Copy Number Variation in Patients with Disorders of Sex Development Due to 46,XY Gonadal Dysgenesis

Disorders of sex development (DSD), ranging in severity from mild genital abnormalities to complete sex reversal, represent a major concern for patients and their families. DSD are often due to disruption of the genetic programs that regulate gonad development. Although some genes have been identified in these developmental pathways, the causative mutations have not been identified in more than 50% 46,XY DSD cases. We used the Affymetrix Genome-Wide Human SNP Array 6.0 to analyse copy number variation in 23 individuals with unexplained 46,XY DSD due to gonadal dysgenesis (GD). Here we describe three discrete changes in copy number that are the likely cause of the GD. Firstly, we identified a large duplication on the X chromosome that included DAX1 (NR0B1). Secondly, we identified a rearrangement that appears to affect a novel gonad-specific regulatory region in a known testis gene, SOX9. Surprisingly this patient lacked any signs of campomelic dysplasia, suggesting that the deletion affected expression of SOX9 only in the gonad. Functional analysis of potential SRY binding sites within this deleted region identified five putative enhancers, suggesting that sequences additional to the known SRY-binding TES enhancer influence human testis-specific SOX9 expression. Thirdly, we identified a small deletion immediately downstream of GATA4, supporting a role for GATA4 in gonad development in humans. These CNV analyses give new insights into the pathways involved in human gonad development and dysfunction, and suggest that rearrangements of non-coding sequences disturbing gene regulation may account for significant proportion of DSD cases

DNA binding-dependent androgen receptor signaling contributes to gender differences and has physiological actions in males and females

We used our genomic androgen receptor (AR) knockout (ARKO) mouse model, in which the AR is unable to bind DNA to: 1) document gender differences between males and females; 2) identify the genomic (DNA-binding-dependent) AR-mediated actions in males; 3) determine the contribution of genomic AR-mediated actions to these gender differences; and 4) identify physiological genomic AR-mediated actions in females. At 9 weeks of age, control males had higher body, heart and kidney mass, lower spleen mass, and longer and larger bones compared to control females. Compared to control males, ARKO males had lower body and kidney mass, higher splenic mass, and reductions in cortical and trabecular bone. Deletion of the AR in ARKO males abolished the gender differences in heart and cortical bone. Compared with control females, ARKO females had normal body weight, but 14% lower heart mass and heart weight/ body weight ratio. Relative kidney mass was also reduced, and relative spleen mass was increased. ARKO females had a significant reduction in cortical bone growth and changes in trabecular architecture, although with no net change in trabecular bone volume. In conclusion, we have shown that androgens acting via the genomic AR signaling pathway mediate, at least in part, the gender differences in body mass, heart, kidney, spleen, and bone, and play a physiological role in the regulation of cardiac, kidney and splenic size, cortical bone growth, and trabecular bone architecture in females.

Identification of a parathyroid hormone in the fish Fugu rubripes

A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system

Minerva Endocrinologica : a journal on endocrinological pathophysiology

<div><p>Background</p><p>Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma <i>In Situ</i> (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments.</p><p>Results</p><p>This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape.</p><p>Conclusion</p><p>The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.</p></div

Display of H3K4me3 and H3K27ac tracks for both NCCIT and TCam-2. (A) <i>SOX17</i>, (B) <i>SOX2</i>, (C) <i>OCT3/4</i> (<i>POU5F1</i>), (D) <i>NANOG</i>.

<p>Arrows indicate direction of transcription. Green boxes indicate markers specific for the histological subtype represented by the cell line. Black boxes  =  no difference between the cell lines; red boxes  =  not a marker for that cell type. Note the different ranges on the y-axis for H3K4me3 and H3K27ac.</p

Heat map of epigenetic markers and gene expression profiles.

<p>Genes with quantified methylation status around their TSS (based on Illumina annotation) and valid (see Materials &amp; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098330#s2" target="_blank">Methods</a>) measurement of their expression level were included (n = 11,620). Log-2 summed peak heights per gene were used as an estimate of histone marker enrichment. Variables scaled between 0 and 1. Hierarchical clustering was performed using complete linkage. Clusters of interest were identified based on a consistent enriched state for one or more of the active histone markers and a hypomethylated state around the TSS. Number of genes in the displayed right panel (zoomed in heat maps, top→bottom): 899/892 (TCam-2) and 1,224/37/308 (NCCIT). Gene expression was allowed to vary within clusters, but clusters with almost absent expression levels (completely red) were not selected. Gene symbols indicate genes that overlap with the analysis of top differentiating genes between the cell lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098330#pone-0098330-g006" target="_blank">Figure 6</a>). Gene symbols are listed alphabetically. An indication of the level for each gene in each column is presented by the color/shade and a blue line (for each column: left = 0, right = 1).</p

Overlap between top differentiating genes (methylation/histone modification/gene expression).

<p>(Hypo) methylation, (high) gene expression and histone marker (enrichment) should be interpreted relative to the other cell line. Criteria for selection are described in the main text. Briefly, significant differential methylation of regions with sufficient probe density was identified by DMRforPairs (frequently, but not necessarily close to, the TSS). The difference in histone modification enrichment was assessed by significant differences in summed peak heights between the cell lines. Finally, a fold difference of 3.65 (boundary of 99% CI) was used as cutoff for differential gene expression. Gene lists are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098330#pone.0098330.s005" target="_blank">Table S1</a>, and overlap was determined based on matching gene symbol.</p

Relation between histone modification level (summed peak heights per gene) and expression level.

<p>Top and bottom right images depict the percentage of highly (&gt;p50) expressed genes calculated for an interval of summed peaks. For example, 5% of genes with a log2(summed peak height) of ≈5.5–7.5 were highly expressed. (B) Relation between CpG methylation (TSS/no TSS) and gene expression.</p