Edition 71 - December 2018 / Subject Review / Uncategorized

Subject Review – Ed. 71

Marco A. Rivarola y Alicia Belgorosky. Servicio de Endocrinología, Hospital de Pediatría Garrahan, Buenos Aires, Argentina. 

For the Subject Review Section of this on-line issue of Endopedonline, we have selected some abstracts of the Plenary Conferences (PL) and Symposia (S) delivered at the European Society for Paediatric Endocrinology (ESPE) 2018, during the 57th ESPE Meeting, in Athens (Greece). These free-access abstracts have been recently published in Hormone Research in Paediatrics, 2018; 90 (Suppl 1).

PL1. Oncofertility: From Bench to Bedside to Babies.
Teresa Woodruff. Northwestern University, Chicago, USA. 

Facing a cancer diagnosis at any age is devastating. However, young cancer patients have the added burden that life-preserving cancer treatments, including surgery, chemotherapy, and radiotherapy, may compromise their future fertility. The possibility of reproductive dysfunction as a consequence of cancer treatment has a negative impact on the quality of life of cancer survivors. The field of oncofertility, which merges the clinical specialties of oncology and reproductive endocrinology, was developed to explore and expand fertility preservation options and to better manage the reproductive status of cancer patients. Fertility preservation for females has proved to be a particular challenge because mature female gametes are rare and difficult to acquire. The purpose of this presentation is to provide a comprehensive overview of how cancer treatments affect the female reproductive axis, delineate the diverse fertility preservation options that are currently available or being developed for young women, and describe current measures of ovarian reserve that can be used pre- and post-cancer treatment.

PL2. Oxytocin and the Healing Power of Love.
Sue Carter. Indiana University, Bloomington, IN, USA

This presentation discussed the hormonal and neural mechanisms that support the beneficial and healing effects of loving relationships. Love is deeply biological and has profound effects on our mental and physical health, pervading every aspect of our lives. Without loving relationships or in isolation, humans fail to flourish, even if all of their other basic needs are met. Two neuropeptides, oxytocin and the related molecule, vasopressin, and their receptors, form an integrated system that is at the heart of the biology of love and attachment. These peptides also help to explain the consequences of positive or negative relationships. The evolution of oxytocin allowed human evolution. Embedded in this system are neuroendocrine processes that regulate a sense of fear or safety across the life cycle. These in turn permit social cognition, social bonding, social support, growth and restoration. Oxytocin and vasopressin interact to regulate the functions of the autonomic nervous system, with effects on vagal and sympathetic pathways. Oxytocin also has direct antioxidant and anti-inflammatory consequences for tissues throughout the body. The oxytocin system is influenced by early experience, and oxytocin can epigenetically alter the expression of its own receptors. The capacity of oxytocin to regulate these systems helps to explain the pervasive adaptive consequences of social experiences for emotional and physical health across the lifespan. Knowledge of the pathways through which oxytocin and vasopressin act offers a new perspective on the healing power of love.

PL5. Dynamic Control of Tissue Glucocorticoids – Lessons for Optimizing Replacement Therapy. 
Brian Walker. Newcastle University, Newcastle upon Tyne, UK.

Although endocrinologists have focused for decades on circulating concentrations of cortisol, arguably the important concentrations are those within tissues which are available at corticosteroid receptors. Tissue concentrations are influenced by plasma proteins binding cortisol, by physicochemical characteristics of the steroid, by active transport across cell membranes, and by steroid metabolism within target tissues. Many of these factors vary between individuals, and within individuals according to nutritional and inflammatory status. For example, common variants at the locus encoding corticosteroid binding globulin (CBG) make a genetic contribution to variations in plasma cortisol, affecting CBG stability and affinity for cortisol and thereby potentially altering the tissue delivery of cortisol. Moreover, cortisol metabolism within tissues varies substantially after feeding and during acute illness; indeed, tissue regeneration of cortisol by 11ß-HSD1 has provided a target for therapeutic manipulation of tissue cortisol levels independently of circulating levels. These variations reinforce the unmet need for monitoring of glucocorticoid effects which extends beyond measurement of blood steroid concentrations. Recently, the authors discovered differences between tissue-specific control of cortisol and corticosterone, the other endogenous glucocorticoid in humans. Using stable isotope tracers they found rapid exchange between free and bound cortisol pools in plasma, and between plasma and brain or liver cortisol pools, but very slow exchange between plasma and adipose tissue, consistent with substantial buffering of ultradian and circadian cortisol rhythms within adipose. They attributed this to tissue-specific expression of ABCB1, an ATP-binding cassette transporter, which exports cortisol from brain but not adipose tissue. However, in adipose tissue they showed that an alternative transporter, ABCC1, exports corticosterone and not cortisol. Consistent with these findings, in Addison’s disease they showed that ACTH suppression is relatively resistant to cortisol while adipose tissue transcript induction is relatively resistant to corticosterone. Development of corticosterone as a novel replacement therapy may therefore allow adequate suppression of ACTH, for example in congenital adrenal hyperplasia, without adverse effects that are mediated in adipose tissue such as obesity and metabolic dysfunction.

PL6. Personalizing Treatments Using Microbiome and Clinical Data.
Eran Segal. Weizmann Institute, Rehovot, Israel.
Accumulating evidence supports a causal role for the human gut microbiome in obesity, diabetes, metabolic disorders, cardiovascular disease, and numerous other conditions. The author presented their research on the role of the human microbiome in health and disease, ultimately aimed at developing personalized medicine approaches that combine human genetics, microbiome, and nutrition. In one project, they tackled the subject of personalization of human nutrition, using a cohort of over 1,000 people in which they measured blood glucose response to >50,000 meals, lifestyle, medical and food frequency questionnaires, blood tests, genetics, and gut microbiome. They showed that blood glucose responses to meals greatly vary between people even when consuming identical foods; devised the first algorithm for accurately predicting personalized glucose responses to food based on clinical and microbiome data; and showed that personalized diets based on this algorithm successfully balanced blood glucose levels in prediabetic individuals. Using the same cohort, they also studied the relative contribution of host genetics and environmental factors in shaping human gut microbiome composition. Notably, although their cohort consists of individuals from several distinct ancestral origins who share a relatively common environment, the found no association between microbiome and genetic ancestry. In contrast, they show that over 20% of the gut microbiome variance can be explained by environmental factors related to diet, drugs and anthropometric measurements. They further showed that 24-36% of the variance of several human traits and disease risk factors can be explained by the microbiome even after accounting for the contribution of human genetics. These results suggest that human microbiome composition is dominated by environmental factors rather than by host genetics. Finally, an algorithm that was devised for identifying variability in microbial sub-genomic regions was presented. They found that such Sub-Genomic Variations (SGV) are prevalent in the microbiome across multiple microbial phyla, and that they are associated with bacterial fitness and their member genes are enriched for CRISPR associated and antibiotic producing functions and depleted from housekeeping genes. They found over 100 novel associations between SGVs and host disease risk factors and uncovered possible mechanistic links between the microbiome and its host, demonstrating that SGVs constitute a new layer of meta-genomic information.

Turner Syndrome: New Insights from Prenatal Genomics and Transcriptomics
Diana Bianchi. National Institutes of Health, Bethesda, Maryland, USA

Prior to the clinical and commercial introduction of noninvasive prenatal testing (NIPT) by sequencing of maternal plasma cell-free DNA in 2011, most fetuses with Turner syndrome were detected by sonographic findings related to lymphedema, or incidentally. NIPT, however, has transformed prenatal genetic screening, and an estimated 4–6 million tests have been performed worldwide. In the maternal plasma sample there is both maternal and placental cell-free DNA. Following a screen positive NIPT result, it is universally recommended to confirm the screening test by a diagnostic procedure such as amniocentesis or chorionic villus sampling (CVS) Health pregnancy test. While NIPT performance is excellent for trisomy 21 (positive predictive value [PPV] w91%), it is less so for 45, X (PPV w25%). The reasons for the relatively high number of false positive results include high rates of confined placental mosaicism, demise of a co-twin, and maternal incidental findings. The mother can have constitutional mosaicism for 45, X, or somatic mosaicism resulting from physiologic X-chromosome loss due to ageing. At present there is a significant knowledge gap as to how to clinically manage pregnant women ascertained through NIPT to have 45, X mosaicism. Transcriptomic analyses of cell-free RNA from living mid-trimester fetuses with 45, X demonstrate a consistent and unique pattern of gene expression. As expected, XIST is significantly down-regulated. Dysregulated genes of interest include NFATC3 (Nuclear Factor of Activated T Cells 3, expressed in 22 tissues, including testis, lung, lymph node and skin), LDLR (Low Density Lipoprotein Receptor; Mutations in this gene cause the autosomal dominant disorder, familial hypercholesterolemia), and IGFBP5 (insulin like growth factor binding protein 5, Broad expression in ovary, endometrium and 14 other tissues). These three genes are involved in perivascular tissue remodeling, hyperlipidemia, and growth and fertility, respectively. Using a dysregulated pathway approach, novel treatments could be developed that could be given ante-nataly to a pregnant woman carrying a fetus known to have 45, X. Prenatal screening for Turner syndrome creates ethical challenges for the fetus and mother, yet it also provides novel opportunities for treatments to prevent infertility, short stature and cardiovascular disease.

Molecular Basis of Pituitary Hormone Deficiency: From Mouse to Man and Back.
Sally Camper. University of Michigan, Ann Arbor, Michigan, USA.

Hypopituitarism is genetically heterogeneous disorder that can affect solely the pituitary gland and its target organs, or it can present with craniofacial, brain, and/or neurosensory abnormalities. Mutations in over thirty genes are reported to cause hypopituitarism and growth insufficiency, yet most cases are unexplained. Mutations in the transcription factor PROP1 are the most common known cause of hypopituitarism in humans. Using Prop1 mutant mice they discovered that PROP1 regulates the transition of pituitary stem cells to hormone producing cells in an epithelial to mesenchymal-like transition process. PROP1 is necessary to maintain stem cell proliferation, migration, and pituitary placode fate identity. To discover novel causes of hypopituitarism, they carried out exome sequencing on a cohort of 26 unrelated patients with hypopituitarism and identified mutations in known genes as well as novel candidate genes. To identify additional individuals with mutations in these novel genes and candidate genes from mouse studies, they developed an efficient, cost-effective method to capture the genomic DNA from 37 candidate genes and 30 known genes. They demonstrated that the method is sensitive and accurate in identifying genetic variants and applied it to over 100 patients and their families. They identified rare, likely deleterious variants in many genes, including HESX1, POU1F1, TGIF1, SIX3, and GH1. They are using cell culture and mouse models to assess the pathogenicity of variants of unknown significance. Their findings reinforce the idea that variants in multiple genes interact to influence the severity of clinical presentation.

Stem Cells in the Pituitary: A Role for Regeneration?
Karine Rizzoti. The Francis Crick Institute, London, UK

During morphogenesis, embryonic progenitors proliferate, differentiate and establish the shape of the future organs and tissues. In the mature organism, a certain degree of plasticity and potential for regeneration is retained as most organs maintain a population of adult stem cells sharing important similarities with embryonic progenitors; they are characterized by the ability to both self-renew and differentiate into the full range of the specialized cell types corresponding to the organ in which they reside. It is important to understand how these cells participate in organ cell turnover and regeneration, particularly because adult tissue stem cells can give rise to cancer stem cells. However, it is also relevant to regenerative medicine because stemcells can be transplanted once differentiated into the desired cell type or be manipulated in vivo to restoremissing cells. In the last 12 years, authors and others, have characterized a population of adult pituitary stem cells. While lineage tracing experiments have firmly established their adult stem cell properties, they have also shown that the cells are relatively quiescent in unchallenged animals. This is perhaps not surprising because the pituitary gland is an organ with a relatively low turn-over. In addition, endocrine cells can divide, and this may be enough to maintain a functional pituitary. Therefore, the role of stem cells during homeostasis remains unclear. In contrast, when the gland is challenged, pituitary stem cells are mobilized. Endocrine cell ablation experiments show that stem cells react by actively proliferating and suggest that they also give rise to new endocrine cells, to replenish the depleted population.  Moreover, pituitary target organ ablations, representing physiological challenges for the gland, have also been shown to stimulate both the proliferative and differentiation potential of the stem cells, demonstrating their regenerative potentialities. Characterization of the molecular mechanisms underlining mobilization of the stemcells is now required to be able to manipulate their fate.

A Novel Role for Vasopressin in Parenting
Andres Bendesky. Columbia University, New York, New York, USA

The extent and quality of parental care that children receive greatly influences their development, impacting their physical and psychological growth, their educational and social achievement, and their disease risk as both children and adults. It is thus remarkable that around 25% of children are physically abused worldwide. Parenting is a complex behavior, and we still know little about the causes and mechanisms by which people differ in their parental behaviors. To learn more about the mechanistic basis of parental care, the author took an evolutionary-comparative approach. He set out to identify the genetic, molecular, and neuronal bases of the evolution of parental care in a pair of closely-related sister species of deer mice (genus Peromyscus) that have naturally evolved dramatically-different parental behaviors. Using quantitative-genetics techniques, he localized 13 genetic regions that contribute to interspecific differences in parental behavior. Remarkably, most of these regions differentially affect maternal and paternal behavior, implying that parental behavior evolves through different genetic routes in the two sexes. In one of these regions, he narrowed in on a specific gene, the neuropeptide vasopressin, and showed that it mediates interspecific differences in parental nest-building behavior. This gene is expressed at 3-fold higher levels in the hypothalamus of the less parental Peromyscus species. Finally, he confirmed the causal role of this neuropeptide by demonstrating that an increase in vasopressin levels in the brains of the more parental Peromyscus species inhibits their parental care. Together, an evolutionary-genetics approach led to the discovery that variation in parental behavior has a different genetic basis in males and females and to finding that the highly conserved neuropeptide vasopressin modulates parental behavior.

The Biology of Germ Cell Tumors in Disorders of Sex Development
Leendert Looijenga. Erasmus Medical Center, Rotterdam, Netherlands.

Germ cell tumors (GCTs) comprise a heterogeneous group of neoplasms. The WHO classification 2016 recognizes three types of testicular GCTs, including the GCNIS (Germ Cell Neoplasia In Situ) GCTs, the malignant seminomas and non-seminomas. They originate from a totipotent embryonic germ cell. GCNIS, seminoma and embryonal carcinoma, are characterized by the pluripotency marker OCT3/4 (POU5F1). Seminomas (and GCNIS) are positive for SOX17 (sulfite oxidase17) and embryonal carcinomas for SOX2. Individuals with part of the Y chromosome (GBY) are at higher risk, likely related to TSPY (testis specific protein Y). This directly relates to risk stratification of individual patients with various types of Disorders of Sex Development (DSD). Detection of KITLG (KIT ligand, or SCF, stem cell factor) is informative for the earliest phase of GCNIS formation. A new level of risk stratification has been provided based on Genome Wide Association studies (GWAS) indicating that a selection of Single Nucleotide Polymorphisms (SNPs) are associated with testicular GCTs. These map to a limited number of genes related to relevant pathways, including gonadal development (DMRT1, doublesex and mab-3 related transcription factor 1), embryonic germ cell proliferation and maintenance (KITLG, SPRY4, TERT, BAK1, etc). Data was presented that was informative in a clinical context. A unifying model was presented in which a delicate interaction between the genomic constitution and (gonadal) micro-environment (GENVIRONMENT) are the actual determinants for the risk of an individual to develop a malignant testicular GCT, including patients with DSD. Functional studies on the role of P53 identified that a selected set of embryonic-specific microRNAs, being the miRs 371-3 and 302/367 clusters are highly expressed in the various types of GCTs, including the precursor lesion GCNIS. The only exception is the fully differentiated element teratoma. These miR are informative as molecular serum markers to identify patients with a malignant GCT at the moment of diagnosis, as well as during clinical follow up. The current data based on a quantitative detection protocol was presented, showing the outperformance of this test compared to the currently used standard biomarkers AFP and hCG.

Disruption of Testicular Development and Function.
Rod Mitchell. University of Edinburgh, Edinburgh, UK.

Male reproductive disorders are common and there is evidence for increasing incidence over recent decades. These disorders may present at birth (hypospadias and cryptorchidism) or in adulthood (infertility, testicular cancer) and can arise as a result of underlying genetic abnormalities or following environmental (e.g. phthalates) and pharmaceutical (e.g. analgesics, chemotherapy) exposures that impact fetal, neonatal of prepubertal testicular development. Understanding the relevance of these perturbations on the testis and subsequent reproductive function in humans is often based on the findings of animal studies, however there are important differences between rodent and human in terms of fetal and postnatal testis development, in particular germ cell development. Therefore, reliable human experimental models that can demonstrate human and clinical relevance are required. They have validated in-vitro and ex-vivo experimental models of human testis development. This includes a ‘hanging drop’ culture system and a human testis xenograft approach that can be utilized to investigate the development and function of the human testis during fetal, neonatal and prepubertal life. These model systems can recapitulate seminiferous cord formation, germ and somatic cell development and hormone production. Furthermore these model systems can be combined to investigate the long-term effects of genetic disruption to model the gonadal effects of Disorders of Sex Development and environmental exposures (e.g. industrial chemicals, pharmaceuticals and chemotherapeutics) on the testis. They have utilized these models to mimic in-utero exposure to analgesics on human fetal testis development and function and the effects of therapeutic analgesic exposures on human fetal germ cell development and testosterone production was described. The effect of knockdown, in the human fetal testis, of known and novel genes implicated in Testicular Dysgenesis Syndrome and Disorders of Sex Development was also presented. The model systems have also been adapted for the investigation of human prepubertal testis development as part of a fertility preservation programme for boys treated for cancer. Studies investigating the effects of chemotherapy exposure on the prepubertal testis was described, in addition to experimental approaches to preserving fertility in childhood cancer.

Diagnosis and Management of Silver–Russell Syndrome: First International Consensus Statement.
Irene Netchine. Hôpital Trousseau, INSERM U938, Sorbonne Université, Paris, France.

The first Consensus Statement on Silver Russell Syndrome has been held in 2015, on behalf of the COST Action BM1208 (European Network for Human Congenital Imprinting Disorders, http://www.imprinting-disorders.eu), ESPE, PES, APPES and SLEP with the participation of five representatives from a parent support group from different countries. It has been published in 2016. (https://www.nature.com/articles/nrendo.2016.138). For dissemination, a patient “friendly” document has been generated and has been or will shortly be translated into seven languages. This consensus summarizes recommendations for clinical diagnosis, investigation and management of patients with Silver–Russell syndrome, an imprinting disorder that causes prenatal and postnatal growth retardation. Considerable overlap exists between the care of individuals born small for gestational age and those with SRS. However, many specific management issues exist and evidence from controlled trials remains limited. SRS is primarily a clinical diagnosis; however, molecular testing enables confirmation of the clinical diagnosis and defines the subtype. A ‘normal’ result from a molecular test does not exclude the diagnosis of SRS. The management of children with SRS requires an experienced, multidisciplinary approach. Specific issues include growth failure, severe feeding difficulties, gastrointestinal problems, hypoglycaemia, body asymmetry, scoliosis, motor and speech delay and psychosocial challenges. An early emphasis on adequate nutritional status is important, with awareness that rapid postnatal weight gain might lead to subsequent increased risk of metabolic disorders. The benefits of treating patients with SRS with growth hormone include improved body composition, motor development and appetite, reduced risk of hypoglycemia and increased height. Clinicians should be aware of possible premature adrenarche, fairly early and rapid central puberty and insulin resistance. Treatment with gonadotropin-releasing hormone analogues can delay progression of central puberty and preserve adult height potential. Long-term follow up is essential to determine the natural history and optimal management in adulthood.

Pediatric Obesity—Assessment, Treatment, and Prevention: An Endocrine Society Clinical Practice Guideline.
Dennis Styne. University of California, Davis, California, USA.

Pediatric obesity remains an ongoing serious international health concern affecting about 17% of children and adolescents in the United States, while worldwide over 41 million children under 5 years are overweight or obese, threatening their adult health and longevity. Pediatric obesity has its basis in genetic susceptibilities influenced by a permissive environment starting in utero and extending through childhood and adolescence. Endocrine etiologies for obesity are rare and usually are accompanied by attenuated growth patterns. Comorbidities are common in obese children and adolescents, as are long-term health complications. Clinicians should screen for obesity comorbidities in a hierarchal, logical manner so as to detect them early and prevent more costly complications. Genetic screening for rare syndromes is indicated only in the presence of specific historical or physical features. The psychological toll of pediatric obesity on the individual and family necessitates screening for mental health issues and counseling if indicated. The prevention of pediatric obesity before onset, via physical activity and a healthful diet and environment, should be a primary goal; once obesity occurs, it is difficult to treat effectively with lifestyle modifications. Although some behavioral and pharmacotherapy studies have reported modest success in preventing and/or treating pediatric obesity, there remains a need for substantial research in these areas. Treating children or adolescents with weight loss medications should be restricted to clinical trials. Increasing evidence indicates that bariatric surgery is effective in the most seriously affected mature teenagers who have failed lifestyle modification; however, this requires experienced teams with resources for long-term follow-up. Despite a significant increase in research in recent years, we need more studies to better understand the genetic and biological factors that cause an obese individual to manifest one comorbidity versus another or to be free of comorbidities. Furthermore, we need continued investigation into the most effective methods of preventing and treating obesity and into methods for changing environmental and economic factors that will lead to worldwide cultural changes in diet and activity. We need to focus particular attention on finding ways to effect systemic changes in food environs and total daily mobility, as well as methods for sustaining healthy BMI changes.

Prevention and Management of Rickets.
Wolfgang Hogler. Kepler Universitaetsklinikum, Linz, Austria.

Calcium and phosphorus represent the main building material for bone and growth plate mineralization and bone stiffness. The supplier of these bone minerals is the hormone calcitriol, which originates from vitamin D, itself made by sunshine in human skin. Requirement for bone mineral supply is highest during phases of rapid growth, such as during pregnancy, infancy and puberty. The body can be deprived of calcium either through low dietary calcium intake and/or low vitamin D. Such calcium deprivation can lead to serious health consequences throughout life, such as hypocalcaemic seizures, dilated cardiomyopathy, and skeletal myopathy, ‘nutritional’ rickets and osteomalacia. These five conditions, often summarized as ‘symptomatic vitamin D deficiency’, are fully reversible but also fully preventable. Asymptomatic and undiagnosed morbidity from vitamin D or calcium deficiency is the subject of intensive research. Calcium deprivation has reached epidemic proportions, not only in the third world, but also in high-income countries – specifically amongst dark-skinned and other at-risk populations. The increasing prevalence of rickets and osteomalacia, and infant deaths from hypocalcaemic cardiomyopathy, demand action from global health care providers. The global consensus for the prevention of management of rickets has provided evidence-based guidance on how such programs can be delivered and recommends vitamin D supplementation for risk groups (min 600 IU/day), pregnant women (min 600 IU/day), and infants (min 400 IU/day). The success of prevention programs is intrinsically linked to policy implementation, including accountability of medical and parental responsibilities. The work outlines substantial differences in supplementation policies and their efficacy in Europe and calls for better policy implementation strategies and also well-designed food fortification with vitamin D.
The quality of a nation’s public health standards can be derived from how it treats and invests in its children and other vulnerable risk groups. The foetus and infant have the human right to be protected against harm. Prevention programs, including vitamin D supplementation and food fortification, should have the same public health priority as vaccinations.

Hypogonadism in Girls after Cancer Therapy: Causes, Diagnosis, and Treatment.
Charles Sklar. Memorial Sloan Kettering, New York, New York, USA

Hypogonadism, both primary and secondary, are well documented following cancer therapy. Primary ovarian insufficiency (POI) has most often been associated with exposure to alkylating agents (dose response) and radiation (RT) that includes the ovary (dose response). Early onset POI occurs commonly in girls exposed to ovarian RT at doses O10 Gy and following high-dose alkylating agents as is given for stem cell transplant. Late onset POI (ie, premature menopause) may be seen following modest doses of alkylating agents, especially exposure to procarbazine and lower doses of ovarian RT (!10 Gy). Childhood cancer survivors treated with doses of RTO30 – 40Gy to the hypothalamic-pituitary axis are at risk for deficits of LHand FSH. Elevated gonadotropins, especially FSH, are the hallmark of POI. It is important to note that levels can fluctuate over time; it is not uncommon for girls treated with chemotherapy alone to demonstrate normalization of gondatropins and spontaneous puberty/menses over time. As there are no long term data correlating AMH levels in children and adolescents and subsequent ovarian function, measurement of AMH is not useful in making a diagnosis of POI. Gonadotropin deficiency needs to be considered in survivors exposed to high-dose hypothalamic-pituitary radiation who demonstrate delayed or arrested puberty associated with low or normal gonadotropins and low levels of estradiol. Treatment of hypogonadismin cancer survivors is, in general, similar to what is done in the non-cancer population. Special considerations are needed for those who have been exposed to chest RT and are at higher risk of developing breast cancer later in life.

Diagnosis and Treatment of Hypogonadism in Male Survivors of Childhood Cancer.
Wassim Chemaitilly. St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

Gonadal dysfunction is among the most commonly reported endocrine late effects in childhood cancer survivors (CCS). The main risk factors include the exposure of the hypothalamic-pituitary-gonadal axis to radiotherapy and treatment with gonadotoxic chemotherapy drugs such as alkylating agents. Individuals may experience gonadal dysfunction because of central (or hypogonadotropic) hypogonadism or as a result of primary gonadal injury. The testes have two distinct functional entities: a reproductive compartment that consists of germ cells and their supporting system (including the Sertoli cells) and a hormone producing compartment that consists of testosterone producing Leydig cells. These compartments vary in their vulnerability to cancer treatments that target the proliferative properties of cancer cells. The rapidly multiplying germ cells are more likely to be decimated by such treatments than the more quiescent Leydig cells.
Therefore, a subset of male CCS exposed to gonadotoxic treatments may experience germ cell failure and infertility despite having spontaneously progressed through puberty and maintained normal testosterone secretion during adolescence and adulthood. This presentation seeks to provide medical care providers with guidance regarding the management and treatment of central or primary hypogonadism in male CCS with emphasis on issues and challenges that are specific to this vulnerable population.

Cardiomyocyte Glucocorticoid and Mineralocorticoid Receptors Antagonistically Regulate Heart Disease.
Robert H Oakley, Diana Cruz-Topete, Bo He, Julie F Foley, Page H Myers, Monte S Willis, Celso E Gomez-Sanchez, Pierre Chambo, John A Cidlowski. NIEHS/NIH, Research Triangle Park, Durham, North Carolina, USA

Stress is increasingly associated with cardiac disease. Glucocorticoids are primary stress hormones that regulate homeostasis through two nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). Cardiomyocytes express both receptors but little is known concerning their specific and coordinated actions in heart physiology and pathology. To examine the in vivo function of glucocorticoid signaling in the heart, we generated mice with cardiomyocyte-specific deletion of GR (cardioGRKO), MR (cardioMRKO), or both GR and MR (cardioGRMRKO). The cardioGRKO mice spontaneously developed cardiac hypertrophy and left ventricular systolic dysfunction whereas the cardioMRKO mice exhibited normal heart function. Surprisingly, the cardioGRMRdKO mice were protected from cardiac disease. Gene expression profiling identified cardioprotective gene changes in the double knockout hearts that limit cardiac hypertrophy and promote cardiomyocyte survival. Re-installation of MR into the cardioGRMRdKO hearts reversed the cardioprotective gene changes and resulted in cardiac dysfunction. These findings demonstrate a deleterious role for cardiac MR signaling when unopposed by GR. Moreover, they reveal the target genes and cellular responses altered by MR that contribute to cardiac pathology. Therapies that shift the balance of cardiomyocyte glucocorticoid signaling to favor more GR and less MR activity may provide an improved approach for treating heart disease.

Immune Regulation by Glucocorticoids.
David Ray. University of Oxford, Oxford, UK

Glucocorticoids (cortisol in humans, corticosterone in rodents) are critical regulators of energy metabolism and immunity. Their secretion by the adrenal gland follows a circadian pattern, with serum concentrations peaking before the active phase (day in humans, night in rodents). Synthetic glucocorticoids are the most potent anti-inflammatory agents known, and are widely used therapeutically, with >1% of the UK population holding a prescription long-term. However, frequent therapeutic use is accompanied by development of severe side effects notably fat accumulation, hyperglycemia, and hepato-steatosis. Inactive GR is bound by ligand in the cytoplasm and undergoes nuclear translocation, where it binds glucocorticoid response elements (GREs) in the genome to either enhance, or repress gene transcription.
Mechanisms to explain how the same molecule can drive gene activation or repression remain under investigation, but likely require an allosteric change induced by DNA target sequence, and/or co-binding with other transcription factors. For gene activation, homodimeric GR recruits co-activator molecules including steroid receptor co-activators (SRC1-3), and histone acetyltransferases (CBP/p300). In contrast, gene repression in the context of anti-inflammation is more complex, with multiple mechanisms of action proposed. Publications report that activated GR binds to, and inhibits the transactivation function of pro-inflammatory transcription factors, notably the RelA component of the NFkB complex. This was suggested to require recruitment of a co-repressor protein, such as the SRC2 co-modulator. This tethering mechanism explains the lack of consensus GR binding sites in the regulatory regions of inflammatory genes, with the mode of action for GR being to bind to DNA-bound NFkB.
However, an alternative mechanism of action has also been proposed, which does not require GR recruitment to the inflammatory genes at all. Here, GR is proposed to act in a conventional manner, to trans activate genes that themselves have anti-inflammatory actions. These includeTNFAIP3, DUSP1, and IkB. The protein products of these genes are proposed to act directly on components of NFkB, thereby opposing recruitment to target genes, and thereby interrupting gene activation. The translational implications of uncertainty likely underpin the difficulties in harnessing the anti-inflammatory power of glucocorticoids in the absence of metabolic off-target effects. While some selective glucocorticoid receptor agonists (SeGRMs) have been developed, they are struggling in the clinic. However, an orally-active, and selective GR ligand would have potential to transform the management of chronic inflammatory diseases.


Chemical Modification of the Glucocorticoid Receptor as a Determinant of Tissue Glucocorticoid Sensitivity: Implications to Circadian Rhythms, Stress Response and Treatment of Pediatric Leukemia.
Tomoshige Kino, Sidra Medicine, Doha, Qatar.

Glucocorticoid hormones virtually influence all human functions both in a basal homeostatic condition and under stress. Thus, many other biological pathways adjust glucocorticoid actions in local tissues (tissue glucocorticoid sensitivity) by targeting the single receptor molecule glucocorticoid receptor (GR) as part of the regulatory loop coordinating complex human functions. Among them, chemical modification of GR, such as by acetylation and phosphorylation, is highlighted as one of the important molecular mechanisms for changing local glucocorticoid actions. For example, the circadian transcription factor CLOCK acetylates GR, and participates in the reverse synchronization of local glucocorticoid sensitivity against circadian fluctuation of circulating cortisol. The cyclin-dependent kinase 5 (CDK5) phosphorylates several serine residues of the GR and modulates local glucocorticoid actions in the brain, and participates in the adaptive stress response and pathophysiology of mood disorders. Further, aberrantly activated v-akt murine thymoma viral oncogene homolog 1 (AKT1) develops glucocorticoid resistance in pediatric acute lymphoblastic leukemia by phosphorylating serine 134 of the GR and by inhibiting its cytoplasmic to nuclear translocation in cooperation with 14-3-3 proteins. The author reviewed recent progress in the research investigating chemical modification of GR as a determinant of tissue glucocorticoid sensitivity and discuss its physiologic or pathophysiologic implications.

Central Hypothyroidism – an Update
Paul van Trotsenburg
Amsterdam University Medical Centers, Amsterdam, Netherlands

Central hypothyroidism can be best defined as lower than desirable thyroid hormone production and secretion because of insufficient stimulation of a normal thyroid gland by a defective pituitary or hypothalamus, resulting in a too low plasma or serum (free) thyroxine (FT4) concentration accompanied by a more or less normal thyrotropin (TSH) concentration. Central hypothyroidism can occur isolated or as part of multiple pituitary hormone deficiency and can be a congenital or acquired condition. Because basal TSH is generally unusable as diagnostic test for central hypothyroidism, its diagnosis heavily relies on the height of the FT4 concentration, making especially isolated central hypothyroidism a challenging diagnosis. In the last six to seven years, three new genetic causes of isolated central hypothyroidism – mutations in the IGSF1 (immunoglobulin superfamily member 1), TBL1X (transducin beta like 1 X-linked) and IRS4 (insulin receptor substrate 4) genes – have been added to the two well-known causes (mutations in TRHR, thyrotropin releasing hormone receptor, and TSHB, thyroid stimulating hormone beta). This speaker presented an overview of the current knowledge about diagnosing, and the genetic causes of (isolated) central hypothyroidism.

Paediatric Differentiated Thyroid Cancer: Outcome and Long Term Effects
Thera Links. University Medical Hospital Groningen, Groningen, Netherlands.

Pediatric differentiated thyroid carcinoma (DTC), which includes papillary (PTC) and follicular thyroid carcinoma (FTC), has an excellent prognosis (15-year survival rates >95%). The initial treatment in children generally consists of a (near) total thyroidectomy, followed by ablation therapy with radioiodine (131-I), nowadays, the latter often depends on risk stratification. TSH suppressive therapy with thyroid hormone has for decades been administered during follow-up to diminish the risk of recurrent disease. However, data about long-term effects of 131-I treatment, long-term TSH suppressive therapy and quality of life in pediatric DTC patients are limited. In the Netherlands a nationwide study in patients with pediatric DTC has been performed regarding presentation, treatment-related complications and outcome of treated and long term effects. Overall survival of 170 patients was 99.4% after a median follow-up of 13.5 (range 0.3–44.7) years. Medical follow-up data were available from 105 patients (83.8% women). At last follow-up, 8.6% patients had persistent disease and 7.6% patients had recurrent disease. Permanent hypoparathyroidism was found in 23.8%, 12.4% patients had recurrent laryngeal nerve injury. Health-related quality of life (RAND 36), fatigue (MFI20), anxiety and depression (HADS) scores of survivors and age, sex, and socioeconomic status survivors and matched controls did not differ significantly.
However, survivors had more physical problems (PZ0.031), role limitations due to physical problems (PZ0.021), and mental fatigue (PZ0.016) than controls. Evaluation of the achievement of psychosocial developmental milestones was evaluated by the course of life questionnaire (CoLQ) and showed similar development on social, autonomy, and psychosexual domains of survivors compared to controls. While systolic function is unaffected, diastolic dysfunction was present in 21.2% of the asymptomatic survivors, which may suggest early cardiac aging. In survivors clinically significant salivary gland dysfunction was found in 35.5%, related to the cumulative 131I activity.

Prof. dr. Thera P. Links, University of Groningen, University Medical Center Groningen, Department of Endocrinology, HPC AA31, P.O. Box 30.001, 9700 RB Groningen, the Netherlands. E-mail: t.p.links@umcg.nl.

Novel CNS Imaging Techniques
Maria Argyropoulou. Department of Radiology, Medical School, University of Ioannina, Ioannina, Greece

These techniques are designed to focus on the assessment of functional tissue characteristics, such as neuronal activity (functional MRI, fMRI), microstructural properties (diffusion tensor imaging-DTI) and tissue perfusion (DSC perfusion, ASL). fMRI reveals brain activation during performance of behavioral tasks, based on the blood oxygen level dependent (BOLD) MRI signal, which is modulated by neural activity via a process of neurovascular coupling. Resting-state fMRI can be performed and correlates brain areas with similar spontaneous fluctuations in the BOLD signal — thereby enabling estimates of ‘functional connectivity’. DTI evaluates brain microstructure and quantifies integrity through metrics such as apparent diffusion coefficient (ADC) and fractional anisotropy (FA). ‘Structural connectivity’ is based on white matter tracts that can be depicted and assessed with tractography. Perfusion MR imaging methods detect signal changes that accompany the passage of a tracer through the cerebrovascular system. A less invasive approach is arterial spin labeling (ASL) that uses arterial water as an endogenous tracer to measure CBF. MR perfusion is applied in the evaluation of brain tumors, neurological diseases and developmental disorders. Clinical applications of novel CNS imaging techniques are expected to expand greatly in the future due to the increasing availability, as well as the continuous advancements in the field of research.

Novel Techniques in Diagnostics of Bone Strength.
Nicola Crabtree. Birmingham Women’s and Children’s NHS Trust, Birmingham, UK

There are several different techniques for assessing bone strength and fracture susceptibility in children namely; dual energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT), plain radiography and magnetic resonance imaging (MRI). The most readily available technique and recommended by the International Society of Clinical Densitometry (ISCD) is DXA. The advantage of DXA is that it is widely available, affords the child a low radiation dose and can assess fracture prone areas of the body with fast scanning times. The main disadvantage of DXA is that it is a two-dimensional technique which measures the ratio of bone mineral content to projected bone area, such that areal bone density is highly dependent on bone size. As a result, reduced bone density in childhood is frequently a reflection of poor growth rather than a true measure of reduced bone strength. Given the limitations of DXA it is not surprising that other imaging techniques are appealing for assessing reduced bone strength and fracture risk.
Techniques such as QCT have the advantage of being able to separately measure cortical and trabecular bone densities. Dedicated peripheral QCT scanners can assess fracture prone long bones with minimal radiation exposure and high resolution QCT scanners now give almost in-vivo bone biopsy type outputs of parameters such as trabecular bone volume, trabecular separation etc. However, small measurement regions, long scanning times and a high susceptibility to movement, keep these techniques predominantly in the research arena rather than for routine clinical assessments. The most recently developed technique is MRI. It can be applied to the peripheral or axial skeleton. The advantage of MRI is that it uses non-ionising radiation to assess bone architecture and muscle structure in multiple planes without repositioning. The disadvantages of MRI are that it is time consuming, expensive, and it has been used only in few research protocols. As with QCT its applicability in clinical practice has yet to be fully assessed. Increasingly, old and new technologies are being combined to fully exploit currently available diagnostic procedures. The improved image resolution of modern DXA scanners has facilitated accurate assessment of osteoporotic vertebral fractures. At the same time, sophisticated intelligent computer software programs have enabled techniques such as textural analysis, shape modelling and finite element modelling to be genuine contenders for the assessment of bone strength in children. How well any of these novel diagnostic tools are incorporated into clinical practice remains to be seen.

It is not Just the Growth Hormone-IGF-I Axis
Ola Nilsson. Orebro University, Orebro, Sweden

For decades, the dominant conceptual framework for understanding short and tall stature was centered on the GH-IGF-I axis. However, recent findings in basic molecular and cellular biology and in clinical genetics have uncovered a vast array of other regulatory systems that control skeletal growth and an accompanying vast array of genetic defects outside the GH-IGF-I axis that can cause disorders of linear growth. As a result, the traditional view of short or tall stature that is centered on the GH-IGF-I axis is now far too narrow to encompass the ever-growing number of defects that cause abnormal linear growth. A much broader conceptual framework can be based on the simple concept that linear growth disorders are necessarily due to dysfunction of the growth plate, the structure responsible for bone elongation and therefore overall body size. Consequently, short stature can more generally be conceptualized as a primary or secondary disorder of the growth plate chondrocytes. The wide array of genetic defects, many newly-discovered, that affect growth plate chondrocyte function and thereby cause childhood growth disorders will be reviewed. A novel concept that has emerged from recent findings is that sequence variants in a single gene can produce a phenotypic spectrum that ranges from a severe skeletal dysplasia to disproportionate or proportionate short stature, to normal variation in height, to tall stature. The recent advances reviewed in this paper are steadily diminishing the number of children who receive the unhelpful diagnoses of severe idiopathic short stature or tall stature.

Fascinating Growth Phenomena: What Causes Individual Catch-up Growth and Population Secular Change?
Jan M Wit. Leiden University Medical Center, Leiden, Netherlands

Catch-up growth (CUG) is characterized by a period of supranormal height velocity following a transient period of growth inhibition. The two classical hypotheses on the mechanism are the neuro-endocrine hypothesis (a central mechanism that would recognize the degree of mismatch between actual size and target size) and the growth plate hypothesis (local regulation of growth according to a preset cellular program of senescence, characterized by decreasing growth proliferation rate). Unilateral CUG in animal experiments argue against the neuro-endocrine hypothesis, and the fast growth in type A CUG argues against the growth plate hypothesis. Recent data indicate that various regulatory factors may be involved in CUG after malnutrition, including Hypoxia-Inducible Factor 1 (HIF1), mTOR, Sirtuins (class III histone deacetylases), microRNAs, the GH-IGF-1 axis, Ghrelin, Leptin and insulin. Positive secular changes in body size and tempo of growth have occurred in most western countries since 1850, generally considered as an indicator of better nutrition, hygiene and health status. Secular increase is most prominent at 4–11 years of age. At the completion of the secular trend in various countries adult height has increased up to 20 cm and is reached >8 years earlier.
However, attained mean adult heights are different, suggesting that besides environmental influences also genetic or geographic factors play a role, and possibly also culturally-engrained nutritional habits. The magnitude of secular trend is relatively strong in individuals of low socio-economic background, reducing social class differences in height. Regarding the mechanism, epigenetic processes (during fetal life or early infancy) seem most plausible. The extensive time interval and magnitude of secular trend, the different stages of secular trend in high income and many low- and middle income countries, on top of the presumed effect of genetic and geographic variations, make it difficult to defend the concept of a ‘global growth standard’.

Interaction between Nutrition, the Endocrine System and the Growth Plate.
Moshe Phillip. Schneider Children’s Medical Center Of Israel, Petah Tikva, Israel

Children’s linear growth is a complex process determined by genetic and environmental factors. It is well known that nutrition influences linear growth, but the precise mechanisms by which nutrition interact with height gain was never fully elucidated. In the present lecture, the way by which nutrition affects linear growth was discussed. Specifically, the effect of nutrition on the GH- IGF-1 axis and its local effect on the chondrocytes of the epiphyseal growth plate of the long bone was discussed. Results from experimental animal studies was presented including the epigenetic changes and the effect nutrition has on leptin and its effect on aromatase expression within the growth plate. Laboratory data and clinical data on the way by which leptin might determine the adult height was presented.


EAP1 Mutations Cause an Impaired Transcriptional Activity on GnRH Promoter that Leads to Self-Limited Delayed Puberty.
Alessandra Mancinia, Sasha R Howarda, Claudia P Cabrerab, Michael R Barnesb, Sabine Hegerc, Leonardo Guastia, Sergio Ojedad, Leo Dunkela
aWilliam Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Centre for Endocrinology, Queen Mary University of London, London, UK; bWilliam Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Centre for Translational Bioinformatics, Queen Mary University of London, London, UK; cInstitute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany; dOregon National Primate Research Center/Oregon Health and Sciences University, Portland, Oregon, USA.

BACKGROUND: The initiation of puberty is orchestrated by the augmentation in the secretion of gonadotropin-releasing hormone (GnRH) from a few thousand neurons located in the hypothalamus. Recent findings identified that the neuroendocrine control of puberty is regulated by a network of transcriptional factors hierarchically organized, but this still remains not fully elucidated. Enhanced at Puberty 1 (EAP1) is one of the main regulators of pubertal onset and it is expressed in the hypothalamus of pubertal rats and non-human primates. Its role has been described as being involved in the initiation of female puberty by transactivating GnRH promoter. Its inhibition in the hypothalamus, causes disrupted estrous cyclicity and delayed puberty in rats. Self-limited delayed puberty (DP) (i.e. constitutional delay of puberty) runs in families with either autosomal dominant or complex inheritance patterns in >70% of families, indicating a strong genetic basis of the trait. However, only a few genes have been identified underlying DP, and to date, no EAP1 mutations have been found in humans. OBJECTIVE, HYPOTHESIS AND METHODS: Whole exome sequencing was performed on DNA from 160 individuals of 67 multi-generational families affected with DP. Variants returned were analyzed to identify rare, potentially pathogenic variants enriched in case versus controls, and relevant to the biological control of puberty. Authors identified two EAP1 variants whose pathogenicity was validated in vitro and tissue expression was examined in mouse hypothalamus. RESULTS: After filtering they identified one in-frame deletion and one rare missense variant in EAP1 in two unrelated families (five affected individuals), and all segregated with the DP trait with the expected autosomal dominant pattern of inheritance. These variants are highly conserved and were predicted to be deleterious by the main prediction software tools (i.e. SIFT and POLYPHEN 2). Expression studies on Eap1 revealed its broad expression in the hypothalamus of peri-pubertal mice and specifically in the nuclei expressing GnRH neurons, such as the arcuate and periventricular nuclei. The pathogenicity of each variant was investigated using a promoter reporter assay in a HEK293T cell line. For verifying the EAP1 transactivating ability on GnRH promoter, they employed a luciferase reporter gene whose expression is driven by the GnRH promoter. EAP1 mutant proteins showed a significantly reduced transcriptional activity compared to wild-type, thus impairing its function on GnRH promoter. CONCLUSIONS: They have identified, for the first time, two human EAP1 mutations leading to a reduced GnRH transcriptional activity resulting in the phenotype of self-limited DP.

Generating a Human Gonadal Cells Model from Terminal Differentiated Fibroblast-Derived Induced Pluripotent Stem Cells.
Daniel Rodrıguez Gutierreza, Wassim Eida,b, Anna Biason-Lauber aUniversity of Fribourg, Fribourg, Switzerland; bUniversity of Alexandria, Alexandria, Egypt.

BACKGROUND: Differentiation of the gonads in men is closely dependent on Sertoli cells maturation. Differences of sex development (DSD) are caused by variations in this process. The study of the mechanisms underlying these complex conditions is crucial for optimal clinical management and Sertoli cells would be an ideal model for this purpose. However, there are two main obstacles for the study of human Sertoli cells. Firstly, mature human Sertoli cells lose their proliferation abilities in culture. Secondly, the currently available models (human NT2D1 and mouse TM4 cells) demonstrated to have limitations due to their origin as mouse or tumor cells. OBJECTIVE AND HYPOTHESIS: To establish a more suitable model to study human testis formation, they differentiated human fibroblasts-derived induced pluripotent stem cells (iPSCs) into human Sertoli-like cells (SLC). METHODS: They reprogrammed human fibroblasts into iPSCs by lentivirus transduction of reprograming factors (Oct4, SOX2, NANOG, LIN28, KLF4 and C-MYC). Subsequently, they guided the differentiation of iPSCs into SLCs by growth factors and characterized this new model by new generation sequencing techniques including 44.946 genes expression analysis. In a more detailed analysis, they selected 20 gene markers for the different stages of Sertoli cell development including SRY-Related HMGBox 9 (SOX9), vimentin (VIM), Cytochrome P450 Retinoid Metabolizing Protein (CYP26B1) and Proto-Oncogene Tyrosine-Protein Kinase Src (SRC). They additionally tested whether SLC are able to create three-dimensional structures in gel matrix and the expression of claudin-11 (CLDN-11) in tight junctions. RESULTS: This approach revealed that SLCs expressed Sertoli cell markers such as SOX9 and VIM, When compared with the other current models (NT2D1 and TM4 cells), SLCs showed a reduction of the germ cell markers SOX2, POU5F1, DPPA2, DPPA4 and NANOG and an increased expression of Sertoli cell markers CYP26B1, SCF and SRC (P

Posted in Edition 71 - December 2018, Subject Review, Uncategorized
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