Superoxide dismutases, lung function and bronchial responsiveness in a general population

Online Data Supplement to paper:
Superoxide dismutases, lung function and bronchial responsiveness
in a general population
Authors: Mateusz Siedliński1, Cleo C. van Diemen1, Dirkje S. Postma2, Judith M. Vonk1
and H. Marike Boezen1
Institutions: 1Department of Epidemiology, 2Pulmonology, University Medical Center
Groningen, University of Groningen, Groningen, The Netherlands
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METHODS
Subjects
From all subjects information was collected on respiratory symptoms, smoking status,
age, and sex by the Dutch version of the British Medical Council standardized
questionnaire. Spirometry was performed using a standardized protocol [1].
Subjects with forced expiratory volume (FEV1) lower than 1.5 L and those who could not
perform a forced expiratory manoeuvre were excluded from responsiveness testing, as
were subjects suffering from heart disease, hypertension, or acute respiratory infections.
DNA extraction and genotyping
Neutrophil depots from centrifuged blood samples were collected and stored at –20°C.
DNA was extracted with a QIAamp DNA blood mini kit (Qiagen, Hilden, Germany) and
checked for purity and concentration with a NanoDrop ND-1000 UV–Vis
spectrophotometer (NanoDrop Technologies, Wilmington, DE). We genotyped DNA
samples of those subjects with more than 1,500 ng of isolated DNA available (n =
1,390).
Genotyping was performed using Applied Biosystems TaqMan® SNP Genotyping
Assays (Nieuwekerk aan de IJssel, The Netherlands) as described previously [1].
Sequences of primers and probes used for genotyping are shown in table S1.
Due to the low fluorescence signal at the end-point measurement and in order to ensure
genotyping quality, SNP rs2842958 in the SOD-2 was genotyped two times. Subjects
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with conflicting (n=18) genotypes for both independent genotyping rounds were
excluded from the further analyzes.
Statistics
Linear Mixed Effects (LME) models were used to investigate the effect of SOD SNPs on
the annual decline in FEV1. Time was defined as the time in years relative to the first
FEV1 measurement and corresponding age. FEV1 measurements were included from
the age of 30 years, because an individual’s maximal achieved lung function is assumed
to have been reached before that age and lung function is considered to be either in the
plateau or decline phase [2]. Variables included in the model were sex, packyears of
smoking, and the first available FEV1 after age 30 years and their interaction with time.
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TABLES
Table S1. Sequences of primers and probes
SNP
SOD-2
Ala16Val
rs4880
SOD-2
C7693T
rs2842958
SOD-3
Ala58Thr
rs2536512
SOD-3
Phe149Cys
Primers
Probe 1*
Probe 2*
Applied Biosystems assay ID: C___8709053_10
Forward:
ATGCCTGTAATCCCAGCTACTTG
Reverse:
CCTCCGCCTTTCAGGTTCAT
Forward:
CTGGCAGGAGGTCATGCA
Reverse:
CCGACGGCTGCACCT
Forward:
GCACCCGGGCGACT
Reverse:
CCGGCGCGGTACCT
SOD-3
Arg213Gly
rs1799895
CTGAGACACAAGAATT
TGAGACACGAGAATT
ACGACGACGGCACG
CGACGACGGCGCG
ACCGCGAAGTTGCCGA
CGCGCAGTTGCCGA
Applied Biosystems assay ID: C___2307506_10
LEGEND:
*Underlined bases indicate specific polymorphic site recognized by each probe
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Table S2. COPD prevalence in selected subgroups
COPD
SNP
MAF
Genotype
total
population,
COPD
P (2 df)*
ever-smokers
group,
n [%]§
n [%]§
Ala/Ala
44 [12.5]
36 [15.1]
Ala/Val
75 [11.7]
Val/Val
42 [13.4]
38 [17.9]
C/C
110 [12.9]
92 [15.6]
C/T
38 [9.6]
T/T
14 [23.0]
14 [29.8]
Ala/Ala
72 [12.8]
58 [14.6]
Ala/Thr
68 [12.4]
Thr/Thr
12 [8.0]
Arg/Arg
154 [12.0]
Arg/Gly
9 [17.6]
P (2 df)*
SOD-2
rs4880, Ala16Val
rs2842958, C7693T
0.49
0.21
0.74
0.01
61 [13.9]
31 [12.1]
0.41
0.008
SOD-3
rs2536512, Ala58Thr
rs1799895,Arg213Gly
0.34
0.02
0.27
58 [16.2]
0.34
11 [10.5]
0.20
128 [14.7]
9 [23.1]
0.17
LEGEND:
§ within
*in
2
each genotype
tests with all genotypes (2 df)
MAF = minor allele frequency in the investigated group
SNP = single nucleotide polymorphism
COPD = Chronic Obstructive Pulmonary Disease
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Table S3. BHR prevalence in selected subgroups
SNP
MAF
Genotype
BHR
P (2 df)*
BHR
total
ever-smokers
population,
group,
n [%]§
n [%]§
Ala/Ala
48 [40.0]
34 [42.5]
Ala/Val
88 [46.8]
Val/Val
47 [53.4]
32 [53.3]
C/C
119 [44.6]
93 [49.7]
C/T
52 [48.1]
T/T
13 [72.2]
10 [71.4]
Ala/Ala
84 [46.9]
70 [54.7]
Ala/Thr
74 [45.1]
Thr/Thr
21 [48.8]
Arg/Arg
184 [46.7]
Arg/Gly
3 [27.3]
P (2 df)*
SOD-2
0.45
rs4880, Ala16Val
rs2842958, C7693T
0.20
0.16
0.07
72 [54.5]
34 [50.7]
0.21
0.29
SOD-3
0.32
rs2536512, Ala40Thr
rs1799895, Arg213Gly 0.01
0.89
51 [49.0]
0.33
13 [40.6]
0.23
135 [50.2]
3 [42.9]
0.72
LEGEND:
§ within
*in
2
each genotype
tests with all genotypes (2 df)
MAF = minor allele frequency in the investigated group
SOD = superoxide dismutase
BHR = bronchial hyperresponsiveness
SNP = single nucleotide polymorphism
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Table S4. BHR prevalence at the last survey in never-smokers according to
Ala40Thr SOD-3 SNP
BHR
SOD-3
never-smokers
P (2 df)
Ala40Thr
group,
genotype
n [%]§
Ala/Ala
14 [27.5]
Ala/Thr
23 [38.3]
Thr/Thr
8 [72.7]
0.02
LEGEND:
§ within
*in
2
each genotype
tests with all genotypes (2 df)
SOD = superoxide dismutase
BHR = bronchial hyperresponsiveness
SNP = single nucleotide polymorphism
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Table S5. LME models analysis on FEV1 decline
Excess annual FEV1
Excess annual FEV1
decline for
SNP
MAF
decline for
p
p
heterozygotes, ml/yr
homozygotes mutant
[95% C.I.]*
ml/yr [95% C.I.]*
SOD-2
rs4880, Ala16Val
0.49
-0.96 [-3.26 – 1.34]
0.42
-0.23 [-2.93 - 2.47]
0.87
rs2842958, C7693T
0.21
0.91 [-1.22 - 3.04]
0.40
2.66 [-1.84 - 7.16]
0.25
rs2536512, Ala40Thr
0.34
0.05 [-2.05 - 2.15]
0.96
0.91 [-2.96 – 4.16]
0.58
rs1799895, Arg213Gly
0.02
4.28 [-1.40 - 9.96]
0.14
-
-
SOD-3
LEGEND:
*compared to homozygotes wild type (reference); negative values indicate faster FEV 1
decline
LME = Linear mixed effect
MAF = minor allele frequency
SOD = superoxide dismutase
FEV1 = forced expiratory volume in one second
SNP = single nucleotide polymorphism
C.I. = confidence intervals
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REFERENCES
(1) van Diemen CC, Postma DS, Vonk JM, Bruinenberg M, Schouten JP, Boezen HM.
A disintegrin and metalloprotease 33 polymorphisms and lung function decline in
the general population. Am J Respir Crit Care Med 2005; 172(3):329-333.
(2) Rijcken B, Weiss ST. Longitudinal analyses of airway responsiveness and
pulmonary function decline. Am J Respir Crit Care Med 1996; 154(6 Pt 2):S246S249.
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