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Technical Briefs |
1-Antitrypsin
1
Department of Laboratory Medicine, Molecular Biology Division, and
2
Department of Internal Medicine IV, Division of Pulmonology, University of Vienna, Medical School, A-1090 Vienna, Austria;
a address correspondence to this author at: Klinisches Institut fuer medizinische und chemische Labordiagnostik, Abteilung fuer Molekularbiologie, Waehringer Guertel 18-20, A-1090 Vienna, Austria
1-Antitrypsin (A1AT), a highly
polymorphic 52-kDa glycoprotein with at least 100 alleles, functions as
the major inhibitor of neutrophil elastase (1).
Two allelic variants, PiZ and PiS,
frequently lead to A1AT deficiency, which can manifest clinically as
emphysema and, less frequently, as liver disease in neonates
(2). Recently, A1AT deficiency has been
recognized to play a role in the development of bronchial asthma
(3). The inflammatory process in bronchial asthma
involves various mechanisms by which the inflammatory response is
perpetuated. These include, for example, macrophage-mediated or mast
cell-mediated recruitment and activation of inflammatory cells as well
as amplification of neutrophil recruitment and activation. The
activated neutrophils release neutrophil elastase, which then can
stimulate production of cytokines (4).
For the deficiency variants of A1AT, PiZ and PiS, reduced elastase inactivation capacity has been demonstrated. A diminished inhibitory activity has also been described for PiM2 and PiM1(ala) (5). The resulting high elastase activity will stimulate cytokine production and can further amplify recruitment of activated neutrophils in the inflamed airway (3)(6)(7). Recently, an association of the variants PiM2 and PiM1(ala) with bronchial asthma has been reported (5)(8)(9). Thus, the measurement of the serum A1AT and the identification of the A1AT phenotype are clinically important.
Traditionally, the A1AT phenotype has been analyzed by isoelectric focusing. This analysis is tedious and prone to difficulties in interpretation (10). PiZ and PiS genotypes may be determined by DNA-based methods such as restriction fragment length polymorphism, allele-specific oligonucleotide hybridization, allele-specific amplification, direct sequencing, dual-color detection by ligase-mediated analysis, temperature or denaturing gradient gel electrophoresis, and PCR-mediated site-directed mutagenesis (11)(12)(13)(14)(15)(16)(17)(18)(19). However, simultaneous DNA-based determination of PiM2, PiM1(ala), PiZ, and PiS has not been reported.
We developed a multiplex PCR assay for the detection of the A1AT variants Z, S, M2, M1(ala), and M1(val) and examined a well-characterized group of 94 Austrian patients with asthma, defined by the standards of the American Thoracic Society, in comparison with 55 control individuals. All control individuals were free of asthmatic symptoms, had concentrations of IgE within the health-related reference intervals, as determined by CAP System IgE fluoroenzyme immunoassay (FEIA) (Pharmacia) and a negative phadiatope® test (CAP System Phadiatope® FEIA; Pharmacia). Informed consent to test for A1AT variants was obtained from all individuals.
PCR amplification of A1AT gene-specific sequences was carried out in a multiplex PCR reaction containing nine oligonucleotide primers. The primers spanned exons II, III, and V and included all regions of interest. For the detection of the S variant in exon III and the Z variant in exon V, the principle of mutagenically separated PCR was adapted (20). Wild-type and mutation-specific forward primers of different lengths (SF and P4WT for the S allele, P1Z and P2WT for the Z allele) and two common reverse primers (ATP2 for the S allele, SDMZR for the Z allele) were used. To prevent interaction of the two allele-specific primers with each other and to adjust the melting temperature, mutations were introduced into the mutant primers at nucleotide positions 9965, 9966, and 9986 in P2WT and 7653, 7654, and 7675 in P4WT. For the amplification of exon II (M2 variant) and exon III (M1 variant), the previously published primers M21, M22 (16), ATP1, and ATP2 (8) were used. The multiplex PCR was performed in a total volume of 50 µL, containing 0.25 mmol/L each of dATP, dGTP, dCTP, and dTTP polymerase (Perkin-Elmer Cetus); 2 mmol/L MgCl2; 10 mmol/L Tris-HCl buffer, pH 8.3; 50 mmol/L KCl; 1.25U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus); and the following amounts of primers (mutated sequences in the primers are underlined):
5 pmol of ATP1 (5'-CCCACCTTCCCCTCTCTCCAGGCAAATGGG-3') and ATP2 (5'-GGGCCTCAGTCCCAACATGGCTAAGAGGTG-3');
20 pmol of M21 (5'-GCAGGACAATGCCGTCTTCTGTCTC-3'), M22 (5'-CCACTAGCTTCAGGCCCTCGCTGAG-3'), and P2WT (5'-CAGGCCGTGATTAAGGCTGTGCTGACCATCTACG-3');
25 pmol of P1Z (5'-CATAAGGCTGTGCTGACCATCGTCA-3') and SDMZR (5'-TTGGGTGGGATTCACCACTTTTC-3');
30 pmol of P4WT (5'-TTCCTGCCTGTAGAGGGGAAACTACAGCACCTAGA-3');
60 pmol of SF (5'-GGGAAACTACAGCACCTGGT-3'); and 20–100 ng of genomic DNA.
All primers were synthesized by MWG Biotech, Ebersberg, Germany, and were HPSF® purified.
The reaction was carried out in a DNA Thermal Cycler 480 (Perkin-Elmer Cetus). Following denaturation at 94 °C for 5 min, 30 cycles at 94 °C, 59 °C, and 72 °C for 1 min each were performed. A final extension step at 72 °C for 7 min concluded the reaction. All PCR products were separated on 6% polyacrylamide gels (Novex) and detected by Sybr Green staining (Molecular Probes). Because both the M1(ala) mutation at codon 213 and the M2 sequence alteration at codon 101 affect restriction enzyme cleavage sites, the identification of M1(ala), M1(val), and M2 could be achieved by digestion of PCR products with restriction enzymes BstEII (MBI Fermentas) and RsaI (Promega). PCR product (3 µL) was incubated in a total volume of 30 µL with 1x buffer B (Boehringer Mannheim), 0.3 µg of bovine serum albumin, 3.6 U of BstEII, and 4.5 U of RsaI at 37 °C for 2 h. The digests were analyzed on 6% polyacrylamide gels, followed by Sybr Green staining.
Electrophoretic analysis of the multiplex PCR products yielded bands of
462 bp (exon II, spanning codon 101), 360 bp (exon III, spanning codon
213), 189 bp (exon V, containing codon 342), and 157 bp (exon III,
containing codon 264; Fig. 1
, top; lanes 1–5). With the PiS variant, a band of
142 bp was generated (Fig. 1
, top; lanes 6 and 9). The PiZ
mutation led to the formation of a 180-bp band (Fig. 1
, top; lane 7 for
the heterozygous PiZ genotype and lane 10 for the homozygous
PiZ genotype). In the presence of arginine at codon 101,
restriction digestion with RsaI led to cleavage of the
462-bp product and the generation of fragments of 383 bp and 79 bp. The
M2 variant, characterized by histidine at codon 101,
remained uncleaved. Heterozygous M1/M2
individuals show three fragments at 462, 383, and 79 bp (Fig. 1
, bottom; lanes 5 and 9).
|
Exon III contains two BstEII recognition sites at codon 213
(in the case of valine) and codon 288. Digestion of the multiplex PCR
product with BstEII led to cleavage of the 360-bp fragment
and the generation of fragments of 228, 83, and 49 bp (Fig. 1
, bottom;
lanes 1, 4, 6, and 9). In the presence of alanine
[M1(ala)] the BstEII recognition site at codon
213 was lost, and only two bands of 311 and 49 bp were observed (Fig. 1
, bottom; lanes 3, 7, and 10). Heterozygous subjects
M1(ala)/M1(val) showed four fragments at 311,
228, 83, and 49 bp (Fig. 1
, bottom; lanes 2, 5, and 8).
The multiplex PCR assay can also be performed with crude cellular DNA obtained by freezing and thawing of 100 µL of peripheral blood, followed by boiling for 10 min and centrifugation at 12 000g for 10 min (21) as well as with DNA extracted from mouthwash samples.
The results for the prevalence of A1AT alleles in 94 patients with
bronchial asthma and 55 control individuals are shown in Table 1
. In agreement with the literature (3), we
observed an increased prevalence of PiMZ (2% of patients;
0% of controls) and PiMS (4% of patients; 2% of controls)
in asthmatic patients. The homozygous PiM2 genotype occurred
twofold more frequently in the patient group (7%) than in the control
group (4%), which corresponds well with data reported by Gaillard et
al. (5). In contrast to observations by these
authors (8) in South African patients, we did not
observe a higher prevalence of PiM1(ala) in our cohort of
asthmatic patients. Whether this can be attributed to differences in
the ethnic background of the study populations, the limited number of
patients, or differences in the definition of asthma remains to be
determined.
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It is generally agreed that PCR methods are useful for the determination of the A1AT deficiency variants PiS and PiZ. The multiplex PCR assay that we developed offers a relatively simple approach for a simultaneous determination of the M2, M1(ala), and M1(val) genotypes. This will allow the evaluation of their role in the development of bronchial asthma in a large number of well-defined patients.
Footnotes
fax 43-1-40400-2097, e-mail christine.mannhalter{at}univie.ac.at
References
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[ISI][Medline]
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1-Antitrypsin deficiency and asthma [Review]. Postgrad Med 1997;101:153-168.
1-protease inhibitor and 2-macroglobulin for asthma patients and control subjects with various 1-protease inhibitor phenotypes. Clin Chem 1993;39:675-679.
1-antitrypsin haplotype. Biochemistry 1987;26:5259-5267.
[Medline]
[Order article via Infotrieve]
1-antitrypsin Z and S mutations [Technical Brief]. Clin Chem 1997;:403-404.
[ISI][Medline]
[Order article via Infotrieve]
1-proteinase inhibitor deficiency alleles Pi*Z and Pi*S by DNA Analysis. Eur J Clin Chem Clin Biochem 1996;34:761-764.
The following articles in journals at HighWire Press have cited this article:
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  S. Andolfatto, F. Namour, A-L. Garnier, F. Chabot, J-L. Gueant, and I. Aimone-Gastin Genomic DNA extraction from small amounts of serum to be used for {alpha}1-antitrypsin genotype analysis Eur. Respir. J., February 1, 2003; 21(2): 215 - 219. [Abstract] [Full Text] [PDF]
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