X-linked non-specific mental retardation (MRX) is a heterogeneous condition in which mental retardation (MR) appears to be the only consistent manifestation. The genetic and phenotypic heterogeneity exclude any possibility of pooling families and, therefore, of fine-mapping the related disease genes. In order to identify genomic critical regions involved in the MRX condition assigned to Xp21.3-22.1 region, we have implemented the PCR screening of non fragile X MR patients for the presence of deletions in this region. The amplification by PCR of 12 markers located between POLA and DXS704 using genomic DNA from 192 MR males led to the identification, in a 9 year old mentally retarded boy, of a microdeletion which extends from DXS1202 to DXS1065. None of the known genes, POLA, MAGE genes cluster, DAX1, GK and DMD, that map in the Xp21.3-22.1 region is affected by this deletion. This approach, which could easily be applied to several other MRX loci, allowed not only a confirmation of the presence of a potential locus in Xp21.3-22.1 involved in non-specific mental retardation, but also a better definition of the genomic critical region corresponding to this locus.
X-linked mental retardation (XLMR) is a common and vastly heterogeneous group of disorders which can be roughly categorized as syndromic or nonspecific (MRX) (1 ,2 ). Families with syndromic disorders usually have a quite distinct phenotypic presentation whereas families with nonspecific disorders present no distinctive somatic features. Despite recent advances in identifying genes such as FMR1, L1CAM, FGD1 and XH2, involved in XLMR conditions, progress in unravelling nonspecific X-linked mental retardation has been slow, partly because of the clinical and genetic heterogeneity which preclude any possibility of pooling families and, therefore, to fine map the related disease genes. Although mapping data showed at least 28 nonspecific loci with an apparent clustering around the pericentromeric region of the X chromosome (1 ), it is almost impossible to evaluate how many MR genes there are in reality, partly because of the broad localisation and the presence of several overlaps between intervals of assignment. Thus, fine mapping and identification of genes implicated in nonspecific X-linked mental retardation will essentially depend on thorough investigation of molecular abnormalities and systematic screening of many probands for mutations in many candidate genes.
Given these difficulties, we thought that the screening of genomic DNA from MR patients for deletions could allow the identification of genomic MRX critical regions. This screening could be carried out by PCR using very dense set of STS markers previously mapped to specific regions of the X chromosome. We have implemented the PCR screening of 192 MR patients for the presence of deletions in Xp21.3-22.1 region in which a non- specific mental retardation locus has been assigned on the basis of (i) deletions associated with contiguous gene syndromes (3 ), (ii) linkage analysis (4 ), (iii) recent data reported by Fryn's group describing the identification of an inherited deletion encompassing the DXS1218 CA-repeat (5 ). In this report, we describe the identification of a microdeletion in Xp21.3-22.1 associated with a severe non specific mental retardation. Also, we demonstrate the value of screening for microdeletion to define MRX loci using an STS PCR-based approach.
DNA samples from 192 MRX patients have been tested with a panel of 12 X-linked microsatellites or STS markers located between POLA and DXS1020 (Fig. 1 A) (6 ). PCR amplification of these 12 markers using genomic DNA from patients led to the identification of one microdeletion which extends from DXS1202 to DXS1065 (Fig. 1 A and B). None of the known genes, POLA, the MAGE genes family, DAX-1, GK and DMD, that map in Xp21.3-22.1 region is affected by this deletion. Deletion of the MAGE genes was ruled out by Southern blot analysis using HindIII, PstI and EcoRI digested DNA hybridized with a probe corresponding to exon 4 of the MAGE-Xp gene, known to be present in a minimum of five copies in a 30 kb interval of Xp23.1 region (7 ). Analysis of the patient's DNA showed qualitatively and quantitatively patterns similar to those of a non-deleted male used as a control (data not shown). In addition, owing to the fact that DXS1074 and DXS319 which map within the 30 kb interval of the MAGE genes family are proximal and at least 300 kb far from DXS7188 (the non deleted proximal marker) (6 ), we can exclude the involvement of these genes in the deletion.
PCR amplification of STSs in the Xp21-3 locus using genomic DNA from 192 non fragile X MR patients led to the identification of one microdeletion which extends from DXS6764 to DXS7188. These results are in line with the inherited microdeletion associated with nonspecific mental retardation reported by Fryn's group during the 7th International Workshop `Fragile X and X-linked Mental Retardation' (5 ). They also confirm the presence of a potential locus in Xp21.3-22.1 involved in nonspecific mental retardation. This specific-MR deletion will enable focus on the genomic critical region involved in this MRX condition and further investigations aiming to identify expressed sequences.
This very simple PCR-based approach could also be applied to: (i) several other regions of the X chromosome exhibiting deletions of contiguous genes with mental retardation, such as, Xp22.3 (10 ), Xq21.1 (11 ) and Xq28 (12 ); (ii) MR-intervals of assignment based on linkage analysis carried out on individual large families; (iii) genomic regions flanking balanced X;autosome translocation breakpoints associated with mental retardation. The screening for deletions which will be enhanced by the increasing number of mapped STS and EST might allow to pinpoint small specific-MR critical regions both in large genetic-intervals of assignment and in contiguous genes syndromes. In MR cases exhibiting balanced X;autosome translocations, the screening for deletion could allow to favour the involvement of X-linked genes in the phenotype rather than autosomal ones.
J. L. Mandel reported a proposal (13 ) based on systematic screening of many probands for mutations in many candidate genes. He proposed a scheme that associates standardized reporting of XLMR families including small ones and deposit of a lymphoblastoid cell line for one proband of each family to an accessible repository. The STS and EST PCR-based screening for deletions is a complementary approach to Mandel's proposal and to Flint's strategy (14 ) based on the screening for subtelomeric DNA rearrangements in patients with idiopathic MR. Ultimately these systematic approaches could speed up identification of critical regions and genes involved in XLMR.
In this study, patients with idiopathic MR were identified by examining the fragile X case record of J. C. Kaplan's molecular diagnosis unit at Cochin Institute. Male patients were selected on the basis of (i) a low IQ or, if too young for psychological assessment, the presence of significant developmental delay; (ii) no recognizable syndrome whether chromosomal, Mendelian, acquired or environmental. Fragile X syndrome was excluded by Southern blot analysis using DNA digested with EcoRI/EagI endonucleases and StB12-3 probe corresponding to FRAXA locus (15 ).
PCR were carried out in microtitre plates using 50 ng of genomic DNA in a final volume of 50 [mu]l. PCR primers and sizes of fragments were previously reported (8 ,10 ). Amplification conditions were: (i) 5 min denaturation step at 94oC, (ii) 35 cycles of 30 s at 94oC, 30 s at 52oC, 1 min at 72oC, (iii) a final 10 min extention step at 72oC. PCR products were analysed by electrophoresis using 2% Nusieve® (FMC) agarose gel and ethidium bromide staining. PCR products corresponding to microsatellite markers were also analysed by Southern blot probed with a (CA)10 oligonucleotide labelled by kination.
Southern blot was performed according to Sambrook et al. (16 ).
We wish to thank J. C. Kaplan for giving access to genomic DNA samples of MR patients, DMD/DMB patients and normal males and J. L. Mandel for providing the StB12-3 probe. This study was supported by grants from the Association Francaise contre les Myopathies (AFM), Institut National de la Sante (INSERM), Centre National de Recherche Scientifique (CNRS), Fondation pour la Recherche Médicale (FRM) and Groupement de Recherche et d'Etudes sur les Génomes (GREG).
jnl.info{at}oup.co.uk
Human Molecular Genetics
Pages
Introduction
Results
PCR screening and molecular investigation of the deletion
Discussion
Materials And Methods
MR patients
STS amplification and Southern blot analysis
Acknowledgements
References
REFERENCES
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