ORIGINAL RESEARCH ARTICLE

Genetic Polymorphisms of Surfactant Protein D rs2243639,Interleukin (IL)-1b rs16944 and IL-1RN rs2234663 in ChronicObstructive Pulmonary Disease, Healthy Smokers,and Non-Smokers

Marianne Samir M. Issac • Wafaa Ashur •

Heba Mousa

� Springer International Publishing Switzerland 2014

Abstract

Background and Objectives Chronic obstructive pul-

monary disease (COPD) is a complex chronic inflamma-

tory disease that involves the activity of various

inflammatory cells and mediators. It has been suggested

that susceptibility to COPD is, at least in part, genetically

determined. The primary aim of this study was to investi-

gate the association between surfactant protein D (SFTPD)

rs2243639, interleukin (IL)-1b rs16944 and IL-1 receptor

antagonist (IL-1RN) rs2234663 gene polymorphisms and

COPD susceptibility, as well as examining the association

between the various IL-1RN/IL-1b haplotypes and pul-

monary function tests (PFT). Secondly, we aimed to

examine the influence of SFTPD rs2243639 polymorphism

on serum surfactant protein D (SP-D) level.

Methods A total of 114 subjects were recruited in this

study and divided into three groups: 63 COPD patients, 25

asymptomatic smokers, and 26 healthy controls. Polymer-

ase chain reaction-restriction fragment length polymor-

phism (PCR-RFLP) was performed for the detection of

SFTPD rs2243639 and IL-1b rs16944 polymorphisms.

Detection of variable numbers of an 86-bp tandem repeat

(VNTR) of IL-1RN was done using PCR. Serum SP-D level

was measured using enzyme linked-immunosorbent assay.

PFTs were measured by spirometry.

Results Carriers of the SFTPD AG and AA polymorphic

genotypes constituted 71.4 % of COPD patients versus 48 %

in asymptomatic smokers, with a statistically significant dif-

ference between the two groups (p = 0.049). Smokers who

were carriers of the polymorphic SFTPD rs2243639 A allele

(AG and AA genotypes) have a 2.708 times risk of developing

COPD when compared with wild-type GG genotype carriers

[odds ratio (OR) 2.708 (95 % CI 1.041–7.047)]. Forced

expiratory flow (FEF) 25–75 % predicted was higher in

IL-1RN*1/*1 when compared with *1/*2 (p = 0.013).

FEF25–75 % predicted in carriers of haplotype IL-1RN *1/IL-

1b T (49.21 ± 10.26) was statistically significantly higher

than in carriers of IL-1RN *2/IL-1b T (39.67 ± 12.64)

[p = 0.005]. Forced expiratory volume in 1 s (FEV1)/forced

vital capacity (FVC) in carriers of haplotype IL-1RN *1/IL-1bT (64.09 ± 6.39) was statistically significantly higher than in

carriers of IL-1RN *2/IL-1bT (59.44 ± 7.71) [p = 0.048].

There was no association between SFTPD rs2243639 geno-

types and serum SP-D level.

Conclusions Smokers who are carriers of the SFTPD AG

and AA polymorphic genotypes may be at a higher risk of

developing COPD when compared with wild-type GG

genotype carriers. IL-1RN rs2234663/IL-1b rs16944 hap-

lotypes influence FEF25–75 % predicted and FEV1/FVC.

SFTPD rs2243639 polymorphism did not influence serum

SP-D levels in our group of recruited subjects.

1 Introduction

Chronic obstructive pulmonary disease (COPD) is a dis-

ease state characterized by chronic airflow limitation

Electronic supplementary material The online version of thisarticle (doi:10.1007/s40291-014-0084-5) contains supplementarymaterial, which is available to authorized users.

M. S. M. Issac (&)

Department of Clinical and Chemical Pathology,

Faculty of Medicine, Cairo University,

El Saray St., El Manial, 11956 Cairo, Egypt

e-mail: mariannesamir@kasralainy.edu.eg;

mar_path@hotmail.com

W. Ashur � H. Mousa

Department of Chest Diseases, Faculty of Medicine,

Cairo University, Cairo, Egypt

Mol Diagn Ther

DOI 10.1007/s40291-014-0084-5

caused by a mixture of small airways disease (obstructive

bronchitis) and lung tissue parenchymal destruction

(emphysema). The relative contributions of these two

pathologic states vary from person to person [1]. Although

the most important risk factor for the development of

COPD is cigarette smoking, only 10–20 % of heavy ciga-

rette smokers develop COPD [2]. It was suggested that

apart from environmental factors [3], susceptibility to

COPD for each individual is, at least in part, genetically

determined [4]. Long-term cigarette smoking is associated

with activation of a cascade of inflammatory responses and

it is suggested that variants in several candidate genes,

which affect individual susceptibility to tobacco smoke

injury, might be implicated in the pathogenesis of COPD

[5].

Pulmonary surfactant, which lines the alveolar epithe-

lium, is highly active in host defense through bacterial

agglutination, opsonization, and viral neutralization [6].

Surfactant protein D (SP-D) is a member of the protein

family of collectins and serves as a pattern recognition

receptor (PRR) by binding selectively to the surfaces of

bacteria, viruses, and fungi, thereby enhancing phagocy-

tosis and intracellular killing [7, 8]. SP-D bridges the innate

and adaptive immune systems, with the ability to bind

several classes of immunoglobulins [9]. SP-D has pro- and

anti-inflammatory signaling functions [10]. In vitro data

have suggested that SP-D, through binding of different

receptors, acts in a dual manner to suppress or enhance

inflammation depending on binding orientation of the SP-D

molecule [11]. SP-D is mainly synthesized in alveolar type

II cells of the lung, and localizes to epithelial cells of

mucosa-associated tissues [12]. The origin of SP-D in the

vascular compartment is not known but it is generally

assumed that SP-D is released from the lung into the

bloodstream [13]. This is consistent with the observation

that the levels of SP-D in the blood increase in association

with certain types of lung injury [14]. Moreover, it has

been shown that expression of SP-D and its serum con-

centrations are substantially influenced by genetic variants

within the gene encoding for SP-D, the SFTPD gene which

is located on 10q22.2–23.1 and contains seven protein-

coding exons [15]. Within the SFTPD gene, the three

functional single nucleotide polymorphisms (SNPs)

Met11Thr, Ala160Thr and Ser270Thr are of potential func-

tional relevance [16]. One of these non-synonymous SNPs,

which is present in exon 4, is SFTPD c.478 G[A

(rs2243639) involving the substitution of alanine to thre-

onine at amino acid 160 (p.Ala160Thr) [17], which affects

collagen domain (disrupting collagen domain mediated

immunological interactions) [18].

Increasing evidence implicates SP-D in the pathogenesis

of COPD. SP-D enhances the clearance of apoptotic

cells [19], a potentially important mechanism in the

pathogenesis of COPD [20]. It was suggested that elevated

serum concentrations of SP-D are a biomarker for COPD

and may ultimately have clinical relevance in the evalua-

tion and design of novel drug therapies [21].

The chronic inflammatory state in COPD suggests an

imbalance between pro- and anti-inflammatory mediators.

Interleukin (IL)-1 and its antagonist may influence the rate

of decline in lung function because of the effects of IL-1 on

neutrophil function and chemotaxis. IL-1 stimulates the

synthesis of IL-8, a potent chemotactic agent for neutro-

phils, and it also induces release of neutrophil elastase [22].

IL-1 promotes the adhesion of neutrophils and other cells

by enhancing the expression of adhesion molecules such as

ICAM-1, VCAM-1, and L-selectin [23]. IL-1b is also

known to increase the synthesis of the collagenase [24] that

was reported to cause the development of emphysema of

mouse lungs [25].

The genes of the IL-1 complex, which are located on

chromosome 2q13, encode for three proteins: IL-1a, IL-1b,

and IL-1 receptor antagonist (IL-1RN). Each of the genes is

polymorphic, and there is evidence that certain alleles are

associated with susceptibility to inflammation [26]. An

SNP (rs16944) has been identified at bp position -511 in

the promoter region of the IL-1b gene with a substantial

influence on its serum level [27]. The IL-1RN gene has a

penta-allelic polymorphic site in intron 2 (rs2234663)

containing variable numbers of an 86-bp tandem repeat

(VNTR) sequence. IL-1RN binds to the IL-1 receptor that

inhibits IL-1a and IL-1b binding. Because the effect of

IL-1b is countered by its endogenous antagonist IL-1RN, it

is also considered to have an important role in inflamma-

tion [26].

In view of the role of SP-D, IL-1b, and IL-1RN in

inflammation, and hence the development and progression

of COPD, the first aim of our study was to investigate the

association between SFTPD (rs2243639), IL-1b (rs16944)

and IL-1RN (rs2234663) gene polymorphisms and COPD.

This includes examining the association between the vari-

ous IL-1RN/IL-1b haplotypes and pulmonary function tests

(PFT). Our second aim was to examine the influence of

SFTPD (rs2243639) on serum SP-D concentrations in

COPD patients, asymptomatic smokers and healthy non-

smokers.

2 Subjects and Methods

2.1 Subjects

Our study included 114 male subjects who were divided

into three groups: group 1 (non-smokers), which included

26 non-smoking healthy controls with normal spirometry;

group 2 (asymptomatic smokers), which included 25

M. S. M. Issac et al.

smokers with no clinical suspicion of COPD and a normal

spirometry; and a COPD group (group 3), which included

63 smokers with COPD. The recruited COPD patients were

randomly chosen from those attending the Chest Outpatient

Clinic and Chest Department of Kasr Al-Ainy Hospital,

Faculty of Medicine, Cairo University, over 14 months.

COPD patients were assigned to the study according to the

following criteria: age above 40 years, smoking history

above 20 pack-years, clinical and radiological examination

fulfilled the diagnostic criteria of COPD [28], a post-

bronchodilator forced expiratory volume in 1 s (FEV1)/

forced vital capacity (FVC) ratio of B0.7 and Global Ini-

tiative for Chronic Obstructive Lung Disease (GOLD)

stage I (FEV1 C80 % of the predicted value), II (FEV1

50–80 % of the predicted value), III (FEV1 30–50 % of the

predicted value), or IV (FEV1\30 % of the predicted value)

[28]. Exclusion criteria included diseases other than COPD

and working in industrial areas. Group 1 (non-smoking con-

trol subjects) and group 2 (asymptomatic smokers; C20 pack-

years) were enrolled if they were aged 40–75 years and

exhibited normal lung function (post-bronchodilator FEV1 of

[85 % of the predicted value and FEV1/FVC of[0.7). All

subjects underwent standardized spirometry following

200 lg (two puffs) Ventolin, salbutamol sulphate, Glaxo-

SmithKline (GSK), with reversible airflow obstruction being

defined as an increase in FEV1 of 15 % and C200 mL [29].

All recruited subjects were subjected to medical history

taking, clinical examination, plain chest x-ray postero-

anterior view, and pre- and post-bronchodilator spirometric

measurements of FEV1, FEV1/FVC and forced expiratory

flow (FEF) 25/75 % of predicted. Normal pulmonary

function test (PFT) was considered as FEV1 % of predicted

[85 %, FEV1/FVC % of predicted [70 %. Bronchodila-

tors were stopped 24 h before PFT. Calculation of smoking

index is based on multiplication of the number of cigarettes

smoked per day 9 total duration smoked in years [30]. To

compare serum SP-D levels between the three groups, 15

healthy control subjects, 25 asymptomatic smokers, and 20

COPD patients (in a stable condition) were randomly

chosen. The study was approved by the local ethical

committee and all patients gave informed consent.

2.2 Laboratory Methods

2.2.1 Blood Sampling

Five milliliters of blood were collected; two milliliters of

blood were collected in a tube containing EDTA as an

anticoagulant for DNA extraction and stored at -20 �C,

while three milliliters of blood were transferred into plain

tubes, allowed to clot for at least 30 min and centrifuged at

1,0009g for 15 min. Serum was then aliquoted and stored

frozen at -20 �C until serum SP-D measurement.

2.2.2 Genotyping

Genomic DNA was isolated from peripheral blood leuko-

cytes by means of a genomic DNA purification kit

according to the manufacturer’s instructions (ThermoSci-

entific, USA).

2.2.2.1 Genotyping of SFTPD Ala160Thr (G[A)

[rs2243639] by Polymerase Chain Reaction-Restriction

Fragment Length Polymorphism (PCR-RFLP) Each

polymerase chain reaction (PCR) was carried out with

100 ng of genomic DNA, 20 pmol of each primer, 12.5 lL

Master Mix (Dream TaqTM, Green PCR Master Mix;

ThermoScientific, USA) in a total volume of 25 lL.

SFTPD genotyping was performed as previously

described [15] with the primer pair (forward, 50-GTT CCT

GTG TGT TCC TTC TTC AGG AGA AGT AGG-30, and

reverse, 50-CCA GCT CTT TCC ACT GCT CAC CTG

CTC ACC CTG-30) [Bioneer, Korea] with an initial

denaturation at 95 �C for 15 min followed by 35 cycles of

94 �C for 30 s, 65 �C for 30 s, and 72 �C for 30 s, and a

final extension step at 72 �C for 10 min. PCR product was

digested by restriction endonuclease DraIII (ThermoSci-

entific, USA) and visualized by electrophoresis on a 3 %

agarose gel stained with ethidium bromide. Alleles were

coded as G: 161 bp, and A: 132 ? 29 bp as shown in

supplementary Figure IA.

2.2.2.2 Genotyping of IL-1b (-511 C[T) [rs16944] by

PCR-RFLP IL-1b-511 C/T genotyping was performed as

previously described [31] with the primer pair (forward, 50-TGG CAT TGATCT GGT TCATC-30, and reverse, 50-GTT TAG GAATCT TCC CAC TT-30) [Bioneer, Korea]

with initial denaturation at 95 �C for 1 min followed by 30

cycles of 95 �C for 30 s, 55 �C for 30 s, and 72 �C for

30 s, with final extension at 70 �C for 7 min using a PCR

Thermal Cycler (ThermoHybaid, UK). PCR products were

digested by restriction endonuclease Aval (ThermoScien-

tific, USA) and visualized by electrophoresis on a 3 %

agarose gel stained with ethidium bromide. Alleles were

coded as T: 304 bp, and C: 190 and 114 bp as shown in

supplementary Figure IB.

2.2.2.3 Genotyping of IL-1RN VNTR (rs2234663) by

PCR IL-1RN genotyping was performed as previously

described [31] with the primer pair (forward, 50-CTC AGC

AAC ACT CCT AT-30, and reverse, 50-TCC TGG TCT

GCA GGT AA-30) [Bioneer, Korea] with initial denatur-

ation at 94 �C for 4 min followed by 32 cycles of 94 �C for

1 min, 50 �C for 1 min, and 72 �C for 1 min, with final

extension at 72 �C for 10 min using a PCR thermal cycler.

PCR products were analyzed by electrophoresis on a 1.5 %

agarose gel stained with ethidium bromide. Alleles 1–5 (IL-

SFTPD, IL-1b and IL-1RN Polymorphisms in COPD

1RN 1 to IL-1RN 5) were detected according to their sizes

relative to a 100-bp DNA ladder: allele 1 (four repeats),

410 bp; allele 2 (two repeats), 240 bp; allele 3 (five

repeats), 500 bp; allele 4 (three repeats), 325 bp; and allele 5

(six repeats), 595 bp as shown in supplementary Figure IC.

2.2.3 Determination of Serum Level of Surfactant Protein

D (SP-D) Using Enzyme-Linked Immunosorbent

Assay

Serum levels of SP-D were measured by operators who were

blind to an individual’s lung disease using a commercially

available enzyme-linked immunosorbent assay test (Bio-

Vendor, Brno, Czech Republic) according to the manufac-

turer’s instructions. Samples were tested at elevenfold dilution

with the dilution buffer supplied by the manufacturer. The

concentration of SP-D in the diluted samples was interpolated

from the standard curve of recombinant human SP-D and then

corrected for the dilution factor.

2.2.4 Statistical Analysis

Data were statistically described in terms of

mean ± standard deviation (SD), or frequencies (number

of cases) and percentages when appropriate. Serum SP-D

levels were transformed to a natural logarithm to achieve

normality of distribution. Odds ratio (OR) and the 95 %

confidence interval (CI) were calculated for all studied

genotypes between COPD and the other groups. Compar-

ison of numerical variables between the study groups was

done using Student’s t-test for independent samples in

comparing two groups when normally distributed. Com-

parison of numerical variables between more than two

groups was done using the one-way analysis of variance

(ANOVA) test with post hoc multiple two-group compar-

isons in normal data. Within-group comparison of numer-

ical variables was done using the Wilcoxon signed rank test

for paired (matched) samples. The observed genotype

frequencies were compared with those expected under

Hardy–Weinberg equilibrium using the Chi-square test.

For comparing categorical data, the Chi-square test was

performed. Exact test was used instead when the expected

frequency was less than 5.

Correlation between various variables was done using

the Pearson moment correlation equation for linear relation

in normally distributed variables. Multivariate regression

analysis models were used to test for the significant inde-

pendent variables affecting the development of COPD as

well as the studied PFTs. p-Values less than 0.05 was

considered statistically significant. All statistical calcula-

tions were done using the computer program SPSS (Sta-

tistical Package for the Social Sciences; SPSS Inc.,

Chicago, IL, USA) version 15 for Microsoft Windows.

3 Results

3.1 Characteristics of the Studied Groups

The characteristics of the studied groups are shown in

Table 1. Smoking history (smoking index and number of

pack-years) did not show a statistically significant dif-

ference when compared between asymptomatic smokers

and COPD patients, which denote that they were equally

exposed to tobacco smoke. Serum SP-D was statistically

significantly higher in asymptomatic smokers when

compared with healthy controls (p = 0.015). There was

no statistically significant difference when comparing

serum SP-D levels between healthy controls and COPD

patients (p = 0.420), and between asymptomatic smok-

ers and COPD patients (p = 0.434). FEV1 %,

FEF25–75 % predicted and FEV1/FVC showed a statis-

tical significant decrease in COPD patients when com-

pared with both healthy controls and asymptomatic

smokers.

3.2 Association of SFTPD rs2243639, IL-1b rs16944,

and IL-1RN rs2234663 Gene Polymorphisms

and Chronic Obstructive Pulmonary Disease

(COPD)

The genotype and allele frequencies of SFTPD

rs2243639, IL-1b rs16944, and IL-1RN rs2234663 did

not show deviation from the Hardy–Weinberg equilib-

rium and were not statistically significantly different

when compared between healthy controls, asymptomatic

smokers, and COPD patients, as shown in Table 2.

However, when the studied subjects were stratified

according to wild-type genotype carriers and carriers of

heterozygous and homozygous polymorphic genotypes,

it was observed that carriers of the SFTPD heterozygous

(AG) and homozygous (AA) polymorphic genotypes

constituted 71.4 % of COPD patients versus 48 % in

asymptomatic smokers, with a statistically significant

difference between the two groups (p = 0.049). More-

over, smokers who were carriers of the polymorphic

SFTPD 160 A allele (AG and AA genotypes) have a

2.708 times risk of developing COPD when compared

with wild-type GG genotype carriers [OR 2.708 (95 %

CI 1.041–7.047)].

3.3 Association of Pulmonary Function Test

Parameters with SFTPD rs2243639, IL-1b rs16944,

and IL-1RN rs2234663 Gene Polymorphisms

in COPD Patients

On comparing each of the pulmonary function test

parameters with SFTPD rs2243639, IL-1b rs16944, and IL-

M. S. M. Issac et al.

1RN rs2234663 gene polymorphisms in COPD patients, it

was observed that FEF25–75 % predicted was higher in

IL-1RN *1/*1 when compared with *1/*2 (p = 0.013), as

shown in Table 3.

3.4 Association of Pulmonary Function Test

Parameters with Haplotypes of IL-1RN rs2234663

and IL-1b rs16944 Gene Polymorphisms in COPD

Patients

The frequencies of haplotypes for both IL-1RN

rs2234663 and IL-1b rs16944 were calculated in the

COPD patients. The four haplotypes were *1/C, *1/T,

*2/C, and *2/T. Both FEV1 % and FVC % did not

show statistically significant difference when compared

between the four haplotypes, while there was a

statistically significant difference on comparing

FEF25–75 % predicted and FEV1/FVC between the

different haplotypes in COPD patients. FEF25–75 %

predicted [mean (±SD) 49.21 ± 10.26] in carriers of

haplotype *1/T was statistically significantly higher

than [mean (±SD) 39.67 ± 12.64] in carriers of *2/T

(p = 0.005). FEV1/FVC [mean (±SD) 64.09 ± 6.39] in

carriers of haplotype *1/T was statistically significantly

higher than [mean (±SD) 59.44 ± 7.71] in carriers of

*2/T (p = 0.048).

3.5 Association of SFTPD rs2243639, IL-1b rs16944,

and IL-1RN rs2234663 Genotypes with Global

Initiative for Chronic Obstructive Lung Disease

(GOLD) Classification for COPD

There was no association between SFTPD rs2243639,

IL-1b rs16944, and IL-1RN rs2234663 genotypes with

GOLD classification for COPD: GOLD I (mild), GOLD II

(moderate), and GOLD III (severe), as shown in supple-

mentary Table 1 (see electronic supplementary material).

However, when the studied subjects were stratified

according to IL-1RN wild-type genotype (*1) carriers and

carriers of polymorphic (*2) genotypes, it was observed

that carriers of the IL-1RN heterozygous and homozygous

(*1/*2 and *2/*2) polymorphic genotypes constituted 56.2 %

of GOLD III versus 14.3 % in GOLD I, but the difference did

not reach statistical significance (p = 0.089).

3.6 Association of SFTPD rs2243639 Gene

Polymorphism with Serum SP-D in the Studied

Groups

There was no association between SFTPD rs2243639

genotypes and serum SP-D level in healthy controls

(p = 0.143), asymptomatic smokers (p = 0.965), and

COPD patients (p = 0.725), as shown in Table 4.

Table 1 Demographic data of

the studied groups

Quantitative data are

represented as mean ± SD;

qualitative data are represented

as frequency (%). N = serum

SP-D was measured in 15

healthy controls, 25

asymptomatic smokers, and 20

COPD patients. Numbers

carrying the same letters do not

show statistically significant

difference

BMI body mass index, FEV1

forced expiratory volume in 1 s,

FVC forced vital capacity, FEF

forced expiratory flow, GOLD

Global Initiative for Chronic

Obstructive Lung Disease, SP-D

surfactant protein D, S steroids,

BD bronchodilators, COPD

chronic obstructive pulmonary

disease

* Comparison between

asymptomatic smokers and

COPD patients

Healthy controls

(n = 26)

Asymptomatic smokers

(n = 25)

COPD patients

(n = 63)

p value

Age (years) 55.65 ± 8.85 54.92 ± 7.12 56.81 ± 9.72 0.947

BMI (kg/m2) 20.5 ± 1.75 20.8 ± 1.8 21.0 ± 1.9 0.552

Smoking history

Smoking index 0 577 ± 218.89 638.10 ± 340.99 0.406*

Number of pack-years 0 29 ± 11 32 ± 17 0.324*

Current smokers 0 23 (92) 49 (77.8)

Ex-smokers 0 2 (8) 14 (22.2)

Serum SP-D (ng/ml)n

[natural log]

3.61 ± 0.49a 4.19 ± 0.57b 3.92 ± 0.70a,b 0.018

FEV1 % predicted 79.4 ± 21.5a 76.24 ± 11.87a 61.62 ± 14.81b \0.001

FVC % predicted 69.7 ± 18.9 64.32 ± 10.08 71.29 ± 16.38 0.182

FEF25–75 % predicted 88.7 ± 28a 92.24 ± 21.12a 45.79 ± 12.13b \0.001

FEV1/FVC 87.55 ± 6.28a 89.08 ± 6.46a 61.87 ± 7.69b \0.001

GOLD classification

I (mild) 7 (11.1)

II (moderate) 40 (63.5)

III (severe) 16 (25.4)

Treatment

S 21 (33.3)

BD 19 (30.2)

BD ? S 8 (12.7)

No treatment 15 (23.8)

SFTPD, IL-1b and IL-1RN Polymorphisms in COPD

3.7 Correlation between Pulmonary Function Test

Parameters with Age, Smoking Index, and Serum

SP-D

There was a significant negative correlation between

FEV1 % and smoking index (r = -0.350; p = 0.005),

FEF25–75 % predicted and age (r = -0.406; p = 0.001);

FEF25–75 % predicted and smoking index (r = -0.411;

p = 0.001), serum SP-D and FEF25–75 % predicted in

COPD patients (r = -2.2; p = 0.04), FEV1/FVC and age

(r = -0.266; p = 0.035), and FEV1/FVC and smoking

index (r = -0.361; p = 0.004).

3.8 Multivariate Logistic Regression Analysis

To examine the contribution of the different variables

[age, smoking history, log serum SP-D, SFTPD

(rs2243639), IL-1b (rs16944), and IL-1RN (rs2234663)

polymorphic genotypes] to the development of COPD,

where both the healthy controls and asymptomatic

smokers acted as the COPD negative cases. This analysis

was carried out on the recruited subjects, in whom serum

SP-D was measured. As shown in Table 5, age and

smoking history contributed significantly to the develop-

ment of COPD.

3.9 Multivariate Linear Regression Analysis

To detect the association between age, smoking history,

natural log of serum SP-D, and the studied PFTs. Age and

smoking history were significantly associated with

decreased FEV1 %, FEF25–75 % predicted, and FEV1/

FVC (p = 0.001, 0.001, and 0.002, respectively, and

p = 0.040, 0.020, and 0.002 respectively).

Table 2 Genotype and allele

frequencies of SFTPD

rs2243639, IL-1b rs16944 and

IL-1RN rs2234663

polymorphisms in the studied

groups

Data presented as frequency (%).

Numbers carrying different letters

show statistically significant

difference

SFTPD surfactant protein D, IL-

1b interleukin-1b, IL-1RN

interleukin-1 receptor antagonist,

COPD chronic obstructive

pulmonary disease, HWE Hardy–

Weinberg equilibrium

Healthy controls

(n = 26)

Asymptomatic smokers

(n = 25)

COPD patients

(n = 63)

p value

SFTPD rs2243639 genotypes

GG (n = 43) 12 (46.2) 13 (52) 18 (28.6) 0.260

AG (n = 57) 11 (42.3) 10 (40) 36 (57.1)

AA (n = 14) 3 (11.5) 2 (8) 9 (14.3)

GG (n = 43) 12 (46.2) 13 (52) 18 (28.6) 0.049

AG ? AA (n = 71) 14 (53.8) 12 (48)a 45 (71.4)b

G allele (n = 143) 35 (67.3) 36 (72) 72 (57.1) 0.136

A allele (n = 85) 17 (32.7) 14 (28) 54 (42.9)

HWE p-value 0.7629 0.9367 0.0816

IL-1b rs16944 genotypes

CC (n = 22) 6 (23.1) 7 (28) 9 (14.3) 0.499

CT (n = 61) 15 (57.7) 11 (44) 35 (55.5)

TT (n = 31) 5 (19.2) 7 (28) 19 (30.2)

CC (n = 22) 6 (23.1) 7 (28) 9 (14.3) 0.218

CT ? TT (n = 92) 20 (76.9) 18 (72) 54 (85.7)

C allele (n = 105) 27 (51.9) 25 (50) 53 (42.1) 0.398

T allele (n = 123) 25 (48.1) 25 (50) 73 (57.9)

HWE p-value 0.1269 0.2301 0.1609

IL-1RN rs2234663 genotypes

*1/*1 (n = 71) 14 (53.8) 19 (76) 38 (60.3) 0.351

*1/*2 (n = 35) 11 (42.4) 5 (20) 19 (30.2)

*2/*2 (n = 8) 1 (3.8) 1 (4) 6 (9.5)

*1/*1 (n = 71) 14 (53.8) 19 (76) 38 (60.3) 0.218

*1/*2 ? *2/*2 (n = 43) 12 (46.2) 6 (24) 25 (39.7)

*1 allele (n = 177) 39 (75) 43 (86) 95 (75.4) 0.274

*2 allele (n = 51) 13 (25) 7 (14) 31 (24.6)

HWE p-value 0.116 0.0902 0.0816

M. S. M. Issac et al.

4 Discussion

COPD is a complex chronic inflammatory disease that

involves the activity of various inflammatory cells and

mediators [32]. Our results show that the frequency of

SFTPD rs2243639 genotypes and alleles were not statisti-

cally significantly different when compared between con-

trols, asymptomatic smokers, and COPD patients; this is in

concordance with the findings of previous studies [33, 34]

in the American and Mexican populations, respectively.

However, we observed that smokers who were SFTPD AG

and AA genotype carriers presented increased risk of

developing COPD when compared with wild-type GG

genotype carriers. This is in discordance with the results of

Shakoori et al. [18] who reported no association of SFTPD

rs2243639 with risk for COPD in the Pakistani population.

Table 3 Association of

pulmonary function test

parameters with SFTPD

rs2243639, IL-1b rs16944 and

IL-1RN rs2234663 gene

polymorphisms in COPD

patients

Data are represented as

mean ± standard deviation.

Numbers carrying the same

initials are not statistically

significantly different

COPD chronic obstructive

pulmonary disease, FEV1

forced expiratory volume in 1 s,

FVC forced vital capacity, FEF

forced expiratory flow, SFTPD

surfactant protein D, IL-1binterleukin-1b, IL-1RN

interleukin-1 receptor

antagonist

* Statistically significant

Pulmonary function test parameter Genotype (n) Value p value

SFTPD rs2243639

FEV1 % predicted GG (18) 63.33 ± 17.67 0.816

AG (36) 61.25 ± 13.70

AA (9) 59.67 ± 14.25

FVC % predicted GG (18) 74.28 ± 19.34 0.664

AG (36) 70.11 ± 15.30

AA (9) 70.00 ± 15.20

FEF25–75 % predicted GG (18) 39.94 ± 15.00 0.082

AG (36) 47.58 ± 10.85

AA (9) 50.33 ± 5.61

FEV1/FVC GG (18) 60.89 ± 9.34 0.459

AG (36) 62.89 ± 6.96

AA (9) 59.78 ± 7.05

IL-1b rs16944

FEV1 % predicted CC (9) 61.37 ± 14.53 0.583

CT (35) 62.91 ± 15.22

TT (19) 57.11 ± 14.51

FVC % predicted CC (9) 71.37 ± 18.47 0.580

CT (35) 72.57 ± 16.40

TT (19) 66.11 ± 11.43

FEF25–75 % predicted CC (9) 44.37 ± 11.04 0.709

CT (35) 46.94 ± 13.25

TT (19) 44.33 ± 10.28

FEV1/FVC CC (9) 63.26 ± 6.76 0.640

CT (35) 61.37 ± 7.81

TT (19) 60.89 ± 9.48

IL-1RN rs2234663

FEV1 % predicted *1/*1 (38) 63.68 ± 15.03 0.239

*1/*2 (19) 56.79 ± 14.32

*2/*2 (6) 63.83 ± 13.56

FVC % predicted *1/*1 (38) 71.63 ± 15.75 0.773

*1/*2 (19) 69.47 ± 18.69

*2/*2 (6) 74.83 ± 14.29

FEF25–75 % predicted *1/*1 (38) 49.18 ± 10.46a 0.015*

*1/*2 (19) 39.58 ± 13.22b

*2/*2 (6) 44.00 ± 12.03a,b

FEV1/FVC *1/*1 (38) 63.05 ± 7.40 0.311

*1/*2 (19) 59.79 ± 8.19

*2/*2 (6) 61.00 ± 7.54

SFTPD, IL-1b and IL-1RN Polymorphisms in COPD

There are several possible reasons for this discrepancy,

among which is the ethnic variation. In the study of

Shakoori et al. [18], the recruited controls included never

smokers and smokers grouped together in one group, with a

smoking history comprising a lower number of pack-years

(18 ± 17 in controls vs. 48 ± 31 in COPD patients),

while the positive finding in our study evolved on com-

paring COPD patients with asymptomatic smokers with

comparable smoking history (29 ± 11 pack-years in

asymptomatic smokers vs. 32 ± 17 in COPD patients).

The SFTPD rs2243639 SNP changes the codon for amino

acid 160 from GCA (Ala) to ACA (Thr) which is located in

the collagen domain of SFTPD [35]. However, association

between SFTPD rs2243639 genotypes and several respi-

ratory diseases was reported with contradictory findings.

The Thr160 allele was associated with severe respiratory

syncytial virus (RSV) disease in young children [35, 36],

while haplotypes containing the Thr160 allele were protective

in premature infants with respiratory distress syndrome (RDS)

[37]. Furthermore, Krueger et al. [38] observed that SFTPD

rs2243639 does not play a major role in the development of

bronchial asthma in German children.

Our results show the lack of association between SFTPD

rs2243639 genotypes and PFT among COPD patients. This

contradicts the findings of van Diemen et al. [39] who reported

that SFTPD rs2243639 showed evidence suggestive of asso-

ciation with FEV1/inspiratory vital capacity (IVC) and con-

cluded that SFTPD may be important in the progression of

COPD in the Genetic Research in Isolated Populations (GRIP)

program that is based in a recently genetically isolated pop-

ulation from the south-western part of The Netherlands. The

GRIP population was different from ours in gender and age

distribution, as well as ethnic background.

Our findings show no association of IL-1b rs16944 and

IL-1RN rs2234663 gene polymorphisms with COPD,

which is in agreement with previous reports from Japan,

the US, The Netherlands, and Egypt [3, 40–42]. In dis-

cordance with our results, in their meta-analysis Mei et al.

[43] concluded that IL-1b polymorphisms may play a role

in the susceptibility to COPD in an ethnicity-specific

manner, where in the Caucasian population, the risk of

COPD was associated positively with the IL-1b-511 T

allele. In contrast, Asada et al. [44] observed that the T

allele at -511 SNP was overrepresented in asymptomatic

smokers, and concluded that the homozygote subjects were

particularly at lower risk for COPD in the Japanese pop-

ulation. Danilko et al. [45] reported no significant differ-

ence in genotype or allele frequency of IL-1b rs16944

between COPD patients and healthy controls, while IL-

1RN *2/*2 was significantly higher in COPD patients when

compared with healthy controls, and the risk of COPD was

increased twofold in carriers of genotype IL-1RN*2/*2 in

the studied Russian patients.

Our results show that among COPD patients,

FEF25–75 % predicted showed a statistically significant

decrease in carriers of IL-1RN *1/*2 when compared with

Table 4 Serum SP-D level in the different SFTPD rs2243639 genotypes in the studied groups

Studied groups SFTPD rs2243639 genotypes p value

GG AG AA

Serum SP-D level (ng/ml) in healthy controls (n = 15) (n = 4) (n = 8) (n = 3) 0.143

3.83 ± 0.48 3.45 ± 0.57 3.76 ± 0.04

Serum SP-D level (ng/ml) in asymptomatic smokers (n = 25) (n = 13) (n = 10) (n = 2) 0.965

4.23 ± 0.64 4.12 ± 0.56 4.24 ± 0.09

Serum SP-D level (ng/ml) in COPD patients (n = 20) (n = 3) (n = 13) (n = 4) 0.725

3.67 ± 0.41 3.98 ± 0.73 3.90 ± 0.85

Natural log serum SP-D is represented as mean ± SD

SP-D surfactant protein D, COPD chronic obstructive pulmonary disease

Table 5 Multivariate logistic regression analysis to examine the contribution of the different variables to the development COPD

Variable p value Odds ratio 95 % CI

Age (years) 0.002 1.242 1.083–1.424

Smoking history (pack-years) 0.023 1.004 1.001–1.007

Natural log serum SP-D (ng/ml) 0.453 1.600 0.469–5.455

SFTPD (rs2243639) polymorphic genotypes 0.854 1.170 0.220–6.213

IL-1b (rs16944) polymorphic genotypes 0.233 0.293 0.039–2.206

IL-1RN (rs2234663) polymorphic genotypes 0.779 0.772 0.127–4.702

COPD chronic obstructive pulmonary disease, SP-D surfactant protein D, IL-1b interleukin-1b, IL-1RN interleukin-1 receptor antagonist

M. S. M. Issac et al.

carriers of the IL-1RN *1/*1 (p = 0.013). FEF25–75 %

predicted is the spirometric variable most commonly cited

as an indicator of small airways obstruction [46]. Small

airways are usually defined as airways less than 2 mm in

internal diameter, which includes airways from the fourth

to fourteenth generation of branching [1]. Small airways

disease is a key factor leading to airway obstruction in

early COPD [46].

Since the genes of the IL-1 family are closely linked and

the polymorphic variants of IL-1RN and IL-1b may have a

combined effect on the synthesis of IL-1b and IL-1RN

[47], we thought it would be reasonable to study the dis-

tributions of haplotype combinations for these two genes.

We observed that FEF25–75 % predicted and FEV1/FVC

in carriers of haplotype IL-1RN *1/IL-1b T were statisti-

cally significantly higher than in carriers of IL-1RN *2/IL-

1b T.

It was observed that carriers of the IL-1RN (*1/*2 and

*2/*2) constituted 56.2 % of GOLD III versus 14.3 % in

GOLD I, but the difference did not reach statistical sig-

nificance. Putting all these data together, this may suggest

that allele *2 of IL-1RN is associated with decreased pul-

monary function. It was previously reported that the IL-

1RN*2 genotype has been associated with pro-inflamma-

tory responses more severe and more prolonged than those

of other IL-1RN genotypes [48–50]. The repeat region of

IL-1RN contains three potential protein-binding sites.

Therefore, the variable copy number of this repeat region

may have functional significance [51].

Hurme and Santtila [52] found that IL-1RN*2 was

associated with higher plasma levels of IL-1RN than IL-

1RN*1, but only in individuals who also had the T allele of

IL-1b. It has been proposed that IL-1RN is subject to

coordinate regulation by IL-1b and IL-1RN [52]. Further-

more, IL-1RN*2 was reported to be associated with

reduced production of IL-1a protein, and enhanced pro-

duction of IL-1b by monocytes in vitro [53, 54]. A pro-

posed explanation was that, in carriers of genotype IL-

1RN*2/*2, genetically predisposed in favor of IL-1RN

production, this protein is produced in quantities exceeding

those required for an adequate inflammatory response,

which causes compensatory production of even larger IL-

1b quantities. This, in turn, leads to a further increase in IL-

1RN production and to a prolonged inflammatory reaction

[52]. Theoretically, increased IL-1RN production should

reduce the IL-1 binding to its receptors, thus contributing

to anti-inflammatory effects. It was postulated that IL-

1RN*2 predominantly increases the level of IL-1b and

therefore leads to an imbalance in the IL-1b/IL-1RN ratio,

resulting in an increased susceptibility or more severe

outcome of inflammatory diseases [41].

Alternatively, it could be hypothesized that the IL-

1RN*2 allele is a marker for a linked disease-associated

locus and may not be a direct disease causing allele [41].

IL-1b and IL-1RN variants may affect the course of

inflammation. In particular, IL-1b-511C and IL-1RN*1

alleles determine adequate production of the respective

cytokines and a balanced functioning of the IL-1 system. In

carriers of the IL-1b -511T allele, who are genetically

predisposed to enhanced IL-1b production, inflammation

takes a more acute course [47].

In discordance with our results, Joos et al. [41] observed

that haplotype IL-1RN*1/IL-1b-511 T was more ‘frequent’

in patients with rapid lung function decline compared with

the IL-1RN*2/IL-1b-511T haplotype.

Our findings show that the differences in the distribution

of IL-1b and IL-1RN genotypes and alleles were not sta-

tistically significant in different GOLD stages of COPD,

which is concordant with the results of Danilko et al. [45].

The discrepancies in the findings of our study compared

with previous studies might be explained by ethnic varia-

tion, differences in study design, and number of recruited

subjects.

The course of an inflammatory process will be determined

by the balance between pro-and anti-inflammatory media-

tors. IL-1b and IL-1RN are logical candidate genes in

inflammatory airway diseases because of the critical role of

IL-1 in inflammation. The actions of IL-1 include activation

of T and B cells, and chemotaxis of neutrophils and macro-

phages. In addition, IL-1 stimulates the production of other

cytokines such as tumor necrosis factor (TNF) and granulo-

cyte macrophage colony-stimulating factor (GMCSF) [55].

Our results show that serum SP-D level was lower in

COPD patients when compared with asymptomatic smok-

ers; however, the difference was not statistically significant

and this is in agreement with the results of Ishii et al. [4].

Our findings are in discordance with previous studies [21,

56, 57] which reported that levels of serum SP-D were

higher in COPD patients compared with asymptomatic

smokers. The serum SP-D was measured in stable COPD

patients in our study, while other studies recruited COPD

patients who showed exacerbations that might increase

serum SP-D levels. Our results show that levels of serum

SP-D were statistically significantly higher in asymptom-

atic smokers when compared with healthy controls, which

is in concordance with previous reports [21, 56]. A twin

study has shown that serum levels of SP-D are elevated by

smoking [58]. In agreement with our results, Lomas et al.

[21] concluded that the largest difference in serum SP-D

levels occurred between non-smokers and asymptomatic

smokers, which marks serum SP-D as a powerful bio-

marker for smoking. Moreover, they added that the dif-

ference in serum SP-D levels between individuals with

COPD and smoker and non-smoker controls is not suffi-

ciently large to use as a screening test to diagnose COPD

[21].

SFTPD, IL-1b and IL-1RN Polymorphisms in COPD

Because SP-D is mainly synthesized within the respi-

ratory tract and approximately 75 % of SP-D is found in

bronchoalveolar lavage fluid [4], its presence in the vas-

cular compartment has, so far, been explained by leakage

from the lung into the bloodstream as a consequence of

increased vascular permeability associated with inflam-

mation [58]. Thus, serum SP-D reflects intrapulmonary

inflammation, which would explain the higher levels in

smokers with and without COPD [21], while it was

reported that bronchoalveolar SP-D was significantly

decreased in smokers [59]. This could imply that either SP-

D is differently regulated in the two compartments or ret-

rograde flooding of SP-D into the lung is somehow inhib-

ited as a consequence of smoking [60].

Our results show the lack of association between SFTPD

rs2243639 gene polymorphism and serum SP-D level in the

recruited healthy controls, asymptomatic smokers, and

COPD patients, and this is in concordance with previous

findings [18, 60, 61]. Furthermore, Leth-Larsen et al. [61]

reported that the SFTPD rs2243639 polymorphism had no

detectable influence on the oligomeric state of SP-D

observed on gel filtration chromatography. However, Hei-

dinger et al. [62] observed a correlation of a single hap-

lotype consisting of six SFTPD polymorphisms including

Met11Thr and Ala160Thr with low plasma levels of SP-D.

Regulation of the serum SP-D concentration is largely

unknown. SP-D is synthesized by endothelial cells, yet it is

unknown if endothelial cells secrete SP-D into the circu-

lation [59]. In addition, SP-D is expressed by a series of

epithelial tissues and by pulmonary Clara cells and type II

cells [58]. Of the three biallelic polymorphisms in the

SFTPD gene (Met11Thr, Ala160Thr, and Ser270Thr), sig-

nificant association of SFTPD Met11 Thr gene polymor-

phism and serum SP-D level was observed [4, 58, 61],

where the homozygous 11Met genotype was significantly

associated with higher SP-D serum levels compared with

the homozygous 11Thr genotype. It was reported that ‘A’

allele at rs3088308 and ‘C’ allele at rs721917 were asso-

ciated with reduced serum SP-D levels [18]. The exact

‘causal alleles’ within SFTPD that influence serum SP-D

are still unknown [58]. It is noteworthy that an SNP in the

promoter region (rs1885551) and an SNP that has a strong

linkage disequilibrium with the promoter region

(rs10887199) have been shown to be more strongly asso-

ciated with the serum SP-D concentration than Met11 Thr

(rs721917) among Caucasians and the Japanese, respectively

[4]. The known promoter sequence in the SFTPD gene indi-

cates that SP-D synthesis contains various gene regulatory

sequences, including AP-1, E-box, and sequences implicated

in modulation of the acute phase response, including the NF-

IL-6 consensus sequence [63, 64]. Therefore, it is speculated

that the promoter region of SFTPD may also affect serum

concentration [4]. Furthermore, Leth-Larsen et al. [61]

demonstrated that polymorphic variations in the N-terminal

domain of SFTPD affect surfactant serum concentrations,

polymerization, and function.

In COPD patients, there was a lack of correlation

between serum SP-D and PFT parameters except for

FEF25–75 % predicted, which showed a significant nega-

tive correlation with SP-D level. Shakoori et al. [65]

reported that FEV1/FVC % predicted, PEF % predicted,

and FEF25–75 % predicted were inversely and signifi-

cantly associated with serum SP-D levels. However, Eng-

stroma et al. [66] concluded that serum SP-D showed no

significant association with any of the PFT parameters in

COPD patients. The discrepancy in results might be due to

differences in the study design, gender, and ethnicity of the

recruited subjects and clinical staging for COPD.

When the risk of COPD was assessed in the presence of

age, smoking history, serum SP-D, and the three studied

polymorphic genotypes using multivariate logistic regres-

sion analysis, age and smoking history were significantly

associated with the risk of COPD (p = 0.002 and 0.023,

respectively). This is similar to previous studies reporting

that advanced age is one of the risk factors for COPD [18],

yet the same study failed to prove that smoking history is a

risk factor for COPD, and they explained this by the

modest sample size or the fact that many of their patients

were ex-smokers with significant smoking history who had

quit smoking [18].

The control groups recruited in previous studies were

variable; they might be healthy controls [45] or smokers

who did not develop COPD [3]. Our study attempted to

investigate the association of the SFTPD, IL-1b and IL-1RN

polymorphisms with susceptibility to COPD recruiting both

healthy controls and asymptomatic smokers, so that COPD

patients would be compared with each of these two groups.

To the best of our knowledge, this is the first study exam-

ining the association of SFTPD gene polymorphism and

serum SP-D levels with COPD susceptibility in the Egyp-

tian population. Hegab et al. [42] previously examined IL-

1b (rs16944) and IL-1RN (rs2234663) gene polymorphisms

in Egyptian COPD patients; however, the novelty in our

study was the investigation of the influence of these poly-

morphisms on the PFT parameters and the examination of

genotype distribution in Egyptian patients with different

clinical stages of COPD. It is noteworthy that the recruited

subjects were all Egyptians living in the same geographical

area, which reduced genetic heterogeneity in our study.

However, our study is not without limitations. First, all

the recruited subjects were men. Although this limits the

general application of our findings, the positive side was

the removal of gender as a confounding factor to our

findings. Second, the recruited sample size was relatively

modest, so we cannot entirely rule out the associations of

the other two polymorphisms with COPD.

M. S. M. Issac et al.

5 Conclusions

Carriers of the SFTPD AG and AA polymorphic genotypes

may be at a higher risk of developing COPD when com-

pared with wild-type GG genotype carriers. IL-1RN

rs2234663/IL-1b rs16944 haplotypes influence FEF25–75 %

predicted and FEV1/FVC. SFTPD rs2243639 genotypes did

not influence serum SP-D levels in our group of recruited

subjects.

Acknowledgments and Disclosures The authors have no conflicts

of interest that are directly relevant to the content of this article.

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