Participants involved: Nijmegen (responsible), Leuven.
Collaborating institutes: Nicosia.

Objectives

1. MRX candidate genes will be identified upon the detection and characterization of X-chromosomal microdeletions in MRX patients. Microdeletions will be detected along two approaches: array-based comparative genomic hybridisation (Array-CGH) and multiplex amplifiable probe hybridisation (MAPH) analysis
2. improvement of the sensitivity of the detection level of chromosomal deletions. The X-chromosomal microarrays used for arrayCGH can be directly used for Cytogenetic Diagnostic testing
3. conditions for implementation of novel genes for DNA-diagnostics

Methodology and study materials

X-chromosomal genomic microarrays will be generated. Two types of arrays will be constructed with increasing sensitivity. The first generation will consist of approximately 350 BAC clones with an average spacing of less than 400 kb, covering 25-30% of the X-chromosome. The next array, containing almost 1500 BAC clones, will cover the entire X-chromosome (participant Nijmegen).

These arrays will be used for arrayCGH analysis. DNA from patients will be fluorescently labeled (Cy-3/Cy-5) according to established protocols and resulting probes will be hybridized to the genomic microarrays. Decreased signal intensities will identify BAC clones corresponding to a (partial) deletion/duplication in a patient. This approach allows the detection of deletions/duplications of >50-100 Kb, which is a significant improvement of the current cytogenetic techniques, which allow detection of only those deletions larger than 2 Mb (participants Nijmegen and Leuven).

The other microdeletion screening protocol, MAPH-analysis, is based on PCR methods. A collection of approximately 800 PCR-amplifiable probes will be generated, evenly spaced across the X-chromosome (average distance 200 kb). These probes will be used for MAPH screening of a representative male patient from each MRX family (participant Nicosia). The MAPH probes that do not hybridize to immobilized patient DNA are indicative for the presence of a microdeletion and will be further investigated.

The Identification of microdeletions, either by arrayCGH or MAPH, will immediately result in novel genes for mental retardation. For this bio-informatic analysis of the deletion regions will be employed.

MRX candidate genes will further tested as described in work package 5.