Disorders of Sex Development and Germ Cell Cancer: genetics and microenvironment

The ultimate purpose of sexual reproduction, which depends on specialized male and female anatomy and physiology, is to enable continuation of a species and introduction of genetic diversity. In mammals the developmental path towards a male or a female is in principle determined at the moment of fertilization, when either a Y- or an X-chromosome is inherited from the father. The subsequent chromosomal constitution, either XY (male) or XX (female) (referred to as chromosomal sex), will eventually drive formation of a testis or an ovary (the so called gonadal sex). This in turn will result in the next step in sex determination (the phenotypic sex), ultimately leading to a phenotypical male or female respectively. Because of the relevance of the general principles related to this phenomenon in understanding the various levels in which pathological gonadal processes can occur, the next paragraphs will explain these issues in more d

Antiandrogens prevent stable DNA-binding of the androgen receptor

The androgen receptor (AR) is essential for development of the male gender and in the growth of the majority of prostate cancers. Agonists as well as most antagonists induce translocation of the receptor to the nucleus, whereas only agonists can activate AR function. Antagonists are therefore used in the therapy of metastasized prostate cancer. To obtain insight into the mechanism by which antagonists block AR function in living cells, we studied nuclear mobility and localization of green fluorescent protein (GFP)-tagged AR in the presence of either the agonist R1881 or the antagonists bicalutamide and hydroxyflutamide. As controls we investigated a non-DNA-binding AR mutant (A573D) and two mutants (W741C and T877A) with broadened ligand specificity. We demonstrate that in the presence of R1881, AR localizes in numerous intranuclear foci and, using complementary fluorescence recovery after photobleaching (FRAP) approaches and computer modelling, that a fraction of AR ( approximately 10-15%) is transiently immobilized in a DNA-binding-dependent manner (individual ARs being immobile for approximately 45 seconds). By contrast, antagonist-bound GFP-AR showed no detectable immobile fraction and the mobility was similar to that of the R1881-liganded non-DNA-binding mutant (A573D), indicating that antagonists do not induce the relatively stable DNA-binding-dependent immobilization observed with agonist-bound AR. Moreover, in the presence of bicalutamide and hydroxyflutamide GFP-AR was homogeneously distributed in the nucleus. Binding of bicalutamide and hydroxyflutamide to GFP-AR(W741C) and GFP-AR(T877A), respectively, resulted in similar mobility and heterogeneous nuclear distribution as observed for R1881-liganded GFP-AR. The live cell studies indicate that the investigated antagonists interfere with events early in the transactivation function of the AR

A multi-exon deletion within WWOX is associated with a 46,XY disorder of sex development

Disorders of sex development (DSD) are congenital conditions where chromosomal, gonad or genital development is atypical. In a significant proportion of 46,XY DSD cases it is not possible to identify a causative mutation, making genetic counseling difficult and potentially hindering optimal treatment. Here, we describe the analysis of a 46,XY DSD patient that presented at birth with ambiguous genitalia. Histological analysis of the surgically removed gonads showed bilateral undifferentiated gonadal tissue and immature testis, both containing malignant germ cells. We screened genomic DNA from this patient for deletions and duplications using an Illumina whole-genome SNP microarray. This analysis revealed a heterozygous deletion within the WWOX gene on chromosome 16, removing exons 6-8. Analysis of parental DNA showed that the deletion was inherited from the mother. cDNA analysis confirmed that the deletion maintained the reading frame, with exon 5 being spliced directly onto exon 9. This deletion is the first description of a germline rearrangement affecting the coding sequence of WWOX in humans. Previously described Wwox knockout mouse models showed gonadal abnormalities, supporting a role for WWOX in human gonad development

Hdac6 deletion delays disease progression in the SOD1G93A mouse model of ALS

Defects in axonal transport are thought to contribute to the pathogenesis of neurodegenerative disease. Because α-tubulin acetylation facilitates axonal transport, inhibition of the α-tubulin deacetylating enzymes, histone deacetylase 6 (Hdac6) and silent information regulator 2 (Sirt2), is thought to be an interesting therapeutic strategy for these conditions. Amyotrophic lateral sclerosis (ALS) is a one such rapidly progressive and fatal neurodegenerative disorder, in which axonal transport defects have been found in vitro and in vivo. To establish whether the inhibition of Hdac6 or Sirt2 may be of interest for ALS treatment, we investigated whether deleting Hdac6 or Sirt2 from the superoxide dismutase 1, SOD1G93A mouse affects the motor neuron degeneration in this ALS model. Deletion of Hdac6 significantly extended the survival of SOD1G93A mice without affecting disease onset, and maintained motor axon integrity. This protective effect was associated with increased α-tubulin acetylation. Deletion of Sirt2 failed to affect the disease course, but also did not modify α-tubulin acetylation. These findings show that Hdac6, rather than Sirt2, is a therapeutic target for the treatment of ALS. Moreover, Sirt2 appears not to be a major α-tubulin deacetylase in the nervous syste

Amino acids 3-13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N-terminal and ligand-binding domain of the androgen receptor

The N-terminal domain (NTD) and the ligand-binding domain (LBD) of the androgen receptor (AR) exhibit a ligand-dependent interaction (N/C interaction). Amino acids 3-36 in the NTD (AR3-36) play a dominant role in this interaction. Previously, it has been shown that a PhixxPhiPhi motif in AR3-36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3-36 can be subdivided into two functionally distinct fragments: AR3-13 and AR16-36. AR3-13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD-AR.LBD complex. AR16-36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic alpha-helix. A second PhixxPhiPhi candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16-36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction

Prevalence of c-KIT mutations in gonadoblastoma and dysgerminomas of patients with disorders of sex development (DSD) and ovarian dysgerminomas

Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent similar to 3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY