Polymerase chain reaction detection of S and Z alpha-1-antitrypsin variants by duplex PCR assay

Lucotte, G.; Sesboüé, Richard

doi:10.1006/mcpr.1999.0264

Cited by 13 publications

(13 citation statements)

References 11 publications

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“…The presence of the most common mutated A1AT alleles, Z and S, was detected by the PSM method, as it was previously described (Lucotte and Sesboü é, 1999). In summary, fragments containing Z and S mutations were coamplified; PCR products were digested with TaqI (New England BioLabs), separated on a 4% agarose gel, and visualized by ethidium bromide staining.…”

Section: Methodsmentioning

confidence: 99%

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

Topić

Stanković²,

Rankov³

et al. 2012

Genetic Testing and Molecular Biomarkers

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Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n = 348), asthma (n = 71), and bronchiectasis (n = 35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1:5519, 1:38, and 1:5519). Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population.

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Section: Methodsmentioning

confidence: 99%

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

Topić

Stanković²,

Rankov³

et al. 2012

Genetic Testing and Molecular Biomarkers

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“…Usually, additional cycles of PCR provide enough product for RFLP analysis. Using a similar protocol to Tazelaar et al [59] , Lucotte and Sesbo ü é managed to genotype products co-amplified for both Pi * S and Pi * Z mutations in one duplex PCR reaction [61] . Modifications of the method initially described by Tazelaar et al are presently used worldwide [50,[62][63][64][65][66][67][68] .…”

Section: Restriction Fragments Length Polymorphismmentioning

confidence: 99%

Molecular diagnostics of α₁-antitrypsin deficiency

Kaczor

Sanak²,

Szczeklik³

2007

Expert Opinion on Medical Diagnostics

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Diagnosis of α1-antitrypsin (AAT) deficiency is based on serum protein level, phenotyping and genotyping. Each of these methods has serious limitations and not enough accuracy to be used alone. Combinations of two different techniques are recommended to increase the reliability of AAT deficiency testing. Changes of AAT serum levels can result from clinical conditions and the inflammatory response may increase protein level. Phenotyping discriminates AAT variants due to the altered isoelectric point of the protein. It can detect numerous mutations, but often fails in the presence of rare, unknown or null alleles. Genotyping assays can detect or exclude specific mutations. The results are reliable and definitive, but restricted to the most common variants. DNA techniques for AAT gene screening and detection of new variants are available, but these are of limited value as sequencing is required to confirm the results. This review describes common approaches used in AAT deficiency testing and discusses their strengths and weaknesses. A novel functional test for the rapid discrimination of patients with AAT deficiency would greatly enhance diagnostic algorithms.

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“…Genotypes were determined by PCR-RFLP assay as described previously 15,17 . The PCR primers for the Ala C. The digestion products were separated on 2.5% agarose gel for the M1 and M2 variants and on 8% acrylamide gel for the S and Z variants.…”

Section: Genotypingmentioning

confidence: 99%