Identification of biologically-active PDE11-selective inhibitors using a yeast-based high throughput screen

Thesis advisor: Charles S. HoffmanThe biological roles of the most recently discovered mammalian cyclic nucleotide phosphodiesterase (PDE) family, PDE11, are poorly understood, in part due to the lack of selective inhibitors. To address this need for such compounds I completed a ~200,000 compound high throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay. Further characterization of lead candidates using both growth-based assays in the fission yeast Schizosaccharomyces pombe and in vitro enzyme assays identified four potent and selective PDE11 inhibitors. I examined the effect of these compounds on human adrenocortical cells, where PDE11 is believed to regulate cortisol levels. One compound, along with two structural analogs, elevates cAMP levels and cortisol production through PDE11 inhibition, thus phenocopying the behavior of adrenocortical tumors associated with Cushing syndrome. These compounds can be used as research tools to study the biological function of PDE11, and can also serve as leads to develop therapeutic compounds for the treatment of adrenal insufficiencies. This study further validates the yeast-based HTS platform as a powerful tool for the discovery of potent, selective and biologically-active PDE inhibitors.Thesis (PhD) — Boston College, 2012.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Whole Exome Sequencing Reveals DYSF, FKTN, and ISPD Mutations in Congenital Muscular Dystrophy Without Brain or Eye Involvement

Background: Congenital muscular dystrophies (CMDs) are a genetically and clinically heterogeneous group of neuromuscular disorders. Several genes encoding extracellular matrix, nuclear envelope, sarcolemmal proteins and glycosylation enzymes have been implicated in CMDs. The large overlap of clinical presentations due to mutations in different genes poses a challenge for clinicians in determining disease etiology for each patient. Objective: We investigated the use of whole exome sequencing (WES) in identifying the genetic cause of disease in 5 CMD patients from 3 families who presented with highly similar clinical features, including early-onset rapidly progressive weakness without brain or eye abnormalities. Methods: Whole exome sequencing was performed on DNA from affected individuals. Potential functional impacts of mutations were investigated by immunostaining on available muscle biopsies. Results: Pathogenic mutations in 3 different genes, DYSF, FKTN, and ISPD were identified in each family. Mutation in DYSF led to absence of dysferlin protein in patient muscle. Mutations in ISPD led to impaired ISDP function, as demonstrated by deficiency of α-dystroglycan glycosylation in patient muscle. Conclusions: This study highlights the benefit of unbiased genomic approaches in molecular diagnosis of neuromuscular disorders with high clinical heterogeneity, such as the phenotypes observed in our patients. Our results suggest that dysferlin deficiency should be in the differential diagnosis of congenital and rapidly progressive muscular dystrophy, and therefore dysferlin antibody should be in the standard immunohistochemistry panel for muscle biopsies in cases with suspected CMD

Evaluating the Clinical Validity of Gene-Disease Associations: An Evidence-Based Framework Developed by the Clinical Genome Resource

Supplemental Data Supplemental Data include 65 figures and can be found with this article online at http://dx.doi.org/10.1016/j.ajhg.2017.04.015. Supplemental Data Document S1. Figures S1–S65 Download Document S2. Article plus Supplemental Data Download Web Resources ClinGen, https://www.clinicalgenome.org/ ClinGen Gene Curation, https://www.clinicalgenome.org/working-groups/gene-curation/ ClinGen Gene Curation SOP, https://www.clinicalgenome.org/working-groups/gene-curation/projects-initiatives/gene-disease-clinical-validity-sop/ ClinGen Knowledge Base, https://search.clinicalgenome.org/kb/agents/sign_up OMIM, http://www.omim.org/ Orphanet, http://www.orpha.net/consor/cgi-bin/index.php With advances in genomic sequencing technology, the number of reported gene-disease relationships has rapidly expanded. However, the evidence supporting these claims varies widely, confounding accurate evaluation of genomic variation in a clinical setting. Despite the critical need to differentiate clinically valid relationships from less well-substantiated relationships, standard guidelines for such evaluation do not currently exist. The NIH-funded Clinical Genome Resource (ClinGen) has developed a framework to define and evaluate the clinical validity of gene-disease pairs across a variety of Mendelian disorders. In this manuscript we describe a proposed framework to evaluate relevant genetic and experimental evidence supporting or contradicting a gene-disease relationship and the subsequent validation of this framework using a set of representative gene-disease pairs. The framework provides a semiquantitative measurement for the strength of evidence of a gene-disease relationship that correlates to a qualitative classification: “Definitive,” “Strong,” “Moderate,” “Limited,” “No Reported Evidence,” or “Conflicting Evidence.” Within the ClinGen structure, classifications derived with this framework are reviewed and confirmed or adjusted based on clinical expertise of appropriate disease experts. Detailed guidance for utilizing this framework and access to the curation interface is available on our website. This evidence-based, systematic method to assess the strength of gene-disease relationships will facilitate more knowledgeable utilization of genomic variants in clinical and research settings

Newborn Sequencing in Genomic Medicine and Public Health

The rapid development of genomic sequencing technologies has decreased the cost of genetic analysis to the extent that it seems plausible that genome-scale sequencing could have widespread availability in pediatric care. Genomic sequencing provides a powerful diagnostic modality for patients who manifest symptoms of monogenic disease and an opportunity to detect health conditions before their development. However, many technical, clinical, ethical, and societal challenges should be addressed before such technology is widely deployed in pediatric practice. This article provides an overview of the Newborn Sequencing in Genomic Medicine and Public Health Consortium, which is investigating the application of genome-scale sequencing in newborns for both diagnosis and screening

Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy

Peer reviewe

Universal germline genetic testing in patients with hematologic malignancies using DNA isolated from nail clippings

Not available

A Three-Tier Diagnostic Test to Assess Pre-Service Teachers' Misconceptions about Global Warming, Greenhouse Effect, Ozone Layer Depletion, and Acid Rain

This study describes the development and validation of a three-tier multiple-choice diagnostic test, the atmosphere-related environmental problems diagnostic test (AREPDiT), to reveal common misconceptions of global warming (GW), greenhouse effect (GE), ozone layer depletion (OLD), and acid rain (AR). The development of a two-tier diagnostic test procedure as described by Treagust constitutes the framework for this study. To differentiate a lack of knowledge from a misconception, a certainty response index is added as a third tier to each item. Based on propositional knowledge statements, related literature, and the identified misconceptions gathered initially from 157 pre-service teachers, the AREPDiT was constructed and administered to 256 pre-service teachers. The Cronbach alpha reliability coefficient of the pre-service teachers' scores was estimated to be 0.74. Content and face validations were established by senior experts. A moderate positive correlation between the participants' both-tiers scores and their certainty scores indicated evidence for construct validity. Therefore, the AREPDiT is a reliable and valid instrument not only to identify pre-service teachers' misconceptions about GW, GE, OLD, and AR but also to differentiate these misconceptions from lack of knowledge. The results also reveal that a majority of the respondents demonstrated limited understandings about atmosphere-related environmental problems and held six common misconceptions. Future studies could test the AREPDiT as a tool for assessing the misconceptions held by pre-service teachers from different programs as well as in-service teachers and high school students

Protective effects of melatonin on female rat ovary treated with nonylphenol

We investigated using histochemistry and immunohistochemistry ovarian damage caused by nonylphenol (NP) and the protective effect of melatonin treatment of NP induced ovarian damage. We used 21 female rats divided randomly into three groups: control, NP and melatonin + NP. Histopathological examination of the ovaries, and counting and classification of follicles were performed using Masson's trichrome staining. Expression of anti-Mullerian hormone (AMH), Bax, Bcl-2 and caspase-3 was detected in the ovaries using immunohistochemistry. Melatonin had an ameliorative effect on NP induced follicular atresia and absence of corpora lutea. More follicles were observed in the ovaries of animals treated with melatonin prior to treatment with NP. AMH immunoreactivity was significantly lower in the NP group than in the melatonin + NP group. NP increased immunostaining for Bax, Bcl-2 and caspase-3. Melatonin significantly reduced the increased expression of Bax, Bcl-2 and caspase-3 due to NP exposure. We found that pretreatment with melatonin is beneficial for protecting the ovaries from damage by NP

Fumarate hydratase c.914T > C (p.Phe305Ser) is a pathogenic variant associated with hereditary leiomyomatosis and renal cell cancer syndrome

BackgroundHereditary leiomyomatosis and renal cell cancer syndrome (HLRCC), caused by heterozygous germline pathogenic variants in the FH, confers an increased risk for cutaneous and uterine leiomyomas and renal cancer.MethodsAbout 13,722 advanced cancer patients, including 560 with renal cell carcinoma, had germline analysis performed in the context of tumor-normal sequencing under an IRB approved protocol.ResultsWe report two unrelated individuals with early onset kidney cancer who both carried the c.914C > T (p.Phe305Ser) germline variant in the FH. Both tumors exhibited loss of FH staining by immunohistochemistry and/or positive 2SC staining. Subsequent familial testing discovered that a daughter of a proband who carried the variant had both cutaneous and uterine leiomyomas.ConclusionThis combination of evidence suggests that the FH c.914C > T (p.Phe305Ser) is pathogenic for HLRCC