Association Study Between Beta2-adrenergic Receptor Gene Haplotype/polymorphisms And Asthma And Clinical Phenotype In Chinese Han Nationality Population

One of the most widely studied candidate genes in asthma genetics istheβ₂-adrenergic receptor gene (ADRB2), which encodes a G protein-coupled receptor. ADRB2 is located on chromosome 5q31-33, a genomicregion that has demonstrated linkage to asthma-related phenotypes inmultiple studies.To date, at least 19 SNPs of the ADRB2 identified and polymorphismsat position 46 and 79 in the coding region have been extensively studiedand reported to cause variation in the encoded amino acids leading tosubstitution of Arg16/Gly16 and Gln27/Glu27. An important SNP at position-47 in promoter of the ADRB2, resulting in either Arg or Cys at amino acidposition-19, was found within the 5'-leader cistron(5'-LC) involved intranslation regulation of theβ₂AR.β₂AR polymorphisms have been associated with a variety of asthma-related phenotypes, but association results have been inconsistent acrossstudies]. This inconsistency may be partly explained by the fact that moststudies were designed to study relationships of single SNP in isolation(mainly SNPs at positions 46 and 79), not taking into account otherβ₂ARSNPs and the relevance of combinations of multiple SNPs or specific haplotypes(adjacent alleles on a single chromosome). The study of Drysdaleet al indicated that unique interactions of multiple SNPs within a haplotypeultimately affect biological and therapeutic phenotype and individual singlenucleotide polymorphisms may have poor predictive power aspharmacogenetic loci. The other problem, not enough appreciated to date,this is a selection method of SNPs genotyping. Most population studies haveused modified polymerase chain reaction (PCR)-based methods that mightresult in some percentage of false genotyping when compared with referencemethod such as DNA sequencing. In addition, other possible contributors tothese inconsistent results include genetic heterogeneity between studypopulations, different ethnic and small sample sizes.Previous analyses of asthma genetics among adults have not, to ourknowledge, linked ADRB2 haplotype to asthma phenotype in Chinese,therefore it is necessary to examine the effects of ADRB2 haplotypes/polymorphisms on asthma-related phenotypes and clinical response toβ₂-agonist in Chinese Han nationality population.Part 1Haplotype/Polymorphisms ofβ₂-Adrenergic Receptor Genein Chinese Asthmatics and Non-Asthmatic SubjectsObjective: The aims of this study were to determine frequencies ofpolymorphisms and haplotypes of ADRB2 and to analyse linkage disequilibrium between ADRB2 SNPs in Chinese asthmatic andnon-asthmatic subjects, and to compare these results with previous studiesdone in other ethnic groups. Methods: Using two methods of allele specificmultiplex PCR and direct DNA sequencing, five SNPs (in positions: -47,-20,46, 79, 252) genotype in the promoter and coding region of ADRB2 weredetermined in a group of 477 unrelated Chinese Han Nationality subjects(201 asthmatics and 276 controls ) and haplotypes were combined.Results: 1. Allele and genotype frequences for five SNPs(in positions: -47,-20, 46, 79, 252) were similar between Chinese asthmatics andcontrols(P＞0.05), there were also no significant diâ–¡erence in the distributionof haplotypes and haplotypes pairs between the two groups(P＞0.05). 2.Srong linkage disequilibrium phenomenon between analysed SNPs ispresent. There were significant linkage disequilibrium between-47 loci(Cys19) in the promoter and 79 loci (Gln27) in coding regions (P＜0.0001),and to lesser extent 46 loci (Arg16) in coding regions(P＜0.0001). When thehaplotypes constroncted from 46 and 79 loci in coding block regions wereanalysed, there were also significant linkage disequilibrium betweenhaplotypes of the polymorphisms in position 46 and 79 and the rest threeloci(P＜0.0001). 3. The frequencies of ADRB2 five SNPs and haplotypes inChinese were similar to those in Japanese and Asians and African Amercians,but were different from those in Caucasians and White Amercians andHispanic-Latinos. Conclusions: Chinese have a unique distribution of ADRB2 SNPs and haplotypes and there is strong linkage disequilibriumbetween the five SNPs. ADRB2 polymorphisms and haplotypes are notgenetic determinants of susceptibility to Chinese asthmatics, however theymay be involved in the pharmacogenetics of asthma and associated withasthma clinical phonetypes. These divergences of different ethnic populationmight imply the need for independent pharmacogenetic studies for ADRB2in each ethnic group.Part 2Beta2-adrenergic receptor gene haplotype/polymorphismsassociate with asthma phenotypeObjective: The aim of this study was to analyse the association betweenADRB2 polymorphisms/haplotypes and asthma phenotypes (lung functionmeasurements, bronchodilator response, total serum IgE and nocturnalasthma). Methods: Using two methods of allele specific multiplex PCR anddirect DNA sequencing, five SNPs (in positions: -47,-20, 46, 79, 252)in thepromoter and coding block regions of ADRB2 were determined in 201asthmatics and 276 controls, and haplotypes were combined. ELISA wasused to measure the total serum IgE of 201 asthmatics. Results: 1. Therewas no significant association between lung function measurements (FEV₁,PEF and FVC) and ADRB2 polymorphic loci (P＞0.05). 2. Significantly higher bronchodilator response(ΔFEV₁) was observed in patients withhomozygotic genotype 46A/A(13.40±3.48ï¼…), compared to those withhomo-(7.25±3.11ï¼…) and hetero-zygotes(7.39±3.14ï¼…) respectively, there wassignificant difference between three groups(P＜0.0001). 3. There was alsosignificant diâ–¡erence in bronchodilator response whenβ₂-AR haplotypeswere analysed (P＜0.0001), Patients with haplotypeâ… (TTACG) had thehighest meanΔFEV₁(13.40±3.48ï¼…) by comparison of those withhaplotypeâ…¡(7.56±3.10ï¼…) and haplotypeâ…¢(6.43±0.55ï¼…); Comparisonof the meanΔFEV₁ values for the six haplotype pairs showed significantdiâ–¡erence (P＜0.0001). 4. Significantly higher total IgE levels were foundin patients with homozygotic carriers of 79C genotypes(P=0.022) andhomozygotic haplotypeâ… (P＜0.0001). 5. The Gly16 allele was highlyassociated nocturnal asthma(P＜0.05), and could increased prevalence ofnocturnal asthma. Conclusions: Although ADRB2 polymorphisms/haplotypes are not genetic determinants of susceptibility to bronchial asthma,our study suggests that depending on examined asthmatic phenotypes eitheran individual ADRB2 polymorphism or haplotype may aâ–¡ect diseasemanifestation. Future studies are required to show whether it will requiretyping of all known ADRB2 polymorphisms to predict associatedphenotypes. Part 3Salmeterol response is not affected by ADRB2 Arg16Glygenotype in subjects with persistent asthmaObjective: The aims of the study were to evaluate the effects of variationArg16Gly in ADRB2 on clinical response to the salmeterol administeredwith fluticasone propionate in subjects with persisant asthma. Methods: 62persistent asthma were administered to twice-daily therapy with fluticasonepropionate/salmeterol (100mg/50mg) for 12weeks, followed by a 2-to 4-dayrun-out period. Using two methods of allele specific multiplex PCR anddirect DNA sequencing, five SNPs in the promoter and coding block regionsof ADRB2 were determined in 62 persisant asthma, and haplotypes werecombined. Results: 1. There was sustained and significant improvement(P＜0.001) over baseline in all measures of asthma control ( PEF, FEV₁,albuterol use and asthma symptom score) in subjects receiving salmeterol,regardless of Arg16Gly genotype. PEF in subjects with the Arg/Arggenotype showed 114.4±21.5 L/min improvement over baseline comparedwith 100.3±14.7 L/min for Gly/Gly subjects and 103.3±23.7 L/min forArg/Gly subjects, however changes were similar for Arg/Arg compared withGly/Gly or Arg/Gly genotypes. 2. Responses did no appear to be modifiedby haplotype pairs. 3. During the run-out period, all subjects had predictableand similar decreases in measures of asthma control, with no differencesbetween genotypes.The measures of the other three asthma control had also similar changes. Conclusions: Response to salmeterol does not varybetween ADRB2 genotypes after chronic dosing with an inhaledcorticosteroid. However, larger prospective clinical pharmacogenetic studieswith higher power to evaluate haplotypes across different ethnic/racialgroups, as well as genetic epidemiologic studies, are clearly needed to helpelucidate this field of great interest.