title: Determination of human beta(2)-adrenoceptor haplotypes by denaturation selective amplification and subtractive genotyping.
abstract: OBJECTIVE
  beta(2)-Adrenoceptor haplotype may be better associated with asthma severity and drug response than a polymorphic variant at any single site. Because present methods of haplotype determination are time consuming and impractical for large population studies, we sought to develop a simple and efficient method of determining haplotype of 3 common polymorphisms at codons -19 (Arg/Cys), 16 (Arg/Gly) and 27 (Glu/Gln).
  
  
  METHODS
  Preliminary studies showed that the C/G base pair of the Arg(-19) allele increases the local melting temperature over the T/A base pair of the Cys(-19) allele by 3.6 degrees C and establishes a new local maximum denaturation temperature. By choosing a suitable denaturation temperature and appropriate primers and coupling them with restriction fragment length polymorphism (RFLP) analysis, we hypothesized that the genotype of one separately amplified allele followed by subtraction from the combined genotype of two alleles would yield the beta(2) haplotype in > 99% of the population.
  
  
  RESULTS
  Haplotype determined by our method was in complete agreement with haplotype determined by cloning and sequencing in 29 samples. The frequencies of haplotype pairs in 78 healthy adults, according to our method, were in agreement with published values that were inferred, and were: RGE/CRQ, 26.9%; CRQ/CRQ, 25.6%; RGE/CGQ, 16.7%; CRQ/CGQ, 10.3%; RGE/RGE, 11.5%; CGQ/CGQ, 7.7%. The haplotype pair in one individual was RRE/CRQ (1.3%).
  
  
  CONCLUSIONS
  Our method of determining beta(2)-adrenoceptor haplotype is simple, accurate and cost effective for haplotyping large populations.
authors.affiliation: Center for Clinical Pediatric Pharmacology, Nemours Children's Clinic, Jacksonville, Florida, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: E B
authors.initials: EB
authors.lastname: Mougey
authors.forename: C J
authors.initials: CJ
authors.lastname: David
authors.forename: L M
authors.initials: LM
authors.lastname: Humma
authors.forename: J A
authors.initials: JA
authors.lastname: Johnson
authors.forename: J J
authors.initials: JJ
authors.lastname: Lima
authors.forename: J E
authors.initials: JE
authors.lastname: Sylvester
mesh_terms: D017931:DNA Primers; D005838:Genotype; D006239:Haplotypes; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation; D012150:Polymorphism, Restriction Fragment Length; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics*
chemical_list: D017931:DNA Primers; D018343:Receptors, Adrenergic, beta-2