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Vol. 9 Supplement 1

September 2011


How Epigenomics Brings Phenotype into Being

Jose Ignacio Martín-Subero, PhD


After sequencing the human genome, it has become clear that genetic information alone is not sufficient to understand phenotypic manifestations. The way the DNA code is translated into function depends not only on its sequence but also on the interaction with environmental factors. It is in this intersection where the science of epigenetics plays a crucial role. Epigenetic mechanisms like DNA methylation and histone modifications are essential for multiple physiological processes like development, establishment of tissue identity, imprinting, X-chromosome inactivation, chromosomal stability and gene transcription regulation. Additionally, environmental factors like nutrition or maternal behavior in early childhood are able to induce epigenetic changes. This short review aims at summarizing the role of epigenetics in multiple aspects of biology and medicine, including development, cancer, non-tumoral diseases, environmentally induced phenotypic changes, and also in inheritance and evolution.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):506-510

Keywords: Epigenomics, DNA methylation, phenotype, environment, disease


How to Analyse Epigenetic Marks?

Ole Ammerpohl, PhD, Reiner Siebert, MD


By adding adaptable information about the activity of genes, epigenetics enables the activation of specific genes depending on the prevalent environmental conditions and individual requirements of a cell. Although epigenetic information is heritable, it is not stored in the sequence of the DNA but mainly in the modification pattern of the chromatin, i.e. the methylation of cytosine residues in the DNA or covalent modifications of the histones. By controlling gene activity and therefore the availability of the final gene product in the cell, epigenetic alterations can have similar effects as classical genetic mutations. Indeed, the recent past epigenetic modifications have become a focus for the clinic for diagnostics, prognostics, as well as therapeutic purposes. This review briefly summarizes the major aspects of epigenetics and presents a comprehensive overview about the fundamental principles of DNA methylation analysis.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):511-514

Key words: epigenetics, chromatin, DNA methylation


Molecular Androgen Memory in Sex Development

Paul-Martin Holterhus, MD


Sex specific development in the human comprises irreversible sexual differentiation of the external genitalia during embryogenesis, sexual maturation of secondary sex characteristics during puberty (e.g., sex specific body proportions, pubertal voice change) and eventually sex specific development of extragenital tissues and organs, including the brain. The presence or absence of androgens acting via the androgen receptor plays a key role therein. At the single cell level, androgens cause reversible short term changes of gene transcription by activating the androgen receptor. From a developmental perspective, this often leads to irreversible long-term changes of anatomy (e.g., sex-specific differentiation of the external genitalia) and function (e.g., sex-specific play behavior in children). These observations are discussed in the context of recent genome–wide gene expression studies and first published experimental data at the epigenome level. In essence, there is evidence for a molecular androgen memory at both the transcriptome and the epigenome level.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):515-518

Keywords: hormonal programming, androgen memory, disorders of sex development, androgen insensitivity syndrome, androgen receptor, epigenomics, methylation, microarray, genital fibroblasts


Tumor Risk and Clinical Follow-Up in Patients with Disorders of Sex Development

Martine Cools1, MD, PhD, Leendert Hj Looijenga2, PhD


A subset of patients with disorders of sex development (DSD) is at risk for malignant germ cell tumors (GCTs). The degree of gonadal differentiation (or “testicularization” in the presence of a specific part of the Y chromosome), in combination with expression of embryonic germ cell markers, and (a) Y specific gene(s) related to cell-cycle control and proliferation, determines this risk. Incompletely matured Sertoli/granulosa cells are insufficiently capable of directing the normal mitotic block / meiotic induction germ cell program, and as a result, embryonic germ cells are delayed or blocked in their normal maturation process. Thereby, they remain pluripotent and gain increased mitotic and survival characteristics, being the first step in the pathogenesis of GCTs. The patient’s underlying genetic defect and phenotype might be informative in assessing the degree of gonadal “testicularization” on a clinical basis. Current knowledge allows development of an informative cancer risk assessment of DSD patients.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):519-524

Key words: Germ cell tumor, DSD, gonadoblastoma – carcinoma in situ, gonadal dysgenesis


Clinical Use of Anti-Müllerian Hormone (AMH)

Determinations in patients with Disorders of Sex Development: Importance of Sex- and Age-specific Reference Ranges

Casper P. Hagen, MD, Lise Aksglaede, MD, PhD, Kaspar Sørensen, MD, PhD, Annette Mouritsen, MD, Anders Juul, MD, DMSc, PhD


Determination of postnatal AMH levels in circulation has been used for decades when evaluating a child with ambiguous genitalia. We describe the age- and gender- specific changes of postnatal AMH serum levels to enable an appropriate clinical use of AMH assessment in pediatric endocrinology. In males, cord blood AMH is measurable at high levels (mean 148 (53-340) pmol/L), whereas AMH is undetectable (54%) or very low (95% CI: <2-16 pmol/L) in female infants. AMH is constant through childhood in both sexes, boys having approximately 35 times higher levels than girls with no overlapping between the sexes until puberty. Ambiguous genitalia due to impaired androgen secretion or action may be a result of various conditions with low, normal or high AMH. Furthermore, low AMH is a marker of premature ovarian failure in Turner Syndrome girls. Measurement of AMH is an important tool in assessing gonadal function in children. In this context, detailed normative data are essential.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):525-528

Key words: AMH, DSD, Testicular function, normal ranges


From GHRH to IGF-1 and Downstream: Clinical Phenotypes and Biological Mechanisms

Roland Pfäffle, MD, Wieland Kies, MD, Jürgen Klammt, PhD


Genetic defects have been observed at almost all levels of the GHRH-IGF-1 axis. The first observations of GH-1 gene deletions date some 30 years ago. Whereas mutations in the GH-1 and GHRHR genes account for the majority of mutations detectable in patients with Isolated Growth Hormone Deficiency (IGHD) resulting in postnatal growth failure, the overall detection of genetic defects in these patients remains low with app. 10-15 %. Similarly, at the lower end of the GHRH-IGF-1 axis the frequency of defects within the IGF-1 and IGF-1 receptor (IGF1R) genes might hardly approach 10% of all cases with intrauterine and postnatal growth retardation. In this article we examine the pathomechanisms involved in the genetic defects at both ends of the GHRH-IGF-1 axis and describe the clinical and biochemical phenotypes involved. Although it seems tempting to increase the detection rate by limiting genetic investigations to patients with phenotypic characteristics described, at present it seems more appropriate to follow a permissive approach for such investigations as we are probably have not envisioned the full spectrum of phenotypic variability.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):529-534

Key words: IGHD type1, type 1 B, IGF-1 Mutations


Growth Hormone Deficiency: New Approaches To The Diagnosis

Gerhard Binder, MD, PhD


International consensus statements based on expert experience recommended guide lines how to diagnose GHD. Most recommendations reached only a low level of evidence. Cut-offs for GH were central part of these recommendations, their definition however was arbitrary. Evidence based cut-offs are needed. Using newborn screening cards from healthy and affected newborns, the GH cut-off to detect severe congenital GHD was reassessed and redefined. A GH cut-off level of 7 μg/L confirmed the diagnosis of severe GHD with 100 % sensitivity and 98 % specificity on the basis of our assay method, if clinical evidence was present. The previous cut-off of 20 μg/L cited in the international consensus statements was based on old GH assays methods not used anymore. For the calculation of an non-arbitrary GH cut-off for biochemical testing in children, we defined an auxological gold standard for GH deficiency: non-familial short stature due to catch-down growth during the childhood phase of growth in combination with an effective catchup growth in response to low-dose GH therapy, after exclusion of alternative growth disorders and other potential confounders of growth velocity (true positives). Reference cohorts were normally growing children with Turner syndrome or SGA short stature having the same age (true negatives). Using our in-house GH RIA, highest diagnostic accuracy was provided at a peak GH cutoff during spontaneous secretion at night of 7.3 μg/L (sensitivity, 96.8%; specificity, 82.4%; AUC = 0.93). For arginine, cut-off with the highest number of true test results was 6.6 μg/L (sensitivity, 84.3%; specificity, 75.5%; AUC = 0.83). Importantly, children diagnosed GHD in the past with GH test values above the new cut-offs showed a lower response to GH. In conclusion, by use of retrospective and prospective cohort studies evidence-based cut-offs for GH levels measured in newborns and children can be calculated. By use of these cut-offs, tests can be improved. Because of the well known intrinsic diagnostic inaccuracy of any GH test, the correct selection of the child to be tested remains of utmost importance. The diagnosis of growth hormone deficiency (GHD) in childhood is guided by recommendations of national and international consensus statements which are based on the experience of experts. Most of these recommendations reach only a low level of evidence. Research on two central topics of these guidelines has recently been published by us and will be reviewed here (1, 2).

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):535-537

Key words: Growth hormone deficiency, newborn, hypogylcemia, cut-off, arginine, night secretion, ROC analysis, auxology


Diagnosis and Management of Disorders of IGF-I Synthesis and Action

J.M.Wit, MD, PhD


After a proper medical history, growth analysis and physical examination of a short child, followed by radiological and laboratory screening, the clinician may decide to perform genetic testing. We recently proposed several clinical algorithms that can be used to establish the diagnosis. GH insensitivity (primary IGF-I deficiency) can be caused by genetic defects in GHR, STAT5B, IGF1, IGFALS, which all have their specific clinical and biochemical characteristics. IGF-I resistance is seen in heterozygous defects of IGF1R. If besides short stature additional abnormalities are present, these should be matched with known dysmorphic syndromes. If no obvious candidate gene can be determined, a whole genome approach can be taken to check for deletions, duplications and/or uniparental disomies (SNP-array) or whole exome sequencing. Children with GHR defects, and presumably STAT5B and homozygous IGF1 defects, can be treated with rhIGF-I. Children with IGF1R defects and mild or heterozygous IGF1 defects respond to GH treatment.

Ref: Ped. Endocrinol. Rev. 2011;9(suppl 1):538-540

Key words: short stature, growth, growth hormone, IGF1, GHR, STAT5B, IGFALS, IGF1R



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