Here you find some short work package summaries about the Progress of the work packages
At the heart of the EuroDSD project is a security-oriented software system: the Virtual Research Environment (VRE). The VRE supports all research aspects of the project. Most notably the VRE provides a secure environment for ethically-driven data sharing between the clinical partners who deal directly with patients with Disorders of Sex Development (DSD), and the biomedical research partners who wish to access and analyse bio samples from these patients to better understand DSD. This VRE comprises a data registry for inclusion of patients with DSD by clinicians and interfaces for biomedical researchers to query and access information on those patients. It is important to note that this information does not include patient-identifying information. Indeed a key aspect of the VRE design has been to ensure that no-patient identifying information is accessible. As well as patient privacy, a key aspect of the work has been to ensure information governance and ethics and strictly adhered to. In this context, a well documented set of standard operating procedures have been identified outlining the process for access to the VRE and subsequent inclusion and usage of clinical/biomedical information. The inclusion of a patient record starts and ends with a question to the data entry person to ensure that they have obtained consent to include this record.
The VRE has been iteratively designed and rolled out to the wider DSD research community. This has resulted in several versions of the VRE – each having increased functionality as required by the wider community. This functionality now covers clinical information and associated characterisation of DSD as well as specific modules for following up patients and the genetic screening they may have undertaken (and mutations that may have been found). To support the security requirements of the project, fine-grained access control is supported where researchers can restrict access to and use of their data sets based on a variety of reasons. For example, a clinician might insist that the data is: only available to researchers at their own site/hospital; only available to researchers in their given country, e.g. due to ethics; available to all EuroDSD researchers, or indeed to recognised DSD researchers around the globe. All of this is completely transparent to the clinicians and biomedical researchers themselves.
At the time of writing, the EuroDSD VRE includes information on 800 patients with different types of DSD. The EuroDSD VRE now represents a globally unique resource for research into DSD. As well as supporting the portfolio of clinical and biomedical researchers associated with the EuroDSD project, a key result of the VRE work is that this resource is now used by a far greater range of researchers around the world. For example, researchers from Argentina, Australia, Brazil, the Czech Republic, Estonia, Jordan, Kuwait, Morocco, Poland, Portugal, Spain, Sudan, Turkey, UK and the USA now have access to the EuroDSD VRE and many are using it for storing of their own clinical data sets. As well as numerous clinical/biomedical research publications, the establishment and support of the VRE has also resulted in a collection of software engineering related publications. These results have published in international conferences and journals. The results of the work are also shaping many other efforts involving the VRE development team. For example, major new EU funded projects based upon the EuroDSD security-oriented access and usage model pioneered in EuroDSD are now starting. These include projects in the area of adrenal tumours and in other rare diseases.
The work package aims to identity new genetic changes that may affect the development of the urogenital system. These alterations may be in genes we already know to be involved in the development of the urogenital system or they may be new genes. To identify these causes we are using two approaches. The first is a new sequencing technology that allows us to rapidly, and at low cost, simultaneously sequence 36 genes known to be involved in sexual development. This involves the development of a microarray (or chip) that contains all the sequence information of the 36 genes. Patient DNA would be compared against this array for changes in DNA sequence. This would greatly improve the speed and efficiency of the genetic diagnosis. The second approach is to study DNA from patients for small rearrangements in their genomes. In this approach, called aCGH (array comparative genomic hybridization) we are looking for deletions or duplications that may be either variants in the general population or they may be associated with the problem of urogenital development.
We have designed a microarray (or GeneChip) that can sequence 36 genes known to be involved in human urogenital development. This includes the regions of the gene that code for the proteins and also regions immediately next to these genes. We have also defined simple and common protocols for the preparation of the DNA specifically for this sequencing approach. We are currently testing the accuracy of this technique compared with existing sequencing technologies. We will publish a report on the effectiveness of this technology and make recommendations for the use of the GeneChip. In the second approach, (aCGH), we are currently analysing more than 150 DNA samples from patients with DSD using three technology platforms in Paris, London and Munster that can detect small deletions or duplications in the genome. We aim to study the DNA of 300 patients using this technique by the end of the current project.
The aCGH approach to detect new deletions or duplications that may cause DSD has proven to be very informative. Preliminary results suggest that in patients where the testis failed to form small deletions or duplications can be found in 25% of cases where other organ systems are affected and in 5.6% of cases where only testis development was affected. Therefore, this aCGH approach is a very useful diagnostic tool in the management of DSD patients.
Male genital development is dependent on the action of androgens (male steroid hormones) targetting a key receptor during a small developmental time window. The androgen receptor (AR) is the key player mediating the hormone signal by protein-protein interactions in responsive cells. We are studying the effects on human development when the AR is faulty. We have also searched for known and so far unknown proteins that interact with the AR in this short but important time slot and analyse their impact on genital formation.
Mutations in the gene that control the androgen receptor have been identified and the effects on hormone targetting analysed. A mouse model is also being developed that will enable us to ‘insert’ an abnormal androgen receptor into developing genital tissues to observe its effects.
The AR also works in concert with other proteins called co-regulators. We have extracted mRNA from the genital tissue of male mouse embryos, collected during the developmental stages when the first important male-specific changes occur. Using a cellular system we searched for proteins interacting with the AR in the presence of hormone. Newly identified interaction partners were confirmed by biochemical, molecular and histological assays.
Many androgen receptor mutations have been characterised and their function compared with the normal receptor. Importantly, we have been able to gather information on patients who have these mutations by studying how they develop at puberty. It is now possible to predict what will happen in later life when a baby is born with a mutation in the androgen receptor and calculate the amount of androgen hormone that may need to be given at puberty.
We have found several new candidate genes for AR binding partners. Candidate genes that play a role in proliferation, cell cycle control and differentiation were further analyzed by biochemical and cellular assays. So far we could identify new proteins that interact with AR and verify a regulatory function in AR-dependent gene transcription. By further experiments we aim to elucidate the exact molecular function of the identified proteins.
The project to insert an abnormal androgen receptor into a mouse cell line has been challenging but it is now progressing well with a ‘cassette’ of the receptor in place. A later project will be to create a whole mouse model with the ‘cassette’ containing examples of some of the receptor mutations we study at puberty.
Sexual differentiation in the human starts during early embryogenesis. The very first developmental steps are under control of genes which differ between males and females depending on the gonosomal chromosome constellation, i.e. XY in males and XX in females. In the normal male, the SRY-gene which is located on the Y-chromosome will initiate development of the sexually bipotent gonads into the testes. The testes produce large amounts of testosterone – the major androgen in males- starting as early as in the 7th week of gestation (pregnancy). In the following weeks, the male embryo’s genitalia will change dramatically: the initial female appearance of the sexually bipotent external genitalia will change irreversibly and forever to the male appearance until the 12th week of gestation.
Our previous research has revealed that this testosterone-induced difference between the male and the female external genitalia is paralleled by programming life-long sex specific programs of genome activity in the genital cells. In other words, these cells appear to have a life-long molecular memory reflecting the early action of testosterone. Further clinical and molecular research by us and by others supports that androgen programming is not restricted to the external genitala but occurs in other organs as well, e.g., in the blood, in the brain. Since the production and the action of testosterone may be defective (either increased or decreased) in different forms of disorders of sex development, long term effects of androgen may influence the long term outcome in DSD patients. Therefore it is very important to understand the molecular basis of androgen memory.
Work package 4 of the EuroDSD consortium investigates the hypothesis whether androgen programming occurs at the level of the methylome. „Methylome“ means the introduction of small molecules called methyl-groups into specific regions of the genes at a genome wide level thus controlling their activity and eventually the specific development of our organs and their functions. New gene chip technology enables us to investigate many different sites where methyl groups are expected in our genes (about 15.000 genes). Indeed, our results support that androgen-memory has an epigenomic background. Our current research analyses this so called „epigenomic signature“ in normal males, females and in different forms of DSD to understand clearer their mechanisms and biological function in sex specific development of males, females and patients having DSD.
Sexual differentiation in the human starts during early embryogenesis. The very first developmental steps are under control of genes which differ between males and females depending on the gonosomal chromosome constellation, i.e. XY in males and XX in females. In the normal male, the SRY-gene which is located on the Y-chromosome will initiate development of the sexually bipotent gonads into the testes. The testes produce large amounts of testosterone – the major androgen in males- starting as early as in the 7th week of gestation (pregnancy). In the following weeks, the male embryo’s genitalia will change dramatically: the initial female appearance of the sexually bipotent external genitalia will change irreversibly and forever to the male appearance until the 12th week of gestation.
Our previous research has revealed that this testosterone-induced difference between the male and the female external genitalia is paralleled by programming life-long sex specific programs of genome activity in the genital cells. In other words, these cells appear to have a life-long molecular memory reflecting the early action of testosterone. Further clinical and molecular research by us and by others supports that androgen programming is not restricted to the external genitala but occurs in other organs as well, e.g., in the blood, in the brain. Since the production and the action of testosterone may be defective (either increased or decreased) in different forms of disorders of sex development, long term effects of androgen may influence the long term outcome in DSD patients. Therefore it is very important to understand the molecular basis of androgen memory.
Work package 4 of the EuroDSD consortium investigates the hypothesis whether androgen programming occurs at the level of the methylome. „Methylome“ means the introduction of small molecules called methyl-groups into specific regions of the genes at a genome wide level thus controlling their activity and eventually the specific development of our organs and their functions. New gene chip technology enables us to investigate many different sites where methyl groups are expected in our genes (about 15.000 genes). Indeed, our results support that androgen-memory has an epigenomic background. Our current research analyses this so called „epigenomic signature“ in normal males, females and in different forms of DSD to understand clearer their mechanisms and biological function in sex specific development of males, females and patients having DSD.
Our work package intends to develop an e.learning webportal module that will give entrance to an interactive learning environment for an up to date program on DSD including normal development, patho-physiological mechanisms and current views on diagnostic and therapeutic intervention with the inclusion of psychological counseling and outcome. Scientists from the DSD Consortium but also teachers anywhere in the world and students at various levels of training will meet each other to gain, share, contribute and develop knowledge in an accessible and flexible way.
This module intends to provide up to date and state of the art information exchange at two levels: core level for medical students and advanced level for residents and fellows. It will offer discussion and meet the expert platforms and provide advice for fellows or local supervisors in complicated matters. Additionally, the program can be expanded to provide advice in matters of education and assessment as part of postgraduate training.
The portal has an editorial board (listed below) that is responsible for the content. Several theory chapters and interactive problem solving cases have been uploaded. Many more are in progress. A Medclopedia offers the user the possibility to search for specific subjects in an alphabetically ordered list with subjects. A forum functionality enables authors and reviewers to post comments and remarks and discuss certain topics. The forum will be further developed for users for specific discussions on a case or study results or knowledge sharing. Further steps include the development of a system for scoring and competence assessment. With expert advice from medical educationalists. Many cases offer opportunities to develop and evaluate several competencies such as medical expert, communicator, collaborator, scholar, professional
The module DSD is part of an ESPE website http://www.espe-elearning.org/ and can be accessed by a login request.
Thus far the following chapters and cases are available:
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