title: Rapid LightCycler assay for identification of the perforin codon 374 Trp --> stop mutation in patients and families with hemophagocytic lymphohistiocytosis (HLH).
abstract: BACKGROUND
  Recently, point mutations in the Perforin gene on chromosome 10q21 have been described to be the cause of hemophagocytic lymphohistiocytosis (HLH) in a subset of patients. Small deletions, missense, or nonsense mutations were found in both coding exons of the gene. One mutation was found apparently independently in different families of Turkish origin. This Trp374stop mutation is located within a cystein-rich epidermal growth factor (EGF) precursor type domain in exon 3 of the Perforin gene.
  
  
  METHODS
  To detect this mutation, we developed a rapid "fluorescence resonance energy transfer" (FRET) assay on the LightCycler based on the different melting behavior of mutated and wild-type sequences.
  
  
  RESULTS
  In seven out of 20 analyzed patients with defined HLH history, this Trp374stop mutation was detectable within the different genotypes. Additionally, one out of 90 alleles tested in 45 healthy Turkish controls had this mutation.
  
  
  CONCLUSIONS
  We present a rapid and reliable test for the most common Perforin (Trp374stop) mutation in HLH suitable for genetic testing and prenatal diagnosis. The mutation was detected in a large proportion of Turkish patients. Furthermore, a rapid and precise diagnosis of HLH will shorten the initiation of therapy in this subset of patients.
authors.affiliation: Department of Pediatric Hematology and Oncology, University Children's Hospital, Hamburg, Germany. zurstadt@uke-hamburg.de
authors.forename: Udo
authors.initials: U
authors.lastname: zur Stadt
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kabisch
authors.forename: Gritta
authors.initials: G
authors.lastname: Janka
authors.forename: E Marion
authors.initials: EM
authors.lastname: Schneider
mesh_terms: D001483:Base Sequence; D002648:Child; D002675:Child, Preschool; D002879:Chromosomes, Human, Pair 10 / Q000235:genetics*; D018388:Codon, Terminator / Q000235:genetics*; D017931:DNA Primers; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods / Q000592:standards; D020022:Genetic Predisposition to Disease / Q000235:genetics; D015616:Histiocytosis, Non-Langerhans-Cell / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010375:Pedigree; D054353:Perforin; D017354:Point Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D052899:Pore Forming Cytotoxic Proteins; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013696:Temperature; D014421:Turkey; D044465:White People / Q000235:genetics
chemical_list: D018388:Codon, Terminator; D017931:DNA Primers; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin