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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 814-817, (2002)
© 2002 American Thoracic Society

Original Articles

Rapid Screening for {alpha}1-Antitrypsin Deficiency in Patients with Chronic Obstructive Pulmonary Disease Using Dried Blood Specimens

Francisco Rodriguez, Rosendo Jardí, Xose Costa, Montserrat Cotrina, Roman Galimany, Rafael Vidal and Marc Miravitlles

Departments of Biochemistry and Pneumology, Hospital Universitario Vall d'Hebron, Barcelona, Spain

Correspondence and requests for reprints should be addressed to Rosendo Jardí, Servicio de Bioquimica, Hospital General Vall d'Hebron, Paseo Vall d'Hebron 119–129, Barcelona 08035, Spain. E-mail: rjardi{at}hg.vhebron.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We describe two reliable methods for high-throughput screening of proteinase inhibitor (PI) S and PI Z {alpha}1-antitrypsin ({alpha}1-AT) deficiency alleles from dried blood spot (DBS) specimens using the LightCycler fluorimetric analyzer. The method was used to study 72 patients with chronic obstructive pulmonary disease. Results were confirmed with DNA sequencing. The {alpha}1-AT concentration in DBS was determined with immune nephelometry. Sixteen patients (22%) showed no PI Z or PI S mutations. Five patients (7%) had a heterozygous genotype consisting of a PI S allele and a normal allele for the Z and S positions (non-S non-Z). Twenty-five patients (35%) had a heterozygous genotype consisting of a PI Z and a non-S non-Z allele. Two (3%) had the PI SS genotype, 2 (3%) the PI SZ, and 20 (28%) the PI ZZ. All patients with two normal {alpha}1-AT alleles and 10 heterozygous carriers of one normal and one deficient allele had {alpha}1-AT levels that fell within the {alpha}1-AT DBS normal range (1.8–3.1 mg/dl). Two patients with the rare PI MMmalton- and PI MMheerlen-deficient variants showed deficient {alpha}1-AT levels; PI S and PI Z were not detected. Processing 32 samples requires only 40 minutes. This single-step, cost-effective technology is optimal for working with small amounts of DNA, as are present in DBS. The method is suitable for large-scale screening, in cases where PI type is important.

Key Words: {alpha}1-antitrypsin deficiency • proteinase inhibitor S allele • proteinase inhibitor Z allele • dried blood spot • LightCycler analyzer


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}1-Antitrypsin ({alpha}1-AT) deficiency is a hereditary autosomal disorder, resulting from a variety of mutations in the {alpha}1-AT gene and associated with a high risk for the development of early-onset pulmonary emphysema (1). {alpha}1-AT is a highly polymorphic protein with more than 70 variants, known as proteinase inhibitor (PI) types. The PI M allele and its serum subtypes are the most common of the normal alleles. In the vast majority of cases, {alpha}1-AT deficiency is caused by homozygosity for the PI Z allele (Glu342Lys, nucleotide 11940 G->A) or by compound heterozygosity for PI Z and PI S (Glu342Lys, nucleotide 11940 G->A and Glu264Val, nucleotide 9628 A->T). Population studies indicate that {alpha}1-AT deficiency is underdiagnosed and that prolonged delays in diagnosis are common (2). Recent recommendations of the World Health Organization advocate screening programs among patients with chronic obstructive pulmonary disease and adolescents with asthma (3).

At the protein level, routine laboratory diagnosis of {alpha}1-AT deficiency is currently based on serum {alpha}1-AT measurement, and in cases with a low {alpha}1-AT concentration, on identification of the {alpha}1-AT phenotype by isoelectric focusing pattern on polyacrylamide gels. We recently described a protocol for {alpha}1-AT deficiency screening that combines measurement of {alpha}1-AT in dried blood spot (DBS) specimens on filter paper by immune nephelometry and identification of the {alpha}1-AT phenotype by isoelectric focusing and/or {alpha}1-AT genotyping with a polymerase chain reaction (PCR) and DNA sequencing assay (4). Although our genotyping method is effective for this purpose, it is quite labor intensive and time consuming.

To facilitate population screening, we modified this method using the new high-speed thermal LightCycler analyzer (Roche Diagnostics, Mannheim, Germany) approach to DNA amplification and simultaneous detection and documentation of genetic polymorphisms, and we tested its applicability to the identification of PI S and PI Z alleles in {alpha}1-AT deficiency screening.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DBS samples were obtained from 72 patients with chronic obstructive pulmonary disease, 50 with unknown {alpha}1-AT genotypes and 22 with known {alpha}1-AT deficiency and genotypes (20 PI ZZ, 1 PI MMmalton, and 1 PI MMheerlen), as established by PCR-DNA sequencing. Patients were recruited from the outpatient clinic of a tertiary hospital and had to fulfill the following inclusion criteria: chronic obstructive pulmonary disease diagnosed according to the Spanish Society of Pneumology criteria (5), basically proven fixed airflow obstruction with a FEV1 of less than 70% predicted and FEV1/FVC of less than 70%, and smokers or ex-smokers of at least 10 pack-years and no previous diagnosis of asthma. Control subjects were patients with known PI ZZ, PI MMmalton, and PI MMheerlen genotypes recruited from the Spanish Registry of {alpha}1-AT Deficiency (5). All patients gave their informed consent for participation in the study.

The clinical and demographic characteristics of the patients are described in Table 1 . In the DBS screening protocol, deficiency was evaluated by combining the results of {alpha}1-AT quantification by immune nephelometry and {alpha}1-AT genotyping by LightCycler PCR. In cases of discordance between the two determinations (DBS {alpha}1-AT levels below normal range and no detection of the PI S and/or PI Z allele), diagnosis of deficiency was performed by {alpha}1-AT genotyping using the PCR-DNA sequencing assay.


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  		TABLE 1. Patient characteristics and pulmonary function*

 
Genotyping of PI S and PI Z {alpha}1-AT Alleles by LightCycler PCR
Two DBS samples were prepared for each patient. DNA from the samples was eluted using a previously described protocol (6), with some modifications. One 3-mm paper DBS disk was cut and placed in a 1.5-ml plastic tube. We added 80 µL of water and incubated at 60°C overnight. Then we added 50 µL of water and incubated at 80°C for 30 minutes. Finally, tubes were centrifuged for 30 minutes to eliminate contaminants and to recover the eluted DNA.

{alpha}1-AT genotyping was performed in the LightCycler analyzer, a combination of thermal cycler and fluorometer that achieves a fast real-time PCR with mutation detection by analysis of the melting point of one of the two fluorescent hybridization probes. The assay is based on the concept that a fluorescence signal is generated when two adjacent fluorescently labeled probes hybridize to the same strand in close proximity. During cycling, the probes hybridize the specific PCR product. After completion of the PCR, the PCR mixture is denatured; the temperature is lowered to 40°C to facilitate binding of the hybridization probes, and it is then slowly increased to 90°C to permit melting of the detection probe, which is monitored by the decline of fluorescence. Melting curves are converted to melting peaks by software, allowing easy distinction of the wild-type from the mutant by the different melting temperature (7). Two PCR assays, one for each polymorphism, were performed. The oligonucleotide sequences for amplification in the PI S genotyping analysis were as follows: PI SF 5'-GGTGCCTATGATGAAGCGTTTAGGC-3' (nucleotide 9488–9512) and PI SR 5'-AGGTGTGGGCAGCTTCTTGGTCA-3' (nucleotide 9725–9703); the size of the amplified fragment was 238 bp. Hybridization was performed with two oligonucleotide probes: 5'-GCACCTGGAAAATGAAC-3' (nucleotide 9620–9636), labeled at the 5' end with a LightCycler red fluorophore LCR 640 (designed to hybridize over the mutation position) and 5'-TTCTTCCTGCCTGATGAGGGGAAACTA-3' (nucleotide 9591–9617), labeled with fluorescein at the 3' end. The oligonucleotide sequences for amplification in the PI Z genotyping analysis were PI ZF 5'-GGTGTCCACGTGAGCCTTGC-3' (nucleotide 11839–11858) and PI ZR 5'-AAAAACATGGCCCCAGCAGCT-3' (nucleotide 11974–11954); the size of the amplified fragment was 136 bp. Hybridization was performed with the 5'-GACCATCGACGAGAAAGGG-3' (nucleotide 11930–11948) probe, labeled at the 5' end with a LightCycler red fluorophore (designed to hybridize over the mutation position) and the 5'-CTCCAGGCCGTGCATAAGGCTGT-3' (nucleotide 11904–11926) probe labeled with fluorescein at the 3' end. Polymorphic positions are shown in italics. The nucleotide numbering corresponds to the human {alpha}1-AT sequence obtained from the Gene-Bank Accession No. K02212. The probes that hybridize over the mutation positions match perfectly, with no mutated sequences. The PCR conditions were identical for both applications: 3 mM of MgCl2, 4 pmol of each hybridization probe, 10 pmol of the two PCR primers, 2 µl of LightCycler Fast Start DNA Master Hybridization probe mix (Roche Diagnostics), and 5 µl of DNA sample, in a total volume of 20 µl. PCR cycling conditions were also identical for both mutations: an initial denaturation step of 94°C for 7 minutes, followed by 55 cycles of denaturation at 95°C for 2 seconds, annealing at 53°C for 12 seconds, and extension at 72°C for 15 seconds. After the amplification, melting curves were generated by denaturation of the reaction at 94°C for 15 seconds, holding the sample at 40°C for 20 seconds, and then slowly heating the sample to 85°C.

Quantitative Determination of {alpha}1-AT: Genotyping by PCR-DNA Sequencing Method
{alpha}1-AT levels and {alpha}1-AT genotyping in DBS samples were determined by an immune nephelometric method and a PCR-DNA sequencing method, respectively, as previously reported (4). The normal range of {alpha}1-AT levels in the DBS specimens was 1.8–3.1 mg/dl.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Figure 1 shows representative examples of the fluorescence profiles and melting curves generated by the LightCycler from control DNA samples with known {alpha}1-AT genotypes. Individuals homozygous for the PI S allele exhibited a single peak at 48.8°C. Those homozygous for the normal sequence at the site of the PI S allele had a single peak at 56°C, and the heterozygous subjects had two peaks (48.8°C and 56°C). Individuals homozygous for the PI Z allele had a single peak at 55.7°C. Those homozygous for the normal sequence at the site of the PI Z allele had a single peak at 62.3°C, and in the heterozygous subjects, two peaks were detected (55.7°C and 62.3°C). On analysis of various samples with different amplification efficacies, the derivative melting curves were highly reproducible. The within- and between-run melting peaks for the same allele differed by less than 0.3°C for both the PI Z and PI S mutations. Thus, melting curve analysis allowed easy and unambiguous assignment of genotyping for PI S and PI Z alleles.



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  		Figure 1. Results of {alpha}1-AT genotyping of control samples by the DBS-LightCycler PCR and by PCR-DNA sequencing. (A1) PI S genotyping by melting peaks in the LightCycler system: Case 1, homozygous PI SS; Case 2, heterozygous PI MS; Case 3, homozygous PI MM; and Case 4, negative (no template) control. The empirical melting points are 48.8°C for the PI S allele and 56.0°C for the normal sequence in the position corresponding to the PI S mutation (non-S). (A2) PI S genotyping by PCR-DNA sequencing method: The sequence where mutation GAA to GTA is shown corresponds to a PI MS heterozygous individual. (B1) PI Z genotyping by melting peaks in the LightCycler system: Case 1, homozygous PI ZZ; Case 2, heterozygous PI MZ; Case 3, homozygous PI MM; and Case 4, negative (no template) control. The empirical melting points are 55.7°C for the PI Z mutant allele and 62.3°C for the normal sequence in the position corresponding to the PI Z mutation (non-Z). (B2) PI Z genotyping by PCR-DNA sequencing method: the sequence where mutation GAG to AAG is shown corresponds to a PI MZ heterozygous individual. A, adenine; T, thymine; Glu, glutamic acid; Val, valine; G, guanine; Lys, lysine; N, *heterozygous A/T and **heterozygous G/A.

 
Among the total of patients, 16 (22%) showed no PI Z or PI S mutations. Five (7%) had a heterozygous genotype consisting of a PI S allele and a normal allele for the Z and S positions (non-S non-Z). Twenty-five patients (35%) had a heterozygous genotype consisting of a PI Z allele and a non-S non-Z allele. Two (3%) had the PI SS genotype, 2 (3%) the PI SZ, and 20 (28%) the PI ZZ. All patients without PI Z or PI S mutations had DBS {alpha}1-AT levels higher than or equal to 1.8 mg/dl (normal range, 1.8–3.1 mg/dl). Levels of DBS {alpha}1-AT were below 1.8 mg/dl in 44 (61%) patients: 1 heterozygous case with the PI S allele and a non-S non-Z allele (1.6 mg/dl), 19 heterozygous cases with the PI Z allele and a non-S non-Z allele (0.8–1.7 mg/dl), 2 PI SS (1.2–1.4 mg/dl), 2 PI SZ (0.7–0.9 mg/dl), and 20 PI ZZ (less than 0.5 mg/dl). The two patients (3%) with the rare PI MMmalton- and PI MMheerlen-deficient variants showed DBS {alpha}1-AT levels of 1.2 and 0.9 mg/dl; PI S or PI Z alleles were not detected. The relationship between {alpha}1-AT genotypes and {alpha}1-AT levels is shown in Figure 2 .



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  		Figure 2. Relationship between {alpha}1-AT levels and {alpha}1-AT genotypes in the DBS obtained from 72 patients with COPD. The cut-off value corresponds to the lower limit of the DBS-{alpha}1-AT normal range.

 
There was total concordance between PI S and PI Z genotyping results obtained from DBS samples with the LightCycler-PCR and with the PCR-DNA sequencing method in the 72 patients studied.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DBS specimens have been assessed as an alternative to serum for {alpha}1-AT screening. This approach, first used by Laurell and Sveger in 1975 (8), would be particularly useful for large-scale screening programs, in which samples from many areas are sent to a central laboratory, frequently located at some distance. Blood collection on filter paper requires a small amount of sample, is minimally invasive and produces a specimen that is inexpensive to ship, and requires no special handling or storage conditions. Nevertheless, for diagnostic purposes in patients, determination of {alpha}1-AT levels in serum and PI typing in patients with low concentrations is still appropriate.

Several assays have been developed for {alpha}1-AT PI S and PI Z genotyping, including PCR-mediated site-directed mutagenesis, single-strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization, PCR restriction fragment length polymorphism analysis, and PCR plus DNA sequencing. All of these methods require relatively large amounts of DNA, and they have multiple manual steps, making them costly and labor intensive (9). Recently, instrumentation and techniques have been developed that combine PCR technology using real-time fluorescence and analysis of differences in the melting temperatures of specific probes, thereby allowing easy polymorphism detection (7). This technology has been applied to DNA and RNA quantification in serum (11) and to the identification of mutations in whole blood specimens (7, 11, 12).

In this work, we developed two new assays for rapid screening of {alpha}1-AT deficiency linked with the PI Z mutation (the most common allele associated with severe {alpha}1-AT deficiency) and/or with the PI S mutation (a common allele associated with mildly reduced plasma {alpha}1-AT concentration) from DBS specimens. These assays have been incorporated into our genotyping system. In this new DBS screening protocol, deficiency is evaluated by combining the results of {alpha}1-AT quantification by immune nephelometry and {alpha}1-AT genotyping by LightCycler-PCR. In cases showing discordance between the two determinations (DBS {alpha}1-AT levels below 1.8 mg/dl and no detection of PI S and/or PI Z allele), diagnosis of hereditary {alpha}1-AT deficiency is confirmed by {alpha}1-AT genotyping using the PCR-DNA sequencing method. This was done in our two patients with the rare {alpha}1-AT deficiency variants, PI MMmalton (characterized by Phe52 deletion), and PI MMheerlen (characterized by Prol369->Leu substitution) (13), in whom DBS {alpha}1-AT levels were below 1.8 mg/dl, and neither the PI S nor the PI Z allele were detected. Once the deficiency is diagnosed by the screening system, a complete study is performed.

Based on population studies, the gene frequency for the PI Z allele in Spain has been determined to be approximately 1.5% (14); that is, in a population of 35-million people, 8,000 patients can be expected to have the deficient homozygous form, PI ZZ. The Spanish Registry of patients with {alpha}1-AT deficiency currently includes 320 patients from 15 of the 17 Spanish regions (5, 14). This number has continued to rise since the foundation of the registry in 1993 but still falls short of the expected number of patients.

Based on the World Health Organization's recommendations and with the aim of increasing the rate of detection of individuals with this deficiency in our country, we are now performing a large-scale screening study of {alpha}1-AT deficiency in collaboration with the Spanish Registry in all chronic obstructive pulmonary disease patients, both smokers and nonsmokers. In the ongoing first phase, we are analyzing samples from seven different areas of the country. Blood is drawn by pinprick from a finger, placed on filter paper, and sent by mail to our reference laboratory for {alpha}1-AT quantification and PI Z and PI S genotyping.

Blood collected on filter paper is simple to process since a DNA extraction method is not required, and DNA elution from DBS specimens costs approximately 80% less per sample than DNA isolation with the kit currently used in our laboratory for whole-blood specimens. However, an important limitation of DBS samples is their small DNA concentration. The LightCycler system overcomes this drawback with its use of fluorescently labeled probes, which make it a highly sensitive technique for detecting minimal amounts of DNA. The LightCycler PCR system provides a simple, rapid, and inexpensive method for {alpha}1-AT genotyping analysis. Post-PCR data analysis is performed using a computer connected to the LightCycler detector without the need for digestion of PCR products with restriction enzymes and/or fragment separation on gels. This minimizes hands-on time, PCR contamination related to sample handling, and the risk of error. The running time for the PCR and mutation identification with simultaneous assay of 32 samples is only 40 minutes. The complete screening test, including {alpha}1-AT quantification by immune nephelometry and {alpha}1-AT genotyping by LightCycler PCR is inexpensive, although the capital cost of the equipment is high. Nevertheless, it must be taken into consideration that this equipment can be used for multiple purposes.

In typical testing, determination of {alpha}1-AT levels would be the first step, with PI typing only for those below normal range. The test described here is both technically and economically effective, and it provides new possibilities for large-scale screening of {alpha}1-AT deficiency to identify susceptible individuals before the onset of disease.


   Acknowledgments
 
The authors thank Dr. Olfert Landt (TIB Mobiol, Berlin, Germany) for synthesizing the LightCycler primers and hybridization probes used in this work and Celine Cavallo for English language advice.


   FOOTNOTES
 
Supported in part by a grant from the Fundació Marató TV3 and a grant from the Alpha One Foundation.

Received in original form March 18, 2002; accepted in final form June 5, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  3. Anonymous. Alpha-1-antitrypsin deficiency: memorandum from a WHO meeting. Bull World Health Organ 1997;75:397–415.[Medline]
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  5. Miravitlles M, Vidal R, Barros-Tizón JC, Bustamante A, Espana P, Casas F, Martinez M, Escudero C, Jardi R. Usefulness of a national Registry of alpha-1 antitrypsin deficiency: the Spanish experience. Respir Med 1998;92:1181–1187.[CrossRef][Medline]
  6. Yan M, Hendrie HC, Hall KS, Oluwole OSA, Hodes ME, Sahota A. Improved procedure for eluting DNA from dried blood spots. Clin Chem 1996;42:1115–1116.[Free Full Text]
  7. Lay MJ, Wittwer CT. Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin Chem 1997;43:2262–2267.[Abstract/Free Full Text]
  8. Laurell CB, Sveger T. Mass screening of newborn Swedish infants for alpha antitrypsin deficiency. Am J Hum Genet 1975;27:213–217.[Medline]
  9. Braun A, Meyer P, Cleve H, Roscher A. Rapid and simple diagnosis of the two common alpha-1 proteinase inhibitor deficiency alleles PI Z and PI S by DNA analysis. Eur J Chem Clin Biochem 1996;34:761–764.
  10. Jardí R, Rodriguez F, Buti M, Costa X, Cotrina M, Valdes A, Galimany R, Esteban R, Guardia J. Quantitative detection of HBV DNA in serum by a new rapid real-time fluorescence PCR assay. J Viral Hepat 2001;6:465–471.
  11. Ahsen N, Oellerich M, Schutz E. Use of two reported dyes without interference in a single tube rapid-cycle PCR: {alpha}1-AT genotyping by multiplex real-time fluorescence PCR with the LightCycler. Clin Chem 2000;46:156–161.[Abstract/Free Full Text]
  12. Ortiz-Pallardo ME, Zhou H, Fischer HP, Neuhaus T, Sachinidis A, Vetter H, Bruning T, Ko Y. Rapid analysis of alpha 1-antitrypsin PI Z genotype by a real-time PCR approach. J Mol Med 2000;78:212–216.[CrossRef][Medline]
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Copyright © 2002 American Thoracic Society