The research program described below is the backbone of the Euro-MRX project, undertaken by the Euro-MRX consortium and partner institutes. You will find information on the general goals, the expected achievements, background information of the field of study, and experimental approach of this project.

The research program consists of a number of work packages, all aimed at reaching the goals listed below. Please refer to the page Work Packages for more details.

General goals

The research program has the following aims:

• Development of a large central database of X-linked mental retardation (MRX) families, which is connected to a repository of patient cell lines.
• Identification of additional MRX genes.
• Elucidation of the biological role of MRX genes, which will lead to an understanding of how mutations give rise to mental retardation in humans.

Expected achievements

The consortium has clinical and molecular data from a collection of approximately 350 families affected by MRX. Of most (but not all) families a cell-line is present. Organising this data into a database and connecting the cell-lines to the database will make the knowledge available to the entire scientific community. This will expedite the identification of new genes involved in MRX.

We expect that at least 10 additional genes will be identified during the course of this research program. Moreover, we will address the question of how a large variety in mutations can lead to the one phenotype of non-specific mental retardation. Leading in our investigations will be the observation by the participants of the consortium, that 3 out of the 16 identified genes are involved in cellular signalling through Rho-GTPases.

Much of our efforts will be dedicated to the refinement of a protocol for clinical classification of MRX patients. Families in which the causative gene has been indentified will be clinically examined in great detail. The results will be compared with those of other families, in order to reveal subtle phenotypic features associated with a specific mutation. The indentified features can then be used for guidance in DNA diagnostic testing.

Background information

One of the three criteria of the definition of mental retardation is defined as a person having an IQ lower than 70. A person with an IQ lower than 50 is considered to be severely handicapped. About 2% of the population is mentally retarded, most of which are males. Despite its high incidence, little is known about this disease.

The diagnosis of MR is complex because a combination of multiple genes and environmental factors may be involved. In 1996 the first MRX gene was identified, called FMR2, which is located adjacent to the fragile X-E (FRAXE) site on Xq28. Up till now, 15 additional MRX genes have been identified. Three of these encode proteins that are involved in signalling by Rho GTPases. The latter have been implicated in control of the actin cytoskeleton and gene expression. It thus seems that disruption of the normal signalling events through the Rho GTPase pathway is detrimental for adequate information processing by the brain.

The other MRX genes include:

• OPHN1 (oligophrenin 1), encoding a Rho GAP protein
• PAK3 (p21 activating kinase), a regulator of the Rho family of small GTPases
• ARHGEF6, encoding αPIX, aRHOGEF

Proteins of the Rho GTPase family are key regulators of the actin cytoskeleton, and have been shown to be important in regulating neurite outgrowth and neuronal connectivity in response to extracellular signals. In addition, by activating of MAP kinase cascades they influence gene expression. The implication of three Rho-associated genes in MRX suggests that Rho GTPases are prominently involved in mechanisms essential for normal information processing.

Among the remaining MRX genes, IL1RAPL1 (IL-1 receptor accessory protein like) and TM4SF2 (transmembrane 4 superfamily) may signal indirectly through Rho GTPases, while RSK2 may act downstream of PAK3. In addition, several genes mutated in MRXS are also directly or indirectly linked to Rho signalling. For example, the gene involved in Aarskog-Scott syndrome, FGD1, encodes a Rho GEF.

With the completion of the Human Genome Project, the sequence of almost the entire X-chromosome and its encoded genes are known. A major challenge will be to establish the involvement of these genes in human X-linked disorders. The current data suggest that most genes only have a minor contribution to the incidence of MRX in the population. Hence, identification of all causative genes, or a significant portion thereof, requires the availability of large cohorts of MRX patients to be analysed.

Once most of the genes involved in MRX have been identified, the major challenge will be to explain how mutations lead to mental impairment. One important question to address is how mutations in so many genes lead to one phenotype: nonspecific mental retardation, i.e. mental retardation without apparent neurological or physical abnormalities. A partial explanation for this may be that mental retardation is the final consequence of any process that leads to subnormal neural functioning.

However, the alternative theory that genes underlying MRX may be involved in the same or similar biological pathways may also be part of the explanation. This notion is strengthened by the observation that three of the known MRX genes are involved in the same signalling pathway of Rho GTPases. For this reason we are studying the role of the Rho GTPases and its interacting proteins in neural development and plasticity, and explore the effect of mutations found in MRX patients.

Experimental approach

The starting point in our investigations is a collection of clinical and molecular data of approximately 350 MRX families, which was established by the Euro-MRX consortium from 1996 till date. This collection has been instrumental in the identification of seven of the eleven MRX genes presently known. These genes have proved to be causative of MRX in only a small percentage of cases (1% on average). Therefore, in order to reach the goal of identifying additional MRX genes, our collection of families needs to be greatly expanded.

The techniques that will be employed in identifying novel MRX genes, include the following:

• a highly standardised strategy for the characterisation of X-chromosomal breakpoints which are associated with MRX
• cDNA microarrays for the detection of abnormal expression of (X-chromosomal) genes in cell lines from MRX patients
• genomic microarrays for the detection of X-chromosomal microdeletions by Comparative Genomic Hybridisation (arrayCGH) and multiplex amplifiable probe hybridisation (MAPH)

Once causative MRX genes are identified, their biological role will be established by the use of neuronal cell systems and knock-out/transgenic mice. These studies will explore the pathways involving Rho-GTPases and their contribution to micro-anatomical abnormalities underlying mental retardation. The results of these studies should allow us to asses the in vivo function of the novel MRX genes in:

1. regulating the cytoskeleton
2. development of neural morphology and connectivity.

The in vivo results will be translated to a molecular level by the use of microarray techniques. These techniques allow us to study the gene expression profiles in cultured neuronal cells and in relevant brain tissue (like the cerebral cortex and hippocampus) in MRX mouse models. Hereby, abnormal gene expression will be revealed in known or even unknown signalling pathways.

Besides molecular and cellular research, clinical studies will be performed on families in which the causative gene has been identified. By comparing these clinical results with those of other MRX families, subtle phenotypic features will be recognised which are associated with a specific mutation. This will refine the clinical classification of MRX, of which the benefits are twofold:

1. improvement of diagnostic testing
2. acceleration of the identification of novel MRX genes

To manage the expanding sets of clinical, molecular and cellular data, a central database is presently under construction, wich can be queried by the entire scientific community.