Original article
Isoelectric focusing phenotyping and denaturing gradient gel electrophoresis genotyping: a comparison of two methods in detection of alpha-1-antitrypsin variants

https://doi.org/10.1016/j.trsl.2008.02.002Get rights and content

Laboratory diagnosis of alpha-1-antitrypsin (AAT) deficiency is routinely performed by phenotyping methods, which include measurement of serum alpha-1-antitrypsin concentration and isoelectric focusing (IEF). Several DNA-based methods are also used for AAT deficiency testing, but they still have not become part of routine diagnostics. The aim of the study was to identify AAT variants using 2 different methods, isoelectric focusing and denaturing gradient gel electrophoresis (DGGE), and to compare obtained results as well as practical application of these 2 methods. The study has encompassed 27 emphysema patients. In all patients, AAT phenotypization was conducted using IEF, whereas genotypization was performed by DGGE. Variations detected by DGGE were characterized by DNA sequencing. Mutations in the AAT gene were detected in 6 patients. Three patients were homozygous for the Z allele, whereas 1 patient was heterozygous. In 2 patients, novel AAT variants, G320R and V321F, were detected. When results obtained by IEF and DGGE were compared, it was observed that IEF results were inconclusive or misinterpreted in 5 cases (18.5%). Both methods proved to be reliable for detection of the Z alleles, whereas discrepancy existed for M4 allele and rare variants. Therefore, the optimal strategy for diagnostics of AAT deficiency should encompass detection of the most common AAT variants by IEF and screening for the less common variants by DGGE in combination with sequencing.

Section snippets

Patients

The study has been approved by the institutions' ethical committees and was carried out according to the principles of the Declaration of Helsinki. This study has encompassed 27 patients (8 women and 19 men; age range, 20–75 years) found to have emphysema. The diagnosis was made based on clinical features, chest x-ray, and pulmonary function tests (spirometry, body plethysmography, and pulmonary diffusion capacity). Pulmonary function tests were performed in 26 patients. The mean ± SD value of

Results

Identification of AAT variants by IEF was successfully performed in 25 emphysema patients, whereas all 27 patients were successfully genotyped by DGGE. The obtained results of IEF corresponded to DGGE results in 22 cases (Table I).

Of the 21 samples in which only normal AAT variants were detected, 7 were phenotyped as M1, 10 as M1M2, 2 as M1M3, and 2 as M2M3. Three patients were ZZ homozygotes, whereas M1Z phenotype was present in 1 case (Fig 1). Two samples could not be phenotyped because they

Discussion

AAT deficiency is caused by mutations in the AAT gene, which lead to absence or dysfunction of the AAT protein. The most common causes of AAT deficiency in a population of European descent are S and Z variants. Beside those variants, at least 15 known mutations can impair the function of the AAT protein, which are estimated to account for 2–4% of all AAT-deficient variants.15, 16

Considerable variability exists in the clinical manifestations of patients with AAT deficiency, with some patients

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Supported by Grant 143051 from the Ministry of Science.

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