Alpha-1 antitrypsin Null mutations and severity of emphysema.
ABSTRACT Alpha-1 antitrypsin (AAT) deficiency is an autosomal-codominant disorder, caused by mutations in the SERPINA1 gene on chromosome 14. Individuals affected by the most common mutations, SZ and ZZ, have serum AAT concentrations of 25% and 15% of normal levels, and present a higher risk of emphysema. Mutations causing total absence of serum AAT (Null mutations) were suggested to be associated with very early onset emphysema but their clinical phenotype is poorly known.
Absence of AAT in Null mutations results in more severe emphysema as compared to ZZ and SZ.
We genotyped all known Dutch subjects (n=12) with absent serum AAT, and compared their lung function values (FEV1 and KCO) with those of individuals with ZZ and SZ genotype, matched for age and smoking history.
All subjects with absent serum AAT presented homozygous Null mutations. In three subjects, a new mutation in exon 2 of the SERPINA1 gene was found. Subjects with Null mutations showed significantly lower lung function values than SZ and ZZ individuals (p=0.000 and 0.001 for FEV1 and KCO, respectively). In all groups, there was a positive correlation between serum AAT and lung function values (p=0.025 and 0.014 for FEV1 and KCO, respectively).
Serum levels of AAT are correlated with the severity of pulmonary phenotype. Subjects with Null mutations should be considered a subgroup at particularly high risk of emphysema within AAT deficiency (AATD). Early detection of carriers of this genotype would be important for preventive and therapeutic interventions.
[show abstract] [hide abstract]
ABSTRACT: alpha(1)-Antitrypsin deficiency (AATD) is an uncommon genetic disease which occurs in 1-2.5% of Americans with chronic obstructive pulmonary disease (COPD). Little is known about current demographics of AATD. This survey study reviews the clinical characteristics of diseased individuals in North America. A survey of members from the mailing lists of US AATD patient support organizations was commissioned with duplicate persons omitted. The survey was mailed to 5,222 unique individuals with AATD. Questionnaires were returned by 1,953 individuals, including 1,810 with severe deficiency, 93 with the carrier state and 41 who were caregivers of others. The majority (81%) of participants reported COPD with symptoms of asthma, chronic bronchitis, and emphysema, usually in combination. The mean age of respondents [53.1 +/- 13.2 (SD) years] is older than the general US population. Lung or liver transplantation was reported by 9% of all respondents (n = 175), including 66 single lung transplants, 68 double lung transplants, and 47 liver transplants. Another 6.6% (n = 128) reported that they were currently on a transplant list. Twenty-one percent of lung transplants report continuing augmentation therapy use. Augmentation use is reported by 75% of those with obstructive lung disease. The majority of patients with liver disease also have COPD. AATD remains a devastating illness for many of those affected as reflected in a high incidence of transplantation for liver and lung disease.Respiration 02/2006; 73(2):185-90. · 2.26 Impact Factor
Article: Alpha1-antitrypsin deficiency. 2: genetic aspects of alpha(1)-antitrypsin deficiency: phenotypes and genetic modifiers of emphysema risk.[show abstract] [hide abstract]
ABSTRACT: The genetic aspects of AAT deficiency and the variable manifestations of lung disease in PI Z individuals are reviewed. The role of modifying genetic factors which may interact with environmental factors (such as cigarette smoking) is discussed, and directions for future research are presented.Thorax 04/2004; 59(3):259-64. · 6.84 Impact Factor
Article: Chronic obstructive pulmonary disease in alpha1-antitrypsin PI MZ heterozygotes: a meta-analysis.[show abstract] [hide abstract]
ABSTRACT: Severe alpha(1)-antitrypsin deficiency, usually related to homozygosity for the protease inhibitor (PI) Z allele, is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD). The risk of COPD in PI MZ heterozygous individuals is controversial. A search of MEDLINE from January 1966 to May 2003 identified studies that examined the risk of COPD in PI MZ individuals and studies that measured forced expiratory volume in 1 second (FEV(1)) in heterozygotes. In 16 studies that reported COPD as a categorical outcome, the combined odds ratio (OR) for PI MZ versus PI MM (normal genotype) was 2.31 (95% CI 1.60 to 3.35). The summary OR was higher in case-control studies (OR 2.97; 95% CI 2.08 to 4.26) than in cross sectional studies (OR 1.50; 95% CI 0.97 to 2.31) and was attenuated in studies that adjusted for cigarette smoking (OR 1.61; 95% CI 0.92 to 2.81). In seven studies that reported FEV(1) as a continuous outcome there was no difference in mean FEV(1) between PI MM and PI MZ individuals. Case-control studies showed increased odds of COPD in PI MZ individuals, but this finding was not confirmed in cross sectional studies. Variability in study design and quality limits the interpretation. These results are consistent with a small increase in risk of COPD in all PI MZ individuals or a larger risk in a subset. Future studies that adjust for smoking and include other COPD related phenotypes are required to conclusively determine the risk of COPD in PI MZ heterozygotes.Thorax 11/2004; 59(10):843-9. · 6.84 Impact Factor
Alpha-1 antitrypsin Null mutations and severity of
Laura Fregonesea,?, Jan Stolka, Rune R. Frantsb, Barbera Veldhuisenb,c
aDepartment of Pulmonology, Leiden University Medical Centre, Leiden, The Netherlands
bDepartment of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands
cDepartment of Medical Statistics, Leiden University Medical Centre, Leiden, The Netherlands
Received 3 September 2007; accepted 12 January 2008
Available online 18 March 2008
Background: Alpha-1 antitrypsin (AAT) deficiency is an autosomal-codominant disorder,
caused by mutations in the SERPINA1 gene on chromosome 14. Individuals affected by the
most common mutations, SZ and ZZ, have serum AAT concentrations of 25% and 15% of
normal levels, and present a higher risk of emphysema. Mutations causing total absence of
serum AAT (Null mutations) were suggested to be associated with very early onset
emphysema but their clinical phenotype is poorly known.
Hypothesis: Absence of AAT in Null mutations results in more severe emphysema as
compared to ZZ and SZ.
Methods: We genotyped all known Dutch subjects (n ¼ 12) with absent serum AAT, and
compared their lung function values (FEV1and KCO) with those of individuals with ZZ and SZ
genotype, matched for age and smoking history.
Results: All subjects with absent serum AAT presented homozygous Null mutations. In
three subjects, a new mutation in exon 2 of the SERPINA1 gene was found. Subjects with
Null mutations showed significantly lower lung function values than SZ and ZZ individuals
(p ¼ 0.000 and 0.001 for FEV1and KCO, respectively). In all groups, there was a positive
correlation between serum AATand lung function values (p ¼ 0.025 and 0.014 for FEV1and
Conclusions: Serum levels of AAT are correlated with the severity of pulmonary
phenotype. Subjects with Null mutations should be considered a subgroup at particularly
high risk of emphysema within AAT deficiency (AATD). Early detection of carriers of this
genotype would be important for preventive and therapeutic interventions.
& 2008 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS
0954-6111/$-see front matter & 2008 Elsevier Ltd. All rights reserved.
?Corresponding author. Tel.: +31715262082.
E-mail address: firstname.lastname@example.org (L. Fregonese).
Respiratory Medicine (2008) 102, 876–884
Alpha-1 antitrypsin (AAT) is the most prevalent serine
protease inhibitor in serum; it is synthesized by hepatocytes
and belongs to the family of serpins (serine protease
inhibitors).1Its main role is that of inhibiting neutrophil
elastase (NE), an enzyme that degrades several components
of the extra cellular matrix in the lungs. Insufficient
inhibition of NE in AAT deficiency (AATD) can cause severe,
early age onset pulmonary emphysema, resulting in high
incidence of lung transplantation and reduced life expec-
AATD is one of the most common autosomal-codominant
genetic disorders in the Caucasian population.1,3To date,
about 100 genetic variants of AAT have been identified.
The normal AAT genotype is Pi MM, (94–96% of the white
population),4–6characterized by protein serum levels of
150–350mg/100ml (20–48mM). The S variant, originated
in the Iberian Peninsula, and the Z variant, arisen in the
Viking population, account for more than 95% of the
mutations in patients with severe AATD.7,8Both variants
are missense mutations in the serpina1 gene (SERPINA1)
on chromosome 14q32.1.9–11In contrast to the S, the Z
phenotype is characterized by polymerization of AAT,
which can result in liver cirrhosis. Individuals with SS, SZ,
and ZZ genotypes have serum AAT concentrations of
approximately 85%, 25%, and 15% of normal levels,
respectively.12Homozygosis for S mutation has not been
associated with disease. A very limited number of studies
investigated the different risk of developing emphysema
between ZZ and SZ genotypes.13,14Most studies concluded
that the SZ genotype is less important than the ZZ in the
development of emphysema and SZ patients develop
emphysema at an older age than ZZ patients.1However, it
is not know whether the different risk is linked to the
different AAT serum levels between the two populations or
to other, unknown, factors.
Among the variants within the SERPINA1, several muta-
tions have been described leading to total absence of AAT
production (Null mutations). Although the number of these
mutations is large, they are rare within the population, thus
very little is known about their clinical phenotype. In 1988,
Cox and Levison showed that lung disease associated with
Null homozygosis (Null Mattawa: Q0mat) was more severe
than that associated with ZZ genotype.15The very small
sample size of that study (three homozygous sisters)
prevents generalizability of the findings; however, this study
and other sparse clinical reports seem to point to a higher
risk and severity of emphysema in subjects with absence of
AAT in serum. If this would be the case, early detection of
the Null homozygous genotype should be promoted and
should lead to stronger educational intervention (e.g.
smoking avoidance) and to a possible use of the replacement
therapy as preventive treatment. Recently, Ferrarotti
et al.16reported lower values of FEV1in subjects with rare
variants of AATas compared to ZZ; however, in their study ZZ
and rare variants had similar AAT plasma levels and the lung
function results were confounded by smoking. Thus, the
authors could not discriminate between the importance of
genetic factors (type of mutation), protein serum levels,
and environmental factors (smoking) in determining the
severity of the disease.
Aim of our study was to analyze whether homozygous Null
genotype is related to severity of emphysema in AATD and
whether protein serum levels are the major determinants of
severity. To this aim, we characterized the SERPINA1
mutations in the Dutch Null population, we compared lung
function of Null subjects with that of ZZ and SZ subjects
matched for age and smoking history, and correlated the
functional data with AAT serum levels.
All subjects included in the study were recruited from the
Alpha-1 International Registry database (AIR, www.aatregistry.
org). AIR is the largest international database of individuals
with AATD, containing lung function data, clinical history,
and AATD phenotype of more than 2600 individuals with
severe deficiency from 21 different countries, collected
in the years 1997–2006. A review with a detailed descrip-
tion of the AIR database development and methodo-
logy has been recently published.17The Dutch part of the
AIR database includes a total of 290 subjects, and one of the
largest Null populations ever detected, composed by 12
subjects from 7 families. All 12 Null subjects were included
in our analysis.
The study had a matched-paired design.18For each Null
case, a subject with ZZ and a subject with SZ genotype were
selected from the AIR Registry, matched on the basis of age
(75 years) and smoking (77 pack-years). Due to the young
age of the Null subjects, only one-to-one matches with
complete lung function data were found within the ZZ and
SZ population. The study was approved by the Ethics
Committee of the Leiden University Medical Centre and
patients gave their written informed consent.
Lung function tests to determine the severity of emphysema
were performed according to the European Respiratory
Society guidelines.19,20All tests were performed after
nebulization of 5mg of salbutamol and 500mcg of ipratro-
pium bromide. Among the lung function measurements,
forced expiratory volume as percentage of predicted
(ppFEV1) and the coefficient of diffusion of carbon monoxide
as percentage of predicted (ppKCO) were included in the
analysis, since they are currently considered as the most
relevant for determining emphysema severity in AATD.21,22
Serum AAT levels
Serum AAT levels were measured using a completely
automated immunoassay, as previously described.23The
lower limit of detection of the assay is 10nM. As confirma-
tion of absence of an AAT band observed in the gel of iso-
electrophoresis of serum samples, all subjects with AAT
serum values lower than 1.5mM were considered as having
ARTICLE IN PRESS
Alpha-1 antitrypsin Null mutations and severity of emphysema877
no serum AAT. Values of AATare presented as micromolar and
gram per liter.
The software package SPSS 11.5 (SPSS inc., Chicago, USA)
was used. Due to their young age, for two Null/Null subjects
it was not possible to find in the database matched ZZ and SZ
subjects with KCO data. Therefore, only 10 subjects per
group were included in the analysis of KCO. Data are
expressed as mean (S.D.) when normally distributed, as
median (range) otherwise. For normally distributed data,
differences within the means of the three groups were
analyzed by the analysis of variance (ANOVA) test. Differ-
ences between two groups at one time were established
using as post-hoc the Student’s t-test. When data were not
normally distributed, Kruskal–Wallis test was used for
comparisons within the three groups and Mann–Whitney
test was applied post-hoc to compare two groups at a time.
Correlations between lung function parameters and AAT
serum levels were tested using the non-parametric Spear-
man’s correlation coefficient. The results were considered
statistically significant for p-values below 5%.
Genotype and haplotype analysis
Genomic DNA was isolated from 10ml of peripheral blood
using standard procedures.24Genotypic analysis was per-
formed in the Null/Null subjects by direct sequencing after
PCR amplification of the all exons (1a, 1b, 1c, and 2–5) of
Primer sequences (obtained from K. Morgan, Nottingham,
UK) and annealing temperatures used for PCR amplification
are shown in Table 1. PCR products were purified using the
Qiagen PCR purification kit (Qiagen Benelux, Venlo, the
Netherlands). Sequencing was performed using the ABI Prism
BigDye Terminator Cycle Sequencing Ready Reaction kit
(Applied Biosystems, Arrington, UK) and products were
analyzed on the ABI 3730 sequencer. Total RNA was
extracted from fresh blood using the RNA Insta-Pure
extraction kit (Eurogentec, Maastricht, the Netherlands).
RNA was obtained from control individuals without A1ATD
and one A1ATD subject to determine the expression of a
novel Null mutation in blood monocytes. RNA from controls
and a homozygous Null patient was sequenced after RT-PCR
amplification using the forward primer of exon 4 and the
reverse primer of exon 5 to determine the allelic expression.
Polymorphic markers (D14S1054, D14S299, D14S1142, and
D14S749) flanking SERPINA1 were used to determine the
haplotype inheritance within a family with a compound
heterozygous Null mutation (family E). Table 2 shows the
primers sequences and conditions for PCR amplification. The
forward primers of D14S1054 and D14S1142 were labeled
with FAM fluorophores; the forward primers of D14S299 and
D14S749 were labeled with HEX. PCR fragments were
analyzed on ABI3700 sequencer.
Characteristics of Null subjects
The characteristics of Null subjects are presented in Table 3.
Genotypes of the Null subjects are presented in Table 4.
Seventy-five percent of the subjects were current or
ARTICLE IN PRESS
Primer sequences and annealing temperatures used for PCR amplification of the coding exons (2–5) of SERPINA1.
Exon Forward primer Reverse primerAnnealing (1C)Size (bp)Coding region (bp)
Primer sequences and annealing temperatures used for PCR amplification of polymorphic markers flanking
Marker Forward primerReverse primerAnnealing (1C) Product size (bp) Location (cM)
D14S299 H-GATCTCAATAAACATTGATACTGG CTGCATGAGCTAAAGCATACTG 55
SERPINA1 located at 98.88cM on chromosome 14 (Decode map).
L. Fregonese et al. 878
ex-smokers. The reason of assessment was lung disease in 9
out of 12 subjects (75%), while the other 3 (25%) were
ascertained because of family screening. Similar results for
ascertainment were found in the SZ subjects (75% lung
disease vs. 25% family screening) while in the ZZ group
reason for ascertainment was lung disease in 10 subjects
(83%) and family screening in 2 (17%). In the Null population,
lung function could vary between siblings from the same
family, even when the subjects had comparable age and
smoking history. In family A, sibling 2 (S2) and sibling 3 (S3)
had similar age and were both non-smokers, nevertheless S3
had almost double ppFEV1than S2. Interestingly, these two
subjects had similar KCOimpairment. Similarly, FEV1was
different between S1 and S2 in family D, with much higher
ARTICLE IN PRESS
Characteristics of the homozygous Null (Null/Null) subjects.
Family MemberAge SexSmoking Pack-
Genomic mutations in individuals with absent serum AAT.
FamilyFamily memberNucleotide changeMutated exons Amino-acid changes Name mutation
Sibling 1 (Null)
Sibling 2 (Null)
Sibling 3 (Null)
BSibling 1 (Null)
Sibling 2 (Null)
C Sibling 1 (Null)
Sibling 2 (Null)
Sibling 1 (Null)
Sibling 2 (Null)
Sibling 1 (Null)
F Sibling 1 (Null)C1195T/C1195T 5/5 P369L/P369L PiM Heerlen
GSibling 1 (Null)C1195T/C1195T 5/5P369L/P369L PiM Heerlen
?Nucleotides are numbered from the translation start site in exon 2.
yAmino-acids are numbered from the signal peptide cleavage site (the first methionine of A1AT is located at position 24).
Alpha-1 antitrypsin Null mutations and severity of emphysema879
FEV1in S2. Discordant impairment of ppFEV1and ppKCOwas
noted in individuals A-S2, B-S2, and G-S1.
Genotype of Null subjects
Genomic mutations of SERPINA1 on chromosome 14 were
characterized in all 12 subjects with absent serum AAT.
All subjects were shown to be homozygous for Null
mutations (Null/Null). Whenever possible, genotypic analy-
sis was performed also in the first’s degree relatives of the
Null/Null subjects. The results are shown in Table 2. In five
families (A, B, D, F, and G), previously published mutations
were identified (PiNull Bellingham and PiM Heerlen).
Interestingly, a new mutation was found in three individuals
from two different families (C and E). A nucleotide
substitution of a C to G in exon 2 at position 548 (counted
from the translation start site of SERPINA1) resulted in a
premature stop codon (Y160X). Both siblings of family C
were homozygous carriers of the novel Null mutation, while
the subject in family E was shown to be a heterozygous
carrier. Haplotype analysis of family E is shown in Figure 1.
One of the haplotypes (gray) of the proband with the
Null/Null genotype (sibling 1: S1) is shared by his father (F)
and brother; the other haplotype (white) is shared by his
mother and sister. Sequence analysis showed that the white
haplotype is associated with the PiM Heerlen mutation in
exon V of SERPINA1 and the gray haplotype is associated
with the novel Null mutation in exon 2. The novel mutation
is named PiNull Bredevoort after the place of residence of
the oldest known carrier (F).
We analyzed the expression of both alleles of the proband
S1 (containing the PiM Heerlen and the PiNull Bredevoort
mutation) in blood monocytes. Part of the DNA and cDNA
(RNA) sequences of exon 5 of SERPINA1 for the proband and
for a control are shown in Figure 2. The proband is
heterozygous for the T nucleotide of the PiM Heerlen
mutation, showing both alleles at DNA level (Figure 2a). At
cDNA level only the T nucleotide of the PiM Heerlen
mutation is observed, while the C-allele associated with
the PiNull Bredevoort mutation is missing (Figure 2b).
Control sequences show only the normal C-allele at both
DNA and cDNA level (Figure 2c and d). Sequence analysis of
exon 2 from father and brother of the proband confirmed
the presence of a heterozygous PiNull Bredevoort mutation
(Figure 2e and f).
Characteristics of Null/Null, ZZ, and SZ subjects
The demographic characteristics of all subjects, their lung
function values and protein serum levels are shown in
Table 5. The mean age difference between groups was 0.5
years (2.7) and the mean difference in smoking history 0.8
pack-years (4.1). Being the groups matched for age and
smoking history, no significant differences were observed in
these parameters (p40.5 for all comparisons).
Severity of emphysema
The comparisons of lung function measurements are
presented in Figure 3. Significant differences among the
groups were detected for both ppFEV1 (p ¼ 0.000) and
ppKCO(p ¼ 0.001). Post-hoc analysis showed lower values of
ppFEV1in Null/Null as compared to ZZ (p ¼ 0.002), and to SZ
subjects (p ¼ 0.000). Interestingly, a trend but not a
statistically significant difference in FEV1 was observed
between the ZZ and SZ group (p ¼ 0.062). Similar results
were obtained from the comparison of ppKco in the three
groups, with lower values in Null/Null as compared to ZZ
(p ¼ 0.000) and to SZ (p ¼ 0.007). No significant difference
was observed between ZZ and SZ (p ¼ 0.16).
Correlation with AAT serum levels
Considering ZZ and SZ as a combined group, we found a
positive correlation between severity of emphysema,
represented by ppFEV1and ppKCO, and serum levels of AAT
(Figure 4). The levels of AAT in the serum of Null/Null
subjects were all so low (o0.25mM) that, although even in
this group there was a positive correlation between lung
function and levels, the authors doubted whether inter-
subject differences in this group could be considered
In this study, we showed that subjects with absent serum
AAT due to homozygous Null mutations present more severe
pulmonary emphysema than subjects with ZZ or SZ muta-
tions. This finding, and the correlation between AAT levels
and lung function in SZ and ZZ that were matched for age
ARTICLE IN PRESS
flanking SERPINA1, and of four intragenic single nucleotide
polymorphisms. The G in exon 2 of SERPINA1 causes the novel
PiNull Bredevoort mutation (gray haplotype); the T in exon 5
causes the PiM Heerlen mutation (white haplotype).
Segregation in family E of four polymorphic markers
L. Fregonese et al.880