Pathophysiological mechanisms and optimization of diagnosing congenital adrenal hyperplasia

Autoreferát 4 Souhrn Vrozená adrenální hyperplazie (CAH) je skupinou autozomáln recesivních onemocn ní, které jsou charakteristické neadekvátní sekreci steroidních hormon k ry nadledvin. Nejčast jší se setkáváme s deficitem 21-hydroxylázy (gen CYP21A2), který vede k nedostatečné sekreci mineralokortikoid a glukokortikoid a zárove je p ítomná nadm rná produkce androgen . Je patrná dobrá korelaci mezi typem mutace a deficitem 21-hydroxylázy a následn klinickým obrazem. Novorozenecký screening CAH (NS CAH) byl zaveden k včasné detekci nejt žší formy deficitu 21-hydroxylázy (salt wasting formy CAH). NS CAH je založen na m ení hladiny 17-OHP ze suché kapky krve pomocí fluoroimunoeseje (Delfia). V ýeské republice byl screening zaveden v roce 2006. Zhodnocením NS CAH za období 2006 - 2011 byla zjišt na senzitivita screeningu 98 %, specificita 99,5 %, nízká pozitivní prediktivní hodnota (PPV) 1,6 % a vysoká četnost falešné pozitivity (FPR) 0,51 % v celé skupin vyšet ených. Díky nízké pozitivní prediktivní hodnot screeningu je u části novorozenecké populace (0,51%) hladina 17-OHP opakovan kontrolována pro mírné p echodné zvýšení 17-OHP nad cut-off limit negativity. K zvýšení pozitivní prediktivní hodnoty m že vést jednak úprava cut-off limit a jednak zavedení druhého stupn NS. Druhý stupe novorozeneckého screeningu...Autoreferát 5 Summary Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive diseases which are characterized by inadequate secretion of steroid hormones of the adrenal cortex. The most common type of CAH is a deficiency of 21-hydroxylase (CYP21A2 gene), which leads to insufficient secretion of mineralocorticoids and glucocorticoids and excessive androgen production. There is apparent good correlation between the type of mutation and a 21-hydroxylase deficiency, and subsequently the clinical presentation. Neonatal screening for CAH was introduced to early and effectively recognize the most severe type of 21-hydroxylase deficiency (salt wasting form of CAH). Neonatal screening CAH is based on the detection 17-OHP level in dried blood spots by fluoroimmunoassay (Delfia). In the Czech Republic NS CAH was implemented to screening program in 2006. During the period of 2006 - 2011 we evaluated the results of NS CAH and we observed sensitivity of 98%, specificity of 99.5%, a low positive predictive value (PPV) of 1.6% and a high false positive rate (FPR) of 0.51% in the whole group examined newborns. Due to the low positive predictive value in the part of neonatal population (0.51%) the levels of 17-OHP are repeatedly checked for transiently elevated levels of 17-OHP above the cut-off limit of...Department of PaediatricsPediatrická klinikaSecond Faculty of Medicine2. lékařská fakult

The molecular biology of hypertensive congenital adrenal hyperplasia

The purpose of this project was to identify the molecular basis of hypertensive congenital adrenal hyperplasia in patients with two forms of the disease; steroid 17α-hydroxylase/17,20 lyase (CYP17) and 11β-hydroxylase (CYP11B1) deficiencies. These enzyme defects account for approximately 1% and 5% of recorded cases of congenital adrenal hyperplasia respectively. CYP17 is encoded by a single gene, CYP17 on chromosome 10. The CYP11B1 gene is located on chromosome 8q22, in tandem with the aldosterone synthase gene, CYP11B2, with which it shares 93% base sequence homology. The hypothesis was proposed that the higher incidence of CYP11B1 deficiency is the result of non-homologous recombination and gene conversion between the duplicated CYP11B genes. Two mutation screening methods were employed and evaluated. The first method involved construction of a genomic DNA library in bacteriophage lambda, isolation of fragments of CYP17 and subsequent sequencing by manual dideoxy chain termination to locate mutations in DNA from a single individual with 17α- hydroxylase deficiency. The second procedure used the polymerase chain reaction (PCR) and single strand conformation polymorphism analysis (SSCP) to screen DNA from several 11β-hydroxylase deficient patients simultaneously and proved to be a much faster and more successful approach to screening for mutations. A single point mutation was found on each allele of the CYP17 gene. In contrast point mutations, small deletions, small duplications and gene conversion events were found in the CYP11B genes, often with several mutations present in each patient. Deletion and duplication events occurred where there were direct repeats of base sequence. Pathological mutations in CYP11B1 were shown not to arise directly from gene conversion or non-homologous recombination in the subjects studied. Other factors, such as differences in the degree of DNA methylation and selection acting strongly against mutation within CYP17, may account for the higher rate of mutation at the CYP11B1 locus as compared to that of CYP17

Molecular Genetics of Human 3beta-Hydroxysteroid Dehydrogenase Deficiency

3beta-hydroxysteroid dehydrogenase (3beta-HSD) catalyses a series of obligatory biosynthetic steps in the synthesis of mineralocorticoids, glucocorticoids and sex steroids. Specifically, it converts the 3beta-hydroxysteroids pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, and androstene 3beta,1713-diol into the respective 3beta4 3-ketosteroids: progesterone, 17alpha-hydroxyprogesterone, androstenedione and testosterone. In rodents at least three genes for 3beta-HSD have been found to be expressed. When analysed on southern blots, human DNA shows evidence of six or more sequences with homology to 3beta-HSD probes. So far, two highly homologous but distinct human genes have been cloned, encoding 3beta-HSD type I expressed principally in the placenta, and 3beta-HSD type II expressed in adrenal, ovary and testis. Though the type I and II enzymes differ in their kinetics in detail, they are both capable of converting the same 3beta5 substrates

Spectroscopic studies on cytochrome P450 11-beta-hydroxylase and model compounds

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996.Includes bibliographical references.by Normand J. Cloutier.Ph.D

Development of Congenic Lines and Application of Physical Mapping Strategies for the Dissection of Blood Pressure Quantitative Trait Loci in the Stroke-Prone Spontaneously Hypersensitive Rat

Human essential hypertension is a complex, multifactorial, quantitative trait under polygenic control. Several strategies have been developed over the last decade to dissect genetic determinants of hypertension. Of these, the most successful have been studies identifying rare Mendelian syndromes in which a single gene mutation causes high blood pressure (BP). The attempts to identify multiple genes, each having a small contribution to the common polygenic form of hypertension, have been less successful. Experimental models of genetic hypertension have been used to develop paradigms for the study of human essential hypertension in order to remove some of the complexity inherent in studying human subjects. Several laboratories, using diverse crosses between hypertensive and normotensive strains, identified several quantitative trait loci (QTLs) for BP regulation. The strategy used to identify BP QTLs is known as a genome scan and involves the determination of the BP in a large segregating F2 population derived by crossing contrasting inbred rat strains, and the genotyping of a large panel of polymorphic microsatellite markers with a thorough coverage of the entire rat genome. The next step is the production of congenic strains and substrains to confirm the existence of the BP QTLs and to narrow down the chromosomal region of interest. The investigations reported in this thesis incorporate the use and validation of a "speed" congenic strategy to dissect two BP QTLs identified previously on rat chromosome 2. We produced 4 congenic strains through introgression of various segments of chromosome 2 from the WKYGla strain into the recipient SHRSPGla strain, and vice versa. Transfer of the region of rat chromosome 2 containing both BP QTLs from WKYGla into an SHRSPGla genetic background lowered both baseline and salt-loaded systolic BP by ~20 and ~40 mmHg in male congenic rats compared with the SHRSP parental strain (F=53.4, p<0.005; F=28.0, p<0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the BP values recorded for the SHRSPcia, indicating that if such heterozygosity exists, its effect on BP is negligible. Reciprocal congenic strain in which one QTL was transferred from SHRSPGla onto the WKYoia background resulted in statistically significant but smaller BP increase. This implicated region contains different candidate genes including the Na+-K+ATPase a1 isoform (Atplal), natriuretic peptide receptor A/Guanylate cyclase A (Gca), angiotensin II receptor type IB (Agtrib), and calcium/calmodulin-dependent protein kinase II delta subunit (Camk2d). Sequencing analysis showed no differences in the coding regions of the Atplal gene between the WKYGla strain and the published sequence. Two transitions were found between WKYGla and SHRSPGla resulting in silent mutations. Therefore, the Atplal gene was not supported as a candidate gene for the BP QTL on rat chromosome 2. Radiation hybrid mapping was performed along with fluorescence in situ hybridisation of rat chromosome 5 due to the discrepancies between our genetic map and other genetic maps of rat chromosome 5. We successfully constructed a radiation hybrid map of rat chromosome 5 using 35 microsatellite markers covering a genetic distance of 78 cM, corresponding to a physical distance of approximately 1,304 cR (17 cR/cM) and comparable to reports from other laboratories. The Anf microsatellite marker was mapped between D5Rat48 and D5Rat47 located at the telomeric end of rat chromosome 5. Fluorescence in situ hybridisation confirmed that the Anf gene is localised to the 5q36.3, which corresponds to the telomeric end of the chromosome 5. Two different physical mapping methods have therefore given identical results and are in agreement with genetic maps published by other groups. We also produced a high resolution radiation hybrid map of the telomeric end of rat chromosome 2 between markers D2MH6 and D2Mghl2. The physical to genetic distance conversion gave us an estimate of 20.8 cM to 31.2 cM for this region and facilitated fine mapping of the two BP QTLs on rat chromosome 2. We constructed congenic substrain SP.WKY.Gla2c* where a small segment of approximately 20 cM was transferred from the normotensive WKYGla strain into the hypertensive SHRSPGla- Phenotyping of the congenic substrain is currently ongoing and will determine if the BP QTL was successfully trapped. Additionally, we produced a high resolution radiation hybrid map of this segment, which will help in the identification of the gene(s) involved in this BP QTL. (Abstract shortened by ProQuest.)

A computational study of the substrate conversion and selective inhibition of aldosterone synthase

When a functional or structural impairment of cardiac output has occurred, the cardiovascular system will attempt to compensate for the reduced blood flow. Unfortunately, many of the resulting processes, such as the renin angiotensin aldosterone system, will progressively weaken the heart, resulting in the condition called heart failure. The renin angiotensin aldosterone regulatory system is currently targeted with medicine for heart failure. Many successes for the prolongation of patient age have been achieved by inhibition of angiotensin II synthesis and action. It has become apparent that this approach is suboptimal. Antagonists of aldosterone have provided better treatment options, however, side-effects are still observed. In the search for an alternative therapeutic application, we have studied a novel treatment involving the selective inhibition of aldosterone biosynthesis. The scope of this study has involved the in silico design and prediction of novel inhibitors, the synthesis of these inhibitors and analogues, and finally the in vitro measurement of their potency. The biosynthesis of aldosterone is performed by two cytochrome p450 enzymes, 11B1 and 11B2, denoted as CYP11B1 and CYP11B2, respectively. From these two family members, only CYP11B2 can perform the final synthesis step that converts 18-hydroxycorticosterone into aldosterone. CYP11B1 performs the synthesis of glucocorticoids that are responsible for metabolic, immunologic and homeostatic functions. Because these glucocorticoid actions should not be inhibited, the newly designed medicine must be CYP11B2 selective. Since CYP11B1 is highly homologous to CYP11B2, we have performed an in silico study that allows us to model the interactions of substrates and inhibitors in both the active sites of CYP11B1 and CYP11B2. Using comparative modelling, we have constructed models for the three dimensional architecture of both proteins. These models have been validated by investigating the torsional properties of the protein backbone and residue side chains, the overall protein packing and the dynamic behaviour of the protein models. Subsequently, the models have been used to evaluate the binding mechanisms and conversion mechanisms for the natural steroidal ligands of CYP11B1 and CYP11B2. A hypothetical binding mode has been proposed for 18-hydroxycorticosterone in CYP11B2, featuring the presence of stabilising hydrogen bonding interactions required for its conversion. Quantum mechanical analyses on the conversion of the steroids involved have shown a favourable conversion for this conformation, thereby supporting our hypothesis. In addition, the quantum mechanical analyses have provided insights on steroid conformations in the active sites during conversion. The suitability of the protein models for inhibitor design has been tested by subjecting the models to a case study with four known inhibitors of CYP11B1 and CYP11B2. Using molecular dynamics and molecular docking, the inhibitor potencies for CYP11B1 and CYP11B2 have been predicted, and their interactions with the proteins have been evaluated. The trends in inhibitor potency found by these computational methods have been confirmed by in vitro inhibition measurements. As a next step, the molecular docking study has been expanded to improve the confidence in the predictive power of the models. Using the protein states evaluated by the molecular dynamics study, the molecular docking results of inhibitor analogues have been investigated and the predictive power of the models has been qualitatively improved. In a final approach, we have performed a ligand-based investigation of the inhibitor analogues to determine which ligand characteristics are important for the potency for CYP11B1 and CYP11B2. To this end, we have conducted decision tree analyses on the physico-chemical properties of inhibitor substituents, resulting in a collection of descriptors that can be used for the prediction and design of novel inhibitors. We have shown that a combination of synthesis, molecular modelling and experimental measurements form a promising approach towards the design of potentially new inhibitors

Resistance mechanisms during endocrine treatment in breast cancer

Prolonged endocrine therapy is the mainstay of treatment for ER+ breast cancer patients. However, resistance develops in many patients which leads to more aggressive disease. Understanding the mechanisms of acquired resistance that emerge as a consequence of prolonged endocrine treatment remains critical. This study aimed to use gene expression profiling to discover induced mechanisms shared by a panel of MCF7-derived acquired resistant cells that underpin endocrine resistant growth. The in vitro panel represents resistance to oestrogen deprivation, tamoxifen or fulvestrant and includes long-term (3year) models to better-mimic clinical endocrine exposure. Affymetrix 1.0ST microarrays detected 572 genes induced in all resistant models versus MCF7. Over-represented ontologies, pathways and functional classification for these genes revealed induction of oxidative phosphorylation (OxPhos) and TCA cycle enzymes in the resistant models, a finding further confirmed by mass spectrometry. Increased oxygen consumption, NADH dehydrogenase and/or cytochrome C oxidase activity was detected in resistant cells, and targeting with OxPhos inhibitors Metformin or Antimycin A confirmed growth-dependency on OxPhos. Western blotting for AMPK (energy sensor) activity and its downstream anabolic targets (ACC, mTOR/P70S6K) showed Metformin reduced fatty acid and protein synthesis in growth-sensitive endocrine resistant cells. In silico analysis inferred clinical relevance since many TCA/OxPhos genes associated with earlier relapse in ER+ and/or tamoxifen treated patients. Monitoring basal glycolysis (extracellular lactate) and growth impact of 2DG or glutamine restriction demonstrated glycolysis and glutaminolysis also contribute to endocrine resistance. The microarrays furthermore revealed that metabolic kinases PCK2, ALDH18A1 and PFKFB2, and components of cell response to Zn were commonly-induced which may additionally help endocrine resistant growth. This study has revealed increased OxPhos arises as a consequence of prolonged endocrine treatment and is a key bioenergetic pathway sustaining resistance. Since resistant growth is Metformin-sensitive, such targeting of this energy pathway (alongside further antihormones or glycolysis/glutaminolysis inhibitors) could help treat resistance

Oceanography and Marine Biology

Oceanography and Marine Biology: An Annual Review remains one of the most cited sources in marine science and oceanography. The ever-increasing interest in work in oceanography and marine biology and its relevance to global environmental issues, especially global climate change and its impacts, creates a demand for authoritative refereed reviews summarizing and synthesizing the results of both historical and recent research. This Volume celebrates 60 years of OMBAR, over which time it has been an essential reference for research workers and students in all fields of marine science. The peer-reviewed contributions in Volume 60 are available to read Open Access via this webpage and on OAPEN. If you are interested in submitting a review for consideration for publication in OMBAR, please email the Editor-in-Chief, Stephen Hawkins ([email protected]) for Volume 61. For Volume 62 onwards, please email the new co-Editors in Chief, Dr Peter Todd ([email protected]) and Dr Bayden Russell ([email protected]). Volume 60 features an editorial on the UN Decade of Ocean Science and goes on to consider such diverse topics as Cenozoic tropical marine biodiversity, blue carbon ecosystems in Sri Lanka, marine litter and microplastics in the Western Indian Ocean, and the ecology and conservation status of the family Syngnathidae in southern and western Africa. This volume also contains a retrospective Prologue on the evolution of OMBAR and pays tribute to one of its early Editors in Chief, Margaret Barnes, by providing an update on her review in OMBAR of the stalked barnacle Pollicipes. Supplementary online videos as well as additional Tables and Appendices are available on the Support Tab of the book's Routledge webpage. An international Editorial Board ensures global relevance and expert peer review, with editors from Australia, Canada, Hong Kong, Ireland, Singapore and the UK. The series volumes find a place in the libraries of not only marine laboratories and oceanographic institutes, but also universities worldwide