AJTR Copyright © 2009-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Am J Translational Res 1(4):406-411,2009

Original Article
Real Time PCR Detection of the PI*Z and PI*S polymorphisms
associated with alpha-1 antitrypsin deficiency

Claudine L. Bartels, Angela L. Marchetti, W. Edward Highsmith, and Gregory J. Tsongalis

Department of Pathology, Dartmouth Medical School and Dartmouth-Hitchcock Medical Center, Lebanon, NH,
USA; Diagnostic Genetic Sciences Program, University of Connecticut, Storrs, CT, USA; Division of Clinical
Biochemistry & Immunology, Department of Laboratory Medicine & Pathology, Mayo Clinic College of Medicine,
Rochester, MN, USA

Received July 27, 2009; accepted August 5, 2009; available online August 10, 2009

Abstract: Alpha-1 antitrypsin (A1AT or AAT) is a serine protease inhibitor (PI) which, when present at low levels,
can cause chronic obstructive pulmonary disease (COPD) and liver disease in both children and adults. Several
mutations within the SERPINA1 gene have been found to cause this deficiency. The most common variants are
PI*Z and PI*S, each caused by a single nucleotide polymorphism (SNP). We describe a real time polymerase
chain reaction (PCR) assay for the rapid genotyping of these polymorphisms. DNA was extracted from fourteen
EDTA-anticoagulated whole blood samples using the Qiagen EZ1 blood extraction kit. SNP genotyping was
performed using primer/probe sets purchased from Applied Biosystems. These were evaluated for performance
and assay conditions on the Applied Biosystems 7500 FAST System. The genotypes of these samples were
compared with their phenotype results from isoelectric focusing assays, which were performed by an
independent reference laboratory. In addition, twenty samples that were previously genotyped at another
laboratory were obtained for accuracy studies. Thirty-four samples were tested; five genotypes were represented
and the assay was able to discriminate these successfully. Only one genotype could not be correlated with its
phenotype result, as the phenotype was reported as an “unidentified allele”. All other genotyping results were
concordant with previously determined genotypes and phenotypes. We describe a rapid real time PCR assay that
is suitable for clinical use in genotyping AAT alleles and which can be used as the initial step in A1AT testing
algorithms. (AJTR907001).

Key words: Real-time PCR, alpha-1 antitrypsin, molecular diagnostics, serine protease inhibitor, chronic
obstructive pulmonary disease (COPD), single nucleotide polymorphism (SNP), genotyping

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Address all correspondence to:
Gregory J. Tsongalis, PhD
Department of Pathology
Dartmouth Hitchcock Medical Center
One Medical Center Drive, Lebanon
NH 03756, USA
Tel: 603-650-5498