The long term objective our research program is to define the basic defect in cystic fibrosis (CF). The approach we have taken initially bypasses the search for a specific defect in the disease. Instead, our aim is to identify DNA markers (probes) closely linked to the disease locus at 7q31 and to then use these markers as reference points to search for the CF gene. Although a number of tightly linked DNA marker have been isolated recently, all available data suggest that there is still a considerable distance between these markers and the CF locus. In this application, we propose to isolate additional DNA markers from the CF region and to use them as probes in combination with pulsed field gel electrophoresis to generate a physical (restriction) map spanning the putative gene locus, from which the CF mutation itself can eventually be identified. Cloned genomic DNA fragments suitable for use as probes will be isolated from the flow sorted chromosome 7-specific library constructed by the Los Alamos and Lawrence Livermore National Laboratories. Each of these probes will be used in hybridization analysis with DNA isolated from a set of human-rodent somatic cell hybrids each containing a subset of human chromosome 7 material spanning the q31 region. A total of 13 DNA probes are now available in our collection. It is estimated that an additional 35-40 DNA probes will be sufficient to saturate the CF region with markers. Restriction fragments length polymorphisms will be identified for each of the probes in the q31 region and studied in a small number of informative families to determine the linkage relationship between these DNA markers and CF. These families have been shown tin our previous studies to contain crossover points near the CF locus. The relative order for those DNA markers that are tightly linked to CF will be further examined by linkage disequilibrium analysis and pulsed field gel electrophoresis. Based on the combined genetic and physical map, it will be possible to initiate chromosome walking from points closet to the CF locus and systematically search for sequences that are expressed in the affected tissues. These experiments will be performed in parallel with other ongoing studies utilizing epithelial cells and tissues that are affected in CF. Specifically, epithelial cell cDNA libraries are being constructed and clones that correspond to genes in the 7q31 region are being identified and tested for their involvement in the disease. Attempts are also being made to establish permanent CF epithelial cell lines in order to develop a functional assay for the CF gene by means of DNA transfection to correct the ion transport defect in these cells. With a combination of these various molecular genetic approaches, the basic defect in CF will be resolved.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK034944-04
Application #
3233185
Study Section
Diabetes and Digestive and Kidney Diseases Special Grants Review Committee (DDK)
Project Start
1985-01-01
Project End
1990-12-31
Budget Start
1988-04-20
Budget End
1988-12-31
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Hospital for Sick Chldrn (Toronto)
Department
Type
DUNS #
208511808
City
Toronto
State
ON
Country
Canada
Zip Code
M5 1X8
Browning, Kirsteen N; Mendelowitz, David (2003) Musings on the wanderer: what's new in our understanding of vago-vagal reflexes?: II. Integration of afferent signaling from the viscera by the nodose ganglia. Am J Physiol Gastrointest Liver Physiol 284:G8-14
Orozco, L; Zielenski, J; Markiewicz, D et al. (1997) Two novel frameshift deletions (1924del7, 2055del9-->A) in the CFTR gene in Mexican cystic fibrosis patients. Hum Mutat 10:239-40
Morral, N; Dork, T; Llevadot, R et al. (1996) Haplotype analysis of 94 cystic fibrosis mutations with seven polymorphic CFTR DNA markers. Hum Mutat 8:149-59
Zielenski, J; Markiewicz, D; Lin, S P et al. (1995) Skipping of exon 12 as a consequence of a point mutation (1898 + 5G-->T) in the cystic fibrosis transmembrane conductance regulator gene found in a consanguineous Chinese family. Clin Genet 47:125-32
Zielenski, J; Markiewicz, D; Chen, H S et al. (1995) Identification of six mutations (R31L, 441delA, 681delC, 1461ins4, W1089R, E1104X) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Hum Mutat 5:43-7
Jarvi, K; Zielenski, J; Wilschanski, M et al. (1995) Cystic fibrosis transmembrane conductance regulator and obstructive azoospermia. Lancet 345:1578
Bozon, D; Zielenski, J; Rininsland, F et al. (1994) Identification of four new mutations in the cystic fibrosis transmembrane conductance regulator gene: I148T, L1077P, Y1092X, 2183AA-->G. Hum Mutat 3:330-2
Zielenski, J; Bozon, D; Markiewicz, D et al. (1993) Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621 + 1G-->T and 711 + 1G-->T mutations. Hum Mol Genet 2:683-7
Tsui, L C (1992) Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report from the Cystic Fibrosis Genetic Analysis Consortium. Hum Mutat 1:197-203
Mornet, E; Chateau, C; Simon-Bouy, B et al. (1992) Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism. Hum Genet 88:479-81

Showing the most recent 10 out of 23 publications