B2 Adrenoceptor Polymorphism

Asthma Free Forever

Asthma Free Forever

Get Instant Access

In many ways, the ^-adrenergic receptor gene (b2AR) is an ideal candidate for the study of genetic variation, biological relevance, and clinical consequences of polymorphisms. The receptor is expressed on most cells, and agonists and antagonists are used in treatment of a number of diseases, such as asthma, COPD, hypertension, heart failure, preterm labor, glaucoma, and migraine. On the other hand, all these diseases are complex and multi-factorial, and most have a substantial environmental component. As such, the influence of aIan P. Hall's research is supported by the Medical Research Council and Asthma U.K. bStephen B. Liggett is supported by the National Heart Lung and Blood Institute Pharmacogenetics Research Network grant U01-HL65899.

a b2AR genetic variant on the disease itself, the response to specific therapy (i.e., b-agonists), the response to other therapies where there is interaction (i.e., corticosteroids), and gene-environment interactions all have to be considered. Thus, studies of the effects of b2AR polymorphisms in asthma and asthma treatment have been somewhat of a "case-study" in the evolution of our understanding of how to study polymorphisms and complex traits.

b-agonists are utilized in asthma and COPD as acute interventions during bronchos-pasm and as preventative therapy. The responses to these agents, as assessed by a number of physiologic or clinical outcomes, show a high degree of interindividual variability. Estimates have indicated that >50% of this variability has a genetic basis (1). Epidemiologic studies have also revealed that "excessive" use of b-agonists is associated with loss of asthma control and also increased morbidity and mortality [see Refs. (2,3) for reviews]. Indeed, b-agonists may predispose to bronchoconstriction through cross talk between b2ARs and other signaling pathways in the airway (4). In addition, there are other classes of drugs that also have therapeutic efficacy in asthma and COPD management. This significant interindividual variability in the response to b-agonists, the potential for adverse effects, and alternative treatments prompted the investigation of the b2AR gene for polymorphisms and their relevance in clinical medicine, so as to tailor therapy based on genetic profiles.

In 1993, the existence of polymorphisms of the human b2AR was published (5). Shown in Figure 1 are the locations within the coding region of those polymorphisms. Subsequent studies have failed to uncover additional, nonsynonymous, single-nucleotide polymorphisms (SNPs) in the coding region. By far the two most common polymorphisms (Table 1) are in the amino-terminus at amino acids 16 and 27 (nucleotides 46 and 79). At

Figure 1 Schematic representation of the human b2 adrenoceptor showing known coding region synonymous and nonsynonymous polymorphisms.
Table 1 Coding Region Variants of the Human ^-Adrenergic Receptor

Nucleotides

Amino acids

Nucleotide position

major/minor

Codon position

major/minor

46

G/A

16

Gly/Arg

79

C/G

27

Gln/Glu

100

G/A

34

Val/Met

491

C/T

164

Thr/Ile

252

G/A

84

Leu (syn)

523

C/A

175

Arg (syn)

1053

G/C

351

Gly (syn)

1098

T/C

366

Leu (syn)

1239

G/A

413

Leu (syn)

Abbreviation: syn, synonymous.

Abbreviation: syn, synonymous.

amino acid 16, either Arg or Gly can be found, the former being the minor allele but for many years considered as the "wild type." At amino acid position 27 Gln or Glu can be found. A rare SNP, localized to a codon within the fourth transmembrane spanning domain, results in Thr or Ile at amino acid 164. An extremely rare variant has also been found at position 34 as shown. It is generally accepted that these jb2AR polymorphisms are not associated with risk for asthma, although there have been a few positive association studies (see next). The main emphasis has been on whether these polymorphisms modify asthma, such as defining certain clinical subsets, or alter the response to b-agonist therapy.

In order to have a better understanding of how each polymorphism affects the pharmacology of the receptor, a number of in vitro studies have been carried out (6-10). Initial work with the amino terminal polymorphisms was performed using trans-fected Chinese hamster fibroblasts, stably expressing each possible combination of the two polymorphisms (7). In membrane-based assays, all the variant receptors had similar functional coupling to adenylyl cyclase. In addition, radioligand-binding studies revealed no differences in agonist- or antagonist-binding affinities. Because the amino-terminus was known to be important in receptor trafficking and regulation by agonists, studies were carried out examining such short-term events as receptor internalization and long-term events, including receptor synthesis and agonist-promoted downregulation (loss of net receptor levels after 24 hours of exposure to agonists in culture). The most obvious phe-notype, which arose from such studies, is summarized in Figure 2A. The Arg16/Gln27 receptor underwent ^26% loss of receptor number under these conditions. The Gly16/ Gln27 receptor had enhanced agonist-promoted downregulation (~41%), while the Arg16/Gln27 receptor showed very little downregulation. As discussed in the following, while the pharmacology of this latter variant is interesting, this combination (haplotype) is very uncommon. The Gly16/Glu27 receptor displayed enhanced downregulation similar to the Gly16/Gln27 receptor. Additional studies were subsequently performed with human airway smooth muscle cells, which were obtained from individuals without lung disease and grown in primary culture (8). The advantages of this system is that it is a cell type of interest, the endogenous promoter drives the expression of the receptor, and the levels of expression are "physiologic." However, there is no way to control the polymorphic variation of other genetic loci in genes associated with the signal transduction pathway of the ¡32AR, and thus these cells have their limitations. Genotyping of positions 16 and 27 (but notably not in the promoter or untranslated regions) provided groups of cells to further examine the effects of polymorphisms at these two positions on

Figure 2 Downregulation profiles for different combinations of the codon 16 and 27 b2 adrenoceptor polymorphisms following overnight exposure to isoproterenol (isoprenaline) in CHW cells (A) or primary cultures of human airway smooth muscle (B).

agonist-promoted downregulation. The results were qualitatively quite consistent with the transfected cell results, again showing the marked increase in downregulation of the Gly16 variant (Fig. 2B).

The SNP at position 164 has not been studied in asthma due to its low allele frequency (2-5% heterozygotes, no homozygous individual has been reported to date). However, its properties suggest that in the few patients who carry Ile164, the overall response to all the available (-agonists (such as bronchodilatation) and the duration of action to the agonist salmeterol may be reduced. In vitro, Ile164 is markedly uncoupled from stimulation of adenylyl cyclase (Fig. 3A) (6). In addition, salmeterol binding to the exosite in transmembrane spanning domain 4 is decreased, such that the duration of action [stimulation of cyclic AMP (cAMP)] is reduced by approximately 50% (Fig. 3B) (10).

As is discussed in the following, multiple studies have found correlations between one or more of the two major coding polymorphisms and an asthmatic phenotype. However, it became clear that there was some variability in a given trait (such as the bronchodilator response) even after stratification of patients by the polymorphisms at positions 16 and 27. This has prompted the examination of the promoter and 5' UTR regions of the (2AR gene for genetic variation (9,11). The gene is intronless, and the promoter has been characterized, to various extents, in several rodent genes as well as in the human (11 -14). To address genetic variability, one group resequenced the (2AR gene in multiple individuals from a reference repository composed of Caucasians, African Americans, Asians, and Hispanic Latinos (9). These results are shown in Table 2. Thirteen SNPs were noted from -1023 to +523. (of note, because further sequencing in the coding region had previously not revealed nonsynonymous SNPs, this region was not further

Figure 3 Effect of albuterol (salbutamol) on adenylyl cyclase activity (A) or cyclic AMP production time course (B) in cell lines expressing the Ile 164 and Thr 164 forms of the human adrenoceptor. Abbreviations: cAMP, cyclic AMP; PBS, phosphate buffered saline.

pursued. And, 3' UTR sequencing was somewhat problematic, and so was not included in the analysis). Of the 213 = 8192 possible combinations of the 13 SNPs, only 12haplotypes were detected. This, of course, does not exclude the possibility of additional SNPs, or hap-lotypes, that are present in the human population (or certain isolated populations). But, given the diversity of the population groups that were studied, we contend that any other SNPs/haplotypes found will be uncommon. It is interesting to note that the number of SNPs for this gene is approximately equal to the number of haplotypes (with frequencies >1%). This has turned out to be true for other G protein coupled receptor (GPCR) genes (15) and also for a large group of non-GPCR genes (16). So although one initially considers that the use of haplotypes could markedly expand the number of total possibilities, many of those combinations do not exist due to linkage disequilibrium between the various SNPs. With regards to the f32AR, some of the haplotypes display significant differences in prevalence based on ethnicity. For example, haplotype 1 is ~40-fold more common in African Americans compared with the Caucasians. Other haplotypes are cosmopolitan, such as haplotypes 4, 6, and 2. Despite the relative short distances between these SNPs, the degree of linkage to disequilibrium is sufficiently low for certain positions (Fig. 4) that it is necessary to genotype multiple loci in order to obtain a sufficient amount of genetic information to assign the haplotype. Depending on whether one chooses to identify even the rare haplotypes, or to group certain rare haplo-types, the number of SNP positions that are required to be genotyped can vary. A grouping strategy requires genotyping about 4-6 SNP loci. In Caucasians, the most common homozygous haplotypes are haplotypes 2 and 4 (also referred to as haplotypes 2/2 and 4/4).

Table 2 Haplotypes of the Human ß2 Adrenergic Receptor

Nucleotide Frequency (%)

Table 2 Haplotypes of the Human ß2 Adrenergic Receptor

Nucleotide Frequency (%)

-1023

-709

-654

-468

-406

-367

-47

-20

46

79

252

491

523

Alleles

G/A

C/A

G/A

C/G

C/T

T/C

T/C

T/C

G/A

C/G

G/A

C/T

C/A

Ca

A-A

As

H-L

Haplotype

1

A

C

G

C

C

T

T

T

A

C

G

C

C

0.7

25.0

12.5

10.0

2

A

c

G

G

c

C

C

C

G

G

G

c

C

48.3

6.3

10.0

26.7

3

G

A

A

C

c

T

T

T

A

C

G

c

c

0.7

0.0

0.0

0.0

4

G

C

A

C

c

T

T

T

A

C

G

c

c

33.0

29.7

45.0

40.0

5

G

C

A

C

c

T

T

T

G

C

G

c

c

1.4

0.0

0.0

0.0

6

G

C

G

C

c

T

T

T

G

C

A

c

A

13.2

31.3

30.0

13.3

7

G

c

G

C

c

T

T

T

G

C

A

T

A

1.0

1.6

0.0

3.3

8

G

c

A

C

c

T

T

T

A

C

A

C

A

0.7

0.0

0.0

0.0

9

A

c

G

C

T

T

T

T

A

C

G

C

C

0.0

4.7

0.0

0.0

10

G

c

G

C

C

T

T

T

G

C

A

C

C

0.7

0.0

0.0

3.3

11

G

c

G

C

C

T

T

T

G

C

G

C

C

0.3

0.0

2.5

0.0

12

A

c

G

G

C

T

T

T

A

C

G

C

C

0.0

1.6

0.0

3.3

Location

5'

5'

5'

5'

5'

5'

AA19 BUP

5'

AA16

AA27

syn

AA164

syn

Cys/Arg

Gly/Arg

Gln/Glu

Thr/lle

in in o

Figure 4 Linkage disequilibrium across the human ¡2 adrenoceptor locus.

These genes were constructed exactly as they occur in nature, in a vector with no other promoters or enhancers. Transfection studies (9) were carried out in human embryonic kidney cells, which express a low level of ¡32AR, and thus we presumed that there are transcription factors that regulate receptor expression. Haplotype 2 expression (both mRNA and protein) was greater than that of haplotype 4. Thus in this model system, there appears to be haplotype-specific directivity of receptor expression. There are a number of differences between haplotypes 2 and 4. In all, there are eight SNP positions, which differ between the two. These involve potential «s-acting elements in the 5' region, including sites for AP-4, C/EBP, NF-1, and CP2, as well as the ¡3AR upstream binding protein in the 5' leader cistron. In addition, haplotypes 2 and 4 differ at the coding polymorphisms at amino acids 16 and 27.

Another group has constructed a large number of 5' upstream haplotypes used to drive the expression of the luciferase gene, and only a few appeared to have an effect

(17). Of note, the haplotypes that had the most dramatic changes in expression of the reporter are rare in the human population. In another study, the majority of the promoter activity for the ¡32AR was found to be from —549 forward (11). Transfection of the two major haplotypes with a luciferase reporter construct into COS-7 cells showed differences in expression. The lower expression was obtained with a construct with a limited haplo-type encompassed within haplotype 2 of Table 2. Another study analyzed receptor expression in peripheral blood mononuclear cells, which endogenously express ¡ 2AR

(18). Haplotypes were constructed based on positions —367, —47, +46, and +79. No differences in expression between the four possible ¡ 2AR haplotypes were noted on these peripheral cells. The basis for the discrepancies in these various reports is not entirely clear but may lie in the fact that the methods utilized by each study are different. In addition, there may be a significant contribution of specific transcription factors found in the host cell, which ultimately sets the phenotype of a given polymorphic ¡ 2AR gene.

Was this article helpful?

0 0
Coping with Asthma

Coping with Asthma

If you suffer with asthma, you will no doubt be familiar with the uncomfortable sensations as your bronchial tubes begin to narrow and your muscles around them start to tighten. A sticky mucus known as phlegm begins to produce and increase within your bronchial tubes and you begin to wheeze, cough and struggle to breathe.

Get My Free Ebook


Post a comment