title: Reliable screening for a pain-protective haplotype in the GTP cyclohydrolase 1 gene (GCH1) through the use of 3 or fewer single nucleotide polymorphisms.
abstract: BACKGROUND
  A haplotype in the GTP cyclohydrolase 1 (dopa-responsive dystonia) gene (GCH1) is associated with decreased persistent pain. The aim of the present study was to develop a screening method for the pain-protective haplotype.
  
  
  METHODS
  Complete genetic information for all 15 GCH1 DNA positions constituting the pain-protective GCH1 haplotype was available from 278 patients. In silico analyses, including discriminant analysis of the most frequent haplotypes, identified distinctive DNA positions that allow detection of the pain-protective haplotype at high sensitivity and specificity with the smallest possible number of DNA positions. Pyrosequencing(trade mark) assays were subsequently developed for these DNA positions, established with 662 DNA samples from healthy volunteers, and prospectively validated with a random selection of DNA samples genotyped for all 15 DNA positions.
  
  
  RESULTS
  Diagnosis of the pain-protective GCH1 haplotype was possible with 100% sensitivity and specificity by screening for just 3 GCH1 genetic variants that span the entire DNA range of the haplotype: c.-9610G>A (dbSNP rs8007267G>A) in the 5' untranslated region, c.343 + 8900A>T (dbSNP rs3783641A>T) in intron 1, and c.*4279 (dbSNP rs10483639C>G) in the 3' untranslated region. Test sensitivity and specificity were still >95% with 2 or even just 1 of these GCH1 DNA positions.
  
  
  CONCLUSIONS
  In silico analysis of complex GCH1 gene haplotypes reduced the requisite number of tested DNA positions from 15 to 3 while maintaining the reliability, specificity, and sensitivity of the genetic diagnosis. This screening method could reduce laboratory diagnostic efforts and facilitate investigations of the pain-protective GCH1 haplotype.
authors.affiliation: pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany. j.loetsch@em.uni
authors.forename: Jörn
authors.initials: J
authors.lastname: Lötsch
authors.forename: Inna
authors.initials: I
authors.lastname: Belfer
authors.forename: Anja
authors.initials: A
authors.lastname: Kirchhof
authors.forename: Bikash K
authors.initials: BK
authors.lastname: Mishra
authors.forename: Mitchell B
authors.initials: MB
authors.lastname: Max
authors.forename: Alexandra
authors.initials: A
authors.lastname: Doehring
authors.forename: Michael
authors.initials: M
authors.lastname: Costigan
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Woolf
authors.forename: Gerd
authors.initials: G
authors.lastname: Geisslinger
authors.forename: Irmgard
authors.initials: I
authors.lastname: Tegeder
mesh_terms: D006136:GTP Cyclohydrolase / Q000235:genetics*; D006239:Haplotypes; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D025202:Molecular Diagnostic Techniques; D010146:Pain / Q000175:diagnosis / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011446:Prospective Studies; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D006136:GTP Cyclohydrolase