U.S. patent application number 09/990596 was filed with the patent office on 2002-08-29 for diagnosing asthma patients predisposed to adverse beta-agonist reactions.
Invention is credited to Boushey, Homer, Chinchilli, Vern, Drazen, Jeffrey M., Fish, James E., Ford, Jean G., Lemanske, Robert, Martin, Richard.
Application Number | 20020119474 09/990596 |
Document ID | / |
Family ID | 25206562 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119474 |
Kind Code |
A1 |
Drazen, Jeffrey M. ; et
al. |
August 29, 2002 |
Diagnosing asthma patients predisposed to adverse beta-agonist
reactions
Abstract
The present invention provides a novel method for identifying
individuals who are likely to have negative responses to regular
administration of .beta.-agonists. The invention also provides kits
useful for this purpose.
Inventors: |
Drazen, Jeffrey M.;
(Winchester, MA) ; Chinchilli, Vern;
(Elizabethtown, PA) ; Martin, Richard; (Englewood,
CO) ; Lemanske, Robert; (Madison, OH) ; Ford,
Jean G.; (Newark, NJ) ; Fish, James E.;
(Gladwyne, PA) ; Boushey, Homer; (San Francisco,
CA) |
Correspondence
Address: |
Brenda Herschbach Jarrell, Ph.D.
Choate, Hall & Stewart
Exchange Place
53 State Street
Boston
MA
02109
US
|
Family ID: |
25206562 |
Appl. No.: |
09/990596 |
Filed: |
November 21, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09990596 |
Nov 21, 2001 |
|
|
|
09276232 |
Mar 25, 1999 |
|
|
|
09276232 |
Mar 25, 1999 |
|
|
|
08811441 |
Mar 3, 1997 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/106 20130101; C12Q 2600/156 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Goverment Interests
[0002] Development of the present invention was supported in part
by National Institutes of Health grants numbered U10 HL 51831, U10
HL 51834, U10 HL 51843, U10 HL 51810, U10 HL 51823, and U10 HL
51845. The United States Government may have certain rights in the
invention.
Claims
1. A kit comprising: a first set of primers selected so that one of
the primers hybridizes to a first portion of a human
.beta..sub.2-adrenergic receptor gene, which first portion includes
a sequence encoding position 16 of said human
.beta..sub.2-adrenergic receptor, in such a manner that, when used
in a polymerase chain reaction, said second set of primers
amplifies said portion when position 16 is Arg but not when
position 16 is Gly; and a second set of primers selected so that
one of the primers hybridizes to a first portion of a human
.beta..sub.2-adrenergic receptor gene in such a manner that, when
used in a polymerase chain reaction, said second set of primers
amplifies said portion when position 16 is Gly but not when
position 16 is Arg, said first and second sets of primers being
provided together in a container.
2. The kit of claim 1 further comprising a component selected from
the group consisting of: amplification buffer, water, DNA
polymerase, first control DNA including a first human
.beta..sub.2-adrenergic receptor gene that encodes Arg at human
.beta..sub.2-adrenergic receptor position 16, second control DNA
including a second human .beta..sub.2-adrenergic receptor gene
allele that encodes Gly at human .beta..sub.2-adrenergic receptor
position 16, instructions for use, and combinations thereof.
3. A kit comprising: a primer set selected to hybridize to a human
.beta..sub.2-adrenergic receptor gene in such a manner that, when
used in a polymerase chain reaction, the primer set amplifies a
portion of said human .beta..sub.2-adrenergic receptor gene, which
portion includes a sequence encoding human .beta..sub.2-adrenergic
receptor residue 16; and reagents for determining the nucleotide
sequence of said amplified portion, said primer set and reagents
being arranged together in a container.
4. The kit of claim 3, wherein said reagents are selected from the
group consisting of: a sequencing primer that hybridizes to a piece
of said amplified portion in such a way that allows extension
across said sequence encoding human .beta..sub.2-adrenergic
receptor residue 16, DNA polymerase, dNTPs, ddNTPs, buffer, and
combinations thereof.
5. The kit of claim 3, wherein said sequencing primer is
fluorescently labeled for use in an automated genetic analyzer.
6. The kit of claim 3 further comprising a component selected from
the group consisting of amplification buffer, water, DNA
polymerase, first control DNA including a first human
.beta..sub.2-adrenergic receptor gene that encodes Arg at human
.beta..sub.2-adrenergic receptor position 16, second control DNA
including a second human .beta..sub.2-adrenergic receptor gene
allele that encodes Gly at human .beta..sub.2-adrenergic receptor
position 16, instructions for use, and combinations thereof.
7. A kit comprising: an oligonucleotide primer that hybridizes to a
portion of a human .beta..sub.2-adrenergic receptor gene, which
portion includes a sequence encoding residue 16 of the human
.beta..sub.2-adrenergic receptor, said oligonucleotide having
higher affinity for said portion when said sequence encodes Arg
than it has when said sequence encodes Gly; and an oligonucleotide
primer that hybridizes to a portion of a human
.beta..sub.2-adrenergic receptor gene, which portion includes a
sequence encoding residue 16 of the human .beta..sub.2-adrenergic
receptor, said oligonucleotide having higher affinity for said
portion when said sequence.
Description
PRIORITY INFORMATION
[0001] This application is a Continuation of U.S. patent
application Ser. No. 09/276,232, filed Mar. 3, 1999, which is a
Divisional of co-pending U.S. Pat. No. 6,156,503, issued Dec. 5,
2000, the entire contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Inhaled medium acting .beta.-agonists are the most commonly
prescribed asthma treatments in the world. .beta.-agonists produce
their effects by stimulating the .beta..sub.2-adrenergic receptors
on cells and thereby activating intracellular pathways that produce
increased levels of cyclic adenosine monophosphate (cAMP). The
increased intracellular cAMP levels in turn produce macroscopic
effects in the cells, relaxing the smooth muscles of the bronchial
airways, increasing ciliary beat frequency, and reducing mucous
viscosity. The effectiveness of .beta.-agonists at dilating
bronchial airways has led to their widespread administration both
as a treatment for acute asthmatic episodes and as a long-term
asthma management therapy.
[0004] Concerns about the safety of .beta.-agonist therapy have
arisen periodically over the years (reviewed in, for example,
Taylor et al., Med. Clin. N. America 80:719, 1996; Giunti et al.,
Eur. Respir. J. 8:673, 1995; Barrett et al., Am. J. Respir. Crit.
Care Med. 151:574, 1995; Devoy et al., Chest 107:1116, 1995;
McFadden, Ann. Allergy Asthma Immunol. 75:173, 1995; Crane et al.,
Thorax 50:S5, 1995; McFadden, J. Allergy Clin. Immunol. 95:41,
1995). Reports of possible associations between .beta.-agonist
administration and increased morbidity, particularly for chronic
.beta.-agonist administration protocols, have spurred much debate
over the safety of .beta.-agonist therapy. There is a need to
resolve this debate and to identify risks of deleterious or
salutory effects associated with administration of .beta.-agonsists
to asthmatics.
SUMMARY OF THE INVENTION
[0005] The present invention resolves the debate over the safety of
.beta.-agonist therapy and identifies a population of asthmatic
patients who are at risk for an adverse reaction to regular
administration of .beta.-agonists. In particular, the present
invention provides the discovery that asthmatics who carry a
particular allele of the .beta..sub.2-adrenergic receptor gene are
more likely to have a negative response to chronic .beta.-agonist
therapy. The present invention provides methods of identifying
individuals at risk for an adverse response to .beta.-agonist
treatment, and also provides diagnostic kits useful in the practice
of such methods.
[0006] In preferred embodiments of the methods of the present
invention, a genomic nucleic acid sample is provided from an
individual, first and second .beta..sub.2-adrenergic receptor gene
alleles are identified within the genomic nucleic acid sample, and
any individual for whom both the first and second
.beta..sub.2-adrenergic receptor gene alleles encode Arg at residue
16 is classified as being at risk for adverse reaction to chronic
.beta.-agonist administration. The particular method by which the
.beta..sub.2-adrenergic receptor gene alleles are identified within
the genomic sample is not intended to limit the scope of the
present invention. However, preferred identification methods
include allele-specific polymerase chain reaction (PCR) techniques
and direct sequencing techniques.
[0007] Preferred kits provided by the practice of the present
invention include reagents useful for performing the inventive
methods, which reagents are assembled together in a container for
ease of use.
DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 depicts the primary amino acid sequence and known
polymorphic sites in the human .beta..sub.2-adrenergic receptor
protein. Nine polymorphic sites are shown; those shown in black
represent different gene alleles that encode the same residue,
whereas those in white that are labeled with alternate amino acids
represent gene alleles that result in residue substitutions.
[0009] FIG. 2 is a photograph of an agarose gel presenting genotype
analyses of six BAGS patients at residue 16 of the .beta..sub.2
adrenergic receptor.
[0010] FIG. 3 has panels A and B, showing the morning and afternoon
peak expiratory flow rates (PEFR), respectively, of asthmatics who
received regularly- scheduled or as-needed albuterol treatments. In
each panel, the results are plotted by .beta..sub.2-adrenergic
receptor genotype. Data from asthmatics who are homozygous for the
.beta..sub.2-adrenergic receptor Arg16 variant are plotted either
as a solid line punctuated with diamonds (those who received
regular treatment) or a gray line punctuated by squares (those who
received as-needed treatment); data from Gly16 homozygotes who
received regular treatment are plotted as a light gray line
punctuated by triangles; data from Arg16/Gly16 heterozygotes are
plotted as light gray line punctuated by Xs.
DESCRIPTION OF THE SEQUENCES
[0011] SEQ ID NO:1 presents an amino acid sequence of the
.beta..sub.2-adrenergic receptor. In SEQ ID NO:1, Arg, Gln, Val,
and Thr are located at positions 16, 27, 34, and 164, respectively.
Known gene polymorphisms produce .beta..sub.2-adrenergic receptors
with Gly, Glu, Met, and Ile, respectively, at these positions (see
FIG. 1).
[0012] SEQ ID NO:2 presents a human .beta..sub.2-adrenergic
receptor gene encoding the protein of SEQ ID NO:1. The
Arg16.fwdarw.Gly polymorphism described above with respect to SEQ
ID NO:1 can be produced by substituting a G for the A at SEQ ID
NO:2 position 1633; the Gln 27.fwdarw.Glu polymorphism can be
produced by substituting a G for the C at 1666.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0013] The human gene for the .beta..sub.2-adrenergic receptor has
been cloned (Kobilka et al., Proc. Natl. Acad. Sci. USA 84:46,
1987) and extensively studied. Nine gene polymorphisms have been
identified in the general population, four of which result in amino
acid substitutions (the other five are silent changes) (see FIG. 1;
see also, Reihsaus et al., Am. J Respir. Cell. Mol. Biol. 8:334,
1993). The present invention relates to the "Arg16.fwdarw.Gly"
polymorphism depicted in FIG. 1.
[0014] Various studies have been undertaken to identify any
significance of the Arg16.fwdarw.Gly polymorphism in asthma (for
review, see Liggett, Chapter 21, The Genetics of Asthma [Liggett et
al., eds], Marcel Dekker, NY, 1996). No general association between
either the Arg16 or the Gly16 allele and asthma has been observed
(Reihsaus et al., Am. J. Respir. Cell. Mol. Biol. 8:334, 1993).
Also, the Arg16 and the Gly16 proteins have been shown to have
equivalent affinities for agonists and antagonists, and to couple
normally to G.sub.s , (Green et al., Biochemistry 33:9414,
1994).
[0015] The only difference observed prior to the present invention
between the Arg16 and Gly16 allele was enhanced down regulation of
the Gly16 allele in response to .beta.-agonist administration
(Green et al., Biochemistry 33:9414, 1994). This finding prompted
one expert in the field to conclude that:
[0016] "the Gly16 variant, which undergoes the greatest degree of
agonist promoted down regulation, would be expected to display an
overall reduced level of expression as compared with [the Arg16
variant]. Under this scenario, basal bronchomotor tone might be
decreased or bronchial hyperactivity increased . . . Responsiveness
to .beta.-agonist may also be depressed in asthmatics harboring
[the Gly16] polymorphism. . . During chronic agonist therapy, the
potential for tachyphylaxis would appear to be greatest with the
Gly16 variant . . . Given reports that suggest a relationship
between `overuse` of .beta.-agonists and adverse outcomes in
asthma, it seems prudent to consider that tachyphylaxis may occur
in some individuals" (citations omitted; Ligget, Chapter 21, The
Genetics of Asthma (Ligget et al., eds), Marcel Dekker, NY, 1996,
pg. 470). Thus, prior to the present invention, the state of
knowledge concerning the Arg16.fwdarw.Gly polymorphism in the human
.beta..sub.2-adrenergic receptor indicated that individuals
carrying the Gly16 allele might suffer more tachyphylaxis in
response to chronic .beta.-agonist therapy, and therefore might be
more susceptible to adverse responses to .beta.-agonist
administration.
[0017] Surprisingly, the present invention provides the discovery
that the opposite is true: individuals carrying the Gly16 allele of
the human .beta..sub.2-adrenergic receptor are actually less
susceptible to adverse responses to .beta.-agonist administration.
According to the present invention, individuals who are homozygous
for the Arg16 allele are more likely to have an adverse response to
.beta.-agonist therapy than either Gly16 homozygotes or Gly16/Arg16
heterozygotes.
[0018] As described in Example 2, we analyzed the
.beta..sub.2-adrenergic receptor genotype in 179 subjects who had
participated in a study testing their response to regular and
as-needed albuterol administration (Drazen et al., New Eng. J Med.
335:841, 1996, incorporated herein by reference; see also Example
1). That study had concluded that "in patients with mild asthma,
neither deleterious nor beneficial effects derived from the regular
use of inhaled albuterol beyond those derived from the regular use
of the drug as needed (Drazen et al., id.). When we examined the
.beta..sub.2-adrenergic receptor genotype in the study
participants, however, we came to a rather different conclusion.
FIG. 2 shows our findings: individuals who carry two copies of the
Arg16 .beta..sub.2-adrenergic receptor allele showed significant
decreases in peak expiratory flow rate (PEFR) after receiving
regular .beta.-agonist therapy for 16 weeks. Gly16 homozygotes and
Arg16/Gly16 heterozygotes did not show this effect. Arg16
homozygotes who received .beta.-agonist treatments on an as-needed
basis showed more modest decreases in PEFR and these decreases were
only temporary.
[0019] By demonstrating a correlation between adverse response to
chronic .beta.-agonist therapy and the presence of (two copies of)
the Arg16 .beta..sub.2-adrenergic receptor allele, the present
invention provides methods for identifying asthmatic patients at
risk of such an adverse response. Quite simply, patients are
screened to identify the .beta..sub.2-adrenergic receptor alleles
they carry; those who are homozygous for Arg16 are identified as
susceptible. Any available method can be used to detect patients'
.beta..sub.2-adrenergic receptor genotype (see, for example,
Examples 2 and 3; see also methods described in Current Protocols
in Human Genetics, John Wiley & Sons, Unit 9, incorporated
herein by reference).
[0020] For example, the relevant region of each patients'
.beta..sub.2-adrenergic receptor gene (both alleles) can be
directly sequenced according to known techniques. Alternatively or
additionally, techniques such as denaturing gradient gel
electrophoresis, allele-specific polymerase chain reaction (PCR),
allele-specific hybridization, allele-specific ligation
amplification (see, for example, English et al., Proc. Natl. Acad.
Sci. USA 9:360, 1994, incorporated herein by reference), single
strand conformation polymorphism analysis, restriction fragment
length polymorphism analysis, or any other available technique
useful to distinguish sequence polymorphisms may be employed.
Allele-specific PCR techniques, such as the amplification
refractory mutation system (ARMS), or amplification followed by
sequencing, are preferred methods of polymorphism detection.
Preferred hybridization methods include hybridization to
oligonucleotides on a silica chip array (see, for example, Hacia et
al, Nature Genetics 14:441, 1996, incorporated herein by reference;
see also Nature Genetics 14:367, 1996).
[0021] The present invention also provides kits for identifying
asthma patients susceptible to adverse responses to chronic
.beta.-agonist administration. Preferred kits comprise reaction
components useful for allele-specific PCR techniques. For example,
particularly preferred kits include primer sets capable of
amplifying and distinguishing the Arg16 and Gly16 alleles, and may
also include buffers, thermalstable reverse transcriptase, control
templates, etc. Alternative preferred kits include amplification
reagents that are not necessarily allele-specific, in combination
with sequencing reagents.
EXAMPLES
Example 1
Analysis of Beneficial and Deleterious Effects of
Regularly-Scheduled and As-Needed Albuterol Administration in
Patients with Mild Asthma
(see Drazen et al., NEJM 335:841, 1996, incorporated herein by
reference)
[0022] Materials and Methods
[0023] Patient Recruitment: Patients with mild asthma, as defined
by the criteria shown in Table 1, were recruited from existing
study populations and by advertising. Eligible patients entered a
six-week single-blind run-in period, during which they used a
placebo inhaler on a regular basis (two inhalations four times a
day) and took supplemental puffs of open-label albuterol as needed.
During the run-in period, patients were evaluated three times at
two-week intervals, at which time asthma control was assessed by
the review of a number of criteria.
1TABLE 1 CHARACTERISTICS USED TO DEFINE MILD ASTHMA* CHARACTERISTIC
ALLOWABLE RANGE FEV.sub.1.dagger. .gtoreq.70% of predicted value
Age 12 to 55 yr PC.sub.20 .ltoreq.16 mg/ml Use of .beta.-agonists 6
to 56 puffs of albuterol/wk; patients using less than 6 puffs of
albuterol/wk at visit 1 had to have a PC.sub.20 of .ltoreq.8 mg/ml
Use of other asthma medications None, no corticosteroids for 6 wk
Other serious medical conditions, Not allowed including pregnancy
Smoking None in past year, maximal history of 5 pack-years
permitted *FEV.sub.1 denotes forced expiratory volume in one
second, and PC.sub.20, the concentration of methacholine required
to decrease the FEV.sub.1 by 20 percent. .dagger.The FEV.sub.1 was
measured after at least eight hours without bronchodilator
medications.
[0024] Patient Selection: Patients were randomly assigned to a
treatment group if over the six-week period their asthma was
clinically stable and they demonstrated their ability to comply
with the study procedures, as indicated by the regular use of the
placebo inhaler (monitored by a Chronology recording device) and
their ability to record their peak flow (twice daily, using a
Mini-Wright peak-flow meter) (Clement Clarke, Columbus, Ohio) and
asthma symptoms once daily in a diary. The treatments assigned
consisted of either inhaled albuterol on a regular basis (two
inhalations four times a day) plus albuterol as needed or inhaled
placebo on a regular basis (two inhalations four times a day) plus
albuterol as needed. Albuterol and placebo inhalers were generously
supplied by Schering-Plough (Memphis, Tenn.). Patients were
instructed to have their regularly scheduled inhalations in the
morning after recording their morning peak flow, at midday, in the
late afternoon, and on retiring to sleep after recording their
evening peak flow. They were instructed to allow at least four
hours between their regularly scheduled inhalation in the late
afternoon and the recording of their evening peak flow.
[0025] Patient Treatment: Over the ensuing 16 weeks, while patients
received blinded treatment, the control of asthma was monitored
daily, through peak flow rates and symptoms recorded by patients,
as well as during clinic visits, which were scheduled every two to
three weeks. At the completion of the randomized-treatment period,
all the patients were switched to single-blind treatment with
inhaled placebo for a four-week withdrawal period; during this time
patients continued to use open-label albuterol as needed.
[0026] Seven outcome indicators were monitored: peak flow, the
symptom record, quality of life, the change in the forced
expiratory volume in one second (FEV.sub.1) in response to an
inhaled bronchodilators, the concentration of methacholine required
to decrease the FEV.sub.1 by 20 percent (PC.sub.20), asthma
exacerbations, and treatment failure. Peak flow, the primary
outcome indicator, was measured twice daily by patients using a
Mini-Wright peak-flow meter; the best of three efforts was
recorded. Patients recorded their asthma symptoms and the number of
puffs of supplemental albuterol used daily. Asthma symptoms were
recorded on a 4-point scale, with 0 representing no symptoms and 3
representing severe symptoms. Asthma-specific quality-of-life
scores were recorded during clinic visits, with an instrument
validated by other investigators (Juniper et al., Thorax 47:76,
1992). To determine the spirometric response to an inhaled
bronchodilator, the difference in the FEV.sub.1 before and 15
minutes after two inhalations of albuterol was measured (and
reported as percent improvement) during clinic visits when
responsiveness to methacholine was not tested.
[0027] Patients refrained from taking their study medications for
at least eight hours before all clinic visits. To measure PC.sub.20
for methacholine, methacholine aerosols were generated with a
nebulizer (model 646, DeVilbiss Health Care, Somerset, Pa.) and a
calibrated dosimeter (S&M Instruments, Dovestown, Pa.). The
PC.sub.20 for methacholine was determined by standard procedures
(Tashkin et al., Am. Rev. Respir. Dis. 145:301, 1992). Asthma
exacerbations were monitored during each clinic visit; patients
were asked about their asthma control, and all asthma exacerbations
were recorded. An asthma exacerbation was defined as an increase in
symptoms of cough, chest tightness, or wheezing in association with
one or more of the following; an increase over the base-line use of
supplemental .beta.-agonist treatments of 8 or more puffs per 24
hours for a period of 48 hours, the use of 16 or more puffs of a
supplemental .beta.-agonist per 24 hours for a period of 48 hours,
or a fall in peak flow of 35 percent or more from the best
three-day average (morning and evening) during the run-in
period.
[0028] Treatment was considered to have failed if patients who had
asthma exacerbations and were treated with increased doses of
.beta.-agonists did not respond adequately--that is, if they
continued to meet the criteria for exacerbation. Such patients were
treated with a short course of prednisone, as determined by their
physicians; their data continued to be collected, and they remained
in the trial (in accordance with the intention-to-treat
method).
[0029] Standardization and Quality-Assurance Techniques: All
clinical laboratory tests--that is, measurements of lung function,
skin testing for allergies, methacholine challenges, and
quality-of-life assessments--were performed at each center with the
use of equipment and procedures that were standardized for the
entire network. Workers participating in the network were tested to
ensure proficiency and uniformity in all network-related skills and
had to pass certification examinations before the data they
gathered could be used in the network. All results of spirometric
testing (Collins Eagle 2 spirometer, Quincy, Mass.), including that
for the methacholine challenge, were confirmed by a single network
member. Peak-flow meters were tested against spirometers during
each clinic visit and were replaced if they failed to meet
previously established performance standards. A distributed
data-entry system allowed each clinical center to submit its data
over the Internet directly to the Data Coordinating Center. The
Data Coordinating Center entered the data a second time to verify
it.
[0030] Compliance: Each patient was given a digital wristwatch with
multiple alarms to improve treatment compliance. In addition,
Chronology recording devices were used with the randomly assigned
metered-dose inhalers to provide an electronic record of the date
and time of inhaler use.
[0031] Statistical Analysis: Morning peak flow was chosen as the
primary outcome variable for the calculation of sample size. A
Minimum of 200 patients made it possible to detect a difference of
25 liters per minute between groups with 80 percent statistical
power. A goal of recruiting 250 randomized patients was established
on the assumption that the dropout rate would be less than 20
percent. This sample size also provided 80 percent statistical
power to detect a difference of 0.19 liter in FEV.sub.1 and 0.70
doubling dilution in the PC.sub.20 values for methacholine.
[0032] Response variables--that is, peak-flow values, medication
use, and asthma symptoms--from the patients' diary cards were
averaged each week. Because of the longitudinal nature of most of
the response variables, a mixed-effects linear model was applied
(Vonesh et al., Biometrics 43:617, 1987; Laird et al., Stat Methods
Med Res 1:225, 1992); this approach allowed all data obtained to be
used, not just the data obtained at a single visit. For each
response variable, a segmented linear model was fitted with an
intercept and with slopes for the last 4 weeks of the run-in
period, the first 5 weeks of the treatment period, the remaining 11
weeks of the treatment period, and the withdrawal period. The
"break point" after five weeks of randomized treatment was chosen
on the basis of rates of asthma exacerbation reported by Sears et
al. (Sears et al., Lancet 336:1391, 1990).
[0033] For each outcome measure, values were calculated from the
models for the end of the run-in period, for the end of the
double-blind-treatment period, and for the end of the withdrawal
period. This statistical model was determined before the start of
the study, and therefore other models were not considered during
data analysis. The groups were compared with respect to rates of
treatment failure with the use of Fisher's exact test. To ensure
patient safety, an interim analysis was conducted after
approximately 40 percent of the randomized patients had completed
the trial or withdrawn consent; as a result of this analysis, the P
value considered to indicate statistical significance was reduced
from 0.05 to 0.03 for the final analyses (Pocock, Biometrics
38:153, 1982; Geller et al., Biometrics 43:213, 1987).
[0034] Results
[0035] Enrollment and Retention: Of the subjects recruited, 255
were eligible for enrollment at the end of the six-week run-in
period and were randomly assigned to receive double-blind treatment
(Table 2). There were no significant differences between the
treatment groups with respect to any of the indexes monitored.
During the period of randomized treatment and withdrawal, 25
subjects dropped out of the trial--10 in the scheduled-treatment
group and 15 in the treatment-as-needed group. Two hundred thirty
patients completed the entire trial.
2TABLE 2 CHARACTERISTICS OF PATIENTS IN THE TWO TREATMENT GROUPS
ALBUTEROL TREATMENT.dagger. REGULARLY AS-NEEDED SCHEDULED ONLY
CHARACTERISTIC* (n = 126) (N = 129) Male sex - no. (%) 57 (45.2) 55
(42.6) Minority racial or ethnic 41 (32.5) 43 (33.3) group - no.
(%).dagger-dbl. Atophy - no. (%) 122 (96.8) 127 (98.4) Age - yr
28.6 .+-. 9.0 29.3 .+-. 9.2 Age <18 yr - no. (%) 16 (12.7) 10
(7.8) Morning peak flow - 418.3 .+-. 100.5 421.6 .+-. 99.8
liters/min.sctn. Evening peak flow - 437.6 .+-. 101.5 440.7 .+-.
99.1 liters/min.sctn. Peak-flow variability - %.sctn..paragraph.
3.9 .+-. 5.7 3.7 .+-. 6.9 Symptom score.sctn..vertline. 0.46 .+-.
0.40 0.38 .+-. 0.34 No. of supplemental puffs of 1.5 .+-. 2.0 1.7
.+-. 2.2 .beta.-agonist per day.sctn. FEV.sub.1 - liters.paragraph.
(% of 3.1 .+-. 0.74 3.15 .+-. 0.68 predicted value)** (89.0 .+-.
12.7) (91.4 .+-. 13.9) Quality-of-life score**.dagger..dagger. 2.28
.+-. 0.82 2.44 .+-. 0.82 PC.sub.20 - mg/m**.dagger-dbl..dagger-d-
bl. 0.64 .+-. 1.82 0.64 .+-. 1.82 FEV.sub.1 response to albuterol
10.5 .+-. 8.3 10.8 .+-. 9.2 inhalation - % change from base
line.sctn..sctn. *FEV.sub.1 denotes forced expiratory volume in one
second, and PC.sub.20 the concentration of methacholine required to
decrease the FEV.sub.1 by 20 percent. .dagger.Plus-minus values are
means .+-. SD unless otherwise indicated. .dagger-dbl.Fifty-nine
percent of the minority patients in the scheduled group were black,
and 65 percent in the as-needed group were black. .sctn.Values
represent averages for the last four weeks of the run-in period.
.paragraph.Peak-flow variability was calculated as ([evening peak
flow - morning peak flow] .apprxeq. evening peak flow) .times. 100.
.vertline.Asthma symptoms were graded by the patient each day, from
0 for no symptoms to 3 for incapacitating symptoms. **This
characteristic was measured from week 6 of the run-in period.
.dagger..dagger.Asthma-specific quality-of-life questionnaires were
completed by the patients during clinical-center visits. A score of
1.0 indicates that asthma had no effect on the overall quality of
life; a score of 2.0, that the patient's life was "a little
limited" by asthma; a score of 3.0, that there was "some
limitation"; and a score of 7.0, that there was "total limitation."
.dagger-dbl..dagger-dbl.Values are medians and interquartile
ranges. .sctn..sctn.Data are the averages from weeks 2 and 4 of the
run-in period.
[0036] Compliance: Compliance with the use of inhaled medication,
either active or placebo, on a regular basis was greater than 80
percent, as indicated by Chronolog treatment records and an
analysis of diary cards. Of the 3172 scheduled visits to patients'
clinical center, 26 were missed, for a rate of compliance of over
99 percent.
[0037] Asthma Exacerbations: Asthma was exacerbated 24 times (11
times in the scheduled-treatment group and 13 times in the
treatment-as-needed group) during the active treatment period and 4
times during the withdrawal period (twice in each treatment group).
The 28 exacerbations occurred in 12 patients in the
scheduled-treatment group and 11 patients in the
treatment-as-needed group.
[0038] Treatment Failures: Treatment was considered to have failed
in 11 patients during the 16-week period of randomized treatment (5
in the scheduled-treatment group and 6 in the treatment-as-needed
group) and in 2 during the withdrawal period (both in the
scheduled-treatment group). There were three visits to the
emergency room for asthma (two in the scheduled-treatment group and
one in the treatment-as-needed group). No patients were
hospitalized for asthma during the trial, and none died. There were
no significant differences in any of the event rates between the
two treatment groups.
[0039] Efficacy Outcomes: Lung function (indicated by morning peak
flow, evening peak flow, peak-flow variability, FEV.sub.1,
.beta.-agonist responsiveness, and PC.sub.20) and asthma symptoms
(determined by the number of uses of the supplemental
.beta.-agonists metered-dose inhalers, diary scores and
quality-of-life scores) as derived from the regression analysis
performed for each patient group are shown in Table 3. Graphic
displays of values predicted by the model as compared with sample
means showed excellent goodness of fit by the statistical model
(data not shown). There were no significant differences in morning
peak flow between the two treatment groups (Table 3). Even though
the average use of albuterol was 9.3 puffs per day in the
scheduled-use group and 1.6 puffs per day in the
treatment-as-needed group, the extra use of medication did not lead
to differences in peak-flow variability, FEV.sub.1, supplemental
albuterol use, asthma symptoms, quality of life, or PC.sub.20.
[0040] Two significant differences were found between the groups.
Once was in the change in evening peak flow from the end of the
treatment period to the end of the withdrawal period; mean evening
peak flow fell 17.7 liters per minute in the scheduled-treatment
group but increased 1.3 liters per minute in the
treatment-as-needed group. The other significant difference was in
the change in bronchodilator responsiveness between the run-in
period and the treatment period (Table 3). The FEV.sub.1 response
to treatment with albuterol increased from a 10.7 percent
improvement to a 12.5 percent improvement in the
scheduled-treatment group and decreased from a 10.7 percent
improvement to a 9.2 improvement in the treatment-as-needed group.
A number of small but statistically significant changes within the
groups were noted among the various treatment periods, as shown in
Table 3. Results of the analysis in which data collected after the
subjects in whom treatment was considered to have failed were
excluded were essentially the same as those derived with the use of
the intention-to-treat method.
3TABLE 3 MODEL ESTIMATES (USING INTENTION-TO-TREAT DATA) FOR THE
END OF THE RUN-IN PERIOD (WEEK 6), THE END OF THE ACTIVE-TREATMENT
PERIOD (WEEKS 22), AND THE END OF THE WITHDRAWAL PERIOD (WEEK 26).*
AFTER RUN-IN PERIOD AFTER TREATMENT PERIOD AFTER WITHDRAWAL PERIOD
OUTCOME SCHEDULED AS NEEDED SCHEDULED AS NEEDED SCHEDULED AS NEEDED
Peak flow (liters/min) Morning 415.9 424.1 414.4 424.5 414.8 427.3
Evening 436.3 441.1 441.3 445.2 433.6 446.5 P = 0.005.dagger-dbl. P
= 0.021.sctn. Peak-flow 4.1 3.2 5.7 4.3 4.0 4.2 variability
(%).paragraph. P <0.001.vertline. P <0.001.dagger-dbl.
FEV.sub.1 (liters) 3.09 313 3.04 3.12 3.06 3.12 Albuterol response
10.7 10.7 12.5 9.2 (%)** P = 0.005.dagger..dagger. Extra albuterol
1.4 1.6 1.3 1.6 1.6 1.6 (puffs/day) P = 0.013.dagger-dbl. Symptom
score.dagger-dbl..dagger-dbl. 0.4 0.4 0.4 0.4 0.4 0.4
Quality-of-life 2.3 2.4 2.3 2.3 2.1 2.2 score.dagger-dbl..dagger-d-
bl. P = 0.003.dagger-dbl. P = 0.008.sctn..sctn. P =
0.006.sctn..sctn. PC.sub.20(mg/ml) 0.73 0.73 0.56 0.72 0.66 0.76 p
= 0.013.vertline. *Values differ from those in Table 2 because
Table 2 contains the mean data rather than estimates from the
model. .dagger.FEV.sub.1 denotes forced expiratory volume in one
second, and PC.sub.20 the concentration of methacholine required to
decrease the FEV.sub.1 by 20 percent. .dagger-dbl.P value is for
the within-group comparison of the response at the end of the
treatment period with that at the end of the withdrawal period.
.sctn.P value is for the comparison between groups of the change in
response from the end of the treatment period to the end of the
withdrawal period. .sctn.Peak-flow variability was calculated as
([evening peak flow -- morning peak flow] .div. evening peak flow)
.times. 100 (Martin et al., Am. Rev. Respir. Dis. 143:351, 1991).
.vertline.P value is for the within-group comparison of the
response at the end of the treatment period with that at the end of
the run-in Period. **Bronchodilator response was last measured
during the run-in period at week 4 and during the active-treatment
period at week 20. .dagger..dagger.P value is for the comparison
between groups of the change in response from the end of the run-in
period to the end of treatment period. .dagger-dbl..dagger-dbl.See
the footnotes to Table 2 for an explanation of the scoring system.
.sctn..sctn.P value is for the within-group comparison of the
response at the end of the run-in period with that at the end of
the withdrawal period.
[0041] These results show that regular use of inhaled albuteral in
patients with mild asthma is not generally associated with a
deleterious effect on asthma control.
EXAMPLE 2
Correlation of .beta..sub.2-Adrenergic Receptor Allele with Outcome
of .beta.-Agonist Administration
[0042] Materials and Methods:
[0043] Generally: The patients analyzed in the present study had
been participants in a .beta.-agonist study, referred to as BAGS,
sponsored by the Asthma Clinical Research Network of the United
States National Heart, Lung and Blood Institute (ACRN). The results
of that study are published in the New England Journal of Medicine
(Drazen et al., NEJM 335:841, 1996, incorporated herein by
reference); relevant portions of the Materials and Methods and
Results sections of that paper are reproduced in Example 1. As
noted above, that study concluded that, overall, no negative
effects were associated with regular administration of albuterol to
mild asthmatics.
[0044] We decided to further analyze the results of the BAGS trial
by investigating the genotype of study participants'
.beta..sub.2-adrenergic receptor gene (encoding residue 16 of the
protein). We were able to obtain materials for determining each
patient's genotype for 179 of the 255 subjects. The remaining
subjects either refused to participate or could not be located for
genotyping.
[0045] Arms Assay: The primers used to detect the
.beta..sub.2-adrenergic receptor gene polymorphism (corresponding
to an A.fwdarw.G substitution at nucleotide 1633 of SEQ ID NO:2)
(A.fwdarw.G) that gives rise to the Arg 16.fwdarw.Gly amino acid
change in the protein were: Wild-type forward primer A1
(5'-GCCTCTTGCTGGCACCCAA-AA-3' [SEQ ID NO:3]) corresponding to
nucleotides 1612-1633, except the penultimate base at the 3' end
(underlined) was changed from T to A, polymorphism-specific forward
primer A2 (5'-GCCTTCTTGCTGGCACCCAAAG-3' [SEQ ID NO:4], differs from
the wild-type primer at the last nucleotide at 3' end (shown in
bold), reverse primer Rev (5'-AGGATAACCTCATCCGTAAGG-3' [SEQ ID
NO:5]) corresponding to nucleotides 2483-2503 on the complementary
strand.
[0046] The primers used to detect the .beta..sub.2-adrenergic
receptor gene polymorphism (corresponding to a C.fwdarw.G
substitution at nucleotide 1666 of SEQ ID NO:2) that gives rise to
the Gln27.fwdarw.Glu amino acid change in the protein were:
Wild-type forward primer B1 (5'-CCGGACCACGACGTCACGCAAC-3' [SEQ ID
NO:6] corresponding to nucleotides 1645-1666, except the
penultimate base at the 3' end (underlined) was changed from G to
A, polymorphism-specific forward primer B2
(5'-CCGGACCACGACGTCACGCAAG-3' [SEQ ID NO:7]), differs from the
wild-type primer at the last nucleotide at 3' end (shown in bold),
and reverse primer Rev.
[0047] Amplification by PCR of the genomic DNA of each sample
included two reactions for each assay separately: one with wild
type primers (A1 and REV) and the other with polymorphic (A2 and
Rev) allele-specific primer set for polymorphism detection at
nucleotide 16633 and wild type primers (B1 and Rev) and the
polymorphic allele-specific primer set (B2 and Rev) for
polymorphism detection at nucleotide 1666. Both polymorphism
detection assays included human .beta.-globin primer sets as
positive controls in the PCR reaction mix. The primers for
.beta.-globin were: Forward primer BG1 (5'-GCTGTCATCACTTAGACCTC-3'
[SEQ ID NO:8] corresponding to nucleotides 43-62 (Genbank accession
no. L48217), reverse primer BG2 (5'-CAGACGAATGATTGCATCAG-3' [SEQ ID
NO: 9]) corresponding to nucleotides 766-785 on the complementary
strand (Genbank accession no. L48217).
[0048] Each PCR reaction contained 5 .mu.l template DNA (buccal
cell lysate or 100-200 ng of blood genomic DNA), PCR buffer II
(Perkin Elmer), 1.5 mM MgCl.sub.2 (Perkin Elmer), 12.5 pmoles of
each primer, 400 .mu.M dNTPs (Perkin Elmer), 0.625 units of Taq
polymerase (AmpliTaq polymerase, Perkin Elmer), 0.05 units of
Perfect Match PCR enhancer (Stratagene) in a total volume of 25
.mu.l. Conditions for PCR were: an initial hot start period of 5
min at 94.degree. C., and temperature was hold at 80.degree. C.
after the hot start during this period dNTPs and Taq polymerase
were added. This was followed by 35 cycles of 1 min at 94.degree.
C., 1 min at 58.degree. C., 1 min at 72.degree. C., with a final
extension time of 5 min at 72.degree. C. Thin walled 96 micro-well
plates (Costar) were used with mineral oil for amplification
reactions, in a PTC-100 thermal cycler (MJ Research, Watertown,
Mass.). After amplification, about 20 .mu.l of reaction mixture was
resolved by electrophoresis on a 2.0% agarose gel and stained with
ethidium bromide for analysis.
[0049] Results
[0050] FIG. 3 presents an example of the results we achieved in our
genotype analysis of BAGS subjects. The overall findings for this
residue are at .beta..sub.2-adrenergic receptor residue 16
summarized below in Table 4:
4TABLE 4 GENOTYPE OF BAGS STUDY PARTICIPANTS AT PROTEIN RESIDUE 16
RACE AA AG GG TOTAL African 7 12 11 30 American Hispanic 2 9 3 14
Other 1 4 4 9 Minority Non-Minority 18 64 44 126 Total 28 89 62
179
[0051] These data indicate an allele frequency of 0.405 for Arg 16,
so that one would expect approximately 16% of the population to be
homozygous for this allele.
[0052] Having determined the .beta.-adrenergic receptor genotype of
BAGS participants, we then re-analyzed the BAGS data, stratifying
it by the genotype of the individuals in the various treatment
groups. Our results are shown in FIG. 3. As can be seen,
individuals who are homozygous for the Arg16 had an adverse
response to the regular use of inhaled .beta.-agonists, as
indicated by a decrease in peak flow of 22.5 LPM between the end of
the placebo "run-in" period and the end of the "withdrawal" period.
By contrast, Arg16 homozygotes who received as-needed
.beta.-agonist treatment had a slight increase in peak flow over
this interval. Gly16 homozygotes and Arg16/Gly16 heterozygotes
treated with regularly-scheduled .beta.-agonist administrations,
did not display any adverse effects. These data indicate that the
genotype at position 16 of the .beta..sub.2-adrenergic receptor
provides strong predictive information about the likely response of
the patient to regularly-scheduled .beta.-agonist treatment. The
change in peak flow rate after regularly-scheduled albuterol
treatment was significantly different over the interval between the
end of the run period and the end of the withdrawal period in
individuals homozygous for the Arg16 allele as compared with those
having the Gly16/Gly16 or Arg16/Gly16 genotypes (p=0.0019 for AM
PEFR; p=0.0009 for PM PEFR).
[0053] We note that all of the .beta.-agonist-sensitive Arg16
homozygotes that we analyzed were also homozygous for Gln27, but
other Gln27 homozygotes did not have adverse responses to
.beta.-agonists. Accordingly, we conclude that Gln27 is not likely
to be an indicator, by itself, of susceptibility to adverse
.beta.-agonist responses.
EXAMPLE 3
Preferred Methods of Detecting .beta..sub.2-Adrenergic Receptor
Alleles
[0054] Generally:
[0055] As will be readily appreciated by those of ordinary skill in
the art, any of a variety of techniques may be used to detect
.beta..sub.2-adrenergic receptor alleles in order to identify
patients susceptible to a negative response to .beta.-agonist
administration in accordance with the present invention. The
present Example is intended only to provide certain preferred
examples of possible methods, and is not intended to limit the
scope of the present invention.
[0056] Temperature Gradient Gel Electrophoresis:
[0057] Generally: A temperature gradient gel electrophoresis method
for detection of .beta..sub.2-adrenergic receptor gene
polymorphisms has been described (Reihsaus et al., Am. J Respir.
Cell. Mol. Biol. 8:334, 1993, incorporated herein by reference).
Basically, a gradient of denaturing solvent in a polyacrylamide gel
is employed to separate nucleic acid fragments that differ in
sequence by only a single base pair (see, for example, Wartell et
al., Nuc. Acids Res. 18:2699, 1990, incorporated herein by
reference).
[0058] Gel Electrophoresis: Temperature-gradient gel
electrophoresis can be carried out with a vertical acrylamide
slab-gel apparatus, modified from a conventional vertical gel
apparatus so that the glass plates containing the acrylamide gel
are sandwiched between two aluminum heating blocks. Channels in the
blocks allow circulating fluid to establish a temperature gradient
from the top to bottom or from one side to the other. The channels
running across the top and bottom are used to establish a
temperature gradient in the same direction as electrophoretic
migration. For a gradient perpendicular to DNA migration the fluid
flows along the sides. Adhesive pipe tape is used to insulate the
surfaces of the blocks not facing the glass. The rear block is
placed against the main vertical support of the gel unit in the
space formed by the overhanging upper buffer chamber. Both heating
blocks rest on U-shaped plexiglass pieces which keep them above the
buffer in the lower electrolyte chamber. Two thermostated fluid
circulators (Haake Inc.) are employed to control the high and low
temperatures.
[0059] The temperature gradient produced by the heating blocks is
preferably checked for linearity and uniformity at least two
temperature settings of the water circulators (e.g., 32.degree.
C./28.degree. C. and 44.degree. C./18.degree. C.). A thermistor
probe (.+-.0.5.degree. C.) can be inserted into a gel to determine
the gel temperatures at different depths and horizontal positions.
For all temperature settings, the gradient in the gel is preferably
linear and uniform within the region covered by the blocks. The
appropriate percent acrylamide gel is determined according to
standard practice. Such gels should be prepared in a denaturing
solvent (e.g., 0.5M TBE, 7.0M urea, 40% formamide u/u) gels are
loaded and according to standard procedures.
[0060] Calculation of Thermal Stability Properties: The model of
the DNA helix-coil transmission can be used to calculate the
thermal denaturation behavior of the DNA fragments (see Wartell et
al., Nuc. Acids Res. 18:2699, 1990, and references cited therein).
In addition to predicting the melting curve for a given DNA
sequence, the calculation can also produce melting profiles for the
base pairs in a DNA sequence. A melting profile displays the
probability that the n.sup.th base pair of the sequence is melted,
.theta.(n), at a given temperature. From a three dimensional
display of melting profiles at a series of temperatures the lengths
and locations of cooperatively melting domains can be visualized.
The calculation of the melting profiles assumes that strand
dissociation is negligible.
[0061] The nearest neighbor stacking parameters can be obtained
from McCampbell et al. (Biopolymers 28:1745, 1989), and Gotoh and
Tagashira (Biopolymers 20:1033, 1981). All other parameters, such
as the loop entropy factor, strand dissociation parameters, etc.
can be obtained from McCampbell et al. (Biopolymers 28:1745, 1989).
When only the first melting domain is of concern the dissociation
parameters and loop entropy terms do not significantly influence
theory-experiment comparisons. Extrapolations may be required to
normalize the calculations to the solvent conditions utilized in
the above-cited parameter references. For example, the Gotoh and
Tagashira parameters, determined in 0.02M Na.sup.+, can be
extrapolated to 0.1 M Na.sup.+ by scaling T.sub.AT and T.sub.GC,
the average T.sub.m's of AT and GC base pairs (Vologodskii et al.,
J Biomole Struct. Dynam. 2:131, 1984).
[0062] Allele-Specific PCR:
[0063] Generally: One preferred method for identifying
.beta..sub.2-adrenergic receptor gene polymorphisms in order to
practice the present invention is to perform polymerase chain
reactions (PCR) using primers whose 3'-most nucleotide is
mismatched with respect to either the Arg 16 allele or the Gly 16
allele (see Newton et al., Nuc. Acids. Res. 17:2503, 1989,
incorporated herein by reference; see also Example 2). The PCR
reaction conditions are then adjusted so that product band is only
produced when the primer and template are matched.
[0064] Useful PCR primers and conditions for detection of the Arg
16 and Gly 16 .beta.2-adrenergic receptor gene alleles have been
described (Turki et al., J. Clin. Invest. 95:1635, 1995,
incorporated herein by reference; see also Example 2). As described
in that article, allele-specific PCR is based on the premise that,
under the appropriate conditions, a match between template and
primer at the most 3' nucleotide is necessary for the generation of
a PCR product (i.e., mismatches result in no product).
Allele-specific PCR reactions can be performed, for example, as
follows:
[0065] Genomic DNA is isolated, for example, from 2 ml of
peripheral blood, by any available technique, such as the a
cetylmethyl ammonium bromide separation technique (Jones et al.,
Nature 199:280, 1963). PCR reactions are carried out in a vol of
100 .mu.l using .about.500 ng of genomic DNA. Preferred primer
pairs that delineate the two polymorphisms at nucleic acid 46
(amino acid 16), include i) 5'-CTTCTTGCTGGCACCCAATA-3'- (sense)
(SEQ ID NO:10) and 5'-CCAATTTAGGAGGATGTAAACTTC-3' (antisense) (SEQ
ID NO:11); ii) or the same antisense primer and
5'-CTTCTTGCTGGCACCCAATG-3- ' (sense) (SEQ ID NO:12). The generated
PCR product size using these primers is 913 bp. The polymerase Vent
exo (-) (New England Biolabs, Inc., Beverly, Mass.) can be used for
these reactions. Reaction buffers are preferably those included
with these polymerases from the manufacturers. Temperature cycling
is preferably 98.degree. C. for 30 s, 66-68.degree. C. for 45 s,
and 72.degree. C. for 45 s for 30 cycles, 20 .mu.l of the PCR
reactions can be electrophoresed on 1% agarose gels and visualized
with ethidium bromide staining and ultraviolet illumination.
[0066] The allele-specific PCR technique can be verified by direct
dideoxy sequencing of PCR products, preferably using sequencing
primers different from those used in the PCR. In addition, plasmids
consisting of wildtype .beta..sub.2AR cDNA or mutated cDNA
corresponding to the polymorphisms can be used as positive and
negative control templates for the allele-specific PCR studies.
[0067] Hybridization Studies:
[0068] Generally: It has long been appreciated that differences in
nucleotide sequence can usually be detected by oligonucleotide
hybridization under appropriate conditions. Recently, Hall et al.
(Lancet 345:1213, 1995, incorporated herein by reference) have
demonstrated that such techniques may reliably be used to detect
sequence differences in the .beta..sub.2-adrenergic receptor gene.
As they teach, samples of genomic DNA containing the
.beta..sub.2-adrenergic receptor gene may be immobilized on a
filter such as, for example, a Hybond.RTM. filter. In preferred
methods, the relevant portion of the .beta.B.sub.2-adrenergic
receptor gene (i.e., a portion that includes nucleotide 46,
encoding residue 16, is amplified by PCR, and the PCR product is
affixed to the filter. The filter is then hybridized with excess
unlabeled primer (to "block" nonspecific reactions). Subsequently,
the filter is exposed to labeled primer under high-stringency
conditions. The primer is designed to hybridized with either the
Gly16 or the Arg16 allele, but not with both under the
hybridization conditions employed. In preferred embodiments, the
filter is subsequently stripped and re-hybridized (or a duplicate
filter is prepared and reacted in parallel) with a primer that
reacts with the other allele.
[0069] Restriction Fragment Length Polymorphism:
[0070] The RFLP technique has long been a popular method for
identifying sequence differences within a population (see, for
example, Unit 2.7 of Current Protocols in Human Genetics, John
Wiley & Sons, incorporated herein by reference). The
.beta..sub.2-adrenergic receptor gene contains a .beta.an/site at
position 523 (corresponding to amino acid 175 in the protein) that
is polymorphic (see Ohe et al., Tharax 50:353, 1995). Although this
polymorphism has not been shown to demonstrate linkage with the
Arg16.fwdarw.Gly polymorphism, standard techniques could readily be
employed to detect such a linkage if it exists, so that utilized in
the practice of the present invention.
[0071] Protein Assays
[0072] The presence of .beta..sub.2-adrenergic receptor
polymorphisms can also be detected through protein assays that can
distinguish the Arg16 and Gly16 versions of the
.beta..sub.2-adrenergic receptor protein. For example, Western
blots could be performed using monoclonal antibodies specific for
either variant. Western blot technologies are well known in the
art.
EXAMPLE 4
Kits for Identification of Individuals Susceptible to Adverse
Responses to Chronic .beta.-Agonist Therapy
[0073] As will be apparent to those of ordinary skill in the art,
reagents useful in the practice of the present inventive methods
can usefully be collected together in kits. For example, primer
sets for allele-specific polymerase chain reaction studies can be
provided together in a single container.
[0074] As described above in Example 3, .beta..sub.2-adrenergic
receptor gene alleles can be distinguished from one another through
use of primers whose 3'-most nucleotides hybridize with one allele
but are mismatched with respect to others. Examples 2 and 3
describe particular useful primer sets, but those of ordinary skill
in the art will readily recognize that variations in precise primer
sequence can be made without departing from the spirit or scope of
the present invention, so long as one primer set produces an
amplification product from one .beta..sub.2-adrenergic receptor
gene allele (e.g., the allele encoding the Arg16 variant), and a
different primer set produces an amplification product from another
allele (e.g., the allele encoding the Gly16 variant). Preferred
allele-specific PCR kits also include other PCR reagents, such as
buffer, salt solutions, dNTPs, control DNA including the Arg16
.beta..sub.2-adrenergic receptor gene allele, control DNA including
the Gly16 .beta..sub.2-adrenergic receptor gene allele, and/or DNA
polymerase. Preferably, the DNA polymerase is thermal-stable. Such
kits may optionally include instructions for use.
[0075] Those of ordinary skill in the art will appreciate that
analogous primer-containing kits may be prepared for ligation
amplification reactions, which are based on the premise that
adjacently-hybridized primers are only ligated together when their
terminal residues are hybridized (see English et al., Proc. Natl.
Acad. Sci. USA 9:630, 1994, incorporated herein by reference).
Additional reagents optionally included in ligantion amplification
kits include buffers, salts, ligase (preferably thermal-stable
ligase), ATP, control DNA including the Arg16
.beta..sub.2-adrenergic receptor gene allele, control DNA including
the Gly16 .beta..sub.2-adrenergic receptor gene allele, and/or
instructions for use.
[0076] Primer-containing kits may also be desirably prepared that
do not contain allele-specific primer sets, but rather contain only
a single set of primers, which primers amplify a region of the
.beta..sub.2-adrenergic receptor gene that encodes residue 16.
Preferred such kits also include PCR reagents and/or sequencing
reagents. Preferably, dideoxy sequencing reagents are employed (see
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989,
Chapter 13, incorporated herein by reference). Preferred dideoxy
sequencing reagents include, for example, a sequencing primer (that
hybridizes either to the .beta..sub.2-adrenergic receptor gene
amplification product or to a vector into which the product may be
cloned), dNTPs, ddNTPs, buffers, salts, and/or instructions. In
preferred embodiments, the dNTPs are provided either singly or in
mixtures that are sets of three dNTPs. Preferred kits may also (or
alternatively) include detection reagents, such as, for example,
radioactive or fluorescent. Particularly preferred kits are
designed genetic analyzers and include fluorescently-tagged
primers.
[0077] Some preferred kits also contain reagents for distinguishing
other .beta..sub.2-adrenergic receptor gene alleles (e.g., alleles
at other positions).
Other Embodiments
[0078] One of ordinary skill in the art will readily recognize that
the foregoing has been merely a detailed description of certain
preferred embodiments of the present invention. Various alterations
and modifications of the procedures, techniques, and compositions
described above will be apparent to those in the art and are
intended to be encompassed by the following claims.
Sequence CWU 1
1
* * * * *