Lap-Chee Tsui - US grants

The long term objective of our research program is to define the basic defect in Cystic Fibrosis(CF). The approach described in this application will initially bypass the search for a specifid defect in CF. Instead, our specific aim is to identify a DNA marker closely linked to the disease locus. The study exploits the DNA sequence heterogeneity prevalent in the human population. Since these hereditary sequence polymorphisms are conveniently detected by using restriction endonucleases, the variable lengths of specifc DNA fragments generated from enzyme digestions have been used as markers in genetic analysis. By following the inheritance of a polymorphic DNA marker in families with CF patients, statistical methods are available to determine whether the disease is linked to the marker. A systematic screening of DNA markers distributed over the entire genome (excluding the sex chromosomes) should lead to the discovery of one such marker closely linked to the CF gene. This approach is expected to yield positive results because it is designed to examine the patient's genome directly rather than measuring parameters which could be due to secondary manifestation of the basic defect. Based on the size of the human genome and recombination frequencies, it has been estimated that approximately 150 to 400 markers mikght be required to map an unknown locus. However, this just sets the upper limit of the number of probes to be analyzed. In fact, a marker closely linked to Huntington Disease was found after screening only 12 random markers. To pursue our study, two-generation families with two or more CF children have been chosen as panels for screening DNA markers. DNA samples are prepared from lymphoblast cell lines established from each family member, digested with appropriate restriction enzymes, size-fractionated by agarose gel electrophoresis, transferred to DNA-binding membranes and hybridized with radioactively labelled DNA probes. The restriction fragment length polymorphisms are then revelaed by autoradiography. To facilitate analysis and storage of data we have developed computer-assisted systems. The discovery of a CF-linked DNA marker will likely provide an opportunity for carrier detection and prenatal diagnosis. More importantly, however, the marker should eventually permit identification of the CF gene. Identification of the basic biochemical defect in CF is a prerequisite for the development of effective therapy for this disorder.

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.

Cystic fibrosis (CF) is the most common severe autosomal recessive disorder in the Caucasian population. In North America, approximately 1 in 2,500 live-births is affected with CF, which is characterized by chronic obstructive lung disease, pancreatic insufficiency, abnormalities of electrolyte, fluid and macromolecule secretion of exocrine glands. The basic biochemical defect is unknown. The identification of the CF gene has provided an opportunity to understand the defect and the pathophysiology of the disease at the molecular level, which will allow the development of rational therapy. On the basis of DNA sequence analysis, the CF gene product (CFTR) is predicted to be a transmembrane protein with 2 ATP-binding domains. Genetic analysis shows that approximately 70% of the CF chromosomes suffer a 3 base pair deletion which corresponds to a single amino acid deletion at position 508 of CFTR. Five specific aims are proposed in this application: (1) Additional mutations in the CF gene will be identified in order to map the functional domains of CFTR; simple detection procedures will be developed for each mutation; a number of mutations have already been identified for the remaining 30% of CF chromosomes. (2) Mutations and "epitope tags" will be introduced into selected regions of CFTR to map its functional domains and topology in relation to the plasma membrane in mammalian cells. (3) The effect on the biosynthesis of CFTR of some of the naturally occurring mutations, especially the nonsense and frameshift mutations that have been detected, will be studied to investigate if true "null" mutations exist in CF. (4) In order to understand the factors governing CF gene expression, the sequence elements responsible for basal promoter activity and tissue-specificity for CFTR will be determined by conventional techniques with the use of reporter gene constructs (CAT); transgenic mice carrying the CF gene promoter and a reporter gene (E. coli lacZ) will also be constructed to study developmental regulation of CFTR. (5) DNA sequences that have been detected and shown in preliminary studies, to be closely related to CFTR in the human genome will be isolated and characterized, with respect to their chromosomal location, expression pattern, and their structural and functional relationships with CFTR. The latter information may provide important insight into the evolution function of the CF gene and other CFTR-like genes. These studies, conducted in parallel with 2 other separately funded approaches (use of yeast and mouse models), constitute a comprehensive research program in elucidating the basic defect in CF.

The goal of this SCOR application is to understand the clinical, biological and biochemical consequences of mutations in CFTR, the gene defective in cystic fibrosis (CF). The program brings together a group of basic scientists and clinician researchers with a broad range of expertise to the common task of analyzing the CF disease from different angles, through studying the CFTR molecular defects in patients, generating, mouse models, mapping of modifier genes, and using cell culture and in vitro systems for the protein. We combine our strengths in the area of CF patient documentation, human and mouse genetics, biochemistry and cell biology. The SCOR is organized into 4 Research Projects (RP), 3 Pilot Projects (PP) and 3 Core Units, grouped into 3 research areas, namely, clinical and genetic studies of patients, mouse models of identification of CF modifier genes, and direct characterization of CFTR. In the first area, RP1 will establish a comprehensive understanding of the spectrum of CF disease phenotype caused by or associated with the primary and secondary genetic determinants of the disease. In the second, RP2 will study the role of ClC-2 chloride channels in mediating epithelial chloride secretion in a mouse model and RP3 will dissect the physiologic and genetic aspects of lung disease in CF mice of a specific genetic background. In addition, a pilot project, PP3, is included to characterize the liver disease recently observed in one of the congenic CF mouse strains. These studies will discover new pathways through which alternative methods may be devised to treat CF. In the third area, RP4 will pursue a detailed analysis at the molecular, cellular and functional levels to establish the consequences of the missense mutations that occur in the first nucleotide binding domain (NBD1) of CFTR and mutations causing carboxyl terminal truncations will be used as probes for these studies. This will be complemented by the two pilot projects: PP1 which will explore a new fluorescence transfer technique for the study of transmembrane segment interactions and PP2 which will examine if purified CFTR an mediate energy-dependent transport of large organic anions such as glutamate and glutathione in a reconstituted system. In addition to the Administration Core, the Patient/Biostatistics Core, and the Mouse Core will serve to support the hove projects. The results from Score should yield novel insights into the molecular mechanisms of CF pathology and should lead to new improved therapeutic approaches.

Lung disease is the major contributor to morbidity and the primary cause of mortality of CF patients. The lung disease is characterized by pulmonary obstruction and tissue damage due to chronic inflammation and opportunistic pathogen colonization. The severity of the CF-associated lung disease, how3ever, is variable, and disease among patients with identical CFTR genotypes, and a higher concordance in monozygotic compared to dizygotic CF twins, suggest the contribution of non-CFTR genetic factors in the disease. Identification of these secondary genetic factors will broaden our understanding of CF disease and possibly led to new treatments. The delineation of the genetic influences on CF lung disease, however, is not feasible through human studies due to environmental variability, genetic heterogeneity and small sample sizes. Mice deficient of CFTR function "CF mice" generally die of intestinal obstruction by the age of 5 weeks without displaying significant lung disease. Amelioration of the intestinal obstructions by weaning onto a liquid diet, however, results in increased lifespan and consequential disclosure of abnormal lung phenotypes. In particularly, congenic C57BL/6J (B6) CF mice, in the absence of pathogens, spontaneously develop signs of inflammatory lung disease, not unlike those seen in CF patients. In contrast, under the same conditions no sign of lung disease is observed in their control subs or other strains of F mice, including the BALB/cJ (Bc) congenic CF animals. The two congenic strains of CF mice thus provide a means to characterize the factors contributing to the CF lung disease as well as map and characterize secondary genetic factors underlying the differences. The proposed study will employ an in-depth physiological characterization of pulmonary milieu neutrophil function, along with controlled breeding experiments and candidate gene and QTL genetic mapping to identify the factors contributing to the differences in lung phenotypes between the B6 and Bc CF mice, and possibly CF patient variability.