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Figure 1.
Schematic showing the 7 transmembranes (TM) spanning the β2-adrenergic receptor with single nucleotide polymorphisms (SNPs) within specified codons causing an amino acid change (nonsynonymous SNP [solid circles]) or no amino acid change (synonymous SNP [open circles]).

Schematic showing the 7 transmembranes (TM) spanning the β2-adrenergic receptor with single nucleotide polymorphisms (SNPs) within specified codons causing an amino acid change (nonsynonymous SNP [solid circles]) or no amino acid change (synonymous SNP [open circles]).

Figure 2.
Representative gel shows the phase assignment (top gray bar over 2 columns represents 1 sample). The bottom Arg and Gly labels indicate the allele-specific primer at codon 16. Bbv I cuts Gln27 into 2 products, but does not cut Glu27. bp indicates base pairs.

Representative gel shows the phase assignment (top gray bar over 2 columns represents 1 sample). The bottom Arg and Gly labels indicate the allele-specific primer at codon 16. Bbv I cuts Gln27 into 2 products, but does not cut Glu27. bp indicates base pairs.

Figure 3.
Component bar graphs of the β2-adrenergic receptor codons 16 (A) and 27 (B) show no differences between subjects with primary open-angle glaucoma (POAG) and control subjects within the white and black African ancestry groups. Statistically significant differences exist between ancestry groups.

Component bar graphs of the β2-adrenergic receptor codons 16 (A) and 27 (B) show no differences between subjects with primary open-angle glaucoma (POAG) and control subjects within the white and black African ancestry groups. Statistically significant differences exist between ancestry groups.

Figure 4.
Component bar graph of the haplotypes shows no difference between subjects with primary open-angle glaucoma (POAG) and control subjects but shows differences between the white and black African ancestry groups. Following the nomenclature of Drysdale et al, haplotype 2 is Gly16/Glu27, 4 is Arg16/Gln27, and 6 is Gly16/Gln27. ADRB2 indicates the gene for the β2-adrenergic receptor gene.

Component bar graph of the haplotypes shows no difference between subjects with primary open-angle glaucoma (POAG) and control subjects but shows differences between the white and black African ancestry groups. Following the nomenclature of Drysdale et al,45 haplotype 2 is Gly16/Glu27, 4 is Arg16/Gln27, and 6 is Gly16/Gln27. ADRB2 indicates the gene for the β2-adrenergic receptor gene.

Table 1. 
Oligonucleotide Primers Used for PCR and Sequencing the Human ADRB2 Gene*
Oligonucleotide Primers Used for PCR and Sequencing the Human ADRB2 Gene*
Table 2. 
ADRB2 Allele Combinations of Codons 16 and 27*
ADRB2 Allele Combinations of Codons 16 and 27*
Table 3. 
ADRB2 Haplotype Combinations
ADRB2 Haplotype Combinations
1.
Quigley  HABroman  AT The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006;90262- 267
PubMedArticle
2.
Wiggs  JLAllingham  RRHossain  A  et al.  Genome-wide scan for adult onset primary open angle glaucoma. Hum Mol Genet 2000;91109- 1117
PubMedArticle
3.
Allingham  RRWiggs  JLHauser  ER  et al.  Early adult-onset POAG linked to 15q11-13 using ordered subset analysis. Invest Ophthalmol Vis Sci 2005;462002- 2005
PubMedArticle
4.
Nemesure  BJiao  XHe  Q  et al. Barbados Family Study Group, A genome-wide scan for primary open-angle glaucoma (POAG): the Barbados Family Study of Open-Angle Glaucoma. Hum Genet 2003;112600- 609
PubMed
5.
Woodroffe  AKrafchak  CMFuse  N  et al.  Ordered subset analysis supports a glaucoma locus at GLC1I on chromosome 15 in families with earlier adult age at diagnosis. Exp Eye Res 2006;821068- 1074
PubMedArticle
6.
Collaborative Normal-Tension Glaucoma Study Group, Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Am J Ophthalmol 1998;126487- 497
PubMedArticle
7.
Gordon  MOBeiser  JABrandt  JD  et al.  The Ocular Hypertension Treatment Study: baseline factors that predict the onset of primary open-angle glaucoma. Arch Ophthalmol 2002;120714- 720
PubMedArticle
8.
Leske  MCHeijl  AHussein  MBengtsson  BHyman  LKomaroff  EEarly Manifest Glaucoma Trial Group, Factors for glaucoma progression and the effect of treatment: the Early Manifest Glaucoma Trial. Arch Ophthalmol 2003;12148- 56
PubMedArticle
9.
Bengtsson  BHeijl  A A long-term prospective study of risk factors for glaucomatous visual field loss in patients with ocular hypertension. J Glaucoma 2005;14135- 138
PubMedArticle
10.
Asrani  SZeimer  RWilensky  JGieser  DVitale  SLindenmuth  K Large diurnal fluctuations in intraocular pressure are an independent risk factor in patients with glaucoma. J Glaucoma 2000;9134- 142
PubMedArticle
11.
Nouri-Mahdavi  KHoffman  DColeman  AL  et al. Advanced Glaucoma Intervention Study, Predictive factors for glaucomatous visual field progression in the Advanced Glaucoma Intervention Study. Ophthalmology 2004;1111627- 1635
PubMedArticle
12.
Lichter  PRGillespie  BWMusch  DCMills  RPGuire  KECIGTS Study Group, Intraocular Pressure as a Predictor of Visual Field Loss in the Collaborative Initial Glaucoma Treatment Study.  New Orleans, La American Academy of Ophthalmology2004;
13.
Chang  TCCongdon  NGWojciechowski  R  et al.  Determinants and heritability of intraocular pressure and cup-to-disc ratio in a defined older population. Ophthalmology 2005;1121186- 1191
PubMedArticle
14.
Klein  BEKlein  RLee  KE Heritability of risk factors for primary open-angle glaucoma: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 2004;4559- 62
PubMedArticle
15.
Levene  RZWorkman  PLBroder  SWHirschhorn  K Heritability of ocular pressure in normal and suspect ranges. Arch Ophthalmol 1970;84730- 734
PubMedArticle
16.
Armaly  MF The genetic determination of ocular pressure in the normal eye. Arch Ophthalmol 1967;78187- 192
PubMedArticle
17.
Charlesworth  JCDyer  TDStankovich  JM  et al.  Linkage to 10q22 for maximum intraocular pressure and 1p32 for maximum cup-to-disc ratio in an extended primary open-angle glaucoma pedigree. Invest Ophthalmol Vis Sci 2005;463723- 3729
PubMedArticle
18.
Gottfredsdottir  MSSverrisson  TMusch  DCStefansson  E Chronic open-angle glaucoma and associated ophthalmic findings in monozygotic twins and their spouses in Iceland. J Glaucoma 1999;8134- 139
PubMedArticle
19.
Kalenak  JWPaydar  F Correlation of intraocular pressures in pairs of monozygotic and dizygotic twins. Ophthalmology 1995;1021559- 1564
PubMedArticle
20.
Kearns  LSMacKinnon  JRHewitt  AW  et al.  Genetic influences on intraocular pressure. Invest Ophthalmol Vis Sci 2006;47E-Abstract 178
21.
Klein  BEKlein  RLinton  KL Intraocular pressure in an American community: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 1992;332224- 2228
PubMed
22.
Viswanathan  ACHitchings  RAIndar  A  et al.  Commingling analysis of intraocular pressure and glaucoma in an older Australian population. Ann Hum Genet 2004;68489- 497
PubMedArticle
23.
Lemij  HGvan Koolwijk  LPardo Cortes  LM  et al.  Heritability and determinants of intraocular pressure in an isolated population in the Netherlands. Invest Ophthalmol Vis Sci 2006;47E-Abstract 190
24.
Glazier  AMNadeau  JHAitman  TJ Finding genes that underlie complex traits. Science 2002;2982345- 2349
PubMedArticle
25.
Duggal  PKlein  APLee  KE  et al.  A genetic contribution to intraocular pressure: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 2005;46555- 560
PubMedArticle
26.
McLaren  NCMoroi  SE Clinical implications of pharmacogenetics for glaucoma therapeutics. Pharmacogenomics J 2003;3197- 201
PubMedArticle
27.
Yablonski  MEZimmerman  TJWaltman  SRBecker  B A fluorophotometric study of the effect of topical timolol on aqueous humor dynamics. Exp Eye Res 1978;27135- 142
PubMedArticle
28.
Coakes  RLBrubaker  RF The mechanism of timolol in lowering intraocular pressure in the normal eye. Arch Ophthalmol 1978;962045- 2048
PubMedArticle
29.
Rettig  ESLarsson  LIBrubaker  RF The effect of topical timolol on epinephrine-stimulated aqueous humor flow in sleeping humans. Invest Ophthalmol Vis Sci 1994;35554- 559
PubMed
30.
Topper  JEBrubaker  RF Effects of timolol, epinephrine, and acetazolamide on aqueous flow during sleep. Invest Ophthalmol Vis Sci 1985;261315- 1319
PubMed
31.
Koskela  TBrubaker  RF Apraclonidine and timolol: combined effects in previously untreated normal subjects. Arch Ophthalmol 1991;109804- 806
PubMedArticle
32.
Nathanson  JA Adrenergic regulation of intraocular pressure: identification of beta 2-adrenergic–stimulated adenylate cyclase in ciliary process epithelium. Proc Natl Acad Sci U S A 1980;777420- 7424
PubMedArticle
33.
Mittag  TWTormay  A Adrenergic receptor subtypes in rabbit iris-ciliary body membranes: classification by radioligand studies. Exp Eye Res 1985;40239- 249
PubMedArticle
34.
Wax  MBMolinoff  PB Distribution and properties of beta-adrenergic receptors in human iris-ciliary body. Invest Ophthalmol Vis Sci 1987;28420- 430
PubMed
35.
Elena  PPFredj-Reygrobellet  DMoulin  GLapalus  P Pharmacological characteristics of beta-adrenergic-sensitive adenylate cyclase in non pigmented and in pigmented cells of bovine ciliary process. Curr Eye Res 1984;31383- 1389
PubMedArticle
36.
Gooch  AJMorgan  JJacob  TJ Adrenergic stimulation of bovine non-pigmented ciliary epithelial cells raises cAMP but has no effect on K+ or Cl− currents. Curr Eye Res 1992;111019- 1029
PubMedArticle
37.
Mittag  TTormay  A Desensitization of the beta-adrenergic receptor-adenylate cyclase complex in rabbit iris-ciliary body induced by topical epinephrine. Exp Eye Res 1981;33497- 503
PubMedArticle
38.
Elena  PPDenis  PKosina-Boix  M  et al.  Beta adrenergic binding sites in the human eye: an autoradiographic study. J Ocul Pharmacol 1990;6143- 149
PubMedArticle
39.
Elena  PPKosina-Boix  MMoulin  GLapalus  P Autoradiographic localization of beta-adrenergic receptors in rabbit eye. Invest Ophthalmol Vis Sci 1987;281436- 1441
PubMed
40.
Kahle  GKaulen  PWollensak  J Quantitative autoradiography with (125I)cyanopindolol of ocular tissues [in German]. Fortschr Ophthalmol 1990;87105- 109
PubMed
41.
Small  KMMcGraw  DWLiggett  SB Pharmacology and physiology of human adrenergic receptor polymorphisms. Annu Rev Pharmacol Toxicol 2003;43381- 411
PubMedArticle
42.
Molenaar  PParsonage  WA Fundamental considerations of beta-adrenoceptor subtypes in human heart failure. Trends Pharmacol Sci 2005;26368- 375
PubMedArticle
43.
Lanfear  DEJones  PGMarsh  S  et al.  β2-Adrenergic receptor genotype and survival among patients receiving β-blocker therapy after an acute coronary syndrome. JAMA 2005;2941526- 1533
PubMedArticle
44.
Brodde  OELeineweber  K β2-Adrenoceptor gene polymorphisms. Pharmacogenet Genomics 2005;15267- 275
PubMedArticle
45.
Drysdale  CMMcGraw  DWStack  CB  et al.  Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000;9710483- 10488
PubMedArticle
46.
Silverman  EKKwiatkowski  DJSylvia  JS  et al.  Family-based association analysis of β2-adrenergic receptor polymorphisms in the childhood asthma management program. J Allergy Clin Immunol 2003;112870- 876
PubMedArticle
47.
Liggett  SB Molecular and genetic basis of β2-adrenergic receptor function. J Allergy Clin Immunol 1999;104S42- S46
PubMedArticle
48.
Xie  HGStein  CMKim  RB  et al.  Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals [published correction appears in Pharmacogenetics. 2001;11:185]. Pharmacogenetics 1999;9511- 516
PubMed
49.
Small  KMRathz  DALiggett  SB Identification of adrenergic receptor polymorphisms. Methods Enzymol 2002;343459- 475
PubMed
50.
Reihsaus  EInnis  MMacIntyre  NLiggett  SB Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. Am J Respir Cell Mol Biol 1993;8334- 339
PubMedArticle
51.
Belfer  IBuzas  BEvans  C  et al.  Haplotype structure of the beta adrenergic receptor genes in US Caucasians and African Americans. Eur J Hum Genet 2005;13341- 351
PubMedArticle
52.
Agresti  A Categorical Data Analysis. 2nd Hoboken, NJ Wiley–Interscience2002;
53.
Weir  BS Genetic Data Analysis II.  Sunderland, Mass Sinauer Associates1996;
54.
R Development Core Team, R: A Language and Environment for Statistical Computing: R Version 2.3.0 ed.  Vienna, Austria R Foundation for Statistical Computing2006;
55.
Güngör  KOzkur  MCascorbi  I  et al.  Beta 2-adrenergic receptor polymorphism and susceptibility to primary congenital and primary open angle glaucoma. Eur J Clin Pharmacol 2003;59527- 531
PubMedArticle
56.
Gungor  KBeydagi  HBekir  N  et al.  The impact of acute dynamic exercise on intraocular pressure. J Int Med Res 2002;3026- 33
PubMedArticle
57.
Fuchsjager-Mayrl  GMarkovic  OLosert  D  et al.  Polymorphism of the β-2 adrenoceptor and IOP lowering potency of topical timolol in healthy subjects. Mol Vis 2005;11811- 815
PubMed
58.
Zimmerman  TJKaufman  HE Timolol, dose response and duration of action. Arch Ophthalmol 1977;95605- 607
PubMedArticle
59.
Inagaki  YMashima  YFuse  N  et al.  Polymorphism of β-adrenergic receptors and susceptibility to open-angle glaucoma. Mol Vis 2006;12673- 680
PubMed
Ophthalmic Molecular Genetics
January 2007

Evaluation of the β2-Adrenergic Receptor Gene as a Candidate Glaucoma Gene in 2 Ancestral Populations

Author Affiliations

Author Affiliations: Departments of Ophthalmology and Visual Sciences (Ms McLaren; Drs Reed, Musch, Richards, and Moroi; and Ms Radenbaugh), Epidemiology (Drs Musch and Richards), and Otolaryngology (Ms Downs), University of Michigan, Ann Arbor; Department of Neuroscience, Harvard University, Boston, Mass (Ms Higashi); and Duke University Eye Center, Duke University, Durham, NC (Drs Santiago and Allingham).

 

JANEY L.WIGGSMD, PhD

Arch Ophthalmol. 2007;125(1):105-111. doi:10.1001/archopht.125.1.105
Abstract

Objective  To determine whether the β2-adrenergic receptor (ADRB2) gene is a glaucoma susceptibility locus.

Design and Methods  The design was an association study stratified by ancestry (white vs black African) and disease (primary open-angle glaucoma vs control subjects). The ADRB2 single nucleotide polymorphisms were determined by sequencing, and the haplotypes of the common single nucleotide polymorphisms affecting codons 16 and 27 were phased by allele-specific polymerase chain reaction and restriction enzyme digestion. We analyzed the association of single nucleotide polymorphisms and haplotypes by ancestry and disease with the Fisher exact test, χ2 test, and standardized Pearson residual.

Results  A total of 583 subjects underwent genotyping (156 white subjects with primary open-angle glaucoma; 143 subjects of black African ancestry with primary open-angle glaucoma; 148 white controls; and 136 controls of black African ancestry). There were no differences in ADRB2 alleles and haplotypes between the primary open-angle glaucoma and control groups, whether analyzed together or by ancestry. Previously described ancestry-based differences in allele frequencies were found. We also found ancestry-based differences in ADRB2 haplotypes.

Conclusion  The ADRB2 gene was not a glaucoma susceptibility locus in our study population.

Clinical Relevance  Because this gene is not a disease locus, we can now study the role of ADRB2 haplotypes in the glaucoma risk factor of intraocular pressure fluctuation and variation in intraocular pressure response to β-blockers.

Glaucoma is an important worldwide public health problem.1 Hence, the identification of clinically informative and inexpensive tests to improve glaucoma diagnostics and treatment outcomes is a high priority. Using glaucoma clinical phenotype ascertainment, Mendelian genetic approaches have led to the identification of 15 genes and 30 loci (http://www.ncbi.nlm.nih.gov/mapview/ using glaucoma as the search term). However, these monogenic forms of glaucoma are not common, and the search for glaucoma genes will increasingly require approaches designed to account for genetic complexity25 and contributions of environmental risk factors.

Another approach is based on the study of important phenotypes that contribute to the pathophysiology of glaucoma such as intraocular pressure (IOP),69 IOP fluctuation,1012 and optic disc factors.13,14 The genetic contribution to IOP is supported by results of family,1517 twin,1820 and epidemiologic studies.13,14,2124 The genome screening used in the Beaver Dam Eye Study25 and the linkage used by Charlesworth et al17 show promise in identifying genes whose products play a role in IOP regulation.

Another potentially relevant approach is to identify genetic markers for variation in IOP response to glaucoma medical treatment as an application of pharmacogenomics.26 Given our understanding of the signaling pathways that regulate aqueous humor dynamics, the polymorphic β2-adrenergic receptor (ADRB2) gene is a logical choice for study because it is clear that the mechanism of action for β-adrenergic receptor antagonists or β-blockers is reducing aqueous flow2731 by binding to β-adrenergic receptors in the ciliary body.3240 The effects of ADRB2 single nucleotide polymorphisms (SNPs) (Figure 1) on pharmacology and physiology have been studied in asthma, hypertension, congestive heart failure, obesity, and acute coronary syndrome.4143 Although the ADRB2 gene is not a susceptibility locus for these diseases,4446 the gene is believed to modify the disease course owing to variations in sympathetic tone and response to β-adrenergic receptor treatments.44 The effects of the common nonsynonymous SNPs on ADRB2 function have been studied by means of site-directed mutagenesis.47 A change from the more commonly expressed or wild-type Arg16 to Gly16 enhances isoproterenol-mediated down-regulation. A change from Gln27 to Glu27 was associated with attenuated isoproterenol-mediated down-regulation when coexpressed with the Arg16. The less common change from Thr164 to Ile164 caused lower binding affinity to isoproterenol.

Based on this evidence in systemic adrenergic receptor–mediated physiology and pharmacology, we hypothesize that the polymorphic ADRB2 gene might modify glaucoma pathophysiology by contributing to IOP fluctuation and to variation in IOP response to β-blocker treatment. Given that investigation of the role of ADRB2 variants in pharmacological studies of glaucoma would be confounded if these variants also played a role in the disease process itself, we set out to determine whether the ADRB2 gene is a potential susceptibility locus for primary open-angle glaucoma (POAG).

METHODS
SUBJECTS

Informed consent was obtained from all subjects according to study protocols approved by the institutional review boards at the University of Michigan, Duke University Medical Center, and Ghana Ministry of Health, Accra, all of which complied with the Declaration of Helsinki. The design was a comparative study with a phenotype assignment for subjects with POAG and control subjects. The phenotype was ascertained by results of the ophthalmologic examination on the basis of slitlamp biomicroscopy, IOP applanation findings, and results of gonioscopy and fundus evaluation including the optic disc. Subjects with POAG underwent visual field testing.

Eligibility criteria for subjects with POAG included being 35 years or older and having open filtration angles, IOP of greater than or equal to 22 mm Hg, glaucomatous optic neuropathy, and glaucomatous field loss. Eligibility criteria for controls included no evidence of glaucoma or ocular hypertension and no family history of POAG. Racial information was ascertained by self-report.

MOLECULAR METHODS FOR SEQUENCING AND HAPLOTYPING

We isolated genomic DNA from blood using a commercially available kit (Puregene; Gentra Systems, Minneapolis, Minn) according to the manufacturer's instructions. The entire ADRB2 gene, including the promoter with the 5′ cistron leader, was amplified using sequence-specific primers A and I (Table 1) designed on the basis of the reference sequence (GenBank entry J02960). The polymerase chain reaction (PCR) analysis was conducted in a 50-μL volume using a 2-μL aliquot of the DNA template (50 ng/μL), 2 U of platinum Taq DNA polymerase (Invitrogen Corp, Carlsbad, Calif), 1.5mM magnesium chloride, 0.2mM of each deoxyribonucleotide triphosphate, 1× Q solution (Qiagen Inc, Valencia, Calif), 0.2μM primers (Table 1), 20mM Tris hydrochloride (pH, 8.4), and 50mM potassium chloride. The optimal PCR cycling settings for this reaction were 55°C for 30 cycles. The quality of the predicted 1.616-kilobase (kb) PCR products was evaluated using agarose gel electrophoresis.

Genotyping was performed by means of fluorescence-based sequencing on the amplified PCR gene product with a commercially available sequencing kit (BigDye ABI terminator; PerkinElmer, Boston, Mass) and semiautomated sequencing technology (Applied Biosystems, Foster City, Calif) using primers designed to span the intronless gene every 300 to 400 bases to ensure a readable sequence (Table 1). The amplified ADRB2 gene and the promoter were sequenced in the forward and reverse directions from an initial 275 subjects, and in the forward direction only in the remaining 308 subjects. Sequences were read at least twice independently without knowledge of the diagnosis and compared with the reference sequence (GenBank entry J02960). All polymorphisms were compared with published SNPs,4850 the allelic variant list in the Online Mendelian Inheritance in Man database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), and the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/).

Given the linkage disequilibrium among the closely spaced SNPs of the ADRB2 gene, there are a limited number of haplotypes for the ADRB2 gene.44,45,51 Twelve haplotypes have been described for the ADRB2 SNPs using the nomenclature described by Drysdale et al.45 The most common haplotypes were 2, 4, and 6. Haplotype 2 corresponds to the CGG SNPs in the β2-adrenergic receptor upstream peptide, codon Gly16, and codon Glu27; haplotype 4, to the TAC SNPs in the β2-adrenergic receptor upstream peptide, Arg16, and Gln27; and haplotype 6, to the TGC SNPs in the β2-adrenergic receptor upstream peptide, Gly16, and Gln27.

To perform phasing in subjects who were double heterozygotes at the 2 common functional SNPs at codons 16 and 27, we used a combination of allele-specific PCR with the sense primer B for the Arg16 allele and the sense primer C for the Gly16 allele (Table 1), the antisense primer E, and restriction enzyme digestion (Figure 2). The PCR settings were conducted in a 50-μL volume using a 2-μL aliquot of a 104 dilution of the amplified 1.616-kb DNA product as template, 2 U of Taq DNA polymerase (Promega Corp, Madison, Wis), 1.5mM magnesium chloride, 0.2mM of each deoxyribonucleotide triphosphate, 0.2μM primers, 10mM Tris hydrochloride (pH, 9.0), 0.1% Triton X-100, and 50mM potassium chloride. For reactions using primer B for the Arg16 allele, the annealing temperature was 70°C for 30 cycles. For primer C based on the Gly16 allele, the same annealing temperature was used for 25 cycles. An 8-μL aliquot of the expected 168-base pair (bp) product was treated with the restriction enzyme BbvI (New England Biolabs Inc, Beverly, Mass) at 2 U per reaction (of 50 μL) in the manufacturer's buffer system at 37°C for 16 hours. Because BbvI recognizes a restriction site that is 8 nucleotides downstream from the Gln27 allele, 2 fragments with sizes of 105 and 63 bp can be detected by gel electrophoresis (Figure 2). In contrast, BbvI does not cut in the presence of the Glu27 allele, so the 168-bp fragment remains intact (Figure 2). Using this simple, reproducible method, we were able to determine the haplotypes for subjects who were double heterozygous with the Arg16Gly and Gln27Glu alleles.

STATISTICS

To evaluate ADRB2 as a possible POAG gene, the sample size was determined from power calculations using previously published ADRB2 allele frequencies.48 If we posit that the homozygous Arg16 allele is predictive of a glaucoma phenotype, then a sample size of 528 subjects is needed, with 132 subjects in each of the 4 groups stratified by ancestry and disease. Alternatively, if we posit that the homozygous Gly16 allele is associated with glaucoma, then 4328 subjects are needed. The power calculations become more complex in consideration of haplotypes.

We analyzed the ADRB2 SNPs in our study population using several approaches. In the first approach, we examined the individual allele combinations for Arg16Arg, Arg16Gly, Gly16Gly, Gln27Gln, Gln27Glu, and Glu27Glu. The total allele frequencies were determined for Arg16, Gly16, Gln27, and Glu27. The resulting allele frequencies were compared between the POAG and control groups within the ancestry divisions, among the controls by ancestry, and among subjects with POAG by ancestry. Observations were statistically tested using the Fisher exact test, χ2 test, and an examination of post hoc standardized Pearson residual.52 For the residuals, we used an absolute value of 3.0 as a cutoff for significance. The allele frequencies were tested to determine whether the Hardy-Weinberg equilibrium held for each group.53 All calculations were performed in R version 2.3 statistical software.54

In the second approach, we assembled the haplotype combination for each subject. Subjects who were double heterozygous at Arg16Gly and Gln27Glu underwent phasing using the method described in the preceding subsection. Haplotype combinations were counted and frequencies were determined for each of the 4 groups. Haplotype combinations were compared between the POAG and control groups within the ancestry groups, among the controls by ancestry, and among subjects with POAG by ancestry using the same statistical tests as described in the preceding paragraph.

RESULTS

A total of 583 subjects met eligibility criteria for this comparative association study stratified by ancestry (white vs black African) and disease (POAG vs control group). The black African ancestry group included 9 subjects with POAG from Ghana and 14 controls from Ghana. Among these 4 groups, there were 156 white subjects with POAG (91 women and 65 men; mean ± SD age, 56.5 ± 12.2 years [1 SD]), 143 subjects with POAG who were of black African ancestry (60 women and 83 men; mean ± SD age, 55.7 ± 11.6 years), 148 white controls (70 women and 78 men; mean ± SD age, 65.7 ± 14.9 years), and 136 controls of black African ancestry (61 women and 75 men; mean ± SD age, 57.8 ± 12.7 years).

In the analysis of the common ADRB2 alleles, we studied the SNP diplotypes encoding codons 16 and 27 and their frequencies (Figure 3 and Table 2). At codon 16, the SNP diplotypes encoded for homozygous Arg16Arg, heterozygous Arg16Gly, or homozygous Gly16Gly. At codon 27, the combinations were Gln27Gln, Gln27Glu, and Glu27Glu. When the total allele frequencies for codons 16 and 27 were examined, there were no differences comparing Arg16, Gly16, Gln27, and Glu27 between the subjects with POAG and controls within the ancestry groups using the Fisher exact test (Table 2). Results of tests for association indicate that the Gly16Gly combination is statistically significant (P = .003) in the comparison of white with black African subjects with POAG (Figure 3A and Table 2). All other ancestry comparisons for codon 16 allele combination frequencies were no different. Statistical testing verifies the observed differences (Figure 3B and Table 2) between white and black African subjects within the POAG (P<.001, χ2 test) and control (P<.001, χ2 test) groups with none of the frequencies, except for Gln27Glu in the POAG group, matching statistical expectations as determined by Pearson residual.

With the exception of the black African POAG group for codon 16 (P = .05), the common allele frequencies were consistent with Hardy-Weinberg equilibrium. This deviation may imply that patients referred to our tertiary care center, as a group, had a skewed ADRB2 genotype that was not observed in the black African controls.

For the other nonsynonymous SNPs at codons 34 and 164 (Figure 1), all subjects were homozygous for the common alleles encoding for Val34 and Thr164. We did not identify any subject who had SNPs encoding for homozygous Met34 or Ile164 or for heterozygous Val34Met or Thr164Ile.

In summary, our analysis of the individual common ADRB2 SNPs showed that there were no differences in allele combinations or total allele frequencies between the subjects with POAG and the controls within the ancestry groups. Thus, the common ADRB2 SNPs were not associated with POAG in our study population. However, there were ancestral differences. We found a statistically significant ancestral difference between the white and black African POAG groups that appears to be localized to the homozygous Gly16Gly allele (Figure 3A and Table 2). At codon 27 (Figure 3B), we found ancestral differences within the POAG and control groups. Among the subjects with POAG, all 3 combined genotypes encoding for Glu27Glu, Gln27Glu, and Gln27Gln were different between the white and black African groups. In controls, the differences appeared between homozygous Gln27Gln and Glu27Glu.

Based on these individual allele combinations, we assembled the ADRB2 haplotypes for each subject using the nomenclature described in the methods by Drysdale et al45 (Figure 4 and Table 3). For subjects with compound heterozygous alleles at codons Arg16Gly and Gln27Glu, we phased these alleles using allele-specific PCR and restriction enzyme digestion (Figure 2) as described in the “Molecular Methods for Sequencing and Haplotyping” subsection of the “Methods” section. In 1 white subject with POAG, we identified a haplotype combination of C in the β2-adrenergic receptor upstream peptide, A for Arg16, and G for Glu27 that had not been previously reported. Thus, for this group, there were a total of 155 subjects in this haplotype combination analysis in contrast to the 156 subjects in the SNP analysis. There were no significant differences in haplotype combinations 2, 4, and 6 between the subjects with POAG and the controls within each of the 2 ancestry groups. Thus, the ADRB2 gene was not associated with POAG in our study population.

However, there were ancestral differences between populations for certain haplotype combinations. Among the subjects with POAG, there were significant differences in haplotypes 2/2 (P<.001), 2/4 (P=.002), and 4/6 (P<.001) between the white and black African ancestry groups. The controls showed significant differences in the same haplotypes (2/2 [P=.001], 2/4 [P=.01], and 4/6 [P<.001]) when the white and black African ancestry groups were compared.

COMMENT

In our association study, the ADRB2 gene is not a susceptibility gene for POAG, whether analyzed by individual common SNPs or haplotypes in each of the 2 populations of white and black African ancestries. In addition, we confirm previously reported differences in the frequencies of the common ADRB2 SNPs between groups by different ancestry48 and identify a novel haplotype not seen before.45 Given these ancestral differences between the common SNPs, it is not surprising that we identified differences in the ADRB2 haplotypes frequencies by ancestry (Table 3 and Figure 4). Genetic studies have not previously identified the ADRB2 gene, localized to 5q31-q32, as a glaucoma locus. Thus, the role of the ADRB2 gene in IOP fluctuation and IOP response to β-blockers can be studied without the confounding effect as a POAG disease susceptibility gene.

A retrospective analysis of our subjects with POAG did not allow us to study the association between IOP response to β-blockers and ADRB2 haplotypes. Among the first 165 subjects with POAG (105 in the white and 60 in the black African ancestry groups) who underwent sequencing, 58 of those with available records had used β-blockers. Even with available records, problems with analysis of the retrospective data included the absence of baseline IOP, lack of information on the peak vs trough effect of β-blockers on IOP, the use of multiple classes of glaucoma medications, and the confounding effect of systemic β-blockers. Thus, the role of ADRB2 haplotypes on β-blocker IOP response variations could not be appropriately studied using the retrospective data available in our population.

Previous studies have investigated the role of ADRB2 SNPs on glaucoma susceptibility and IOP. Güngör et al55 reported no differences in the common ADRB2 allele frequencies between 30 cases of congenital glaucoma, 105 cases of POAG, and 92 controls in a Turkish population. Güngör et al56 also investigated the potential effect of ADRB2 SNPs on IOP in 19 healthy young men with a mean ± SD age of 22.6 ± 2.8 years. They found that recovery from exercise-induced lowering of the IOP was prolonged in those who were homozygous for the Gly16 allele compared with those who were heterozygous for the Arg16Gly allele. The implication of their study is unclear because of the small sample size, the unclear role of β2-adrenergic receptors on exercise-induced lowering of the IOP, the use of individual SNPs as opposed to haplotypes, and the study design in young subjects.

In another study, Fuchsjager-Mayrl et al57 screened 270 healthy subjects and identified 24 subjects with homozygous combinations for Arg16/Gln27, 18 with homozygous combinations for Gly16/Gln27, and 47 with homozygous combinations for Gly16/Glu27. All 3 groups showed a comparable IOP response to 0.5% timolol, which led them to conclude that factors other than ADRB2 SNPs caused the “intersubject variability seen with timolol in glaucoma subjects.”57 However, this conclusion may be premature because their findings in young healthy men should not be extrapolated to explain pharmacodynamic variation in an older population with glaucoma, and they did not design a clinical pharmacology study for efficacy30 to measure peak and trough responses to timolol.58

More recently, Inagaki et al59 reported that the Gly16 allele was associated with a younger age at diagnosis, and that the Glu27 allele was associated with higher IOP at diagnosis in Japanese subjects with open-angle glaucoma compared with controls. They suggest that these ADRB2 polymorphisms may influence the pathophysiology of glaucoma in Japanese subjects.

CONCLUSIONS

The ADRB2 gene was not a POAG susceptibility gene in our study population. Thus, we can focus on the role of the ADRB2 SNPs and haplotypes in glaucoma without the confounding effects as a glaucoma disease susceptibility locus. Future association studies may be undertaken to determine whether the ADRB2 gene modifies the pathophysiology of glaucoma by contributing to the known risk factor of IOP fluctuation.1012 The role of this polymorphic gene and the roles of other biologically relevant polymorphic genes involved in the regulation of aqueous humor dynamics may be studied to determine their relative contribution to IOP fluctuation. Furthermore, pharmacogenomic studies may be designed to study the potential association between the ADRB2 haplotypes and IOP response to β-blockers in patients with POAG. Such studies will lead to the identification of clinically informative and inexpensive tests to improve glaucoma diagnostics and treatment outcomes that will lead to personalized treatment for glaucoma.26

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Article Information

Correspondence: Sayoko E. Moroi, MD, PhD, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall St, Ann Arbor, MI 48105 (smoroi@umich.edu).

Submitted for Publication: June 30, 2006; final revision received August 23, 2006; accepted August 25, 2006.

Financial Disclosure: None reported.

Funding/Support: This study was supported by grants EY00353 (Dr Moroi), EY11671 (Dr Richards), EY015543 (Dr Allingham), and EY014939 (Dr Allingham) from the National Institutes of Health (NIH); University of Michigan Vision Core grant EY07003 from the NIH; Midwest Eye-Banks and Transplantation Center (Dr Moroi); a Career Development Award (Dr Moroi) and an unrestricted departmental research grant (Dr Musch) from Research to Prevent Blindness; the University of Michigan Office of the Vice President for Research (Dr Moroi); and the Glaucoma Research Foundation (Dr Moroi).

Acknowledgment: We thank Samir Shah, MD, and Neetaben Patel, MD, for extensive medical chart reviews on some of these subjects.

References
1.
Quigley  HABroman  AT The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006;90262- 267
PubMedArticle
2.
Wiggs  JLAllingham  RRHossain  A  et al.  Genome-wide scan for adult onset primary open angle glaucoma. Hum Mol Genet 2000;91109- 1117
PubMedArticle
3.
Allingham  RRWiggs  JLHauser  ER  et al.  Early adult-onset POAG linked to 15q11-13 using ordered subset analysis. Invest Ophthalmol Vis Sci 2005;462002- 2005
PubMedArticle
4.
Nemesure  BJiao  XHe  Q  et al. Barbados Family Study Group, A genome-wide scan for primary open-angle glaucoma (POAG): the Barbados Family Study of Open-Angle Glaucoma. Hum Genet 2003;112600- 609
PubMed
5.
Woodroffe  AKrafchak  CMFuse  N  et al.  Ordered subset analysis supports a glaucoma locus at GLC1I on chromosome 15 in families with earlier adult age at diagnosis. Exp Eye Res 2006;821068- 1074
PubMedArticle
6.
Collaborative Normal-Tension Glaucoma Study Group, Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Am J Ophthalmol 1998;126487- 497
PubMedArticle
7.
Gordon  MOBeiser  JABrandt  JD  et al.  The Ocular Hypertension Treatment Study: baseline factors that predict the onset of primary open-angle glaucoma. Arch Ophthalmol 2002;120714- 720
PubMedArticle
8.
Leske  MCHeijl  AHussein  MBengtsson  BHyman  LKomaroff  EEarly Manifest Glaucoma Trial Group, Factors for glaucoma progression and the effect of treatment: the Early Manifest Glaucoma Trial. Arch Ophthalmol 2003;12148- 56
PubMedArticle
9.
Bengtsson  BHeijl  A A long-term prospective study of risk factors for glaucomatous visual field loss in patients with ocular hypertension. J Glaucoma 2005;14135- 138
PubMedArticle
10.
Asrani  SZeimer  RWilensky  JGieser  DVitale  SLindenmuth  K Large diurnal fluctuations in intraocular pressure are an independent risk factor in patients with glaucoma. J Glaucoma 2000;9134- 142
PubMedArticle
11.
Nouri-Mahdavi  KHoffman  DColeman  AL  et al. Advanced Glaucoma Intervention Study, Predictive factors for glaucomatous visual field progression in the Advanced Glaucoma Intervention Study. Ophthalmology 2004;1111627- 1635
PubMedArticle
12.
Lichter  PRGillespie  BWMusch  DCMills  RPGuire  KECIGTS Study Group, Intraocular Pressure as a Predictor of Visual Field Loss in the Collaborative Initial Glaucoma Treatment Study.  New Orleans, La American Academy of Ophthalmology2004;
13.
Chang  TCCongdon  NGWojciechowski  R  et al.  Determinants and heritability of intraocular pressure and cup-to-disc ratio in a defined older population. Ophthalmology 2005;1121186- 1191
PubMedArticle
14.
Klein  BEKlein  RLee  KE Heritability of risk factors for primary open-angle glaucoma: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 2004;4559- 62
PubMedArticle
15.
Levene  RZWorkman  PLBroder  SWHirschhorn  K Heritability of ocular pressure in normal and suspect ranges. Arch Ophthalmol 1970;84730- 734
PubMedArticle
16.
Armaly  MF The genetic determination of ocular pressure in the normal eye. Arch Ophthalmol 1967;78187- 192
PubMedArticle
17.
Charlesworth  JCDyer  TDStankovich  JM  et al.  Linkage to 10q22 for maximum intraocular pressure and 1p32 for maximum cup-to-disc ratio in an extended primary open-angle glaucoma pedigree. Invest Ophthalmol Vis Sci 2005;463723- 3729
PubMedArticle
18.
Gottfredsdottir  MSSverrisson  TMusch  DCStefansson  E Chronic open-angle glaucoma and associated ophthalmic findings in monozygotic twins and their spouses in Iceland. J Glaucoma 1999;8134- 139
PubMedArticle
19.
Kalenak  JWPaydar  F Correlation of intraocular pressures in pairs of monozygotic and dizygotic twins. Ophthalmology 1995;1021559- 1564
PubMedArticle
20.
Kearns  LSMacKinnon  JRHewitt  AW  et al.  Genetic influences on intraocular pressure. Invest Ophthalmol Vis Sci 2006;47E-Abstract 178
21.
Klein  BEKlein  RLinton  KL Intraocular pressure in an American community: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 1992;332224- 2228
PubMed
22.
Viswanathan  ACHitchings  RAIndar  A  et al.  Commingling analysis of intraocular pressure and glaucoma in an older Australian population. Ann Hum Genet 2004;68489- 497
PubMedArticle
23.
Lemij  HGvan Koolwijk  LPardo Cortes  LM  et al.  Heritability and determinants of intraocular pressure in an isolated population in the Netherlands. Invest Ophthalmol Vis Sci 2006;47E-Abstract 190
24.
Glazier  AMNadeau  JHAitman  TJ Finding genes that underlie complex traits. Science 2002;2982345- 2349
PubMedArticle
25.
Duggal  PKlein  APLee  KE  et al.  A genetic contribution to intraocular pressure: the Beaver Dam Eye Study. Invest Ophthalmol Vis Sci 2005;46555- 560
PubMedArticle
26.
McLaren  NCMoroi  SE Clinical implications of pharmacogenetics for glaucoma therapeutics. Pharmacogenomics J 2003;3197- 201
PubMedArticle
27.
Yablonski  MEZimmerman  TJWaltman  SRBecker  B A fluorophotometric study of the effect of topical timolol on aqueous humor dynamics. Exp Eye Res 1978;27135- 142
PubMedArticle
28.
Coakes  RLBrubaker  RF The mechanism of timolol in lowering intraocular pressure in the normal eye. Arch Ophthalmol 1978;962045- 2048
PubMedArticle
29.
Rettig  ESLarsson  LIBrubaker  RF The effect of topical timolol on epinephrine-stimulated aqueous humor flow in sleeping humans. Invest Ophthalmol Vis Sci 1994;35554- 559
PubMed
30.
Topper  JEBrubaker  RF Effects of timolol, epinephrine, and acetazolamide on aqueous flow during sleep. Invest Ophthalmol Vis Sci 1985;261315- 1319
PubMed
31.
Koskela  TBrubaker  RF Apraclonidine and timolol: combined effects in previously untreated normal subjects. Arch Ophthalmol 1991;109804- 806
PubMedArticle
32.
Nathanson  JA Adrenergic regulation of intraocular pressure: identification of beta 2-adrenergic–stimulated adenylate cyclase in ciliary process epithelium. Proc Natl Acad Sci U S A 1980;777420- 7424
PubMedArticle
33.
Mittag  TWTormay  A Adrenergic receptor subtypes in rabbit iris-ciliary body membranes: classification by radioligand studies. Exp Eye Res 1985;40239- 249
PubMedArticle
34.
Wax  MBMolinoff  PB Distribution and properties of beta-adrenergic receptors in human iris-ciliary body. Invest Ophthalmol Vis Sci 1987;28420- 430
PubMed
35.
Elena  PPFredj-Reygrobellet  DMoulin  GLapalus  P Pharmacological characteristics of beta-adrenergic-sensitive adenylate cyclase in non pigmented and in pigmented cells of bovine ciliary process. Curr Eye Res 1984;31383- 1389
PubMedArticle
36.
Gooch  AJMorgan  JJacob  TJ Adrenergic stimulation of bovine non-pigmented ciliary epithelial cells raises cAMP but has no effect on K+ or Cl− currents. Curr Eye Res 1992;111019- 1029
PubMedArticle
37.
Mittag  TTormay  A Desensitization of the beta-adrenergic receptor-adenylate cyclase complex in rabbit iris-ciliary body induced by topical epinephrine. Exp Eye Res 1981;33497- 503
PubMedArticle
38.
Elena  PPDenis  PKosina-Boix  M  et al.  Beta adrenergic binding sites in the human eye: an autoradiographic study. J Ocul Pharmacol 1990;6143- 149
PubMedArticle
39.
Elena  PPKosina-Boix  MMoulin  GLapalus  P Autoradiographic localization of beta-adrenergic receptors in rabbit eye. Invest Ophthalmol Vis Sci 1987;281436- 1441
PubMed
40.
Kahle  GKaulen  PWollensak  J Quantitative autoradiography with (125I)cyanopindolol of ocular tissues [in German]. Fortschr Ophthalmol 1990;87105- 109
PubMed
41.
Small  KMMcGraw  DWLiggett  SB Pharmacology and physiology of human adrenergic receptor polymorphisms. Annu Rev Pharmacol Toxicol 2003;43381- 411
PubMedArticle
42.
Molenaar  PParsonage  WA Fundamental considerations of beta-adrenoceptor subtypes in human heart failure. Trends Pharmacol Sci 2005;26368- 375
PubMedArticle
43.
Lanfear  DEJones  PGMarsh  S  et al.  β2-Adrenergic receptor genotype and survival among patients receiving β-blocker therapy after an acute coronary syndrome. JAMA 2005;2941526- 1533
PubMedArticle
44.
Brodde  OELeineweber  K β2-Adrenoceptor gene polymorphisms. Pharmacogenet Genomics 2005;15267- 275
PubMedArticle
45.
Drysdale  CMMcGraw  DWStack  CB  et al.  Complex promoter and coding region β2-adrenergic receptor haplotypes alter receptor expression and predict in vivo responsiveness. Proc Natl Acad Sci U S A 2000;9710483- 10488
PubMedArticle
46.
Silverman  EKKwiatkowski  DJSylvia  JS  et al.  Family-based association analysis of β2-adrenergic receptor polymorphisms in the childhood asthma management program. J Allergy Clin Immunol 2003;112870- 876
PubMedArticle
47.
Liggett  SB Molecular and genetic basis of β2-adrenergic receptor function. J Allergy Clin Immunol 1999;104S42- S46
PubMedArticle
48.
Xie  HGStein  CMKim  RB  et al.  Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals [published correction appears in Pharmacogenetics. 2001;11:185]. Pharmacogenetics 1999;9511- 516
PubMed
49.
Small  KMRathz  DALiggett  SB Identification of adrenergic receptor polymorphisms. Methods Enzymol 2002;343459- 475
PubMed
50.
Reihsaus  EInnis  MMacIntyre  NLiggett  SB Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. Am J Respir Cell Mol Biol 1993;8334- 339
PubMedArticle
51.
Belfer  IBuzas  BEvans  C  et al.  Haplotype structure of the beta adrenergic receptor genes in US Caucasians and African Americans. Eur J Hum Genet 2005;13341- 351
PubMedArticle
52.
Agresti  A Categorical Data Analysis. 2nd Hoboken, NJ Wiley–Interscience2002;
53.
Weir  BS Genetic Data Analysis II.  Sunderland, Mass Sinauer Associates1996;
54.
R Development Core Team, R: A Language and Environment for Statistical Computing: R Version 2.3.0 ed.  Vienna, Austria R Foundation for Statistical Computing2006;
55.
Güngör  KOzkur  MCascorbi  I  et al.  Beta 2-adrenergic receptor polymorphism and susceptibility to primary congenital and primary open angle glaucoma. Eur J Clin Pharmacol 2003;59527- 531
PubMedArticle
56.
Gungor  KBeydagi  HBekir  N  et al.  The impact of acute dynamic exercise on intraocular pressure. J Int Med Res 2002;3026- 33
PubMedArticle
57.
Fuchsjager-Mayrl  GMarkovic  OLosert  D  et al.  Polymorphism of the β-2 adrenoceptor and IOP lowering potency of topical timolol in healthy subjects. Mol Vis 2005;11811- 815
PubMed
58.
Zimmerman  TJKaufman  HE Timolol, dose response and duration of action. Arch Ophthalmol 1977;95605- 607
PubMedArticle
59.
Inagaki  YMashima  YFuse  N  et al.  Polymorphism of β-adrenergic receptors and susceptibility to open-angle glaucoma. Mol Vis 2006;12673- 680
PubMed
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