease subphenotype (p=0.003, OR 2.64 and T-allele, p=0.009, OR 1.70. In this study, bi-directional haplotypic contribution to susceptibility in UC (with protective and susceptible haplotypes) was observed. This led to a more rigorous analysis of this gene by the application of the novel 'gene-wide' haplotype tagging approach, confirming a more significant association with UC (p=4.22xl0"7) but not CD (p=0.22). The strongest association was with the sub-phenotype; extensive UC (p= 1.7 X 10"7), and critically dependent on one tSNP rs3789243 (p= 3.2 x 10"7- 3.6 x 10" 12). This catalysed further rational approaches to study the genetic variations of the genes involved in xenobiotic-metabolism and epithelial transport such as the ATP-binding cassette (ABC) efflux transporters and respective transcriptional regulators in our cohort. Further significant and replicable association of haplotypic variations of the ABCC3/MRP3 gene (also involved in epithelial barrier defence) with IBD (p=0.00004, 1200 IBD and 700 controls) was also shown. The characterisation of these genes in a hitherto unknown pathway regulated by key transcriptional regulators such as PregnaneX receptor (PXR) has now implicated this novel class of transport proteins as important regulators of mucosal defence and as critical in determining susceptibility to inflammatory bowel disease

Genetics and Gene Expression Involving Stress and Distress Pathways in Fibromyalgia with and without Comorbid Chronic Fatigue Syndrome

In complex multisymptom disorders like fibromyalgia syndrome (FMS) and chronic fatigue syndrome (CFS) that are defined primarily by subjective symptoms, genetic and gene expression profiles can provide very useful objective information. This paper summarizes research on genes that may be linked to increased susceptibility in developing and maintaining these disorders, and research on resting and stressor-evoked changes in leukocyte gene expression, highlighting physiological pathways linked to stress and distress. These include the adrenergic nervous system, the hypothalamic-pituitary-adrenal axis and serotonergic pathways, and exercise responsive metabolite-detecting ion channels. The findings to date provide some support for both inherited susceptibility and/or physiological dysregulation in all three systems, particularly for catechol-O-methyl transferase (COMT) genes, the glucocorticoid and the related mineralocorticoid receptors (NR3C1, NR3C2), and the purinergic 2X4 (P2X4) ion channel involved as a sensory receptor for muscle pain and fatigue and also in upregulation of spinal microglia in chronic pain models. Methodological concerns for future research, including potential influences of comorbid clinical depression and antidepressants and other medications, on gene expression are also addressed