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).
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 E-mail: email@example.com