Objectif
A new procedure was implemented to clone some of the genes included in microdeletions if the chromosome X present in patients with contigous gene deletion syndromes.
This procedure relied on the use of a modification of the PERT technique, the PERT reassociated sequences being selected through several cycles of nonspecific polymerase chain reaction (PCR). To optimize the conditions for sequence enrichment, several modifications of the PERT protocol were designed.
The following areas have been mastered:
the conditions for reassociation of chromosome X-enriched deoxyribonucleic acid (DNA) by PERT;
the specific amplification of the products obtained in these experiments (this required to test the efficacy of various adapter molecules and the conditions of PCR);
the experimental conditions for Alu-PCR amplification of somatic cell hybrids having retained the human X chromosomes.
The main results are presented by the identification of candidate genes for the recessive forms of dystrophic epidermolysis bullosa and epidermolysis bullosa simplex by the localization on the X chromosome of a new form of hypophosphatemic rickets and by the identification of the gene for autosomal dominant Hirschsprung disease (HSCR) on chromosome 10.
The RET protooncogene was recognized as being responsible for HSCR. While the mutations of RET protooncogene observed in HSCR patients ar spread through the different domains of the gene, 3 other inherited disorders namely multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familiar medullary thyroid carcinoma (FMTC) are characterized by mutations in specific domains of RET. As an example a single mutation in exon 16 of RET is present in 95% of the patients with MEN 2B.
The tasks of the project are essentially the following:
To identify new polymorphic DNA markers on the X chromosome. A newly discovered humanspecific and ubiquitous polymorphic core sequence is being used in one of the two participant laboratories to screen a humanhamster cosmid library retaining the X as its sole human cmponent. This should lead to the identification and cloning of a number of hypermorphic marker loci throughout the chromosome. Such markers will be physically mapped and subsequently used for genetic studies in CEPH pedigrees and in families segregating for X-inked disease genes.
In addition screenings for such new hypermorphic markers will be performed in particular regions of interest from which YAC clones have already been isolated such as the Xq13 region (where the gene for Menkes disease has been mapped), the Xq25 region (where the gene for Duncan syndrome has been mapped) and the Xq28 region (where several disease genes, among them the Emery Dreifuss muscular dystrophy gene, have been localized). Cell lines from patients and other members of pedigrees with recurrence of such disorders are already available for linkage studies.
To implement new techniques allowing the cloning of the genes included in microdeletions of the X chromosome present in patients with a contiguous gene syndrome. Two approaches will be developed. The first is based on a modification of the PERT (Phenol Enhanced Reassociation Technique) which will be improved in different ways: the starting material will be enriched in Xspecific sequences by chromosome sorting and the PERTreassociated sequences will be selected through several cycles of nonspecific PCR. With the second approach, a modified reassociation technique will be developed starting from genomic DNA from patients bearing a microdeletion in the X chromosome and cDNA derived from a tissue in which the disease gene of interest is normally expressed.
These approaches are already being developed in both laboratories through preliminary experiments aimed at perfecting the techniques. To this end, a large deletion from patients with Duchenne muscular dystrophy, glycerolkinase deficiency and adrenal hypoplasia will be used. Additional microdeletions are available in the two laboratories from patients with Menkes disease, Duncan syndrome, Norrie disease and Xlinked agammaglobulinemia combined with isolated growth hormone deficiency.
To construct an X chromosomespecific library in the bacteriophage P1, which allows the cloning of DNA fragments as large as 100 Kbp. This will make it possible to develop physical maps corresponding to regions of interest, making use of the DNA markers generated as described under 1.
Champ scientifique (EuroSciVoc)
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CORDIS classe les projets avec EuroSciVoc, une taxonomie multilingue des domaines scientifiques, grâce à un processus semi-automatique basé sur des techniques TLN. Voir: Le vocabulaire scientifique européen.
- sciences naturelles sciences biologiques génétique ADN
- sciences médicales et de la santé médecine fondamentale neurologie dystrophie musculaire myopathie de duchenne
- sciences naturelles sciences biologiques génétique mutation
- sciences naturelles sciences biologiques génétique chromosome
- sciences naturelles sciences biologiques génétique génome
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