Characterization of Genetic Causes of Pediatric Cardiomyopathies – Analysis of PRDM16 as a Major Disease Gene

Primary cardiomyopathies (CMPs) are genetically heterogeneous disorders with a large number of disease-causing genes. The underlying genetic mechanisms and early pathological events of children with primary CMP are poorly characterized. This work aimed to identify genetic causes of childhood CMP, and to understand the development of early-onset and severe heart muscle disease. Mutation of PR/SET domain 16 (PRDM16) was shown to be associated with two types of CMP, dilated CMP (DCM) and left ventricular non-compaction CMP (LVNC). PRDM16 encodes for a transcriptional regulator with a previously unknown function in the heart. Eighty unrelated index patients with pediatric primary CMP underwent genetic testing with a panel-based next generation sequencing (NGS) approach of 89 genes. Adverse events such as heart transplantation (HTX) and death were higher for individuals affected by DCM and restrictive CMP than for other types of CMP. Patients with a higher number of variants of interest (VOI) had an increased risk to experience an adverse event. At least one pathogenic or likely pathogenic variant was identified in 30/80 (38%) index patients. In all CMP subgroups, patients carried most frequently VOI in sarcomere genes suggesting them as a major contributor in pediatric primary CMP. Protein and transcript level analysis on heart biopsies from individuals with homozygous mutation of troponin I3, cardiac type (TNNI3) revealed that the TNNI3 protein was absent and associated with upregulation of the fetal isoform troponin I1, slow skeletal type (TNNI1). These observations support the clinical importance of sarcomeric mutation in primary CMP. TNNI3 is the third most important disease gene in pediatric CMP. The mutational spectrum of PRDM16 in an extended cohort of 285 patients with different types of heart muscle diseases revealed 16 VOI in total. These alterations occurred in six different cardiac diseases and comprised missense, splice site, frame shift, and stop gain variants. Pathogenic PRDM16 variants were only found in LVNC patients, confirming the important role of PRDM16 in LVNC pathogenesis. By prediction of posttranslational modification sites, structural features and functional interaction sites of the PRDM16 protein were identified. Functional and biochemical characterization of PRDM16 showed different subcellular localization of CMP variants and the influence of CMP variants on the protein stability of PRDM16. Patient specific CMP variants altered the cellular distribution of PRDM16, with condensed signals differing between the cytoplasm and the nucleus. In conclusion, the spectrum and number of genetic alteration in pediatric primary CMP determine clinical outcome. PDRM16 is a frequent CMP gene and translational approaches for potential therapies need to be developed.Primäre Kardiomyopathien (CMP) sind heterogene, genetisch determinierte Erkrankungen mit einer Vielzahl von krankheitsverursachenden Genen. Die zugrundeliegenden genetischen Mechanismen und früh vorhandenen pathologischen Vorgänge sind bei Kindern mit primärer CMP wenig untersucht. Diese Arbeit beschäftigt sich mit der Identifizierung von genetischen Ursachen der Kardiomyopathie des Kindesalters, insbesondere mit dem Auftreten von früh einsetzenden und schweren Krankheitsverläufen. Genetische Veränderungen von PR/SET domain 16 (PRDM16) führen zu zwei verschiedenen Arten von Kardiomyopathien, der dilatativen CMP (DCM) und der linksventrikulären Noncompaction CMP (LVNC). PRDM16 kodiert für einen transkriptionellen Regulator mit bis dahin unbekannter Funktion im Herzen. Achtzig nicht verwandte Indexpatienten mit pädiatrischer primärer CMP wurden mit einem panel-basierten next generation sequencing (NGS) Ansatz einer genetischen Testung auf Veränderungen in 89 Genen untersucht. Unerwünschte Ereignisse wie Herztransplantation und Tod waren bei Individuen mit DCM und restriktiver CMP höher als bei anderen CMP. Patienten mit einer höheren Anzahl von genetischen Varianten von Interesse (VOI) hatten ein höheres Risiko für ein unerwünschtes Ereignis. Bei 30/80 (38%) Indexpatienten wurde mindestens eine wahrscheinlich pathogene oder pathogene Variante identifiziert. Bei allen CMP Subtypen waren VOI in Sarkomergenen am häufigsten und sind somit für die Entstehung der pädiatrischen primären CMP hauptverantwortlich. Auf Protein- und mRNA Ebene von Herzbiopsien von Individuen mit homozygoter Variante konnte gezeigt werden, dass TNNI3 nicht nachweisbar war und stattdessen eine Hochregulation der fetalen Isoform TNNI1 stattfand. Diese Beobachtungen unterstützen die klinische Bedeutung von genetischen Veränderungen in Sarkomergenen bei primärer CMP. TNNI3 ist das dritthäufigste Krankheitsgen bei pädiatrischer CMP. Das Spektrum der VOI bei PRDM16 in einer erweiterten Kohorte von 285 Patienten mit verschiedenen Herzmuskelerkrankungen ergab insgesamt 16 verschiedene VOI. Diese Veränderungen traten bei sechs verschiedenen Herzerkrankungen auf und umfasste Punkt-Varianten, Spleiß-Varianten, Frameshift-Varianten und Stopp-Varianten. Pathogene PRDM16 Varianten wurden nur bei Patienten mit LVNC gefunden, was die Bedeutung von PRDM16 in der Entstehung der LVNC unterstreicht. Durch die Vorhersage von Stellen für posttranslationale Modifikationen wurden strukturelle Eigenschaften und funktionelle Interaktionsstellen des PRDM16 Proteins identifiziert. Die funktionelle und biochemische Charakterisierung von PRDM16 zeigte unterschiedliche subzelluläre Lokalisationen von CMP Varianten und auch die Stabilität vom PRDM16 wurde unterschiedlich durch die jeweiligen CMP Varianten beeinflusst. Patientenspezifische CMP Varianten veränderten die zelluläre VIII Verteilung von PRDM16 dahingehend, dass kondensierte Signale zwischen Zellkern und Zytoplasma jeweils differierten. Abschließend fanden wir, dass das Spektrum und die Anzahl der genetischen Veränderungen bei pädiatrischer primärer CMP für den klinischen Ausgang verantwortlich sind. PRDM16 ist ein häufiges Krankheitsgen für CMP und translationale Ansätze für mögliche Therapien sollten entwickelt werden

Induction of tetraploidy in zebrafish Danio rerio and Nile tilapia Oreochromis niloticus

The goals of this thesis were to: 1) induce tetraploidy in zebrafish; 2) characterize single-female spawning in Nile tilapia for egg collection; 3) design and construct hatching systems for tilapia eggs; 4) induce tetraploidy in Nile tilapia. Tetraploidy was induced in zebrafish Danio rerio, by applying a 41 C heat shock to eggs for 2 min at 11, 13, 15, 17, or 20 min after fertilization (AF). A trend of increasing percent tetraploid yield occurred the later the shocks were applied. Shocks applied at 11 min AF yielded 1% tetraploidy and shocks applied at 20 min AF yielded 10%. There were no significant differences (P = 0.20) in induction among treatments. This is the first report on induction of tetraploidy in zebrafish. Stocking of single-female Nile tilapia Oreochromis niloticus in ten 80-L aquaria yielded a reliable supply of eggs for polyploidy research conducted in this study. In one year, 72 spawns were recorded, yielding ~90,000 eggs. Peak spawning occurred in summer. The two egg hatching systems for artificial incubation of tilapia eggs proved to be effective, with 75% of control fish surviving to hatch. Methylene blue and ultraviolet sterilization reduced losses of eggs to disease and use of glass test tubes as hatching vessels allowed for constant rolling of the eggs and easy viewing and access. The systems occupied little space (\u3c 1 m2) and could accommodate up to 30 treatments of eggs. The use of egg baskets allowed for rapid and uniform heat shocking of multiple treatments of Nile tilapia eggs for polyploidy induction experiments. Tetraploidy and triploidy were induced by application of 42.8 ± 0.2 C heat shocks to eggs for 3 min at 22, 24, 26 and 28 min AF. Shocks applied at 24 min AF resulted in the highest tetraploid yield (10%) while shocks applied at 22 min AF resulted in the highest triploid yield (7%). This is the first study to report the simultaneous induction of tetraploidy and triploidy in Nile tilapia within treatments from a single spawn and raises questions about technical, maternal and genetic effects on synchrony of zygotic development in this species

On Simulating the Proton-Irradiation of O2_2 and H2_2O Ices Using Astrochemical-type Models, with Implications for Bulk Reactivity

Many astrochemical models today explicitly consider the species that comprise the bulk of interstellar dust grain ice-mantles separately from those in the top few monolayers. Bombardment of these ices by ionizing radiation - whether in the form of cosmic rays, stellar winds, or radionuclide emission - represents an astrochemically viable means of driving a rich chemistry even in the bulk of the ice-mantle, now supported by a large body of work in laboratory astrophysics. In this study, using an existing rate equation-based astrochemical code modified to include a method of considering radiation chemistry recently developed by us, we attempted to simulate two such studies in which (a) pure O$_2$ ice at 5 K and, (b) pure H$_2$O ice at 16 K and 77 K, were bombarded by keV H$^+$ ions. Our aims are twofold: (1) to test the capability of our newly developed method to replicate the results of ice-irradiation experiments, and (2) to determine in such a well-constrained system how bulk chemistry is best handled using the same gas-grain codes that are used to model the interstellar medium (ISM). We find that our modified astrochemical model is able to reproduce both the abundance of O$_3$ in the 5 K pure O$_2$ ice, as well as both the abundance of H$_2$O$_2$ in the 16 K water ice and the previously noted decrease of hydrogen peroxide at higher temperatures. However, these results require the assumption that radicals and other reactive species produced via radiolysis react quickly and non-diffusively with neighbors in the ice.Comment: ApJ, accepted. 30 pages, 5 figure

Bipolar jets produced by a spectroscopic binary

We present evidence that the spectroscopically identified bipolar jets of the pre-main sequence binary KH 15D are a common product of the whole binary system, rather than being launched from either star individually. They may be launched from the innermost part of the circumbinary disk (CBD) or may result from the merging of two outflows driven by the individual stars. This evidence is based on high-resolution H-alpha and [OI] 6300A line profiles obtained during eclipse phases of this nearly edge-on system. The occultation of star A (the only currently visible star) by the disk strongly suppresses the stellar H-alpha and continuum emission and allows one to study the faint redshifted and blueshifted emission components of the bipolar jets. The strongest evidence for jet production by the whole binary system comes from the observed radial velocity symmetry of the two jet components relative to the systemic velocity of the binary, in combination with current accretion models from the CBD onto a binary system.Comment: ApJ Letters, in press [6 pages

Natural Coronagraphic Observations of the Eclipsing T Tauri System KH 15D: Evidence for Accretion and Bipolar Outflow in a WTTS

We present high resolution (R $\sim$ 44,000) UVES spectra of the eclipsing pre-main sequence star KH 15D covering the wavelength range 4780 to 6810 {\AA} obtained at three phases: out of eclipse, near minimum light and during egress. The system evidently acts like a natural coronagraph, enhancing the contrast relative to the continuum of hydrogen and forbidden emission lines during eclipse. At maximum light the H$\alpha$ equivalent width was $\sim$2 {\AA} and the profile showed broad wings and a deep central absorption. During egress the equivalent width was much higher ($\sim$70 {\AA}) and the broad wings, which extend to $\pm$ 300 km/s, were prominent. During eclipse totality the equivalent width was less than during egress ($\sim$40 {\AA}) and the high velocity wings were much weaker. H$\beta$ showed a somewhat different behavior, revealing only the blue-shifted portion of the high velocity component during eclipse and egress. [OI] $\lambda\lambda$6300, 6363 lines are easily seen both out of eclipse and when the photosphere is obscured and exhibit little or no flux variation with eclipse phase. Our interpretation is that KH 15D, although clearly a weak-line T Tauri star by the usual criteria, is still accreting matter from a circumstellar disk, and has a well-collimated bipolar jet. As the knife-edge of the occulting matter passes across the close stellar environment it is evidently revealing structure in the magnetosphere of this pre-main sequence star with unprecedented spatial resolution. We also show that there is only a small, perhaps marginally significant, change in the velocity of the K7 star between the maximum light and egress phases probed here

Effects of Social Policy Reforms and the Economy on Welfare Participation and Employment Among Single Mothers

Child care subsidies and the Earned Income Tax Credit (EITC) are vital government tools for increasing employment and reducing poverty among low-income families. This dissertation, therefore, explores many features of these policies, including their evolution, correlates of participation, and impacts on employment. Chapter 1 provides an overview of child care subsidies and the EITC, focusing on recent policy developments, labor supply incentives, and a critical review of the empirical employment literature. Chapter 2 explores why, despite substantial growth in funding, participation in child care subsidy programs remains comparatively low. Results suggest that although 30 percent of households with children are eligible for child care subsidies, take-up is 14 percent. The low take-up rate is driven by several factors: eligible non-recipients differ from recipients in ways that make subsidies unnecessary or undesirable; the practice by states to trade-off generosity in eligibility for additional generosity in benefits; and the practice by states to ration benefits according to specific household characteristics. Chapter 3 examines the effects of child care costs and net-of-taxes wages on the employment of single mothers. Although a substantial literature estimates separately the impact of prices and taxes, no study has created a modeling framework that accounts for both factors simultaneously. Merging empirical techniques from previous child care and EITC studies yields employment elasticities of -0.174 and 0.711, respectively. An implication of this finding is that price-effects are considerably smaller than those reported elsewhere, while tax-effects accord with previous estimates. Results also suggest that single mothers became less responsive to prices and more responsive to taxes throughout the 1990s, especially after expansions to subsidy programs and the EITC. Chapter 4 investigates heterogeneous employment effects of social policy reforms across varying economic conditions. Allowing the effects of policy reforms on single mothers to vary with the economy leads to several interesting results. Policy "carrots" are more likely to reveal heterogeneous effects at low intensity work margins, while policy "sticks" show significant variation at increasingly demanding margins. However, all policies produce the largest employment effects in favorable economic conditions, implying that a strong economy reinforces the incentives created by social policy reforms

The Performance of Health Workers

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