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Reliable Screening for a Pain-Protective Haplotype in the GTP Cyclohydrolase 1 Gene (GCH1) Through the Use of 3 or Fewer Single Nucleotide Polymorphisms

¹ pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
² National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD.
³ Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA.

^aAddress correspondence to this author at: pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor Stern Kai 7, 60590 Frankfurt am Main, Germany. Fax 49-69-6301-7636; e-mail: j.loetsch{at}em.uni.

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^TM 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.

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