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(Chest. 2000;117:415-419.)
© 2000 American College of Chest Physicians

Distribution of {alpha}1-Antitrypsin Alleles in Patients With Bronchiectasis*

Antoine Cuvelier, MD; Jean-Francois Muir, MD, FCCP; Marie-France Hellot, ScD; Daniel Benhamou, MD; Jean-Pierre Martin, MD, PhD; Jacques Bénichou, MD, PhD and Richard Sesboüé, MD

* From the Respiratory and Intensive Care Department (Drs. Cuvelier, Muir, and Benhamou), Unité INSERM 295 (Drs. Cuvelier, Martin, and Sesboüé), and the Department of Biostatistics (Drs. Hellot and Bénichou), University Hospital, Rouen, France.

Correspondence to: Antoine Cuvelier, MD, Respiratory and Intensive Care Department, Hôpital de Bois-Guillaume, CHU de Rouen, 76031 Rouen CEDEX, France; e-mail:antoine.cuvelier{at}chu-rouen.fr


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Bronchiectasis has been reported in a few patients with homozygous {alpha}1-antitrypsin (AAT) deficiency, but the distribution of AAT alleles among bronchiectatic patients is not known.

Patients and design: Two hundred two patients, 104 men and 98 women, with a mean age (SD) of 63.7 ± 15.4 years, had bronchiectasis diagnosed by CT scan alone (n = 178), bronchography with or without CT scan (n = 17), or radiography alone (n = 7). AAT phenotypes (classified according to the protease inhibitor [PI] system) were determined by isoelectric focusing in blood samples obtained from all patients. Bronchiectasis was primary in 121 cases and secondary in 81 patients. Allele and phenotype frequencies were compared retrospectively between bronchiectatic patients and healthy blood donors living in the same geographic area.

Results: The PI phenotype frequencies among patients were the following: MM, 81.18%; MS, 11.88%; MZ, 3.46%; IZ, 0.49%; IM, 0.49%; SS, 1.48%; SZ, 0.49%; and ZZ, 0.49%. The allelic frequencies among patients were the following: M, 89.1%; S, 7.67%; Z, 2.72%; and I, 0.49%. There was no difference in the distribution of alleles or phenotypes either between patients and control subjects or between patients with secondary and primary bronchiectasis. A significant difference was found between bronchiectatic patients with and without coexisting emphysema (p = 0.028). This difference was caused by an overrepresentation of PI*Z alleles in bronchiectatic patients with coexisting emphysema.

Conclusions: Our results do not support a physiopathologic implication of the AAT genes in the development of bronchiectasis. We suggest that bronchiectasis may be a consequence of emphysema in PI*Z patients rather than a primary effect.

Key Words: {alpha}1-antitrypsin • bronchiectasis • COPD • emphysema


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human {alpha}1-antitrypsin (AAT) is a highly polymorphic protein, the synthesis of which is governed by a 12.2-kilobase gene organized in seven exons and located on the long arm of chromosome 14 at position q31–31.2. Most of the variants of AAT in humans are classified according to the protease inhibitor (PI) system, as defined by isoelectric focusing of plasma in polyacrylamide at an acidic pH. Greater polymorphic variation has been recognized by using restriction fragment length and direct DNA sequence analysis. These techniques now are able to identify > 75 distinct alleles of the AAT gene.1 The AAT gene product is a 54-kd serine proteinase inhibitor (serpin) and the major inhibitor of neutrophil elastase in serum and tissue. Very few alleles are associated with a decreased protein concentration in serum. Some patients with pulmonary emphysema have infrequent phenotypes (null-null, null-Z, ZZ, or SZ) associated with AAT serum concentrations of < 20% of normal values. An unrestricted elastase activity has been suggested as an explanation for pulmonary parenchymal degradation. However, an increasing recognition of asymptomatic ZZ siblings suggests that a cofactor, particularly tobacco smoke, is necessary to develop emphysema in AAT-deficient patients.

A few case reports have suggested a putative association between bronchiectasis and AAT deficiency in the absence of emphysema.2 3 4 5 More recently, investigations using CT scans have demonstrated the presence of bronchiectasis among patients with AAT deficiency.6 7 8 The aim of our study was, therefore, to compare AAT gene distribution between patients with bronchiectasis and control subjects and to assess AAT gene distribution among several clinical subgroups of bronchiectatic patients.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Control Population
We retrospectively studied all the clinical files in our department between January 1991 and June 1996. Two hundred forty patients were classified as having bronchiectasis according to the World Health Organization International Classification of Diseases.9 All these subjects were hospitalized or were seen at the outpatient clinic of our department. AAT phenotype analysis was already available for a few patients or was retrospectively determined in as many patients as possible. Twenty-five patients had died, 9 patients were lost to follow-up, and 4 patients declined to participate in the study. Finally, 202 patients were included in the study. All patient files were reviewed to confirm the diagnosis of bronchiectasis and to collect clinical data.

The control population consisted of 1,030 unrelated healthy blood donors of both sexes living in the same geographic area, ie, the Rouen area in France.10

AAT Phenotype Determination
All patients and control subjects had AAT phenotype determined by isoelectric focusing of blood samples in polyacrylamide gels. The same methodology has been in use in our laboratory for > 20 years. Briefly, isoelectric focusing was performed on a multiphor apparatus (model LKB 2117; Pharmacia; Uppsala, Sweden) connected to a power supply (model LKB 2103; Pharmacia). Each gel was made to a final concentration of acrylamide 5% (w/v), carrier ampholytes 1% (w/v; ampholytes 4.2 to 4.9; Pharmacia), and sucrose 12% (w/v). Polymerization was achieved with riboflavin 0.04% (volume/volume) under ultraviolet light for 1 h. After 1 h of premigration (5 W, 5 mA, 500 V), samples were applied by means of 5 x 1 mm No. 3 filter paper (Whatman; Clifton, NJ). Focusing was performed for 2 h with the following maximal conditions: 10 W, 10 mA, and 1,600 V. Fixation, staining, and destaining of the gels were conducted as described elsewhere.10

Because AAT serum concentrations are of low value for screening patients with intermediate AAT deficiency, these data are not routinely assessed in our department and were only partly available from patient records in this study.

CT Scan
CT scans were performed with 10-mm collimation at 10-mm intervals through the chest during suspended inspiration with the patient in the supine position. For some patients, thin-section images were obtained at 1-mm intervals. A diagnosis of bronchiectasis was made on the basis of the presence of a bronchus with a diameter greater than that of its accompanying pulmonary artery branch. Patients with bronchial thickening without any dilatation of the lumen of the airways were not included in the study.

Statistical Analysis
For control subjects and patients, or patient subgroups, PI allele frequencies were calculated by gene counting according to the Hardy-Weinberg law, and the observed phenotype numbers were compared with expected values. Given the low figures occasionally observed in some classes, the comparisons of AAT phenotype distributions and gene frequencies between groups were performed with the extended Fisher’s Exact Test (StatXact 3.1; Cytel Software Corporation; Cambridge, MA) whenever possible; otherwise, a method based on the Monte Carlo procedure11 was used. All tests were two-sided and were considered significant for a p value < 0.05. Calculations were performed with computer software (StatView; Abacus Concepts; Berkeley, CA; and CHIRXC; provided by Dr. Dimitri Zaykin, PhD; Institute of Marine Biology, 1980; Vladivostok, Russia).


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Patients’ Data
The study population involved 202 patients (104 men and 98 women) with a mean age (SD) of 63.7 ± 15.4 years (range, 17 to 90 years). One hundred ninety-seven patients (97.5%) were white, 4 were African (2.0%), and one was Asian (0.5%). One hundred thirteen patients were nonsmokers (55.9%), 58 were ex-smokers (28.7%), and 31 were active smokers (15.3%). Diagnosis of bronchiectasis was assessed by CT scan alone in 178 patients, CT scan and bronchography in 16 patients, bronchography alone in 1 patient, and radiography alone in 7 patients. Because of typical radiographic and clinical patterns, these last patients were included in the study.

Bronchiectasis was limited to one lobe in 49 patients (24.3%). Bronchiectasis was unilateral in 65 patients (32.2%) and bilateral in 137 patients (67.8%). The following possible causes of bronchiectasis were suggested in 81 patients: posttuberculosis (n = 57); rheumatoid arthritis (n = 8); respiratory infections during childhood (n = 8); Ig deficiency (n = 3); cystic fibrosis (n = 2); pulmonary fibrosis (n = 2); and chronic lymphocytic leukemia (n = 1). Historical, clinical, and radiologic data were compatible with idiopathic bronchiectasis in 121 patients (59.9%). Emphysema was present on CT scan or radiography in 42 patients (20.8%). Bronchorrhea was the main clinical pattern (85.1%), followed by dyspnea (75.2%). Digital clubbing was present in 38% of patients, and hemoptysis was recorded in 39.8%. Respiratory insufficiency was present in 43.6% of the 202 patients, indicating the severity of the disease in our study population.

AAT Phenotype Distribution Among Patients
The distribution of AAT phenotypes among patients with bronchiectasis and control subjects (Table 1 ) showed a remarkably similar number of MM, MS, and MZ phenotypes. Nine patients were heterozygous and only one patient was homozygous for the Z gene. The apparent overrepresentation of rare and deficient phenotypes (IZ, 1 patient; SZ, 1 patient; and ZZ, 1 patient) in patients was not statistically significant (p = 0.1077).


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  		Table 1.. AAT Phenotype Distribution in Patients With Bronchiectasis and in Control Subjects

 
AAT Gene Distribution Among Patients
The overall distribution of the various PI alleles among patients with bronchiectasis did not differ significantly from that observed in the control population (p = 0.5179; Table 2 )


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  		Table 2.. AAT Gene Distribution in Patients With Bronchiectasis and in Control Subjects

 
Patients with primary bronchiectasis (n = 121) were subgrouped according to the following nine main clinical criteria: bronchiectasis localization (extension and lateralization); coexistence of emphysema; respiratory symptomatology (bronchorrhea, digital clubbing, dyspnea, hemoptysis); and respiratory insufficiency (Table 3 ). The ATT gene distribution in these subgroups was not statistically different from that observed in control subjects. We also performed the same analysis on all patients with primary or secondary bronchiectasis (n = 202). The only significant difference was found between bronchiectatic patients with and without coexisting emphysema (p = 0.028). This was caused by an overrepresentation of PI*Z and PI*I alleles in bronchiectatic patients with coexisting emphysema.


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  		Table 3.. AAT Gene Distribution in Patients With Primary Bronchiectasis*

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our study indicates that AAT phenotype distribution and gene frequencies are not different between patients with bronchiectasis and control subjects. We observed the presence of one PI*Z homozygous and two PI*I heterozygous individuals in the subgroup of bronchiectatic patients with emphysema. Although our observation regarding the Z allele is not surprising, inasmuch as this allele is associated with an increased incidence of emphysema, it is more surprising regarding the I allele, the defective pattern of which is more controversial. Our patient population was an unselected population, and a comparative study was performed. Blood donors tend to be healthier than people in the general population. However, it is unlikely that deficient individuals could be underrepresented in the blood donor population because most PI*Z subjects are asymptomatic.12 Despite the availability of polymerase chain reaction techniques in our laboratory, we used isoelectric focusing for gene determination because this technique may recognize the rarer AAT alleles, ie, I, F, or others. However, isoelectric focusing has some limitations when assessing the PI*M subtypes, as PI*M2 stains poorly on gel and PI*M3 is discriminated with difficulty from PI*M1 and PI*M2. This is why we did not differentiate these three M alleles. This study has several other limitations inasmuch as a few clinical data were not available because of the retrospective design of the study. The number of included patients is probably too low, and a higher number of inclusions may have reinforced the significance of Z and I allele excess. In fact, statistical significance because of only one patient (one PI*Z patient in this study) may have led to an overestimation of the risk factor and to a false conclusion. However, this negative study may be caused by a lack of statistical power inasmuch as we perhaps had an insufficient number of cases. The prevalence of abnormal AAT gene frequency was almost identical to that seen for healthy control subjects in the normal population.

Several case reports describing patients with coexisting bronchiectasis and AAT deficiency have been published since the 1970s.2 3 4 5 However, isolated case reports cannot provide any causal link between these two conditions.13 Moreover, some of these previously reported patients had clinical conditions potentially leading to bronchiectasis, eg, severe childhood pneumonia, cystic fibrosis, or remote tuberculosis. Three recent studies assessed the incidence of bronchiectasis by CT scan in patients with AAT deficiency. Shin and Ho6 described seven patients with homozygous AAT deficiency and negative sweat test and without Ig deficiency. Four patients had chronic bronchitis, and all patients were smokers or ex-smokers. All seven patients had severe emphysema, especially in the lower lobes. Three patients had bronchiectasis (43%). Guest and Hansell7 reported CT scan data in 17 AAT-deficient patients. Bronchial dilatations were noted in six cases (35%) and bronchial wall thickening in one case. These authors reported one patient with cystic bronchiectasis and suggested that the deficiency "may directly affect the airways in addition to the lung parenchyma." Finally, King et al8 studied 14 patients with AAT deficiency using thoracic CT scan. Only one patient had AAT deficiency diagnosed by electrophoresis, and one patient had PI*SZ phenotype diagnosed. Six patients (43%) had evidence of multilobar bronchiectasis on CT scan. Interestingly, bronchiectasis was more prevalent in lobes in which the emphysema score was higher. The incidence of bronchiectasis in patients with AAT deficiency is remarkably similar in these three studies, with a common overall estimate of about 40%. Inasmuch as these authors could not determine whether bronchiectasis is a consequence of AAT deficiency or a parallel occurrence, their data do not provide convincing proof of a causal relationship between the two conditions.

The best approach to demonstrate a potential link between AAT deficiency and bronchiectasis is to compare the AAT allelic distribution in an unselected and large population of bronchiectatic patients with that of a control population. To our knowledge, only a few studies were designed to that end.14 15 16 From our point of view, these studies had several methodological limitations: diagnosis of bronchiectasis was usually assessed by methods other than CT scan (chest radiography or bronchography), and most of these studies made no allowance for pathologic conditions that may lead to the development of bronchiectasis.17 Moreover, not all the patients had blood samples analyzed by isoelectric focusing; this is a very critical point because AAT serum concentrations may be influenced by sex or inflammatory conditions and have been found to slightly increase in deficient individuals. Inasmuch as AAT is an acute-phase reactant protein, its serum concentration may increase in patients with stable bronchiectasis as compared with control subjects.16 18 Therefore, because it is not possible to use AAT serum concentrations as a screening method for the diagnosis of AAT deficiency, we only included patients with electrophoretic analysis. In our opinion, only the study of Varpela et al15 can be considered an unselected and controlled study. In 60 consecutive patients with bronchiectasis, these authors did not find any homozygous Z patients but found a significant increase of PI*MZ patients as compared with control subjects. These data are critical because only phenotype distribution, not gene distribution, was evaluated. Groups other than MZ patients were not individualized.

As previously reported in a recent article,19 our study highlights the importance of bronchiectasis-associated emphysema. Emphysema is a confounding factor that may lead to bronchiectasis, but the presence of emphysema in bronchiectatic patients also is associated with an abnormal distribution of AAT alleles. Our data show that bronchiectatic patients without emphysema do not have a different AAT distribution as compared with control subjects in contrast to bronchiectatic patients with emphysema. This suggests that bronchiectasis may be a consequence of emphysema in PI*Z patients rather than a primary cause. The incidence of bronchiectasis in a population of emphysema patients without AAT deficiency is unknown, and it would be interesting to assess the incidence of bronchiectasis in a population of PI*Z patients without emphysema. Shin and Ho6 suggested that the sequential clinical expression of AAT deficiency may be emphysema alone, emphysema with chronic bronchitis, and emphysema with chronic bronchitis and bronchiectasis. Indeed, most occurrences of bronchiectasis in patients with emphysema are thought to be secondary to matrix destruction and pressure loss. However, the same authors also described a 21-year-old AAT-deficient woman who developed bronchiectasis early in life before emphysematous changes appeared. Bronchiectasis may appear in AAT-deficient patients who are exposed to repeated pulmonary infections, even before the development of emphysema.6 Therefore, bronchiectasis in PI*Z patients may be caused by chronic bronchitis that is seen in emphysematous patients. Because approximately 50% of patients with AAT deficiency have symptoms of airway disease, especially chronic sputum production,12 it would be interesting to assess the improvement of such clinical symptoms in AAT-deficient patients receiving AAT substitution.

In conclusion, our comparative study does not provide any evidence that AAT deficiency may play a role in the pathophysiology of bronchiectasis.


   Acknowledgements
 
The authors thank Luis Carlos Molano and Richard Medeiros for their advice in editing the manuscript.


   Footnotes
 
Abbreviations: AAT = {alpha}1-antitrypsin; PI = protease inhibitor

Received for publication January 20, 1999. Accepted for publication September 30, 1999.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  3. Longstreth, G, Weitzman, SA, Browning, RJ, et al (1975) Bronchiectasis and homozygous alpha-1-antitrypsin deficiency. Chest 67,233-235[Abstract/Free Full Text]
  4. Scott, J, Anderson, CL, Shankar, PS, et al (1977) Alpha-1-antitrypsin deficiency with diffuse bronchiectasis and cirrhosis of the liver. Chest 71,535-538[Abstract/Free Full Text]
  5. Rodriguez-Cintron, W, Guntupalli, K, Fraire, AE (1995) Bronchiectasis and homozygous (PiZZ) {alpha}1-antitrypsin deficiency in a young man. Thorax 50,424-425[Abstract]
  6. Shin, MS, Ho, K-J (1993) Bronchiectasis in patients with alpha 1-antitrypsin deficiency: a rare occurrence? Chest 104,1384-1386[Abstract/Free Full Text]
  7. Guest, PJ, Hansell, DM (1992) High resolution computed tomography (HRCT) in emphysema associated with alpha-1 antitrypsin deficiency. Clin Radiol 45,260-266[CrossRef][ISI][Medline]
  8. King, MA, Stone, JA, Diaz, PT, et al (1996) {alpha}1-Antitrypsin deficiency: evaluation of bronchiectasis with CT. Radiology 199,137-141[Abstract/Free Full Text]
  9. The tenth revision of the international statistical classification of diseases. Geneva: World Health Organization, 1992
  10. Charlionet, R, Sesboüé, R, Morcamp, C, et al (1981) Genetic variants of serum alpha-1 antitrypsin (Pi types) in Normans. Hum Hered 31,104-109[CrossRef][ISI][Medline]
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  13. El-Kassimi, FA (1996) Bronchiectasis and homozygous (PiZZ) {alpha}1-antitrypsin deficiency [letter]. Thorax 51,228
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