Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of wheezing, chest tightness and coughing, which vary in severity and frequency from person to person (Suki et al., 2003). It is a condition in which the airways of the lungs become either narrowed or completely blocked, impeding normal breathing. However in asthma this obstruction of the lungs is reversible, either spontaneously or with medication. Currently three main asthma treatments are available (Jeffrey et al., 2000): (a) Inhaled glucosteroids (Martin, 2003; Settipane et al., 2003) (b) beta 2-agonists (Eh et al., 2003) and (c) leukotriene inhibitors (Bjermer et al., 2002). In the patients suffering from asthma with an apparently identical phenotype, response to drug treatment may be remarkably variable (Drysdale et al., 2000). β2AR agonists are recommended for first-line use as bronchodilator therapy in asthma (National Asthma Education and Prevention Program (1997) Expert Panel Report II). Short and long acting β 2 agonists exhibit protective effects against a variety of direct and indirect bronchoconstrictor stimuli (Cockcroft et al., 1996). The beta-adrenergic receptor has been subdivided into at least three distinct groups: β1, β2, β3 classically identified in cardiac, airway smooth muscle, and adipose tissue, respectively (Johnson M, 1998). There is a 65-70% homology between β1, β2 and β3 receptors. There is now good evidence that beta adrenoreceptors exist in activated and inactivated forms and under resting conditions these two forms are in equilibrium with the inactivated state being predominant (Johnson M, 1998). The β2AR is in the activated form when it is associated with the a subunit of the G protein, together with a molecule of guanosine triphosphate (GTP), and it is through this a subunit that the receptor is coupled to adenylate cyclase. The replacement of the GTP by GDP catalyzes the conversion of ATP to cAMP by the enzyme and dramatically reduces the affinity of the a subunit for the receptor, causing dissociation and the receptor to return to its low-energy, inactivated form (Johnson M, 1998). The β2 adrenergic receptor is the key target for the β2 agonist drugs used for bronchodilation in asthma. The β2AR is a G protein-coupled, and has an extracellular amino terminus, seven transmembrane spanning domain, three intracellular and three extracellular loops, and an intracellular carboxyl terminus, that is widely distributed throughout the body especially in smooth muscle cells of bronchi, and mediates the action of catecholamines in various tissues and organs. The β2AR is composed of 413 amino acid residues of approximately 46,500 Dalton (Da) (Drysdale et al., 2000). The β2 A R is encoded by an intronless gene on chromosome 5q31-32 (Kobilka et al., 1987). Johnson M., (1998) have reported several single nucleotide polymorphisms (SNPS) in the coding block of the β2 AR gene that lead to significant genetic variability in the structure of the β2 AR protein in the human population (GenBank Accession Numbers AF022953.1 GI:2570526; AF022954.1 GI:2570528; and AF022956.1 GI:2570532). These SNPs are located at nucleotides 46 (A or G), 79 (C or G), and 491 (C or T) of the β2 AR coding sequence, and result in variation that occurs in the amino-terminus of the receptor at amino acids 16 (Arg or Gly) and 27 (Gln or Glu) and in the fourth transmembrane spanning domain at amino acid 164 (Thr or Ile), respectively. These amino acid variants have clear phenotypic differences as demonstrated by recombinant cell studies (Green et al., 1994), primary cultures of cells (CHW-1102) endogenously expressing these variants (Green et al., 1995), and transgenic mice overexpressing the Thr164 or Ile164 receptors in the heart (Turki et al., 1996). Besides, a synonymous polymorphism of C or A at nucleotide 523 in the coding sequence has been reported to be associated with altered responsiveness to salbutamol in Japanese families (Ohe et al., 1995). In addition to the above polymorphisms in the coding block, several SNPs in the 5′ promoter region have recently been identified and are located at nucleotides −1023 (A or G), −654 (G or A), −468 (C or G), −367 (T or C), −47 (C or T) and −20 (T or C) (Scott et al., 1999). Recently two more SNPs at −709(C or A) and −406(C or T) are reported by Drysdale et al (2000). Thus, thirteen polymorphic sites have previously been identified in the region of the β2AR gene located between nucleotides 565 and 2110 of GenBank Accession No. M15169.1. Different groups have suggested associations between some of the above β2AR amino acid variants and increased susceptibility to various conditions, including: high blood pressure (Gly16 variant, Hoit et al., 2000); atopy (Gly16 variant, Dewar et al., 1998); nocturnal asthma (Gly16 variant, Turki et al., 1995); response to treatment for obesity (Gly16 variant, Sakane et al., 1999); myasthenia gravis (Arg16 variant, Xu et al., 2000); childhood asthma (Gln27 variant, Dewar et al., 1997); obesity (Glu27 variant, Large et al., 1997); and mortality from congestive heart failure (Ile164 variant, Liggett et al., 1998).
It has also been suggested that some of the β2AR gene polymorphisms discussed above may act as disease modifiers in asthma or may be the basis for the known interindividual variation in the bronchodilating response to β agonists (Drysdale et al., 2000). Indeed, Martinez et al (1997) have reported that individuals homozygous or heterozygous for the Arg16 variant are more likely to respond to albuterol than individuals homozygous for the Gly16 variant. Interestingly, another group has reported bronchodilator desensitization in asthmatics homozygous for the Gly16 variant following continuous therapy with the beta-agonist formoterol (Tan et al., 1997). At the same time, however other studies failed to demonstrate any correlations between adverse drug response and regular treatment with beta-agonists (Lipworth et al., 1999).
Asthma is one of the most common diseases worldwide. There are 15-20 million asthmatics in India and 6% of the children in India suffer from asthma (Chabra S. K., 1998). Asthma is a complex, multifactorial disorder, involving many genes as well as some environmental factors (Suki et al., 2003). Genetic factors have yet to be fully elucidated for the Indian population. A lot of irrational drug prescription occurs due to lack of knowledge of the individual and inter-racial variations in the drug response to most of the currently prescribed drugs for asthma leading to wrong treatment. This could prove to be fatal in certain acute cases. These situations can be avoided using prior knowledge of the individual's response to the drug prescribed based on pharmacogenomic rationale. There are also varied side effects due to irrational drug prescription like tremor, palpitation, trachycardia and tolerance to the efficacy (O'Connor et al., 1992, Dennis et al., 2000). The allelic variants of β2AR gene at nucleotide position 46 (A/G), disclosed in the present invention, have been found to be the dictator marker for the bronchodilatory response of the beta agonist drugs particularly in the Indian population. It has been observed that sometimes the patients suffering from asthma do not respond to salbutamol, and it takes long time (days to months) to identify that a particular patient is not responding to the medication. During this time it is very difficult to provide symptomatic relief for the patient. If the physician can identify the responders or the non-responders at the beginning of the treatment, the dose titration time will be saved and the patient would get timely treatment with other alternative therapeutics. In case of an emergency, correct and timely treatment can be given to the non-responders, which may be life-saving.
Drysdale et al (2000) in the US patent application no. 811286 have disclosed a method wherein three SNPs at positions −654 (G/A), 46 (A/G) and 252 (G/A) of the β2AR gene determine the response of beta agonist drugs in the Caucasian population. The method and diagnostic kit claimed by Drysdale et al (2000) is more time consuming, expensive (due to use of three sets of probes and primers and related fine biochemicals). Further their method is restricted for use to the Caucasian population. Hence a need exists to develop an inexpensive, rapid and specific diagnostic method and kit for screening the Indian population for drug response to beta agonists as there are 15-20 million asthmatics in India and 6% of the children in India suffer from asthma. The SNP disclosed in the invention has been found to be associated with the biologic and therapeutic phenotype and has a strong predictive power as an indicator of drug response of individual patient.
Asthma is a complex disease with a phenotype that has been clinically difficult to define. Inhaled beta-adrenergic agonists are the most commonly used medications for treatment of asthma. Polymorphisms of the β2AR can affect regulation of the receptor. The novelty of the present invention is in providing strong association of one single nucleotide polymorphism as pharmacogenetic locus determining the drug response towards beta agonists in Indian asthmatics. The novelty of the present invention is in providing a method for prediction of bronchodilatory response by detecting allelic variants of β2AR gene at position 46 (A/G). This single nucleotide polymorphism has been found to be solely associated with the drug response in the Indian asthmatics. Moreover, this SNP has been found to be a dictator marker for the drug response in the Indian population. Drysdale et al., found three SNPs together contributing the drug response in the Caucasian population whereas in Indian population these three SNPs are found to be unlinked and therefore we observed that taking these three SNPs together in Indian population is less significant than one SNP (A→G).
The invention also provides specific novel probes and primers and diagnostic kit for screening the Indian population for responders to the β2 agonist.
The invention further provides a cheaper and faster method for predicting drug response of the Indian asthmatics to β2 agonist. This polymorphism in β2AR gene has great commercial value both as a cheaper diagnostic reagent and for developing new treatments for this disease.