Edition 74 - July 2019 / Bibliographic Reviews

Bibliographic Reviews – Ed. 74

Marco A. Rivarola y Alicia Belgorosky. Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

For this issue of Endocrinología Pediátrica On Line, we have selected to comment on the following publications:

J Clin Endocrinol Metab. 2019 May 28.
The Low-Dose ACTH test: Usefulness of Combined Analysis of Serum and Salivary Maximum Cortisol Response in Paediatrics. Vaiani E1, Lazzati JM1, Ramirez P1, Costanzo M1, Gil S1, Dratler G1, Zaidman V1, Chaler E2Belgorosky A1,2.

1Servicio de Endocrinologia, Hospital de Pediatria Garrahan, Buenos Aires, Argentina. 2Laboratorio Central, Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

CONTEXT: The low-dose1-µgACTH test (LDT) is widely used to assess central adrenal insufficiency (CAI); however, serum cortisol cut-off values are controversial. Salivary cortisol (SC) could be a more accurate measurement for CAI. OBJECTIVE: to assess a new maximum cut off value of serum cortisol after LDT in paediatric patients, taking into account the measurement of both serum and salivary cortisol. DESIGN AND SETTING: prospective study in a paediatric tertiary referral center. WORKING HYPOTHESIS: the combine analysis of serum and salivary cortisol response to LDT might improve LDT for CAI diagnosis. PARTICIPANT AND OUTCOME MEASUREMENT: 145 paediatric patients underwent LDT. Serum and salivary cortisol levels were measured. Sufficient response (CAS) was established accordingly to the reference serum cortisol cut-off value ≥497 nmol/L. RESULTS: LDT study showed CAS in 72 and CAI in 73 patients. Considering the lower quartile of maximum salivary cortisol value (21 nmol/L) in CAS group (Gr), an intermediate (InCAI) and a real (RCAIGr) were defined within the CAIGr. Regarding the median maximum value of serum cortisol levels in the InCAIGr, a new serum cortisol cut-off value of 450 nmol/L was established. Furthermore, 91 % of the patients in the RCAIG were below this cut-off value. CONCLUSION: The combined evaluation of maximum serum and salivary cortisol levels to LDT might be useful to define an InCAIGr and avoid unnecessary hormone replacement therapy. However, rigorous patient follow-up is required.

Selected Authors’ Comments and Descriptions Out of This Publication:
The diagnosis of adrenal insufficiency is challenging because of its unspecific clinical signs and the limitations of diagnostic tests of adrenal reserve. Indeed, reliability of dynamic tests of the hypothalamic-pituitary adrenal axis have been challenged. Moreover, in pediatric patients, insulin tolerance test (ITT) and metyrapone test, are considered of risk. The current accepted maximum total serum cortisol (TC) cut-off of 497 nmol/L after ACTH test, for a central adrenal insufficiency (CAI) diagnosis, has been revised in the latest years, principally for the LDT. Maximum serum TC levels response to LDT between 441.4-606.9 nmol/L was considered as an indeterminate area, and CAI could not be defined.
Salivary cortisol is mostly in the free form and is in equilibrium with plasma free cortisol (FC), therefore, it is a surrogate for the concentration of serum FC. Its measurement correlates well with both, total and biological active serum FC. Therefore, authors have hypothesized that the combine analysis of the maximum serum TC and SC response to LDT might improve the diagnosis of CAI in the pediatric population.
In this study, a new LDT cut-off value of serum TC level (≥450 nmol/L) was defined taking into account the measurement of both serum TC and SC in a pediatric population. In addition, an intermediate group was defined, to allow the avoidance of unnecessary hormone replacement therapy, but rigorous patient follow-up is recommended.

Clin Epigenetics. 2018 Jul 18;10(1):96.
The epigenetic clock and pubertal, neuroendocrine, psychiatric, and cognitive outcomes in adolescents.
Suarez A1,2, Lahti J1, Czamara D3, Lahti-Pulkkinen M1, Girchenko P1, Andersson S4, Strandberg TE5, Reynolds RM6, Kajantie E7,4, Binder EB3,8, Raikkonen K9.
1Department of Psychology and Logopedics, University of Helsinki, Haartmaninkatu 3, PO Box 21, FI-00014, Helsinki, Finland. 2Helsinki Collegium of Advanced Studies, University of Helsinki, 00014, Helsinki, Finland. 3Department of Translational Research in Psychiatry, Department of Psychiatry and Behavioral Sciences, Max-Planck Institute of Psychiatry, 80804, Munich, Germany. 4Children’s Hospital, Helsinki University Central Hospital and University of Helsinki, 00029, Helsinki, Finland. 5Center for Life Course Health Research, University of Helsinki, Geriatrics, Helsinki University Hospital, University of Oulu, 00029, Helsinki, Finland. 6BHF Centre for Cardiovascular Science, Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, EH16 4TJ, UK. 7National Institute for Health and Welfare, Helsinki and Oulu, 00271, Helsinki, Finland. 8Department of Psychiatry and Behavioral Sciences, School of Medicine, Emory University, Atlanta, 30322, USA. 9Department of Psychology and Logopedics, University of Helsinki, Haartmaninkatu 3, Helsinki, Finland.

BACKGROUND: Molecular aging biomarkers, such as epigenetic age predictors, predict risk factors of premature aging, and morbidity/mortality more accurately than chronological age in middle-aged and elderly populations. Yet, it remains elusive if such biomarkers are associated with aging-related outcomes earlier in life when individuals begin to diverge in aging trajectories. We tested if the Horvath epigenetic age predictor is associated with pubertal, neuroendocrine, psychiatric, and cognitive aging-related outcomes in a sample of 239 adolescents, 11.0-13.2 years-old. RESULTS: Each year increase in epigenetic age acceleration (AA) was associated with the following outcomes: a) 0.06 SD units higher weight-for-age, b) 0.08 SD units taller height-for-age, c) -0.09 SD units less expected adult height, d) 13 and 16% higher odds respectively, for each stage increase in breast/genitals development on the Tanner Staging Questionnaire and pubertal stage on the Pubertal Development Scale, e) 4.2% higher salivary cortisol upon awakening, and f) 18 to 34% higher odds for internalizing and thought problems on the Child Behavior Checklist (p values <  0.045). AA was not significantly associated with cognition. CONCLUSIONS: These findings suggest that already in adolescence, AA is associated with physiological age acceleration, which may index risk of earlier aging. AA may identify individuals for preventive interventions decades before aging-related diseases become manifest.

Selected Authors’ Comments and Descriptions Selected from This Publication:
STRENGHTS. The strengths of this study relate to a well-characterized cohort and availability of a number of aging-related phenotypes that they measured decades before the aging-related diseases become manifest. They were also able to account for a number of early life adversities and their proxies, such as maternal smoking and alcohol use during pregnancy, maternal age and BMI at delivery, mode of delivery, and the adolescent’s birth weight and gestational age. They also accounted for maternal glycyrrhizin in licorice use during pregnancy, which is a potent inhibitor of the placental glucocorticoid barrier enzyme (11-beta hydroxysteroid dehydrogenase type 2) and commonly consumed by young Finnish women. This may result in fetal overexposure to maternal circulating glucocorticoids. The study had been originally designed to examine its association with offspring developmental outcome. None of these early life factors were significantly associated with epigenetic AA in this sample, which is contrary to what they expected based on previous findings showing that these factors may exert adverse consequences on offspring neurodevelopment and HPA-axis functioning. With regard to maternal glycyrrhizin use, they have previously shown that high maternal intake of glycyrrhizin during pregnancy (> 500 mg/week) is associated with slightly shorter length of gestation, poorer performance in neurocognitive tests at ages 8 and 11–13 years, and higher odds for having borderline clinically significant externalizing psychiatric problems at same ages.
Limitations The limitations of this study are the narrow age range of the sample, and hence, the small magnitude of the correlation between DNAm (DNA methylation) age and chronological age. The small magnitude of this correlation is, however, similar to the other two previous childhood epigenetic age studies. The narrow age range also limits generalizability from our findings to samples that differ in age from ours as DNAm changes with age. These findings are also limited to DNAm in one tissue type and the study precludes generalizations beyond Finnish children. Further, the study design was cross-sectional which precludes testing developmental changes and causal inferences. They can neither address the possibility of selection bias resulting from sample attrition to genetic analyses. Also measuring genome-wide methylation with the most recent Illumina EPIC array that lacks 16 (4.5%) of the 353 CpG sites originally needed for Horvath for DNAm age calculation should be kept in mind when interpreting the study findings. Finally, while they measured psychiatric problems with a standardized, validated, and widely used mother-report, they cannot rule out potential information-bias embedded in the mother-report. Hence, future studies are needed to confirm whether this study findings on psychiatric problems also pertain to clinical diagnoses. Conclusions The study shows that among 11.0–13.2-year-old adolescents, AA is associated with a number of markers that index risk for earlier aging, namely, more advanced physical growth and development, higher salivary cortisol upon awakening, and psychiatric problems. Our findings are consistent with the life history theory and lend credence to the proposition that AA may be used as a biomarker of aging already early in life.


Internat J Pediatr Endocrinol, 2018:9.
Poor growth response during the first year of growth hormone treatment in short prepubertal children with growth hormone deficiency and born small for gestational age: a comparison of different criteria. Saartje Straetemans1,2,3*, Muriel Thomas3, Margarita Craen3,4, Raoul Rooman3†, Jean De Schepper3,4,5† and BESPEED3
1Department of Pediatric Endocrinology, Maastricht University Medical Center, 2 NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht University,

BACKGROUND: There is no consensus on the definition of poor growth response after the first year of growth hormone (GH) treatment. Authors determined the proportion of poor responders identified by different criteria in children with GH deficiency (GHD) and born small for gestational age (SGA). The second aim was to analyze the IGF-1 response in poor growth responders. METHODS: First-year height data of 171 SGA and 122 GHD children who remained prepubertal during the first GH treatment year were retrieved from the BESPEED database, and analyzed. Criteria for poor first-year response/ responsiveness were: change in height (ΔHt) SDS< 40%. CONCLUSIONS: The different response criteria yield high but comparable percentages poor responders, but identify different patients. This study does not provide evidence that one criterion is better than another. A limited IGF-1 generation is not the major reason for a poor growth response in the first year of GH treatment in SGA and GHD children.

Selected Authors’ Comments and Descriptions Out of This Publication:

Background Growth hormone (GH) deficiency (GHD) and short stature as a consequence of a small size at birth (SGA) are the most frequent indications for GH therapy in children in Europe. Although in general a substantial fraction of the height deficit is already recovered during the first year of GH treatment in these growth disorders, a high proportion has a poor growth response in the first year of GH therapy. This first year growth response is paramount since it is the major determinant of the gain during the subsequent treatment years and correlates with the final height outcome. Traditionally the growth response during the first year of GH treatment is evaluated by auxological parameters, such as the gain in height SDS (ΔHt SDS), the observed height velocity (HV) expressed in cm/year or in SDS, or the increase in HV (ΔHV) compared to the pre-treatment year. A number of definitions of poor first-year growth response have been proposed in clinical trials and consensus statements, such as a gain in height < − 2 SD and a height < − 2.5 SD at the age of 4 years and at onset of therapy. Prepuberty was defined as having a testicular volume less than 4 ml for boys and Tanner breast stage 1 for girls. In the GHD group, patients born SGA were excluded. In the SGA group, patients with severe GHD (peak GH < 5 μg/L) were also excluded. An additional exclusion criteria for all groups was age ≥10 years for girls.

PLoS Genet. 2013;9(9):e1003796. doi: 10.1371/journal.pgen.1003796.
Meta-analysis of genome-wide association studies identifies six new Loci for serum calcium concentrations.
O’Seaghdha CM1, Wu H, Yang Q, Kapur K, Guessous I, Zuber AM, Köttgen A, Stoudmann C, Teumer A, Kutalik Z, Mangino M, Dehghan A, Zhang W, Eiriksdottir G, Li G, Tanaka T, Portas L, Lopez LM, Hayward C, Lohman K, Matsuda K, Padmanabhan S, Firsov D, Sorice R, Ulivi S, Brockhaus AC, Kleber ME, Mahajan A, Ernst FD, Gudnason V, Launer LJ, Mace A, Boerwinckle E, Arking DE, Tanikawa C, Nakamura Y, Brown MJ, Gaspoz JM, Theler JM, Siscovick DS, Psaty BM, Bergmann S, Vollenweider P, Vitart V, Wright AF, Zemunik T, Boban M, Kolcic I, Navarro P, Brown EM, Estrada K, Ding J, Harris TB, Bandinelli S, Hernandez D, Singleton AB, Girotto G, Ruggiero D, d’Adamo AP, Robino A, Meitinger T, Meisinger C, Davies G, Starr JM, Chambers JC, Boehm BO, Winkelmann BR, Huang J, Murgia F, Wild SH, Campbell H, Morris AP, Franco OH, Hofman A, Uitterlinden AG, Rivadeneira F, Völker U, Hannemann A, Biffar R, Hoffmann W, Shin SY, Lescuyer P, Henry H, Schurmann C; SUNLIGHT Consortium; GEFOS Consortium, Munroe PB, Gasparini P, Pirastu N, Ciullo M, Gieger C, März W, Lind L, Spector TD, Smith AV, Rudan I, Wilson JF, Polasek O, Deary IJ, Pirastu M, Ferrucci L, Liu Y, Kestenbaum B, Kooner JS, Witteman JC, Nauck M, Kao WH, Wallaschofski H, Bonny O, Fox CS, Bochud M.
1National Heart, Lung, and Blood Institute’s Framingham Heart Study and Center for Population Studies, Framingham, Massachusetts, United States of America; Renal Division, Massachusetts General Hospital, Boston, Massachusetts, United States of America.

Calcium is vital to the normal functioning of multiple organ systems and its serum concentration is tightly regulated. Apart from CASR (calcium sensing receptor), the genes associated with serum calcium are largely unknown. They conducted a genome-wide association meta-analysis of 39,400 individuals from 17 population-based cohorts and investigated the 14 most strongly associated loci in ≤ 21,679 additional individuals. Seven loci (six new regions) in association with serum calcium were identified and replicated. Rs1570669 near CYP24A1 (P = 9.1E-12), rs10491003 upstream of GATA3 (P = 4.8E-09) and rs7481584 in CARS (P = 1.2E-10) implicate regions involved in Mendelian calcemic disorders: Rs1550532 in DGKD (diacylglycerol kinase delta) (P = 8.2E-11), also associated with bone density, and rs7336933 near DGKH/KIAA0564 [VWA8 (KIAA0564) von Willebrand factor A domain containing 8] (P = 9.1E-10) are near genes that encode distinct isoforms of diacylglycerol kinase. Rs780094 is in GCKR. They characterized the expression of these genes in gut, kidney, and bone, and demonstrate modulation of gene expression, in bone in response to dietary calcium in mice. These results shed new light on the genetics of calcium homeostasis.

ENDO2019 PLENARY LECTURE Session PL02 – Gene Expression in Early Development: Gene Editing, Imprinting and Epigenetic Regulation.
PL02-2. Imprinting and Epigenetic Gene Regulation During Development and Under Adverse Environmental Conditions.
Marisa Bartolomei, PhD. University of Pennsylvania, Philadelphia, PA, USA.

Imprinted genes, which are unique to mammals, are monallelically expressed in a parent-of-origin specific manner. Most imprinted genes reside in clusters that are located throughout the mammalian genome. The clusters contain an imprinting control region (ICR), which harbors allele-specific methylation and governs the imprinting of the entire domain. Although most imprinted clusters use long non-coding RNAs to regulate imprinted gene expression, a few are regulated by CTCF (CCCTC binding factor) and allele-specific insulator function. One such cluster harbors the H19 and Igf2  imprinted genes, and is controlled by an ICR that contains multiple CTCF binding sites. Gain of maternal methylation and loss of paternal hypermethylation of the H19/IGF2 ICR are associated with the human growth disorders Beckwith-Wiedemann Syndrome (BWS) and Silver-Russell Syndrome (SRS), respectively. Using gene targeting and genome editing, authors have generated cell lines and mice to study imprinting mechanisms. They have also studied imprinting perturbations in a mouse model under adverse environmental conditions such as EDC (endocrine disruptive chemicals) exposure and techniques used in assisted reproductive technologies (ART).

Int J Dev Biol. 2014;58(2-4):291-8. Epigenetics and imprinting in human disease.
Kalish JM1, Jiang C, Bartolomei MS.

Most genes are expressed from both parental chromosomes; however, a small number of genes in mammals are imprinted and expressed in a parent-of-origin specific manner. These imprinted genes play an important role in embryonic and extraembryonic growth and development, as well as in a variety of processes after birth. Many imprinted genes are clustered in the genome with the establishment and maintenance of imprinted gene expression governed by complex epigenetic mechanisms. Dysregulation of these epigenetic mechanisms as well as genomic mutations at imprinted gene clusters can lead to human disease.

Selected Comments
The murine genome contains ~150 imprinted genes. Importantly, imprinting is well-conserved across mammals, with many imprinted genes, and most imprinting mechanisms conserved between mouse and human. Most imprinted genes are present in distinct clusters that are about 1 Mb in length and contain both maternally and paternally expressed genes. In addition to protein-coding genes in these clusters, there are typically long noncoding RNAs (ncRNA), some of which regulate the imprinting of the nearby genes. Regulation of the clustered genes is coordinated through short DNA sequences called imprinting control regions (ICRs). All ICRs identified thus far are differentially methylated regions (DMRs) in which DNA is methylated on one parental allele.
Another growing patient population that questions our understanding of the maintenance and establishment of imprinting is assisted reproductive technologies (ART) conceptions. The timing of ART coincides with both the establishment and maintenance of imprinting. In ART, the egg donor undergoes hormonal hyper-stimulation to facilitate release of multiple oocytes; this is the time at which the oocyte is in its growth phase and is being reprogrammed. With respect to imprinting, mouse studies have shown that maternally methylated ICRs are methylated during oocyte growth, although these ICRs are not methylated simultaneously. The subsequent in vitro fertilization, embryo culture and transfer to mothers also occur when the embryo is undergoing extensive reprogramming. In this case, the embryo undergoes a post-fertilization extensive loss of DNA methylation together with changes in post-translational histone modifications, which prepare the embryo for cleavage divisions and subsequent lineage differentiation. Thus, ART manipulations take place during sensitive periods of mammalian development. Several small studies have suggested increased incidence of BWS and AS following ART; however, large studies to confirm the true incidence have not been completed to date. A recent meta-analysis attempting to correlate the results of 8 studies of ART and BWS summarized that 6 of the studies found a positive correlation between BWS and ART and calculated an overall relative risk of 5.2 (Vermeiden and Bernardus, 2013). In several of the individual studies, when decreased fertility in the parents was taken into account, the increased incidence of imprinting disorders in ART was not significant. Increased incidences of RSS, AS, and PWS were not seen but the overall incidences of these disorders are much lower than BWS. It should be noted that the vast majority of ART-associated cases of BWS and AS involve loss of ICR methylation. This is especially interesting for AS, where loss of methylation is extremely rare in the population. Animal models have confirmed that techniques used in ART can cause epigenetic perturbations at imprinted (and other) loci. The animal models have the added attraction that infertility is not a confounding factor. Animal models have tested hormonal hyperstimulation, IVF, embryo culture and transfer, all of which have been associated with aberrant imprinting, including loss of imprinting and loss of ICR methylation. Bovine models demonstrate that ART leads to increased large offspring syndrome with macrosomia, macroglossia, and abdominal wall defects and biallelically expressed imprinted genes seen in BWS. Interestingly, the ART conceptuses show a much greater imprinting perturbation in the placentas than in embryonic tissues. While there are a number of possible explanations for this result, one of the most compelling explanations is that imprinted genes have redundant mechanisms to maintain parental-specific imprinting, including DNA methylation and post-translational histone modifications, in the embryonic lineages, whereas extraembryonic tissues are less likely to employ both sets of epigenetic machinery in the maintenance of imprinted gene expression.
In summary, establishment and maintenance of imprinted gene expression is integral for normal embryonic and extraembryonic development. Misregulation of this process can occur at many levels and leads to clinical disease. The role of individual genes in each of these imprinted clusters is still being uncovered. Further understanding of the regulation of imprinted genes may lead to improvements in ART and improved management of human imprinting disorders.

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