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Real time PCR detection of the PI*Z and PI*S mutations associated with alpha-1 antitrypsin deficiency
Insights
A new real-time PCR assay rapidly genotypes Alpha-1 antitrypsin (A1AT) deficiency variants PI*Z and PI*S. This method accurately identifies common A1AT genetic mutations, aiding clinical diagnosis of COPD and liver disease.
Area of Science:
- Genetics
- Molecular Biology
- Clinical Diagnostics
Background:
- Alpha-1 antitrypsin (A1AT) deficiency is linked to COPD and liver disease.
- SERPINA1 gene mutations, particularly PI*Z and PI*S, cause A1AT deficiency.
- Accurate genotyping is crucial for diagnosing and managing A1AT-related conditions.
Purpose of the Study:
- To develop and evaluate a rapid real-time PCR assay for genotyping common A1AT deficiency-causing SNPs.
- To assess the assay's performance and accuracy against established methods.
Main Methods:
- DNA extraction from whole blood samples.
- Real-time PCR using specific primer/probe sets for PI*Z and PI*S SNPs.
- Comparison of genotyping results with isoelectric focusing phenotypes and previous genotyping data.
Main Results:
- The real-time PCR assay successfully discriminated between five represented genotypes.
- High concordance was observed between the new assay's results and existing phenotype and genotype data.
- One genotype showed a discrepancy due to an unidentified allele in the phenotype.
Conclusions:
- A rapid and accurate real-time PCR assay for A1AT genotyping has been developed.
- This assay is suitable for clinical application as an initial step in A1AT testing.
- The assay facilitates timely diagnosis and management of A1AT deficiency.
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.
