FLOW KARYOTYPING: OPTIMIZATION AND CLINICAL EVALUATION

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The overall objective of this project is to develop and evaluate improved analytical cytologic techniques for prenatal detection of disease-linked cytogenetic abnormalities. Emphasis will be on a) improving the speed and sensitivity with which the loss or gain of chromosomal DNA can be detected and quantified during analysis of metaphase chromosomes, b) detection of common cytogenetic abnormalities in interphase cells and c) developing techniques for cytogenetic analysis using fetal cells isolated from maternal blood. These objectives will be pursued by: 1) Applying bivariate flow karyotyping to quantification of the extent of chromosome deletions and duplications as small as 1 Mb. Effort during this project period will be devoted to further increasing the sensitivity of the technique and applying it in a series of collaborative studies to characterize subtle structural aberrations of clinical or molecular biological importance. 2) Optimizing fluorescence in situ hybridization with chromosome specific probes for reliable detection and characterization of numerical aberrations involving chromosomes 21, 18, 13, x and y; segmental duplications (initially 21q22.3 for Down syndrome) and deletions (initially 15q11-13 for Prader-Willi syndrome) in metaphase spreads and in interphase nuclei. 3) Developing chromosome-specific probes to support the fluorescence hybridization studies. Whole-chromosome DNA and ssRNA probes depleted in repetitive sequences will be generated from chromosome-specific cosmid libraries. Composite probes for 21q22.3, 15q11-13 and elsewhere will be generated by starting with "seed" probes already mapped to these regions and "walking" outward in chromosome-specific cosmid libraries to expand the regions covered by the composite probes. 4) Continuing efforts to isolate fetal cells from maternal blood for interphase cytogenetic analysis. Specific projects include: a) determination of the gestational age within the first trimester at which the fetal cells are present at highest frequency in maternal blood,b)determination of the origin of the fetal cells (e.g. leukocytes or cytotrophoblasts) and c) development of procedures for enrichment of the fetal cells.