title: Quantitation of circulating wild-type alpha-1-antitrypsin in heterozygous carriers of the S and Z deficiency alleles.
abstract: BACKGROUND
  Alpha-1-antitrypsin (A1AT) deficiency disease results from mutations in the A1AT gene. Controversy exists in regards to treatment of heterozygous carriers of the S and Z deficiency alleles. Quantitation of allelic expression has not been possible with standard laboratory methods. Here we show that the recently described method for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of A1AT tryptic peptides can differentiate between mutated (S and Z) and wild-type (non-S and non-Z) proteins allowing for quantitation of circulating allelic expression in heterozygous patients.
  
  
  METHODS
  Serum (244 M/M, 61 M/Z, and 63 M/S) was combined with isotopically labeled peptide standards, digested with trypsin, and quantitated by LC-MS/MS. Total and allele-specific A1AT quantitation was performed by comparison of peptide peak height ratios to a standard curve for each peptide. Linear regression was used to compare results and central 95(th) percentile intervals were calculated using parametric analysis.
  
  
  RESULTS
  Quantitation of circulating wild-type A1AT based on the proteotypic and allelic (non-S and non-Z) peptides was validated in M/M patients. Proteotypic peptide concentrations correlated linearly with quantitation by non-Z and non-S peptides [slopes (Spearman correlation coefficient) of 1.09 (0.89) and 0.98 (0.80), respectively]. Allele-specific quantitation showed significant differences in wild-type protein expression in M/Z and M/S patients. Although average total A1AT concentration was lower for M/Z patients, the percentage of wild-type protein in M/Z patients was significantly higher at 82 % (55- > 95 %) compared to 63 % (43-83 %) for M/S heterozygotes. In a cohort of M/Z patients with sufficient total A1AT (≥80 mg/dL), half had insufficient wild-type protein that could have clinical implications for pulmonary dysfunction.
  
  
  CONCLUSIONS
  For the first time, a method to quantitate A1AT allele protein expression is described. Given the wide range of circulating wild-type protein observed in heterozygous patients, this method has the potential to reveal correlations between allele concentration and development and/or severity of clinical symptoms.
authors.affiliation: Department of Laboratory Medicine & Pathology Mayo Clinic, Mayo Clinic, 200 First St. SW, 55905, Rochester, MN, USA. donato.leslie@mayo.edu.
authors.forename: L J
authors.initials: LJ
authors.lastname: Donato
authors.affiliation: Present address: University of Minnesota, Minneapolis, MN, USA. robinkarras@gmail.com.
authors.forename: R M
authors.initials: RM
authors.lastname: Karras
authors.affiliation: Department of Laboratory Medicine & Pathology Mayo Clinic, Mayo Clinic, 200 First St. SW, 55905, Rochester, MN, USA. katzmann@mayo.edu.
authors.forename: J A
authors.initials: JA
authors.lastname: Katzmann
authors.affiliation: Department of Laboratory Medicine & Pathology Mayo Clinic, Mayo Clinic, 200 First St. SW, 55905, Rochester, MN, USA. Murray.David@mayo.edu.
authors.forename: D L
authors.initials: DL
authors.lastname: Murray
authors.affiliation: Department of Laboratory Medicine & Pathology Mayo Clinic, Mayo Clinic, 200 First St. SW, 55905, Rochester, MN, USA. snyder.melissa@mayo.edu.
authors.forename: M R
authors.initials: MR
authors.lastname: Snyder
mesh_terms: D000483:Alleles; D015415:Biomarkers / Q000097:blood; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006579:Heterozygote; D006801:Humans; D008297:Male; D053719:Tandem Mass Spectrometry / Q000379:methods; D000515:alpha 1-Antitrypsin / Q000097:blood* / Q000235:genetics; D019896:alpha 1-Antitrypsin Deficiency / Q000097:blood* / Q000235:genetics
chemical_list: D015415:Biomarkers; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin