The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD1, OMIM 158900) is caused by contraction of the D4Z4 repeat array on 4qter. We show that this contraction causes marked hypomethylation of the contracted D4Z4 allele in individuals with FSHD1. Individuals with phenotypic FSHD1, who are clinically identical to FSHD1 but have an unaltered D4Z4, also have hypomethylation of D4Z4. These results strongly suggest that hypomethylation of D4Z4 is a key event in the cascade of epigenetic events causing FSHD1.

The main characteristic of the postnatal development of the stress system in the rat is the so-called stress hypo-responsive period (SHRP). Lasting from postnatal day (pnd) 4 to pnd 14, this period is characterized by very low basal corticosterone levels and an inability of mild stressors to induce an enhanced ACTH and corticosterone release. During the last years, the mouse has become a generally used animal in stress research, also due to the wide availability of genetically modified mouse strains. However, very few data are available on the ontogeny of the stress system in the mouse. This study therefore describes the postnatal ontogeny of peripheral and central aspects of the hypothalamic-pituitary-adrenal (HPA) axis in the mouse. We measured ACTH and corticosterone in blood and CRH, urocortin 3 (UCN3), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) transcripts in the brain at postnatal days 1, 2, 4, 6, 9, 12, 14 and 16. Our results show that we can subdivide the postnatal development of the HPA axis in the mouse in two phases. The first phase corresponds to the SHRP in the rat and lasts from right after birth (pnd 1) until pnd 12. Basal corticosterone levels were low and novelty exposure did not enhance corticosterone or ACTH levels. This period is further characterized by a high expression of CRH in the paraventricular nucleus (PVN) of the hypothalamus. Expression levels of GR in the hippocampus and UCN3 in the perifornical area are low at birth but increase significantly during the SHRP, both reaching the highest expression level at pnd 12. In the second phase, the mice have developed past the SHRP and were now exhibiting enhanced corticosterone basal levels and a response of ACTH and corticosterone to mild novelty stress. CRH expression was decreased significantly, while expression of UCN3 and GR remained high, with a small decrease at pnd 16. The expression of MR in the hippocampus was very dynamic throughout the postnatal development of the HPA axis and changed in a time and subregion specific manner. These results demonstrate for the first time the correlation between the postnatal endocrine development of the mouse and gene expression changes of central regulators of HPA axis function.

Facioscapulohumeral muscular dystrophy (FSHD) progressively affects the facial, shoulder, and upper arm muscles and is associated with contractions of the polymorphic D4Z4 repeat array in 4q35. Recently, we demonstrated that FSHD alleles are hypomethylated at D4Z4. To study potential relationships between D4Z4 hypomethylation and both residual repeat size and clinical severity, we compared the clinical severity score with D4Z4 methylation in unrelated FSHD patients. Correcting the clinical severity score for age at examination improves the parameter to define clinical severity and provides further support for hypomethylation of FSHD alleles. However, a linear relationship between repeat size and clinical severity of the disease cannot be established. Interestingly, FSHD can be separated in two clinical severity classes: patients with residual repeat sizes of 10 to 20 kb are severely affected and show pronounced D4Z4 hypomethylation. In contrast, patients with repeat sizes of 20 to 31kb show large interindividual variation in clinical severity and D4Z4 hypomethylation. Because the majority of familial FSHD cases are represented in this interval and considering the overt variation in clinical severity in these familial cases, it thus is imperative to develop comprehensive allele-specific assays monitoring total D4Z4 methylation to investigate whether interindividual variation in D4Z4 methylation can be translated into a prognostic factor for clinical severity.

A close contact between the dam and the litter is essential for the normal development of the hypothalamic-pituitary-adrenal (HPA) axis in rats and mice. Maternal signals, as licking and feeding, have been shown to sustain the HPA axis of the pups in a hypo-responsive state. Disruption of this mother-pup interaction by 24 h of maternal deprivation activates the otherwise quiescent stress system of the neonates, resulting in an enhanced adrenal sensitivity to adrenocorticotropic hormone (ACTH) and a decreased expression of central HPA markers, such as corticotropin-releasing hormone (CRH). However, the dynamics of these central and peripheral changes over the 24h period are largely unknown. In this study, we examined the time course of some of the central and peripheral indices of HPA activity during 24 h of maternal deprivation. We measured corticosterone and ACTH in the blood as well as CRH, mineralocorticoid and glucocorticoid receptor expression in the brain. Our results demonstrate that each of the components of the HPA axis responds to maternal deprivation at different time points following removal of the mother and with a very specific time course. The main activation of the HPA axis occurred between 4 h and 8 h of maternal absence. By contrast, during the second half of the deprivation period, negativefeedback mechanisms restrained the further increase in ACTH and corticosterone release. We conclude that maternal deprivation triggers a cascade of sequential changes at the various levels of the stress system, and that measuring only one aspect of the system at one time point does not accurately reflect the dynamic alterations of the HPA axis.

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.