Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-04-10
2002-05-14
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024330, C536S024310
Reexamination Certificate
active
06387615
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to diagnosis of asthma and to materials and methods relating thereto.
2. Description of the Related Art
Asthma is a disease which is becoming more prevalent and is the most common disease of childhood (1). Most asthma in children and young adults is initiated by IgE mediated allergy (atopy) to inhaled allergens such as house dust mite and cat dander However, not all asthmatics are atopic, and most atopic individuals do not have asthma. Thus, factors in addition to atopy are necessary to induce the disease (2,3). Asthma is strongly familial, and is due to the interaction between genetic and environmental factors. The genetic factors are thought to be variants of normal genes (“polymorphisms”) which alter their function to predispose to asthma.
Asthma may be identified by recurrent wheeze and intermittent air flow limitation. An asthmatic tendency may be quantified by the measurement of bronchial hyper-responsiveness in which an individual's dose-response curve to a broncho-constrictor such as histamine or methacholine is constructed. The curve is commonly summarised by the dose which results in a 20% fall in air flow (PD20) or the slope of the curve between the initial air flow measurement and the last dose given (slope).
In the atopic response, IgE is produced by B-cells in response to allergen stimulation. These antibodies coat mast cells by binding to the high affinity receptor for IgE (Fc&egr;RI). When a multivalent allergen binds to an IgE-coated mast cell, the cross-linking of adjacent IgEs by allergen initiates a series of cellular events leading to the destabilisation of the cell membrane and release of inflammatory mediators. This results in mucosal inflammation, wheezing, coughing, sneezing and nasal blockage.
Atopy can be diagnosed by (i) a positive skin prick test in response to a common allergen; (ii) detecting the presence of specific. serum IgE for allergen; or (iii) by detecting elevation of total serum IgE.
Genetic associations with atopy have been demonstrated. WO 95/05481 discloses that variants of the gene encoding the &bgr;-subunit of the high-affinity receptor for IgE (Fc&egr;RI&bgr;) are associated with atopy. It teaches a method for diagnosing atopy which is based upon the demonstration of the presence or absence of one of two variants in a specific portion of the DNA sequence of the gene encoding Fc&egr;RI&bgr;, located near the commencement of exon 6 of the Fc&egr;RI&bgr; gene on chromosome 11. A further variant has also been found in which the unusual variant sequence is in the coding sequence for the C-terminal cytoplasmic tail of Fc&egr;RI&bgr; (11).
The known polymorphisms do not account for all of the genetic factors which predispose to asthma. In particular, asthma is not necessarily an atopic disease. Identification of further genetic polymorphisms linked to asthma will allow the identification of children at risk of asthma before the disease has developed (for example immediately after birth), with the potential for prevention of disease.
Tumour necrosis factor (TNF, also known as TNF&agr;) is a potent proinflammatory cytokine that is found in increased concentration in asthmatic airways (4) and in lavage fluid from asthmatic lungs (5). Increased secretion of TNF by peripheral blood lymphocytes or monocytes has also been established in association with the HLA-DRB1*03 genotype (8). The TNF locus is located within the major histocompatibility complex (MHC) between the MHC class III genes and HLA-B. Located downstream of the TNF gene and in tandem arrangement with it is the lymphotoxin &agr; gene (LT&agr;, which was originally designated TNF&bgr;). Unlike the highly polymorphic class I and class II HLA genes, the coding portions of the TNF&agr; and LT&agr; genes only show a very low degree of polymorphism.
The TNF&agr; and LT&agr; loci have been investigated in association with autoimmune diseases such as systemic lupus erythematosus (SLE). It has been suggested that an increased level of TNF secretion is associated with allele 1 of a NcoI polymorphism in the LT&agr; gene (9) and with allele 2 of a TNF promoter variant at position −308 (10). These polymorphisms are known as LT&agr; NcoI*1 and TNF −308*2 respectively. However, there has been some doubt about the significance of these polymorphisms in particular, it has been suggested that the TNF&agr; −308 polymorphism is not relevant to TNF&agr; gene regulation (15, 16).
Several polymorphic microsatellite sequences within the human TNF/LT locus have also been mapped and characterised (12) and used in a cell typing study (13).
It has now been discovered that, surprisingly, unusual genetic variants in or linked to the TNF&agr; gene are predictive of asthma Furthermore, it has been found that the unusual variants are predictive of bronchodilator and inhaled or oral steroid usage in asthmatic individuals. The variants are therefore useful to predict the clinical course of disease (e.g., severe as opposed to mild) or the response to particular treatments, as well as in a diagnostic tool. This information will be of use in relation to both individuals and population, and will be of interest to the insurance industry as well as to the healthcare and pharmaceutical industries.
These unexpected findings make possible new diagnostic and therapeutic strategies.
SUMMARY OF THE INVENTION
The present invention therefore provides a method for diagnosing an individual as being asthmatic, or of having a predisposition to asthma, which method comprises demonstrating in the individual the presence or absence of an unusual variant form of a polynucleotide sequence in the MHC region of chromosome 6p, said unusual variant form associated with an increased secretion of TNF. In particular, the variant may be located in the TNF&agr;/LT&agr; locus, including regulatory regions for the TNF&agr; and LT&agr; genes.
FIG. 1
shows a physical map of the human TNF&agr;/LT&agr;, locus and the locations of various polymorphisms using nomenclature as it appears in the literature The variant may be for example allele 1 of the NcoI polymorphism in the LT&agr; gene, or it may be allele 2 of the TNF promoter polymorphism at position −308. Both of these variants have been described in detail previously in references 10 and 14, the contents of which are incorporated herein by reference. The method according to the invention may involve identifying the presence or absence of two or more such variants.
The sequence difference between the two alleles of the TNF −308 polymorphism is a single base change at position −308 relative to the transcription start site of the gene. For allele 1 the nucleotide at position −308 is a guanine “G”. For allele 2 the nucleotide at position −308 is an adenine “A”.
The sequence difference between the two alleles of the NcoI polymorphism in the LT&agr; gene is a single base change at position +252 relative to the transcription start site. For allele 1 the nucleotide at position +252 is a G which forms part of a restriction site which the NcoI enzyme cuts. For allele 2 the nucleotide at position +252 is an A and the NcoI enzyme does not cut.
Suitable techniques for identifying the presence or absence of the variants are described herein. However, the invention is not limited to these specific methods. Modified versions of these methods, and alternative techniques, will be known to those skilled in the art.
A technique may be employed which uses one or more complementary nucleic acid sequences which are specific for the unusual variant and not for the wild-type. The complementary nucleic acid sequence may simply be used as a probe specific for the unusual variant. Alternatively, one or more suitable complementary nucleic acid sequences may be used as primers in a detection system comprising a polynucleotide amplification technique such as PCR or ARMS. DNA or RNA-based amplification methods may be employed. Thus the amplification may be carried out based on mRNA or cDN
Cookson William Osmond Charles Michael
Moffatt Miriam Fleur
Einsmann Juliet C.
ISIS Innovation Limited
Jones W. Gary
Wenderoth , Lind & Ponack, L.L.P.
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