We propose to perform molecular genetic studies to identify the location of the gene(s) that contribute to the development of asthma, with the goal of determining the complete nucleotide sequence of an 'asthma' gene. We will focus on atopic ('extrinsic') asthma, where there is evidence for a causal relationship between the expression of symptoms and exposure to allergen(s) implicated in the disease. We hypothesize that atopic asthma is controlled by a limited number of major genes, along with numerous environmental and polygenic factors. We postulate; (a) a major asthma-specific gene(s) independent of atopy; (b) a gene(s) controlling the overall genetic propensity of the individual to synthesize IgE; (c) specific immune response (Ir) genes to relevant inhaled allergens, particularly 'indoor allergens.' We will study approximately 30 Caucasian and 30 African- American families, ascertained through multiplex sibships, and totaling about 600 people. We will use genetic linkage analysis to test for the existence of major 'asthma gene(s),' and of asthma-associated genes that may control bronchial hyperreactivity, eosinophil levels, IgE responsiveness, etc. We will combine both 'forward' and 'reverse' molecular genetic strategies, utilizing PCR methodology with fluorescence- based, semi-automated analysis of microsatellite and minisatellite DNA repeat sequences to investigate these genes. In the forward approach, we will analyze polymorphic markers associated with 'candidate' genes implicated in atopic asthma. For reverse genetics, we will initially study one or two polymorphic markers per chromosomal arm (two for the larger chromosomes). The markers for the forward approach will be included in the design of this array, making a total of about 60 markers. We will test for linkage between the polymorphic genetic markers with regressive models of inheritance, in which allergen levels in the homes of the asthma families will be important covariates. If nor linkage is found, we will extend the studies to about 200 markers (ca 15-20 Cm apart). After establishing a linkage between an asthma gene and a polymorphic marker, we will perform additional mapping studies with closely linked markers in the region. We will then identify the gene using a combination of chromosomal 'jumping' and 'walking' strategies, constructing CDNA expression libraries, as well as cytogenetic analysis and studies of homologous mammalian genes, and sequencing. Identification of the alleles controlling risk to develop asthma may allow the generation of transgenic models for asthma, which would be invaluable for the testing of new pharmaceuticals. The availability of closely linked markers, and the subsequent identification of asthma gene(s), should allow predictive diagnosis of individuals predisposed to asthma. The predicted amino acid sequence may well provide an insight into the function of the encoded protein, which will direct clinical investigators toward novel therapies for asthmatics, including gene therapy.
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