INTRODUCTION

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

Over the past eight years, interest has grown in identifying the genetics of a number of diseases, some of which may be unigenic in their transmission and hence adhere to Mendelian principles. Other diseases, which are complex and require the elucidation of major genes, pose a considerable challenge in identifying multiple loci and their interaction. The value of such studies is highlighted by our understanding of the genetic basis of diseases such as cystic fibrosis, the most common Mendelian-based genetic disease in man, as well as more complex diseases such as Alzheimer's disease. In studying the genetics of asthma, it is important to identify the contribution of multiple sets of genes that interact in producing the asthmatic response to protein allergens, chemical sensitizers, or viral and bacterial products. It would be critical to identify the populations at greater risk at different times in their lives and/or development (1). Therefore, in a complex syndrome like asthma, the sole influence or predominance of a single gene or a single set of genes is very unlikely. A more credible scenario would involve the interaction of multiple sets of genes, each contributing a small amount to the pathophysiology of asthma. We are just at the beginning of this process and have only uncovered a few candidate genes at present (human leukocyte antigen [HLA], T cell receptor (TcR), chromosome 5q-cytokines, chromosome 11q-FcRI, etc.). With the identification of other candidate genes we may better understand the pathogenesis of the asthmatic syndrome and design new therapeutic options.

	GENERAL APPROACHES

The development of techniques to actually sequence the human genome as outlined by the Human Genome Project, and worldwide attempts to collaborate with the genome project through efforts in Western Europe, have made the genetic analysis of complex disease much more attractive over the past five or six years (6). In fact, the original approach of functional candidate gene cloning followed by chromosomal localization and case control and family studies to look for the association of a particular gene and its product with a disease has been more or less replaced by the positional cloning approach, looking at disease manifestations and identifying by linkage analysis and eventually DNA sequencing the potential association of diseases with a gene. So, while in the initial analysis of the genetic contribution to a disease, the idea of functional cloning and candidate genes was attractive, the more recent development of positional cloning to link phenotypes and map a gene to a distance site or marker has become the experimental standard. Positional cloning would use better and better markers for linkage within the genome, followed by fine physical mapping and eventual identification of an asthma-linked gene. Once a gene is identified, it can be sequenced and the function of its protein analyzed. A third approach, inspirational cloning, speeds the process of positional cloning by saturating the area around an attractive candidate gene within the linkage map; it combines the power of positional cloning and the candidate gene approach.

Whole-genome screen utilizing significant sets of markers in the analysis of the association of genetics of asthma with atopy and/or other markers has only begun (7, 8). The approach is to look at usual populations at great risk or minimal risk for asthma, as well as restricted populations, including those of restricted island access who are predisposed or protected from asthma. Significant information on this process will be forthcoming in the next year or two. Confirmation of hot spots and identification of candidate genes within the hot spots will undoubtedly be uncovered by this kind of approach (Table 1). For the candidate gene approach, there have been four major chromosomes where associations with asthma have been made: chromosomes 5, 6, 7, and 11 (Table 2). The initial reports on the genetics of asthma linked to human chromosome 6 genes associated some HLA markers by linkage to allergen reactivity in asthmatic populations (9). Recently, information concerning potential association of markers linked to chromosome 7 and presumably the alpha chain of the T-cell receptor in asthmatic atopic populations has also been reported (10). Likewise, reports from England indicate that a marker on chromosome 11q13 is associated with asthma and atopic sensitivity (11, 12). Although the initial reports were not confirmed and this area remains controversial, the identification of potential polymorphisms within the beta chain of the high-affinity IgE Fc receptor as a marker or a functional consequence of this association remains of interest. More recently, there have been reports that bronchial hyperresponsiveness, atopy, and asthma may be linked to genes within the cytokine gene cluster on human chromosome 5q31-33 (13, 17). Within this cluster are the genes for many of the Th2-like cytokines involved in IgE production and allergic inflammation, and different markers within this region have been linked to asthma and atopy in various populations (12, 18). The first publication of a genome-wide search for quantitative trait loci underlying asthma by the Cookson group in Oxford confirmed previously identified sites in the major histocompatibility complex (MHC) and related them to tumor necrosis factor (TNF) on human chromosome 6, the  chain of the T-cell receptor on chromosome 7, the previously described association with chromosome 11q, and previously unassociated linkages to markers on chromosomes 13, 16, and 4 (11). What the candidate genes might be for these new areas are not yet clear. There are significant candidates in all areas that may prove to be informative. The six linkages identified by Cookson's group, however, may be false positives; Monte Carlo simulation suggests that at least 1.8 out of the 6 linkages are false positive linkages.

	MOLECULAR MECHANISMS

Further molecular approaches to this question of asthma genetics have focused on two areas. First, the 11q approach has identified the beta chain of the high-affinity IgE Fc receptor as a potential candidate gene. The first polymorphism within this cluster that was identified as potentially being linked to IgE involved a nonhydrophobic switch in the transmembrane region of the molecule. More recently, a different polymorphism that would identify and alter the potential hydrophobicity of the signaling part of the tail of the IgE high-affinity receptor signaling molecule has been identified, and the functional characteristics of this change are being addressed (12). The second molecular linkage to asthma has been the association of the -adrenergic receptor with specific polymorphisms in the structure of that receptor and asthma, including nocturnal asthma (19). The third approach to functionality regarding candidate genes has been an approach taken by our group to examine the expression of promoter polymorphisms within the chromosome 5 gene cluster to identify markers that may link to transcriptional dysregulation in asthmatic kindred. We have identified polymorphisms in the 5' promoter region of the cytokine genes, including IL-3, IL-9, and IL-4 (14). The IL-4 5' region was of great interest because of the relationship of IL-4 to the IgE isotype switch in allergic inflammation, and we have identified a C to T exchange at -590 base pair (bp) from the open reading frame within the IL-4 gene promoter (16). Within asthmatic kindred, this C to T exchange is associated with significant increases in the mean total IgE level. There are also associations with in vitro IL-4-mediated activity. For example, the ability of polymorphism oligonucleotide to bind nuclear transcription factors from allergen-stimulated or Jurkat human T cells is associated with greater transcription factor binding, as well as alteration in electrophoretic mobility shift assay. Further identification of the transcription factors identifies a complex that contains at least NFAT-1 as the mediator of this transcriptional upregulation of IL-4 (20). Transfection experiments using whole promoters or promoter fragments also show greater gene report associated with the C to T polymorphism within the promoter (19). Our studies indicate a unique binding site for NFAT-1 within an inverted palindrome from -603 to -588a weak NFAT-1 consensus (GGAGAA), stabilized by an adjacent ets-like consensus (TTCC) in the polymorphism configuration (Table 3). Hence, the functional association of a polymorphism within the IL-4 gene promoter seems to correlate with enhanced IL-4 activity in vivo and hence may be significantly linked to asthma. Further clarification of the molecular mechanism of this polymorphism may raise the potential for new pharmacologic interventions.

Interleukin-10 has been defined as a cytokine synthesis inhibitory factor that shares genetic similarities in mouse and human to viral gene products involved in immune escape. For this reason, IL-10 became a very unique cytokine by its capability of shutting down the synthesis of cytokines and other immunologically relevant molecules. In the human, the gene for the IL-10 cytokine is located on chromosome 1, and IL-10 has been studied intensively for its role in immunobiology and in generation of tolerance.

Interleukin-10 was initially identified in mice, where it is a Th2 lymphocyte product that inhibits both Th1 proliferation and production of interferon (IFN)- and IL-2. Subsequent studies have extended these activities to humans, showing that IL-10 is also a product of Th1 lymphocytes and downregulates IL-4 and IL-5 expression by Th2 lymphocytes. Interleukin-10 is also produced by cytotoxic T cells, B lymphocytes, and mast cells. In contrast to mice, in humans monocytes are the major source of IL-10. In addition to its effect on Th1 and Th2 cytokines, IL-10 inhibits the production of IL-1, IL-6, IL-8, IL-12, and tumor necrosis factor (TNF)- by mononuclear phagocytes and IFN- and TNF- by natural killer (NK) cells. Interleukin-10 also inhibits monocyte MHC class II, B7.1/B7.2, and CD23 expression and accessory cell function. Inhibition of B7 expression may be the primary mechanism of IL-10 inhibition of accessory cell function. Accessory signals mediated by the B7 molecules through CD28 on the surface of T signals are essential for optimal T-cell activation. In the absence of B7, T lymphocytes do not proliferate and do not produce cytokines in response to antigen, and the cells are rendered irreversibly nonresponsive (tolerant). This may be the mechanism by which IL-10 inhibits cytokine production by Th1 and Th2 lymphocytes. Expression of IL-10 by antigen-presenting cells therefore represents an established pathway for induction of antigen-specific tolerance, including tolerance to allergens (22). In summary, the constitutive production of IL-10 in the healthy airway contributes to the lack of expression of B7 on alveolar macrophages and the inability of these cells to function as antigen-presenting cells. Interleukin-10 thereby protects the airway from developing inflammatory responses to inhaled allergens (22). Support for a modulating role for IL-10 in human allergic diseases is further derived from observations that IL-10 inhibits eosinophil survival and IL-4-induced IgE synthesis. Administration of IL-10 to sensitized mice abrogates antigen-induced airway inflammation and TNF- generation.

The inhibitory effects of IL-10 contrast with its effect on B lymphocytes, where it functions as an activating factor stimulating cell proliferation, immunoglobulin secretion, and the  isotype switch. Interleukin-10 also functions as a growth factor for immature T cells and is a differentiating factor for cytotoxic T cells. Thus, IL-10 inhibits cytokines associated with cellular immunity and allergic inflammation while stimulating humoral and cytotoxic immune responses.

The lungs of healthy individuals are characterized by constitutive IL-10 secretion, while those with inflammatory lung disorders such as cystic fibrosis and interstitial lung diseases have diminished IL-10 production. We have studied the expression of the IL-10 gene in normal and asthmatic subjects to define its role in allergic inflammation (21). By in situ hybridization, we demonstrated that alveolar macrophages are the cell source for IL-10 in bronchoalveolar lavage (BAL)-derived pellets, confirming the previous observations that mononuclear phagocytic cells are the primary source of IL-10 in human lungs. We then characterized the expression of IL-10 via the RNA-based polymerase chain reaction in BAL cell pellets derived from normal and asthmatic subjects. Hybridization was detected via autoradiography and quantified by densitometry with data normalized to the expression of -actin cDNA. By densitometric analysis, the concentration of the PCR product was significantly less in the nine asthmatic subjects than in the six nonasthmatics. Using an IL-10 ELISA, we extended our observations on IL-10 production in normal and asthmatic subjects to BAL fluid protein concentrations. Our data demonstrated constitutive secretion of IL-10 into BAL fluid of normal, nonasthmatic subjects (130 ± 61 pg/ml; n = 8). Asthmatic subjects' BAL fluid was characterized by diminished concentrations of IL-10 (9 ± 18 pg/ml; n = 8; p < 0.01 compared to normal subjects).

In order to study polymorphisms of the IL-10, polymorphic DNA from kindred subjects with asthma was extracted, amplified by polymerase chain reaction (PCR), and subjected to DNA sequencing. This polymorphism is located at bp -571 from the transcription initiation site (TIS) and represents a C to A base substitution. It is located between consensus sequences for binding to members of the ets family and SP-1. The prevalence of this polymorphism is displayed in Table 4. The data must be interpreted with caution because these families were recruited through an asthmatic proband; therefore, if this base exchange influences the development of allergic inflammation, it would be overrepresented. Support for a physiological role for the C to A base exchange at bp -571 in the IL-10 promoter is provided by data derived from our case-control linkage studies. We have recruited and extensively phenotyped approximately 150 individuals from 40 families defined by the presence of an asthmatic proband, and an additional 10 families with neither allergies nor asthma. Each individual was evaluated via spirometry, reversibility of spirometry in response to ₂-agonist nebulization, methacholine reactivity, total IgE, allergy skin-test reactivity, and eosinophilia. The most significant linkage was observed to total IgE (p < 0.0006) (21).

Further studies have demonstrated diminished promoter strength in constructs containing the A form of the promoter. We have complemented these promoter data with functional studies comparing IL-10 production in individuals homozygotic for both forms of the IL-10 promoter (21). Peripheral blood mononuclear cells were cultured in the presence or absence of IFN- for 48 h, supernatants collected, and ELISAs performed. These experiments are summarized in Table 5. The subjects containing the A form of the promoter demonstrated significantly less IL-10 synthesis than others. This suggests a potentially important molecular genetic mechanism underlying the development and severity of inflammatory disorders in these subjects. Stimuli that induce IFN-, including allergic inflammation or viral infections, would be expected to lead to the synthesis of IL-10. In normal individuals this will lead to resolution of the inflammatory response orin allergen challenge modelsresolution of the late asthmatic response. However, individuals homozygotic for A will have persistent inflammation leading to prolonged and severe asthma episodes.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION CONCLUSION REFERENCES

The study of atopy and asthma genetics is in its infancy regarding identification of significant numbers of genes that may be dysregulated in asthma. The unique set of genes involved in IgE and atopy and bronchial hyperresponsiveness alone makes this a complex disease, whose precise molecular mechanisms for dysregulation may be multiple and daunting in terms of identifying one key gene product, or even several gene loci, that could be targeted for therapeutic alteration. More likely, a concentrated approach for knocking out and altering the function of multiple genes within the sets of different genes expressed in the complex disease of asthma will be the ultimate therapeutic goal.

Even if a single gene, as in cystic fibrosis, or a small set of genes, as in diabetes and multiple sclerosis, is not identified in asthma, to identify the contribution of any of the loci of importance as candidate genes will be very useful. Diagnostic screening tests could then be developed and new regulatory mechanisms for the pathogenesis of asthma may lead to new therapeutic interventions.