Our overall goals in this project are to develop comparative genomic hybridization (CGH) for detection of whole chromosome or segmental aneusomies, to evaluate its utility for pre- and neonatal diagnosis and to apply it to refine genotype-disease phenotype correlations associated with deletions of portions of chromosome 5p associated with cri-du-chat syndrome.
Specific aims are to: (1) Further develop and evaluate the utility of CGH combined with FISH for detection of whole chromosome and segmental aneusomies located anywhere in the genome. In this approach, CGH with differentially labeled test and normal reference DNA to normal metaphase spreads (termed CGHx) will be used to identify aneusomies. Aberrations suggested by CGHx will be confirmed using FISH. Our goal is to reliably detect segmental chromosome aneusomies involving greater than 10Mb of the genome using DNA extracted from the hundreds to thousands of cells that can be obtained without cell culture from a few milliliters of amniotic fluid. This will require: a) Further development of CGHx procedures and analysis software; b) Development/acquisition of a set of well mapped large insert, P1 clones to chromosomes 13q, 18q, 21q, 15q11-q13, 4p16, and 5p; c) Determination of the false positive and false negative rates associated with combined CGHx + FISH analysis through analysis of 50 uncultured amniotic samples. (2) Improve the reliability and sensitivity of CGH for detection of segmental aneusomies by replacing normal metaphase spreads as the target for CGH analysis with an array of well mapped, cloned DNA sequences. This is termed CGHa to indicate that abnormalities are mapped onto an array of clones. Specific developments will include: a) New DNA labels to allow quantitative analysis of the intensity of hybridization to membrane bound DNA. Newly developed chelates of Terbium (Tb) and Europium (Eu) will be evaluated first since they will allow spectral and temporal discrimination against membrane fluorescence; b) A system to measure Tb:Eu emission intensity ratios after CGHa hybridization; c) Production of arrays of P1 clones; d) Evaluation of the utility of CGHa for detection of common whole chromosome and segmental aneusomies through analysis of 50 uncultured amniotic samples. (3) Further validate CGHa and use it to precisely define the associations between 5p deletions and clinical phenotypes by analysis of samples from 106 patients with 5p deletions supplied by Drs. Erik Niebuhr and Lars Bolund. Deletions of 5p determined in this project will be correlated with clinical phenotypes provided by Dr. Niebuhr.
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