National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2015 Annual Report of the Division of Intramural Research

Program on Developmental Endocrinology and Genetics

Director: Forbes D. Porter, MD, PhD

The primary mission of the Program on Developmental Endocrinology and Genetics (PDEGEN) is to carry out basic, translational, and clinical research focusing on endocrine and genetic disorders. Research conducted in the PDEGEN includes projects designed to improve our understand and treatment of childhood growth disorders, glycogen storage diseases, disorders of puberty and reproduction, lysosomal storage diseases, disorders of drug metabolism, inborn errors of cholesterol synthesis, endocrine tumors, and childhood obesity. A second goal of the PDEGEN is to provide training in these areas of research in order to help educate the next generation of scientists. The education is provided both to clinical science trainees participating in an accredited pediatric endocrinology fellowship training program and to basic science trainees in a variety of disciplines.

Jeffrey Baron's Section on Growth and Development investigates the cellular and molecular mechanisms governing childhood growth and development. The basic research performed in this Section focuses on the mechanisms that allow rapid cell proliferation and body growth in young children and that subsequently suppress proliferation, causing body growth to slow and eventually halt by adulthood. The group discovered that juvenile growth deceleration is attributable in part to a multi-organ genetic program, which involves the down-regulation of a large set of growth-promoting genes. Recent studies focused on the molecular mechanisms orchestrating this program. Elucidating such growth-limiting mechanisms not only provides insight into childhood growth disorders but also has broader medical applications because disruption of these regulatory systems contributes to oncogenesis; conversely, transient therapeutic suspension of these regulatory systems in adult cells might be used to achieve tissue regeneration. For the Section’s clinical research, one major focus involves the use of high-throughput sequencing and other molecular-genetic approaches to study patients with growth disorders, including both growth failure and overgrowth.

Janice Chou's Section on Cellular Differentiation conducts research to delineate the pathophysiology of glycogen storage disease type Ia (GSD-Ia), deficient in G6Pase-alpha (or G6PC), GSD-Ib, deficient in the glucose-6-phosphate transporter (G6PT or SLC37A4), and glucose-6-phosphatase-beta (G6Pase-beta or G6PC3) deficiency, and to develop gene therapies for these disorders. Chou's group showed that neutrophils express the G6PT/G6Pase-beta complex and that inactivation of G6PT or G6Pase-beta leads to the enhanced neutrophil apoptosis that underlies the neutropenia in G6Pase-beta deficiency and in GSD-Ib. Chou's group further showed that G6Pase-beta is essential for energy homeostasis in neutrophils and macrophages. G6Pase-beta deficiency prevents recycling of glucose from the endoplasmic reticulum to the cytoplasm, leading to neutrophil/macrophage dysfunction. More recently, Chou's group showed that the mechanism of neutrophil dysfunction in GSD-Ib arises from activation of the HIF-1alpha/PPAR-gamma pathway. Chou's Section also developed mouse models of GSD-Ia, GSD-Ib, and G6Pase-beta deficiency. Using GSD-Ia mice, the Section developed an adeno-associated virus (AAV) vector–mediated gene transfer that corrects hepatic G6Pase-alpha deficiency and prevents chronic hepatocellular adenoma formation. The group further show that AAV–treated mice expressing 3% or more of normal hepatic G6Pase-alpha activity do not develop age-related obesity or insulin resistance. The study suggests that full restoration of normal G6Pase-alpha activity would not be required to confer significant therapeutic benefits in GSD-Ia by gene therapy. The AAV vector developed by Chou's group is the leading candidate in clinical trials for the treatment of human GSD-Ia.

Angela Delaney's Unit on Genetics of Puberty and Reproduction investigates the mechanisms responsible for pubertal onset in children. In collaboration with the Reproductive Endocrine Unit (REU) at the Massachusetts General Hospital, and under the mentorship of William Crowley, one of the world's leading experts on disorders of gonadotropin-releasing hormone (GnRH) secretion, Delaney is conducting translational research on the neuroendocrine and genetic control of GnRH secretion and its regulation of gonadotropin secretion and gonadal physiology. The collaboration aims to phenotypically and genetically characterize subjects with isolated hypogonadotropic hypogonadism (IHH) and other, more common disorders of puberty. The Unit is using insights gained from the investigation of this clinically and genetically heterogeneous group of patients to explore the biological pathways that may contribute to the reactivation of GnRH secretion at puberty. In collaboration with the REU, a variety of molecular techniques are used to further characterize the known genetic defects causing IHH, as well as to identify new genes responsible for the regulation of pubertal onset. Such approaches will help define the developmental physiology of pubertal development in order to gain a deeper understanding of human disorders of puberty and reproduction. Additional clinical studies are aimed at exploring neurocognitive and other outcomes of delayed exposure to sex hormones at or before the normal time of puberty.

Maria Dufau’s Section on Molecular Endocrinology investigates the molecular basis of hormonal regulation of gonadal function, focusing on: (1) modes of transcriptional repression and derepression of receptors for human luteinizing hormone (LHR); (2) functions of novel short prolactin receptor (PRLR) inhibitory forms, identified by the Section, in physiological regulation and cancer and modalities of transcriptional regulation/expression of PRLR in breast cancer; (3) mechanisms involved in Leydig cell function and intracrine and paracrine androgen actions in the progression of spermatogenesis, and the regulation/functions of GRTH/DDX25, an androgen-regulated RNA helicase, essential for spermatogenesis and discovered by this group. The Section revealed the essential role of Positive Coactivator 4 (PC4), which associates with Sp1 at the LHR promoter, in the formation/assembly of the pre-initiation complex in LHR transcription. The Section found that histone 3 (H3) is recruited to the PC4 complex during the activation state of the receptor and that, in the complex, H3 is in an acetylated form. The sites of H3 acetylation and the impact of the PC4:acH3 complex on chromatin structure are under study. Other work demonstrated conformational determinants required for the inhibitory action of the PRLR short form (SF) on prolactin-induced signaling through the long form (LF). Studies revealing the essential role of the D1 domain of the PRLR on the SF configuration for its inhibitory action on LF–mediated function provide the basis for developing drugs of potential use in the treatment of advanced breast cancer. Also, the group provided direct evidence for local actions of prolactin independent of estradiol, with participation of the estrogen receptor in up-regulation of PRLR transcription/expression in breast cancer cells, which is of relevance to states that are refractory to therapy with aromatase inhibitors. Gonadotropin-regulated testicular helicase (GRTH/Ddx25), present in Leydig and meiotic/haploid germ cells, is a multifunctional protein that participates in nuclear transport of specific messages essential for the progress of spermatogenesis and protects Leydig cells from gonadotropin-mediated overstimulation of androgen. The group established the first connection between paracrine actions of androgen and two relevant germ cell genes essential for spermatogenesis: Germ Cell Nuclear Factor (GCNF) and GRTH/Ddx25. A transgenic animal model developed in the laboratory offers insights for the development of a male contraceptive based on the indirect blockade of the actions of androgens on GRTH expression in germ cells without affecting other aspects of androgen action.

Anil Mukherjee's Section on Developmental Genetics conducts both laboratory and clinical investigations into those hereditary neurodegenerative lysosomal storage disorders (LSDs) mostly affecting children. Current research in this Section focuses on the molecular mechanism(s) of pathogenesis of a group of neurodegenerative LSDs called neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease. Mutations in at least 13 different genes (called CLNs) cause various types of NCLs. At present, there is no effective treatment for any of the NCL types. The infantile NCL (or INCL) is an autosomal recessive LSD caused by mutations in the CLN1 gene, which encodes the lysosomal depalmitoylating enzyme palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes the cleavage of thioester linkage in palmitoylated (S-acylated) proteins (constituent of ceroid), facilitating their degradation in lysosomes. Thus, PPT1 deficiency causes accumulation of ceroid in lysosomes, leading to INCL. Recently, Mukherjee and his colleagues concluded a bench-to-bedside clinical trial using a combination of cysteamine bitartrate and N-acetylcysteine. More recently, the group identified a thioesterase-mimetic small molecule, N-tert-butyl hydroxylamine, which arrests neuropathology and extends lifespan in a mouse model of INCL and is thus a potential drug for treating INCL. To conduct these studies, the Section uses gene knock-out and knock-in technologies as well as biochemical, molecular, and neurobiological techniques. Mechanism-based translational research is emphasized.

Ida Owens' Section on Genetic Disorders of Drug Metabolism studies the biology of the endoplasmic reticulum (ER)–based UDP-glucuronosyltransferase (UGT) isozymes. The Section's studies have shown that UGT isozymes convert lipophilic endogenous and exogenous substrates such as dietary aromatic-like therapeutics, environmental pro-carcinogens, and contaminants derived from pyrolysates to water-soluble excretable, non-toxic glucuronides; neurotoxic bilirubin is the most important endogenous substrate, followed by genotoxic catechol estrogens and elevated levels of dihydrotestosterone (DHT). Previously, the group discovered that each UGT isozyme examined requires protein kinase C–based phosphate signaling at an Src (a tyrosine kinase that is an inhibitor of apoptosis) active site that controls broad chemical detoxification of various chemical subtypes. Using Src-free cells, group discovered that the human prostate luminal-cell UGT-2B15, with IYG at amino-acid positions 98–100, is also constantly undergoing Src–based phosphorylation signaling to prevent ER–based apoptosis. By contrast, the prostate basal cell UGT-2B17, with the rare TYS at amino-acid positions 98–100, undergoes increased activity in Src–free cells. Exchange of IYG and TYS between the two isozymes at amino-acid positions 98–100 leads to the exchange of these activities. Hence, caspase-based loss of Src activity at position 98–100 leads to apoptosis for the resident luminal cells. Transfection studies indicate that the IYG Src site in UGT-2B15 elicits apoptosis more easily than the consensus IYA Src site. Human cancerous PC3 prostate cells transfected with UGT-2B15 were far less able to generate apoptotic activity than normal COS-1 cells transfected with UGT-2B15 or transfected LNCaP cells.

Forbes D. Porter's Section on Molecular Dysmorphology studies a group of human and mouse malformation syndromes attributable to inborn errors of cholesterol synthesis. The most common of these disorders is the Smith-Lemli-Opitz syndrome (SLOS). The Section studies both basic science and clinical aspects of SLOS, with the goal of developing and testing therapeutic interventions for SLOS. The Section also studies basic and clinical aspects of Niemann-Pick disease, type C (NPC). The group has maintained an ongoing Natural History trial for NPC and SLOS since 2006 and 1998 respectively. The NPC Natural History trial was designed to investigate biochemical markers and clinical aspects of NPC that could be used as outcome measures in a future clinical trial. In collaboration with the National Center for Translational Medicine, the Section is completing a Phase 1/2 trial of intrathecal 2-hydroxypropyl-β-cyclodextrin in NPC1 subjects and is now involved in a multicenter, multinational Phase 2b/3 trial to establish clinical efficacy in a controlled trial. In collaboration with extramural investigators, the Section was awarded a U01 grant to evaluate the safety and efficacy of histone deacetylase inhibitor therapy in NPC1 subjects.

Constantine Stratakis' Section on Endocrinology and Genetics investigates the genetic and molecular mechanisms leading to disorders affecting the pituitary gland and adrenal cortex, with emphasis on those that are developmental, hereditary, and associated with other conditions. The laboratory has made significant discoveries over the last two decades. The first studies led to the identification of the main regulator of the cAMP signaling pathway, regulatory subunit-type 1A (R1a) of protein kinase A (PKA, encoded by the PRKAR1A gene on chromosome 17q22-24), as responsible for primary pigmented nodular adrenocortical disease (PPNAD) and the Carney complex, a multiple endocrine neoplasia (MEN) whose main endocrine manifestation is PPNAD. Stratakis then described isolated micronodular adrenocortical disease (iMAD), a disorder likely to be inherited in an autosomal dominant manner and unrelated to the Carney complex or to other MENs. In 2006, a genome-wide association (GWA) study led to the identification of mutations in the phosphodiesterases (PDE) PDE11A, a dual specificity PDE, and PDE8B, a cAMP–specific PDE (encoded by the PDE11A and PDE8B genes, respectively) in iMAD. Stratakis also studied primary macronodular adrenocortical hyperplasia (PMAH) and, as part of this work, a new gene was identified (ARMC5), which, when mutated, causes more than a third of the known PMAH cases. Members of the laboratory are now characterizing mouse, fruit fly, and fish models of ARMC5. Recently, the laboratory identified genes encoding two other subunits of PKA as involved in endocrine tumors: PRKACA in various forms of bilateral adrenocortical hyperplasia and PRKACB in a form of Carney complex that is not associated with PRKAR1A mutations. Animal model studies are essential for the investigation and confirmation of each of the identified new genes in disease pathogenesis. Cells from tumors or other lesions from animals with R1a deficiency showed elevated beta-catenin expression and/or aberrant Wnt signaling and similarities to adult stem cells or cancer stem cells in other models of dysregulated Wnt signaling. The laboratory continues to investigate the pathways involved in early events in tumor formation in the pituitary gland and the adrenal cortex and/or the tissues affected by germline or somatic defects of the cAMP/PKA and related endocrine signaling defects, employing animal models and transcriptomic and systems biology analyses. The group continues to accrue patients under several clinical protocols, identify unique patients and families with rare phenotypes such as Carney Triad, and/or explore (mostly on a collaborative basis) various aspects of endocrine and related diseases. Paramount to these investigations is the availability of modern genetic tools such as copy number variation (CNV) analysis, comparative genomic hybridization (CGH), whole-exome sequencing (WES), and DNA sequencing (DSeq). As part of these clinical protocols, much clinical research is also being done, consisting mostly of observations of new associations, description of novel applications or modifications, and improvements in older diagnostic methods, tests, or imaging tools. The most recent discovery of the laboratory was the identification of the defect that explains the vast majority of cases of early pediatric overgrowth or gigantism: Stratakis and his group identified the gene GPR101, which encodes a G protein–coupled receptor, which was overexpressed in patients with elevated growth hormone (GH). Patients with GPR101 defects have a condition that Stratakis named X-LAG for X-linked acrogigantism caused by Xq26.3 genomic duplication and characterized by early-onset gigantism resulting from excess GPR101 function. Employing a variety of molecular techniques and animal models, ongoing work focuses on GPR101 ligands and molecular targets.

Jack Yanovski's Section on Growth and Obesity studies metabolic and behavioral factors involved in body weight regulation and body composition during childhood in an effort to develop etiology-specific prevention and treatment approaches for pediatric obesity. The Section has conducted multiple randomized clinical trials in adults and children involving pharmacotherapy and behavior as well as investigations into genetic causes of obesity. The Section’s use of laboratory feeding paradigms has allowed it to conduct quantitative assessments of psychological constructs such “loss of control over eating” and “eating in the absence of hunger” in a reproducible fashion so that treatments directed at improving specific behaviors can be studied. The Section's recent work on the roles of appetite-regulating genes has focused on the melanocortin 3 receptor and brain-derived neurotrophic factor, elucidating the roles of these factors for human obesity by studying individuals with rare genetic abnormalities, such as the WAGR Syndrome, individuals with non-syndromic obesity, and murine models of gene dysfunction. Ongoing studies attempt to identify factors that predispose children to hyperphagic behaviors, including binge eating and related disorders, and to use this knowledge to develop rational, defect-specific prevention and treatment strategies for pediatric obesity.

In addition to research groups, PDEGEN also supports the Pediatric Endocrine Fellowship Program led by Constantine Stratakis and Maya Lodish. The fellowship in Pediatric Endocrinology is a three-year ACGME–accredited program providing comprehensive training in clinical patient management and guidance in the development of research skills. The NICHD program is based at one of the largest and most sophisticated research institutions in the United States, the NIH Clinical Center, which maintains clinical research protocols investigating the treatment of adrenal and pituitary tumors, congenital adrenal hyperplasia, precocious puberty, idiopathic juvenile osteoporosis, Cushing's syndrome, obesity, and others. Other institutions that participate in this training program include The Johns Hopkins University (JHU) Department of Pediatrics, Division of Pediatric Endocrinology; The Children's National Medical Center (CNMC), Division of Pediatric Endocrinology; and the co-sponsoring institution, Georgetown University (GU), Department of Pediatrics. The facilities make available to the fellows pediatric endocrine, diabetes, oncology, metabolic, bone disorders, and other pediatric subspecialty clinics and consult services, as well as general pediatric inpatient and intensive care units.

Top of Page