Combined Pharmacogenetic Effect of ADCY9 and ADRB2 Gene Polymorphisms on the Bronchodilator Response to Inhaled Combination Therapy

Journal of Clinical Pharmacy and Therapeutics (2011) 36, 399–405 doi:10.1111/j.1365-2710.2010.01196.x

PHARMACOGENETICS Combined pharmacogenetic effect of ADCY9 and ADRB2 gene polymorphisms on the bronchodilator response to inhaled combination therapy

S. H. Kim PhD,Y.M.YeMD,H.Y.LeeMS,H.J.SinMS and H. S. Park MD PhD Department of Allergy and Rheumatology, Ajou University School of Medicine, Suwon, Korea

ADCY9 Ile772Met and ADRB2 Arg16Gly gene SUMMARY polymorphisms in terms of percent change in

What is known and objective: Inhaled combina- FEV1 after 8 and 12 weeks of therapy (P=0Æ002 tion therapy composing of long-acting b2-agonist and P=0Æ027 respectively). and corticosteroid has been widely applied in the What is new and conclusion: Our results suggest management of asthma, but observed treatment that ADCY9 gene polymorphisms may alone, and responses vary. The aim of this study was to in combination with ADRB2 gene polymor- evaluate the pharmacogenetic effect of the ade- phisms, contribute to individual response to nylyl cyclase type 9 (ADCY9) gene polymorphism combination therapy in mild to moderate on combination therapy. asthmatics. Materials and methods: Eighty-six mild to moderate Korean asthmatics were enrolled in this Keywords: adenylyl cyclase type 9, asthma, bron- clinical trial. After the 2-week ‘run-in’ period, chodilator response, gene polymorphism inhaled patients received budesonide (an inhaled corti- corticosteroid, long-acting b2-agonist costeroid) and formoterol (long-acting b2-agonist) during the following 12-week active treatment WHAT IS KNOWN AND OBJECTIVE period. Forced expiratory volume in 1 s (FEV1) and maximum mid-expiratory flow (MMEF) Combination therapy with long-acting b2-agonist levels were measured at all visits as primary (LABA) and inhaled corticosteroid (ICS) is effec- outcome. ADCY9 (Ile772Met, 150127 C ⁄ T, 150130 tive for managing asthma by improving lung C ⁄ T, 150397 C ⁄ T, 150479 C ⁄ T, TTTA 5 ⁄ 4) and function and reducing exacerbations in adult b2-adrenergic receptor (ADRB2, Arg16Gly) gene asthmatics (1, 2). Combination therapy provides polymorphisms were genotyped. an additive therapeutically useful effect as Results: Significant associations were observed compared with mono ICS therapy. Furthermore, between the ADCY9 single nucleotide polymor- synergistic interaction between ICS and LABA phisms and percent changes in FEV1 (Ile772Met may play an important role in increasing the T ⁄ C, P =0Æ030) and MMEF (150397 C ⁄ T, therapeutic efficacy of combination therapy, P =0Æ016) after 8 weeks of combination therapy. because corticosteroid has an anti-inflammatory Haplotype associations were also observed with effect and increases airway response to b2-agon- respect to percent changes in FEV1 after 8 weeks ists by up-regulating b2-adrenergic receptor of therapy (Ht3[TTCC], P =0Æ017). Additive (b2AR) expression and increasing cyclic AMP therapeutic effect was observed in those with the production by airway epithelial cells in response to b2-agonists, which augments bronchodilatory response to b2-agonists (3–5). Furthermore, b2AR Received 02 November 2009, Accepted 26 May 2010 induces not only bronchodilation but also Correspondence: Hae-Sim Park, MD, PhD, Department of enhances anti-inflammatory effects in the pres- Allergy and Rheumatology, Ajou University School of Medicine, Ajou University, Suwon, Korea San 5, Wonchun-dong, Yeong- ence of ICS (6, 7). tong-gu, Suwon 443-721, Korea. Tel.: 82-31-219-5196; fax: 82-31- However, degrees of responsiveness to b2-ago- 219-5154; e-mail: [email protected] nist plus ICS vary and approximately 60–80% of

2010 The Authors. JCPT 2010 Blackwell Publishing Ltd 399 400 S. H. Kim et al. inter-individual variations may be due to genetic airway obstruction after inhaled b2-agonist (at least factors, and thus, several pharmacogenetic studies 12% increase in FEV1); and airway responsiveness have been conducted to investigate the genetic to methacholine (provocative concentration caus- basis of therapeutic response heterogeneity (8, 9). ing a 20% drop in FEV1 <25 mg ⁄ mL). All subjects Several clinical investigations have demonstrated that participated in this study were ethnic Koreans that ADRB2 gene polymorphisms contribute to and all provided written informed consent prior to different bronchodilator responses to LABA (10– study commencement. The study protocol was 13). Moreover, the ADRB2 and adenylyl cyclase approved by the institutional review board of Ajou type 9 gene (ADCY9) genes are suspected to be University Hospital. central regulators of b2-agonist responsiveness, During the treatment period, all patients were because of the critical role adenylyl cyclase activity asked to use a single inhaler containing both plays in b2 adrenergic signal transduction (14). budesonide 160 lg and formoterol 4Æ5 lg, twice a

Tantisira et al. found an association between the day and two puffs in a time. FEV1 and MMEF were ADCY9 non-synonymous single nucleotide poly- assessed three times after 0, 8 and 12 weeks of morphism (SNP), ADCY9 Ile772Met, and increased treatment. bronchodilator response to the short-acting b2-agonist, albuterol (salbutamol), in childhood Selection of SNPs and genotyping asthmatics, especially in those receiving inhaled corticosteroid both in vitro and in vivo (14). Genomic DNA from all participants was extracted Although LABA alone is no longer recommended from whole blood using Puregene DNA Purification as a maintenance therapy, combination inhaler Kits (Gentra, Minneapolis, MN, USA). To examine treatments, which include LABA, have been used functional SNPs in the ADCY9 gene, the as a main maintenance therapy for several years, 3¢-untranslated region was sequenced in 86 samples the influence of different ADCY9 gene polymor- using the following primer pair; 5¢-GGAGAA- phisms on relations between bronchodilatory CAAGTGCCTTCAGGG-3¢ (forward primer) and response and combination therapy have not been 5¢-GAGACATCTGGTTACCTTCCTGG-3¢ (reverse previously investigated. primer). Finally, four SNPs (150127 C ⁄ T, 150130 C ⁄ T, In this study, we conducted a 12-week trial of 150397 C ⁄ T, 150479 C ⁄ T) and one tetranucleotide inhaled combination therapy in Korean adult repeat (TTTA 5 ⁄ 4), found to be polymorphic, were asthmatics to investigate the pharmacogenetic genotyped by direct sequencing. Genotypings of the effect of ADCY9 gene polymorphisms and inter- ADCY9 SNPs Ile772Met and 150127 C ⁄ T and of active influence of genetic polymorphisms in ADRB2 Arg16Gly were performed using the SNaP- ADRB2 and ADCY9 gene on the bronchodilatory shot Primer Extension Kit (Applied Biosystems, response to combination therapy. Foster City, CA, USA).

Statistical analyses METHODS Linkage disequilibrium in ADCY9 gene was Subjects investigated using Haploview ver. 3.32 (http:// This study was a 12-week retrospective clinical www.broadinstitute.org/haploview/haploview). trial, which consisted of a 2–4 week ‘run-in’ period Lung functions, including percentage changes in with medium dose of inhaled corticosteroid predicted FEV1 and MMEF values were compared monotherapy followed by a 12-week active treat- using the Mann–Whitney U-test and general linear ment period with combination therapy. Eighty-six model for adjusting baseline lung function (SPSS patients with mild to moderate persistent asthma 12.0, Chicago, IL, USA). P-values of £0Æ05 were based on the Global Initiative for Asthma (GINA) regarded as signiﬁcant. To determine the ability of guidelines were recruited from Ajou University the study to detect signiﬁcance effects, power Hospital, Suwon, Republic of Korea. Patients were analysis was performed using G*power software required to have: a history of typical, recurring (http://www.psycho.uni-duesseldorf.de/aap/ symptoms of asthma; evidence of reversibility of projects/gpower/).

2010 The Authors. JCPT 2010 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 36, 399–405 ADCY9 and ADRB2 gene polymorphisms and improved lung function on combination therapy 401

gene were examined during the study. Their RESULTS AND DISCUSSION genotype distributions were in Hardy–Weinberg This study consisted of 86 Korean adult asthmatics, equilibrium. Of these polymorphisms, four on 43% of whom were female, with mild to moderate 3¢UTR (150127 C ⁄ T, 150130 C ⁄ T, 150397 C ⁄ T and asthma of mean age 39Æ0±11Æ6 years. 29Æ1% had a 150479 C ⁄ T) showed strong linkage with each other family history of asthma and 61Æ6% had atopy. (Fig. 1). Four ADCY9 haplotypes were constructed Participants had a mean serum total IgE level of using these four SNPs. 324Æ0 ± 339Æ9 (IU ⁄ mL). A signiﬁcant association for percent change in

Five SNPs (exon 7: Ile772Met, 3¢UTR: 150127 FEV1 (DFEV1%) was found for ADCY9 Ile772Met C ⁄ T, 150130 C ⁄ T, 150397 C ⁄ T, 150479 C ⁄ T) and one (C ⁄ T) after 8 weeks of treatment (Table 1). Speciﬁ- tetranucleotide repeat (TTTA)4 ⁄ 5 in the ADCY9 cally, patients with the CT or CC genotypes of the

Fig. 1. Linkage disequilibrium among the ADCY9 gene polymorphisms was examined using conﬁ- dence bounds colour scheme. Four genetic polymorphisms (150127, 150130, 150397 and 150479) in the 3¢UTR of ADCY9 showed strong evidence of LD.

Table 1. Genetic association between the ADCY9 gene polymorphisms and percent changes in FEV1 and MMEF

After 8 weeks After 12 weeks

SNP n DFEV1% P DMMEF% P DFEV1% P DMMEF% P rs2230739 Ile772Met T ⁄ CTT 310Æ7±9Æ6 0Æ03 1Æ6±18Æ60Æ273 2Æ3 ± 12 0Æ609 1Æ7±16Æ90Æ935 CT or CC 52 4Æ0±9Æ64Æ0±13Æ82Æ4±8Æ61Æ9±15Æ0 rs1045475 150127 C ⁄ TCC311Æ1±9Æ10Æ349 2Æ1±15Æ10Æ483 )0Æ6±6Æ30Æ067 0Æ0±13Æ80Æ339 CT or TT 55 3Æ9±9Æ84Æ1±15Æ83Æ8±11Æ02Æ7±16Æ3 rs1045476 150130 C ⁄ TCC311Æ1±9Æ10Æ349 2Æ1±15Æ10Æ483 )0Æ6±6Æ30Æ067 0Æ0±13Æ80Æ339 CT or TT 55 3Æ9±9Æ84Æ1±15Æ83Æ8±11Æ02Æ7±16Æ3 rs879619 150397 C ⁄ TCC464Æ2±8Æ70Æ149 7Æ5±15 0Æ016 3Æ0±9Æ60Æ246 3Æ9 ± 14 0Æ237 CT or TT 40 1Æ4±10Æ5 )1Æ3±14Æ91Æ3±10Æ0 )0Æ8±16Æ7 rs710893 150479 C ⁄ TCC592Æ2±9Æ70Æ497 4Æ3±16Æ20Æ521 1Æ3±9Æ60Æ301 1Æ2±15Æ50Æ617 CT or TT 27 4Æ4±9Æ31Æ6 ± 14 4Æ3±10Æ12Æ9±15Æ5 – TTTA 5 ⁄ 455493Æ8±8Æ60Æ299 6Æ4±15Æ30Æ068 2Æ9±9Æ40Æ28 3Æ0±14Æ10Æ534 45 or 44 37 1Æ6±10Æ8 )0Æ6±15Æ11Æ4±10Æ4 )0Æ1±17Æ1

Bold characters indicate signiﬁcance.

2010 The Authors. JCPT 2010 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 36, 399–405 402 S. H. Kim et al.

Ile772Met polymorphism showed a significant We also genotyped the ADRB2 Arg16Gly poly- improvement in predicted FEV1% (4Æ0±9Æ6%)as morphism and examined gene to gene interactions. compared with TT genotype carriers (0Æ7±9Æ6%) Such an interaction was observed between ADCY9 after 8 weeks of treatment (P =0Æ030). Further- Ile772Met and ADRB2 Arg16Gly gene polymor- more, the ADCY9 150397 C ⁄ T polymorphism was phisms with respect to changes in predicted found to be significantly associated with a percent FEV1% (Fig. 2). Specifically, patients with the CT change in MMEF (DMMEF%). As shown in or CC genotype of ADCY9 Ile772Met (C ⁄ T) and the Table 1, a remarkable improvement in predicted AA genotype of ADRB2 Arg16Gly (A ⁄ G) showed

MMEF% was shown in carriers with the CC more predicted FEV1% improvement (8Æ4±7Æ5%) genotype of 150397 C ⁄ T(7Æ5±15Æ0%), whereas than those with the TT genotype of ADCY9 minimal changes were noted in those with the CT Ile772Met and AG or GG genotype of ADRB2 or TT genotypes ()1Æ3±14Æ9%, P =0Æ016) after Arg16Gly A ⁄ G(0Æ4±9Æ1%) after 8 weeks of treat- 8 weeks of treatment. A signiﬁcant association was ment (P =0Æ002). Even after adjusting for baseline observed between DMMEF% and haplotype 3 lung function, the bronchodilatory effect of the [TTCC], which was constructed using four SNPs ADCY9 polymorphisms including 150397 C ⁄ T, located on 3¢UTR (Table 2). Carriers with haplo- ht3[TTCC], and the ADCY9 Ile772Met – ADRB2 type 3 [TTCC] showed greater improvement in Arg16Gly combination remained to be signiﬁcant, predicted MMEF% than those without (11Æ6±16Æ5 but the bronchodilatory effect of Ile772Met T ⁄ C did vs. 1Æ2±14Æ6, P =0Æ017), again after 8 weeks of not survive. In addition, power analysis showed treatment. that all comparisons showed above 77% power.

Table 2. Genetic associations between the ADCY9 haplotype and percent changes in FEV1 and MMEF

After 8 weeks After 12 weeks

Haplotype n DFEV1% P DMMEF% P DFEV1% P DMMEF% P ht1[CCCC] +72 2Æ9±9Æ80Æ824 3Æ6±16Æ40Æ623 2Æ2±10Æ50Æ57 1Æ5±16Æ30Æ927 )14 2Æ6±8Æ52Æ5±10Æ22Æ3±4Æ73Æ0±9Æ2 ht2[CCTC] +21 1Æ6±9Æ10Æ52 1Æ0±13Æ10Æ32 )0Æ6±6Æ10Æ128 )1Æ4±13Æ60Æ212 )65 3Æ3±9Æ84Æ2±16Æ23Æ1±10Æ52Æ7±15Æ9 ht3[TTCC] +18 5Æ7±10Æ60Æ279 11Æ6±16Æ5 0Æ017 5Æ7±12Æ60Æ097 5Æ5±15Æ90Æ302 )68 2Æ1±9Æ21Æ2±14Æ61Æ3±8Æ80Æ7±15Æ2 ht4[TTCT] +18 5Æ0±5Æ10Æ267 5Æ0±9Æ80Æ734 2Æ3±6Æ80Æ751 2Æ9±12Æ30Æ817 )68 2Æ3±10Æ43Æ0±16Æ72Æ2±10Æ51Æ4±16Æ2

Bold character indicates signiﬁcance.

Fig. 2. Changes in predicted FEV1% (a) and MMEF% (b) pre- sented for the ADCY9 Ile772Met T ⁄ C and ADRB2 Arg16Gly A ⁄ G polymorphisms (mean ± standard error).

The potential functional effect of the ADCY9 addition, the pharmacogenetic effects of the Ile772Met polymorphism on improved therapeutic ADCY9 Ile772Met and ADRB2 Arg16Gly gene response to combination therapy observed in the polymorphisms on response to 3 months of ICS present study is supported by previous functional therapy have been previously investigated, and no studies (14, 15). The amino acid substitution of Ile significant genotypic effects were noted for FEV1 or to Met in ADCY9 results in loss of function as MMEF predicted values in the Korean population compared with wild-type ADCY9 (15), and ADCY9 (17). Therefore, the pharmacogenetic effects of the Met 772 has lower basal and b2-adrenergic recep- ADCY9 Ile772Met and ADRB2 Arg16Gly gene tor-mediated adenylyl cyclase activities than the polymorphisms on mono ICS therapy can be wild type (15). However, in the presence of corti- excluded. However, the present study suggests costeroid, albuterol-stimulated adenylyl cyclase that the interaction between the ADCY9 and activities in ADCY9 Met 772 transfected cells were ADRB2 gene polymorphisms contributes to bron- increased versus the wild type, and this was found chodilator response to b2-agonist in combination to contribute to increased bronchodilating response therapy. in childhood asthmatics (14). However, the present study possesses several Genetic polymorphisms in the 3¢UTR of ADCY9 limitations. First, the number of study subjects was may influence gene function by altering mRNA limited, and thus, study subjects were allocated to stability. Accordingly, we screened this 3¢UTR for two groups using dominant genetic modelling to functional polymorphisms. We found significant examine the effects of genotypes on clinical out- associations between an improvement in predicted comes. Study cohort and clinical trial design are MMEF% and the ADCY9 150397 C ⁄ T polymor- important aspects of studies on pharmacogenetic phism and haplotype 3 [TTCC], which was con- effects. Therefore, we are currently performing a structed using four SNPs located on the 3¢UTR of prospective study with a larger cohort size and a ADCY9. However, no associations were between genotype-stratified design to confirm the effect the tetranucleotide repeat (TTTA)5 ⁄ 4 located in the of the ADCY9 polymorphism on pharmacologic 3¢-untranslated region of the ADCY9 gene and response to inhaled combination therapy. Second, outcome. This tetranucleotide repeat was first SNP genotyping of the ADCY9 gene was not pre- identified in a Japanese population (16), but this is formed comprehensively during the present study, the first report in asthma even though the clinical and an investigation of other functional SNPs in the relevance was not found. promoter region of ADCY9 that influence gene In the present study, we also examined the inter- function by regulating promoter activity is needed. action between the ADCY9 Ile772Met and ADRB2 Third, in this study, we used FEV1% and MMEF% Arg16Gly A ⁄ G polymorphisms, because the ADRB2 predicted values as primary outcomes, but Arg16Gly A ⁄ G polymorphism has been reported to secondary outcomes, such as, variability of peak affect receptor function and contribute to bron- expiratory flow rates and quality of life scores were chodilator response to b2-agonist. Furthermore, not investigated, and these should be incorporated its protein, b2AR is closely related to adenylyl in future studies. cyclase activity. In the present study, we found that the gene to gene interaction between the WHAT IS NEW AND CONCLUSION ADCY9 Ile772Met and ADRB2 Arg16Gly gene polymorphisms had an additive effect on bron- We found that ADCY9 gene polymorphisms chodilator response to b2-agonist in combination probably affects therapeutic response to LABA therapy. plus ICS combination therapy, and that bronchod- We previously reported that the ADRB2 ilator response to b2-agonist in combination ther- Arg16Gly polymorphism has an effect on combi- apy may be improved by a gene to gene interaction nation therapy outcomes in Korean asthmatics, between the ADCY9 Ile772Met and ADRB2 and we demonstrated that Korean asthmatics Arg16Gly gene polymorphisms. Furthermore, this carrying the ADRB2 Arg ⁄ Arg (A ⁄ A) genotype latter relation may predict individual response to showed better response to combination therapy combination therapy. The combined genotypes of than those carrying other genotypes (13). In the ADCY9 Ile772Met and ADRB2 Arg16Gly gene

2010 The Authors. JCPT 2010 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 36, 399–405 404 S. H. Kim et al. polymorphisms may provide information regard- Additional authors contributed as follows: Seung- ing individual response to combination therapy. Hyun Kim drafted the manuscript with supervi- sion of data manipulation and statistical analyses; Young-Min Ye designed the study and recruited FINANCIAL AND COMPETING INTERESTS the study subjects; Hyun-Young Lee drafted the DISCLOSURE manuscript and statistical analyses; Hye-Jung Sin This study was supported by a grant from the involved in sample preparation and experimental Korea Health 21 R&D Project of the Ministry of analyses. All authors contributed to the final Health & Welfare, Republic of Korea (A070001). paper. The authors have no any relevant commercial associations that might pose a conflict of interest. REFERENCES 1. O’Byrne PM, Bisgaard H, Godard PP et al. (2005) EXECUTIVE SUMMARY Budesonide ⁄ formoterol combination therapy as both Inhaled combination therapy composing of long- maintenance and reliever medication in asthma. acting b2-agonist and corticosteroid has been American Journal of Respiratory and Critical Care Med- widely applied in the management of asthma, but icine, 171, 129–136. bronchodilating responses to combination therapy 2. Rabe KF, Pizzichini E, Stallberg B et al. (2006) showed inter-individual variations. We report the Budesonide ⁄ formoterol in a single inhaler for maintenance and relief in mild-to-moderate asth- pharmacogenetic effects of ADCY9 and ADRB2 ma: a randomized, double-blind trial. Chest, 129, gene polymorphism with using combination ther- 246–256. apy based on Korean asthma cohort. 3. Mak JC, Nishikawa M, Barnes PJ (1995) Glucocorti- costeroids increase beta 2-adrenergic receptor tran- KEY FINDINGS scription in human lung. American Journal of Physiology, 268, L41–L46. • Significant associations of ADCY9 gene poly- 4. Adcock IM, Stevens DA, Barnes PJ (1996) Interac- morphisms (Ile772Met and 150397 C ⁄ T) with tions of glucocorticoids and beta 2-agonists. European

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2010 The Authors. JCPT 2010 Blackwell Publishing Ltd, Journal of Clinical Pharmacy and Therapeutics, 36, 399–405