title: Misclassification of an apparent alpha 1-antitrypsin "Z" deficiency variant by melting analysis.
abstract: BACKGROUND
  Alpha-1 antitrypsin (AAT) is a protease inhibitor that protects the lungs from degradation by neutrophil elastase. AAT deficiency is associated with both lung and liver diseases. AAT deficiency is diagnosed using a combination of genetic and biochemical tests. Here we demonstrate how polymorphisms in the AAT gene can lead to genotype/phenotype discrepancies in common AAT assays.
  
  
  METHODS
  Total AAT was measured using an immunoturbidimetric assay. AAT phenotype was determined using isoelectric focusing. Genotypic identification of Z and S AAT alleles was performed by melt curve analysis using a LightCycler. Genotype/phenotype discrepancy was amended using gene sequencing.
  
  
  RESULTS
  Genotype and phenotype analysis produced conflicting results as a consequence of a polymorphism located in the probe designed to detect the Z allele. Sequencing revealed that the polymorphism had previously been reported as a rare P allele. The probe that caused the discrepancy was designed to match the WT sequence. A new probe was designed to specifically detect the Z allele, eliminating the possibility of future discordance.
  
  
  CONCLUSIONS
  Laboratories utilizing melt-curve analysis to diagnose patients should be aware of the potential for false positive results caused by polymorphisms located in the binding region of the genotyping probes. Alternatively, the probes should be designed to be specific to the mutation, rather than to the WT sequence.
authors.affiliation: Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, UT, USA.
authors.forename: Dina N
authors.initials: DN
authors.lastname: Greene
authors.forename: Melinda
authors.initials: M
authors.lastname: Procter
authors.forename: David G
authors.initials: DG
authors.lastname: Grenache
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Bornhorst
authors.forename: Rong
authors.initials: R
authors.lastname: Mao
mesh_terms: D016172:DNA Fingerprinting / Q000379:methods*; D014644:Genetic Variation / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D009691:Nucleic Acid Denaturation; D010641:Phenotype; D011110:Polymorphism, Genetic / Q000235:genetics; D013696:Temperature; D000515:alpha 1-Antitrypsin / Q000235:genetics*
chemical_list: D000515:alpha 1-Antitrypsin