Objectives

• Design and validate a DSD GeneChip and and use this tool to identify novel genetic markers associated with anomalies of genitourinary development (task 1 and 2)
• Identify deletion/duplication events in the human genome that are associated with DSD using array-based comparative genomic hybridisation (aCGH) technologies (task 3, 4 and 5)

Workpackage Description

Task 1: Design of a DSD GeneChip

Sequencing by hybridization on microarrays offers the opportunity to simultaneously screen many genes without the need for time-consuming and laborious conventional sequencing technology. Consortium members will identify and agree upon a series of genes involved in DSD. Cooperation between basic scientists and clinicians within the consortium is essential to define a common set of genes that will be of interest to the wider community. Technical design of a GeneChip corresponding to these genes will be performed by the subcontractor.

Task 2: Production and validation of DSD GeneChip

The DSD GeneChip will be initially validated by the subcontractor by the analyses of DNA from patients identified from the database developed in WP01, as well as samples obtained directly from consortium members. The accuracy of the chip will be checked by automated capillary sequencing at the Institute Pasteur, UCL Institute of Child Health, London and University of Münster. The validation process is essential to ensure the development of a robust common protocol that could eventually be translated into a diagnostic tool.

Task 3: Patient selection for array CGH Analysis from DSD database

International cooperation between the basic research centres and clinical centres, through the pooling of rare phenotypes and their subsequent analysis, is essential to achieve this objective. Consortium members will agree on a group of patients for genome analysis by array CGH technologies. The analysis will be performed on patients that will have been registered in the DSD database and that have explicit prior informed consent. DNA samples will be analysed in an anonymous manner by the 3 research centres (Paris, London, Münster) and the array CGH data will be sent back to the database for genotype/phenotype correlations at consortium meetings.

Task 4: Array-based comparative genomic hybridization

The microarray CGH analysis will be equally divided between the specialized centres of Paris, London and Münster. Chromosome or regional-specific high-resolution CGH arrays will be used to further refine the exact location of deletion/duplication events that may be associated with the phenotype. The expression profile of novel genes identified by these procedures will be further investigated using standard procedures (University of Rotterdam). These will include analysis on both the RNA and protein level. As well as relying on WP01 for the identification of patients for the CGH analysis, it is anticipated that this part of the work-package could have a significant impact on WP04 and WP03.

Task 5: Genotype/phenotype correlations and interactions with other WPs (all consortium members)

The relationship between the aCGH data and the patient phenotype will be discussed at consortium meetings. At this time the phenotype will be matched with the aCGH datasets. Novel genes identified by this approach will be further analysed by screening patients with a similar phenotype and an apparently intact chromosome structure. This is a key strength of pooling the data centrally by cooperation between the different participating centres.