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Articles |
1-Antitrypsin Deficiency: MZ Individuals with Chronic Obstructive Pulmonary Disease May Have Lower Lung Function Than MM Individuals
1
Department of Clinical Biochemistry, Herlev University Hospital, DK-2730 Herlev, Denmark.
2
Department of Respiratory Medicine, Hvidovre University
Hospital, DK-2650 Hvidovre, Denmark.
3
The Copenhagen City Heart Study, Bispebjerg University
Hospital, DK-2400 Copenhagen NV, Denmark.
a Address correspondence to this author at: Department of Clinical Biochemistry, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. Fax 45-35452524;
at-h{at}rh.dk.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin deficiency affects lung function in the
population at large. Methods: We performed spirometry [forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC)] and genotyping of 9187 individuals from the adult general population of Copenhagen, Denmark.
Results: As expected, the frequencies of individuals with MM, MS, SS, MZ, SZ, and ZZ genotypes were 0.891, 0.054, 0.001, 0.052, 0.001, and 0.001, respectively. Genotype interacted with clinically established chronic obstructive pulmonary disease (COPD) on the percentage of the predicted FEV1 (P = 0.004): the percentage of the predicted FEV1 was reduced in MZ compared with MM individuals among those with clinically established COPD, but not among those without COPD. Furthermore, SZ compound heterozygotes had lower FEV1/FVC ratios than MM individuals (P <0.05), and ZZ homozygotes had lower percentages of the predicted FEV1 and FEV1/FVC ratios than MM, MS, SS, and MZ individuals (all Ps <0.01). Reduced lung function in SZ and ZZ vs MM individuals could be demonstrated in current and ex-smokers, but not in nonsmokers. Compared with MM individuals in the same groups, FEV1 was reduced 655 mL in MZ individuals with clinically established COPD, 364 mL in SZ current smokers, and 791 mL in ZZ current smokers.
Conclusions: In the population at large, MZ was
associated with reduced pulmonary function in individuals with
clinically established COPD, whereas SZ and
ZZ were associated with reduced pulmonary function in
smokers. The presence of the 1-antitrypsin
MZ genotype may in certain circumstances produce marked
aggravation of airway obstruction in individuals prone to develop
COPD.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin deficiency
(1)(3). 1-Antitrypsin
is a protease inhibitor that protects lung parenchyma from destruction
by neutrophil elastase. When 1-antitrypsin is
deficient, lung tissue is slowly destroyed, ultimately leading to
pulmonary emphysema and/or early death (3).
Intermediate and severe 1-antitrypsin
deficiency is almost entirely caused by the Z and
S alleles as opposed to the wild-type M allele in
the 1-antitrypsin gene:
individuals with the six different genotypes, ZZ,
SZ, MZ, SS, MS, and
MM, have relative plasma
1-antitrypsin concentrations of 
16%, 51%,
83%, 93%, 97%, and 100%, respectively (4). A
deteriorating effect of severe deficiency (ZZ genotype) on
lung function has been known for many years; however, this effect may
have been overestimated because mainly patients with COPD have been
studied. The role of intermediate deficiency (MZ and
SZ genotypes) in COPD is less clear
(1)(3)(5)(6)(7)(8)(9)(10)(11).
We tested the hypotheses that both intermediate and severe
1-antitrypsin deficiency affects lung function
in the population at large. For this purpose, we genotyped 9187 white
women and men from a Danish general population sample, thus avoiding
bias by selecting from a specific patient population.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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20 years, were selected at random after age
stratification in 10-year age groups from among 90 000 residents of
Copenhagen (14). Of the 17 180 individuals invited, 10 049
participated, 9259 gave blood, and 9187 were genotyped; of these, 9069
individuals had spirometry performed. Less than 1% were non-Caucasian,
and 99% were of Danish descent. All subjects gave informed consent.
The study was approved by the ethics committee for the City of
Copenhagen and Frederiksberg (number 100.2039/91). From 1991 to 1999, 1588 (22%) nonresponders died compared with 1352 (13%) responders. The mean age of nonresponders at the time of examination was 60 years compared with 58 years in responders. The number of nonresponders who died from respiratory disease [International Classification of Diseases, 8th revision (15), disease classification 460-519; International Classification of Diseases, 10th revision (16), disease classification J00-J99] was 249 (16% of all deaths) vs 176 (13%) in responders.
Participants filled out a self-administered questionnaire, which was validated by the participant and an investigator on the day of attendance. All subjects reported whether they were current smokers, ex-smokers, or life-long nonsmokers, and an estimate of lifetime tobacco exposure (in pack-years) was calculated as: daily tobacco consumption (grams) x duration of smoking (years) divided by 20 (grams/pack). Chronic bronchitis was defined as bringing up phlegm at least 3 months continuously every year. Hospitalization for COPD was assessed via the Danish National Hospital Discharge Register and the International Classification of Diseases, 8th revision (disease classification 490-492) (15); clinically established COPD was taken as previous hospitalization for COPD.
We measured forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) with a dry wedge spirometer (Vitalograph) that was calibrated daily with a 1-L syringe. Three sets of values were obtained, and as a criterion for correct performance of the procedure, at least two measurements of FEV1 and FVC differing by <5% had to be produced. The highest set of FEV1 and FVC values were used in the analyses as absolute values and as the percentage of predicted values, using internally derived reference values based on a subsample of life-long nonsmokers (17). Airway obstruction was defined as FEV1 <80% of predicted and FEV1/FVC <0.7 (18).
Total genomic DNA was extracted from frozen whole blood
(19). The Z (342Glu
Lys) and S
(264GluVal) mutations in the
1-antitrypsin gene were
identified by multiplex PCR (20) using an Omnigene
Temperature Cycler (Hybaid). Primer pairs to diagnose the Z
and S mutations were as follows: Z, sense
(5'-ATAAGGCTGTGCTGACCATCGTC-3') and antisense
(5'-TTGGGTGGGATTCACCACTTTTC-3'); S, sense
(5'-TGAGGGGAAACTACAGCACCTCG-3') and antisense
(5'-AGGTGTGGGCAGCTTC-TTGGTCA-3'). We added 3 pmol of each primer
and 0.5 U of Taq DNA polymerase (Life Technologies) to 100 µg of
DNA in 30 µL (final volume) of a solution containing 20 mM Tris-HCl,
pH 8.4, 50 mM KCl, 1.5 mM MgCl2, and 200 µM
each dNTP. Temperature cycling conditions were as follows:
(a) initial 5-min denaturation at 94 °C; (b)
35 cycles of 1 min at 94 °C, 1 min at 55 °C, and 2 min at
72 °C; and (c) a final extension for 10 min at 72 °C.
The presence of either mutation destroyed a Taq1 site
in the respective PCR products. After Taq1 digestion at
65 °C for 120 min, fragments of 157 bp + 22 bp (wild-type
allele) or 179 bp (Z allele), and 100 bp + 21 bp (wild-type
allele) or 121 bp (S allele) were separated on a 3% agarose
gel (SeaKem LE; FMC BioProducts), stained with ethidium bromide, and
visualized on a ultraviolet transilluminator. Individuals with
SS, SZ, and ZZ genotypes were retested
to confirm the diagnosis. The numerous other non-deficiency alleles are
not detected by this method.
statistical analysis
Statistical analyses were performed with SPSS (21);
P <0.05 in a two-sided test was considered
significant. Differences in the percentage of the predicted
FEV1, the percentage of the predicted FVC, and
the FEV1/FVC ratio according to
1-antitrypsin genotypes were compared using
ANOVA; the Kruskal–Wallis ANOVA was used in case of unequal
variances. The Levene test examined differences in variance among the
six genotypes. To approach gaussian distribution,
FEV1 was square-root-transformed and
FEV1/FVC was cubed before statistical analyses,
but the data shown in Tables 1–5
and Figs. 1
and 2
are untransformed
values. The Student t-test was used as the post hoc test for
two-genotype comparisons. Interactions between genotype and other
covariates (age, gender, smoking, long-term occupational exposure to
dust or fumes, common respiratory infections in childhood, chronic
bronchitis, and COPD) on the percentage of the predicted
FEV1, the percentage of the predicted FVC, and
the FEV1/FVC ratio were examined by introducing
two-way interaction terms between the genotype and the covariate
examined, one at a time, in an analysis of covariance (ANCOVA). To
estimate the average reduction in lung function in MZ,
SZ, and ZZ individuals vs MM
individuals, ANCOVA allowing for age, gender, height, and smoking was
used; the F-statistic determined whether genotype
contributed significantly.
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The Student t-test and 
2 likelihood
ratio test were used for univariate analyses. Logistic regression
analysis assessed 1-antitrypsin genotypes as
predictors of airway obstruction and chronic bronchitis. Interactions
between genotype and age, gender, smoking, occupational exposure to
dust or fumes, or common childhood respiratory infections in predicting
airway obstruction and chronic bronchitis were tested using two-factor
interaction terms, with the likelihood ratio test as a measure of
significance. Multifactorial logistic regression analysis was used to
adjust for age, gender, smoking, long-term occupational exposure to
dust or welding fumes, and common respiratory infections in childhood.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-Antitrypsin genotype frequencies in
this white, Danish general population sample were 0.891, 0.054, 0.001,
0.052, 0.001, and 0.001 for MM, MS,
SS, MZ, SZ, and ZZ,
respectively. Genotype frequencies did not differ from those predicted
by the Hardy-Weinberg equilibrium (2, 0.1
< P < 0.2). Distribution of gender and smoking did
not differ significantly among the six genotypes (Table 1
).
fev1 and fev1/fvc
Genotype interacted with clinically established COPD on the
percentage of the predicted FEV1 (ANCOVA,
P = 0.004). The interaction was caused by decreases in
the percentage of the predicted FEV1 and the
FEV1/FVC ratio in MZ heterozygotes
compared with MM individuals among subjects with clinically
established COPD, but not in those without COPD (Table 2
). Among subjects with clinically established COPD,
MZ heterozygotes had an average reduction of 655 mL in
FEV1, compared with MM individuals,
after adjustment for age, gender, height, and smoking (Table 3
).
In the total general population sample, the percentage of the predicted
FEV1 and the FEV1/FVC ratio
differed among the six genotypes (Fig. 1
; ANOVA, P = 0.02 and P = 0.002,
respectively). In post hoc Student t-tests, SZ
compound heterozygotes had lower FEV1/FVC ratios
than MM individuals (P <0.05). Furthermore,
ZZ homozygotes had lower percentages of the predicted
FEV1 and FEV1/FVC ratios
than MM, MS, SS, and MZ
individuals (all Ps <0.01). When these analyses were
stratified by smoking status, reductions in lung function in
SZ and ZZ individuals vs MM
individuals were statistically significant only among ex-smokers or
current smokers, but not among nonsmokers (Fig. 1
). The percentage of
the predicted FVC did not differ among the six genotypes (data not
shown).
With increasing extent of smoking, the percentage of the predicted
FEV1 did not decrease more in MZ
individuals than in MM individuals, whereas such a trend was
observed for SZ and ZZ individuals (Fig. 2
). Among current smokers, SZ compound heterozygotes
had an average reduction in FEV1 of 364 mL
compared with MM individuals after adjustment for age,
gender, and height (Table 3
). Compared with MM individuals,
ZZ homozygotes had an average reduction in
FEV1 of 791 mL among current smokers.
airway obstruction
In the total general population sample, airway obstruction
characterized by pulmonary function studies was more common in subjects
with SZ and ZZ genotypes than in subject with the
MM genotype, whereas frequency of airway obstruction was
unaffected in subjects with the MZ genotype (Table 1
).
Chronic bronchitis was more common in ZZ than in
MM individuals, but not in any other genotype.
The odds ratios for airway obstruction were 5.4 (95% confidence
interval, 1.5–19) and 8.0 (95% confidence interval, 1.6–40)
for SZ and ZZ vs MM individuals, and
1.1 (95% confidence interval, 0.9–1.5) for MZ vs
MM individuals (Table 4
); an odds ratio equal to 1 indicates that the risk of disease
is not significantly different in probands vs controls, whereas an odds
ratio >1 indicates an increased risk for disease among probands vs
controls. Thus, the estimated risk for developing airway obstruction
was 5 and 8 times higher among SZ and ZZ compared
with MM individuals, whereas risk of airway obstruction was
unaffected overall in MZ carriers. The odds ratio for
chronic bronchitis was 6.3 (1.3–31) for ZZ vs
MM individuals, whereas no other genotype had increased risk
of chronic bronchitis in unifactorial logistic regression.
After adjustment for age, gender, smoking, exposure to occupational
dust or fumes, and common respiratory infections in childhood, odds
ratios for airway obstruction were 5.3 (1.0–26) and 18 (2.9–114) for
SZ and ZZ individuals vs MM
individuals (Table 4
), whereas the odds ratio for chronic bronchitis
was 9.6 (1.7–53) for ZZ vs MM
individuals.
characteristics of zz homozygotes
The six ZZ homozygotes were 44, 44, 49, 61, 72, and 85
years of age compared with a mean age of 57 years in MM
individuals (Wilcoxon, P = 0.93; Table 5
). Although three ZZ individuals fulfilled the
spirometric criteria for airway obstruction, only one had previously
been hospitalized for COPD and was on medication for respiratory
disease.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The role of MZ heterozygosity in COPD has been controversial
(1)(3)(5)(6)(7)(11). The
present study of 476 MZ individuals compared with 8184
MM individuals is very large and is not biased by selection
from a specific patient population: the MZ genotype was not
overrepresented among individuals with airway obstruction, but among
subjects with clinically established COPD, MZ heterozygotes
had reduced lung function compared with MM individuals.
Because MZ heterozygosity modifies the course of disease
only among individuals with clinical COPD, it appears to be a
susceptibility rather than a causative mutation for COPD. This suggests
that MZ heterozygosity works only in certain contexts, i.e.,
only when other, as yet unknown, predisposing factors are present. This
observation may explain previous contradicting findings in different
studies of the MZ genotype. Chronic airway inflammation in
COPD patients may increase the oxidative burden in the lung,
accelerating 1-antitrypsin inhibition
(22). This together with a higher release of neutrophil
elastase as a result of inflammation could push a subtle
antiprotease/protease balance in MZ individuals toward
higher proteolytic destruction of lung tissue. Thus, it seems plausible
that a 17% decrease in 1-antitrypsin
concentrations attributable to the MZ genotype
(4) will not affect lung function in the average individual,
but only in those with preexisting COPD.
It is well known that severe 1-antitrypsin
deficiency reduces protection of lung tissue from neutrophil elastase,
thus leading to progressive destruction of lung tissue and finally to
overt COPD (1)(3). The present demonstration
that intermediate 1-antitrypsin deficiency in
SZ individuals, when identified in the population at large
leads to reduced pulmonary function and a fivefold increase in risk of
airway obstruction, is therefore mechanistically conceivable. Our
finding is in agreement with some (8), but not all previous
results (9). That SZ compound heterozygosity
causes less severe airway obstruction than ZZ homozygosity
is in agreement with earlier findings
(6)(8)(11).
Because it seems well established that severe
1-antitrypsin deficiency, i.e.,
ZZ homozygosity, leads to COPD and early death, particularly
in smokers (1)(3), we expected to find reduced
numbers of ZZ homozygotes in this general population sample
with an average age of all participants of 58 years. However, we
detected 1 ZZ homozygote in 1500, the highest frequency
detected in any population
(3)(23)(24). Furthermore, our sample
appeared to be in Hardy-Weinberg equilibrium with expected and observed
numbers of ZZ homozygotes of seven and six, respectively.
This suggests that although ZZ homozygosity may be a very
serious condition for some individuals (3), a substantial
fraction of ZZ homozygotes, when identified in the
population at large, at most have relatively mild forms of lung
disease. This is supported by the fact that, on average, the
percentages of the predicted FEV1 were 93% and
59% in nonsmokers and smokers with the ZZ genotype in our
sample, whereas in a previous Danish study of ZZ individuals
ascertained in patients with COPD, the equivalent values at the same
age were 25% and <10%, respectively (25).
In the present study, bias caused by investigators’ knowledge of disease or risk-factor status seems unlikely because we selected from a general population and genotyped our samples without knowledge of disease status or lung function test results. Selection bias was possible if severe lung disease in some SZ or ZZ individuals prevented them from participating in our study; however, the expected and observed numbers of these genotypes according to Hardy-Weinberg equilibrium were similar. Nevertheless, if such a bias exists, we may have underestimated the effect of SZ and ZZ genotypes on lung function. It should also be pointed out that our results are based on very small numbers of SZ and ZZ individuals. Misclassification of genotypes is unlikely because the diagnosis of MZ and MS included a control site for restriction enzyme digestion and because all subjects with a SS, SZ, or ZZ genotype were reanalyzed to confirm the diagnosis.
From the odds ratios for airway obstruction in SZ and
ZZ individuals as well as genotype frequencies in this
study, it can be calculated (26) that the fraction of airway
obstruction attributable to the SZ or ZZ genotype
in the general population is 0.4% and 1.5%, respectively. This is
a relatively small fraction, and because many ZZ and
SZ individuals from the present study at most seem to have
modest lung disease, screening for
1-antitrypsin deficiency in the population at
large can be questioned. However, screening for
1-antitrypsin deficiency among patients with
COPD could be warranted: the major reason would be to identify COPD
patients with a genetic background for the disease, allowing additional
screening of siblings of the patients for this disease. Today, clinical
treatment does not differ among MZ, SZ, and
ZZ individuals if they have similar clinical symptoms;
however, future therapies, such as protease inhibitors aimed at
ZZ patients, could differ among these three groups.
Our data support that molecular diagnostics rather than measurement of
plasma concentrations may be used in the future to detect individuals
with 1-antitrypsin deficiency. Although this
may be particularly suitable for a Scandinavian population, where the
great majority of deleterious alleles are Z alleles, it is
less suitable for North American populations, where 5% of
1-antitrypsin deficiency is attributable to
non-Z alleles, and it is certainly not suitable for Asian
populations, where the deficiency is always associated with
non-Z alleles.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-Antitrypsin deficiency. Scriver C Beaudet A Sly W Valle D eds. The metabolic and molecular bases of inherited disease, 7th ed 1995:4125-4157 McGraw-Hill New York. .
1-antitrypsin standard to establish ranges for the common normal and deficient 1-antitrypsin phenotypes. Chest 1991;100:703-708.1-Antitrypsin Pi-types in 965 COPD patients. Chest 1986;89:370-373.1-antitrypsin deficiency. Am J Respir Crit Care Med 1996;154:1718-1725.[Abstract]
1-antitrypsin deficiency PiSZ: a risk factor for pulmonary emphysema?. Respir Med 1998;92:241-245.[Web of Science][Medline]
[Order article via Infotrieve]
1AT deficiency. Crystal RG eds. 1-Antitrypsin deficiency. Lung biology in health and disease 1996;88:245-257 Marcel Dekker New York. .
F508 heterozygosity in cystic fibrosis and susceptibility to asthma. Lancet 1998;351:1911-1913.[Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin Z and S mutations. Clin Chem 1997;43:403-404.1-antitrypsin, rendering it ineffective as an inhibitor of neutrophil elastase. J Clin Invest 1987;80:1289-1295.
-1-antitrypsin (Pi) phenotypes in Denmark determined by separator isoelectric focusing in agarose gel. Hum Hered 1986;36:19-23.[Web of Science][Medline]
[Order article via Infotrieve]
1-Antitrypsin deficiency in Europe: geographical distribution of Pi types S and Z. Respir Med 1998;92:367-377.[Web of Science][Medline]
[Order article via Infotrieve]
1-antitrypsin deficiency. Lung 1990;168(Suppl):579-585.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), SZ (
),
and ZZ (
) vs MM (•) individuals.