Disease Markers

Volume 35 (2013), Issue 6, Pages 633‒640

http://dx.doi.org/10.1155/2013/932356

Clinical Study

Association of a FGFR-4 Gene Polymorphism with Bronchopulmonary Dysplasia and Neonatal Respiratory Distress

Milad Rezvani, Juliane Wilde, Patricia Vitt, Beena Mailaparambil, Ruth Grychtol, Marcus Krueger, and Andrea Heinzmann

Centre for Pediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany

Received 1 May 2013; Accepted 1 October 2013

Academic Editor: Ross Molinaro

Copyright © 2013 Milad Rezvani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Background. Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease of premature birth, characterized by impaired alveolar development and inflammation. Pathomechanisms contributing to BPD are poorly understood. However, it is assumed that genetic factors predispose to BPD and other pulmonary diseases of preterm neonates, such as neonatal respiratory distress syndrome (RDS). For association studies, genes upregulated during alveolarization are major candidates for genetic analysis, for example, matrix metalloproteinases (MMPs) and fibroblast growth factors (FGFs) and their receptors (FGFR). Objective. Determining genetic risk variants in a Caucasian population of premature neonates with BPD and RDS. Methods. We genotyped 27 polymorphisms within 14 candidate genes via restriction fragment length polymorphism (RFLP): MMP-1, -2, -9, and -12, -16, FGF receptors 2 and 4, FGF-2, -3, -4, -7, and -18, Signal-Regulatory Protein α (SIRPA) and Thyroid Transcription Factor-1 (TTF-1). Results. Five single nucleotide polymorphisms (SNPs) in MMP-9, MMP-12, FGFR-4, FGF-3, and FGF-7 are associated () with RDS, defined as surfactant application within the first 24 hours after birth. One of them, in FGFR-4 (rs1966265), is associated with both RDS () and BPD (). Conclusion. rs1966265 in FGF receptor 4 is a possible genetic key variant in alveolar diseases of preterm newborns.

1. Introduction

Great progress has been made in neonatal care over the last few decades, reflected by improving survival rates and clinical outcomes of preterm infants. Despite these advances, 45 years after its first description [1], bronchopulmonary dysplasia (BPD) remains a major complication of premature birth, causing ongoing morbidity and mortality: it is the most common neonatal chronic lung disease, affecting around 25% to 35% of VLBW neonates (very low birth weight, <1500 g) [2], and is associated with increased risk for rehospitalization [3, 4], cognitive delay, and neurosensory deficits [5].

Initially described by Northway et al. in 1967, the “old BPD” mainly affected modestly premature newborns suffering from respiratory distress and therefore mechanically ventilated with high levels of supplemental oxygen [1]. With the introduction of surfactant treatment, prenatal maternal use of glucocorticoids, improved nutrition, and ventilator strategies the clinical course and pathology of BPD have changed considerably. Unlike the original description, today’s “new BPD” is mainly regarded as a disruption of distal lung growth [6, 7]. The underlying etiology is multifactorial. Thus, influenced by both genetic susceptibility [8, 9] and environmental factors [7] on the immature lung, the pathophysiology is characterized by inflammation, abnormal microvascularization, and impaired alveolarization [10].

Alveolar formation of the primitive saccules is a complex process of epithelial morphogenesis, capillary growth, and coordinated extracellular matrix (ECM) remodelling. At this, fibroblast growth factor (FGF) signalling and matrix metalloproteinase (MMP) activity play eminent roles.

MMPs are zinc-dependent proteolytic enzymes degrading all forms of ECM-components [11]. Some MMPs are upregulated in inflammatory environment and yet are involved in pulmonary host defense [11]. There is evidence for some MMP isoforms being important determinants for alveolarization, especially MMP-2 [12], -9 [13], and -16 [14]: MMP-2 deficient mice showed fewer and larger alveoli with thinner interstitial tissue [12]. Hadchouel et al. demonstrated an increase of MMP-16 activity during the alveolar stage and moreover found two SNPs within the MMP-16 gene being associated with lower tracheal MMP-2 and -16 activity and to protect from BPD [14]. Prospecting further potential biomarkers for BPD, also MMP-9 shows some promise; for example, Harijith et al. highlighted a MMP-9-dependent lung injury pathway in an IFN-mediated animal model of BPD. Mice with a partial MMP-9 deficiency showed a reversal of IFN-induced lung injury during hyperoxia [13].

MMPs, particularly MMP-2 and -9, activate fibroblast growth factors (FGFs) by cleavage in the ECM, especially during angiogenesis [15]. In turn activated FGFs upregulate MMP expression [15]. FGFs are secreted glycoproteins involved in interactions between epithelium and mesenchyme regulating cell migration and proliferation in embryonic development, especially in fetal pulmogenesis [16, 17]. Their signalling depends on membrane-located receptors (FGFRs) with a tyrosine kinase domain, encoded by four different genes (FGFR 1–4) [18–20]. They are all translated in developing lungs and are suggested to play major roles in modifying distal lung patterns during alveolarization [21]; for example, FGFR-3-FGFR-4 double-knockout mice show no alveolarization [22].

It has been assumed that heritable determinants contribute significantly to both BPD [8, 23] and RDS [24]. On this account, we were interested in identifying genetic risk factors in a Caucasian population of premature newborn with BPD and RDS. We genotyped 27 polymorphisms within fourteen candidate genes for BPD: MMP-1, -2, -9, -12, and -16, FGF receptors 2 and 4, FGF-2, -3, -4, -7, and -18, signal-regulatory protein α (SIRPA), and thyroid transcription factor-1 (TTF-1). We also included SIRPA because of the known effect on surfactant proteins [25] and inhibition of macrophages [26], as well as TTF-1 due to its effect on lung differentiation [27].

2. Material and Methods

2.1. Subjects

We recruited preterm neonates (≤28 weeks of gestation) born between January 1996 and September 2010 at the Centre for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Germany. To provide an ethnically homogenous population, all infants were of Caucasian origin. Twins and siblings were excluded from the study as were children with chromosomal aberrations, congenital heart defects, or other major congenital malformations. DNA was collected by buccal swabs or by routine blood sampling, between 2 weeks up to 2 years of age.

Medical charts of all recruited infants were reviewed and clinical data were recorded. This included gestational week, number of days with supplemental oxygen, need of mechanical ventilation and positive airway pressure, and need of surfactant therapy. As described previously [28], the subdivision of our BPD study population was based on the analysis by Lavoie et al. about the heritability of BPD [9] according to the consensus defined by the National Institute of Health [6]: the BPD population included all infants with moderate and severe BPD, that is, supplemental oxygen for at least 28 days plus need of oxygen and/or positive pressure at 36 weeks of gestation, whereas the control population consisted of all preterm neonates with no or mild BPD.

Recruiting neonates for the RDS population was targeted on severe cases of respiratory distress by including only newborns depending on surfactant within the first 24 hours after birth (see Supplementary Material available online at http://dx.doi.org/10.1155/2013/932356). At our Neonatal Intensive Care Unit (NICU) the following approach has been applied regarding the treatment with surfactant: avoiding of intubation independent of the gestational week. Therefore, even very premature infants are only intubated if they show failure of ventilation and/or need of supplemental oxygen above 40%. Once they required intubation during the immediate postnatal period, they receive surfactant within 2 hours. This practice was consistent during the whole study period.

2.2. Genotyping

DNA was extracted by standard procedures as previously described [28]. Genotyping was carried out by RFLP following polymerase chain reaction amplification (PCR). Studied SNPs are summarized in Table 1. We included a minority of polymorphisms that already had been tested for other pathologies (rs1799750 in MMP1 [29] and rs2276109 and rs652438 in MMP12 [30]).

Table 1: Studied polymorphisms. Genotyped polymorphisms, their corresponding reference SNP ID number, nucleotide substitution, and gene region. SNPs in exon regions are indicated as their amino acid substitution.

Gene
Polymorphism
Alleles
Gene region

MMP-1
rs1799750
G/GG
Promotor

rs7125062
C/T
Intron

MMP-2
rs7201
A/C
3′UTR

rs243865
C/T
Promotor

rs17301608
C/T
Intron

MMP-9
rs20544
C/T
3′UTR

rs3918242
C/T
Promoter

rs17576
A/G
279 Gln/Arg

MMP-12
rs2276109
A/G
Promoter

rs652438
A/G
357 Asn/Ser

MMP-16
rs2664352
C/T
Intron

FGFR-2
rs2981579
A/G
Intron

rs1219648
A/G
Intron

FGFR-4
rs376618
A/G
136 Leu/Pro

rs1966265
A/G
10 Ile/Val

FGF-2
rs11938826
C/G
Intron

FGF-3
rs11263591
G/C
Intron

rs10796856
T/C
Intron

FGF-4
rs3740639
C/T
3′UTR

FGF-7
rs16962440
C/T
Intron

FGF-7, C15 or f33
rs4316697
G/A
Intron

rs10519230
C/G
Intron

FGF-18
rs4559013
G/A
Intron

SIRPA
rs3828016
A/G
Intron

rs4814734
A/T
Intron

TTF-1
rs11628131
A/C
Promoter

rs999460
C/T
Downstream

For PCR reactions, genomic DNA was initially denatured at 94°C for 5 minutes and underwent 35–40 cycles of denaturation (94°C for 30 seconds), annealing (1 minute, corresponding temperatures displayed in Table 2), extension reaction (72°C for 1 minute), and a final extension step at 72°C for 8 minutes. In Table 2 primers, annealing temperatures, and restriction enzymes (RE) are shown. Some primers contain intended single nucleotide mismatches (mutagenic primers) to create sites for restriction enzymes.

Table 2: PCR conditions: primers, annealing temperatures, number of cycles and restriction enzymes (RE) for RFLP analysis.

Gene
Polymorphism
Forward-primer
Reverse-primer
PCR
RE

MMP-1
rs7125062
5′-ATC TCT GTC GGC AAA TTC GT-3′
5′-AGC AAC AAG AAG GAG CTG GA-3′
56.5°C, 35 cycl.
BseG1

MMP-2
rs7201
5′-TGC CCA AGA ATA GAT GCT GA-3′
5′-CTG TGA GCC ACA GAA GGT TG-3′
59.1°C, 35 cycl.
MboI

rs243865
5′-CTG ACC CCC AGT CCT ATC TG-3′
5′-GAG CTG AGA CCT GAA GAG CCA AA-3′
59.1°C, 40 cycl.
BstXI

rs17301608
5′-GCA GTG TAG ACA CTA GAG GAA GGA T-3′
5′-GTC ACC AAG GCC TGT ACC AC-3′
59.1°C, 40 cycl.
DpnII

MMP-9
rs20544
5′-AGT GCC CTG AGG ACT AGA G-3′
5′-CAT ACA TGC ATA CAT ACG TGC ATA C-3′
54.2°C, 40 cycl.
SacI

rs3918242
5′-GCC TGG CAC ATA GTA GGC CC-3′
5′-CTT CCT AGC CAG CCG GCA TC-3′
67°C, 40 cycl.
XceI

rs17576
5′-AAT TCA CCC TCC CGC ACT CT-3′
5′-GTT TTG GGG GCC AAT ACA TGA-3′
56.5°C, 35 cycl.
SmaI

MMP-16
rs2664352
5′-GGT AGG CCT GCT TTG GTT CT-3′
5′-GCC TTC CAA ACT CCA CTC ACA T-3′
56.5°C, 35 cycl.
NlaIII

FGFR-2
rs2981579
5′-CCA CTC AAG TCC CTG CTT GT-3′
5′-GTG GGG ACT GAT CAG AGG AA-3′
56.5°C, 40 cycl.
AccI

rs1219648
5′-GCA CGC CTA TTT TAC TTG ACA CGC-3′
5′-CAT TGT GGT GAT CCT TCA CG-3′
59.1°C, 40 cycl.
HhaI

FGFR-4
rs376618
5′-GGG GGT AAC TGT GCC TAT TCTA-3′
5′-TAG CAG GGA GTG AAG GGA TG-3′
56.5°C, 40 cycl.
XbaI

rs1966265
5′-CAA AGG TGC ACG TGT AGC AG-3′
5′-TCC CAC CTC AGA AGC CAT AC-3′
56.5°C, 40 cycl.
BamHI

FGF-2
rs11938826
5′-GAT GAT TTC TCA GGG CCA AA-3′
5′-GCT TCA CAT GCC TAT TTG CTT-3′
59.1°C, 40 cycl.
AluI

FGF-3
rs11263591
5′-GGT GTG TGG GCT GAA GAG AT-3′
5′-CAC CCT GAC ACT CCT GGT CT-3′
61.8°C, 40 cycl.
TaqI

rs10796856
5′-CTT CCC CTC CTA GGC TTC AG-3′
5′-GAA TGC CAG GTT AGC AAA GG-3′
59.1°C, 40 cycl.
HhaI

FGF-4
rs3740639
5′-AAA AAC ACA CCC GCA GAA CT-3′
5′-TGA AGG TCA CCC ACT TCC TC-3′
59.1°C, 40 cycl.
HinfI

FGF-7
rs16962440
5′-TGT CGA ACA CAG TGG TAC CTG-3′
5′-GCA AAA GGA AGA TGA GAA AAG AT-3′
56.5°C, 40 cycl.
EcoRV

FGF-7, C15 or f33
rs4316697
5′-TGG CTC TTT CCA GCA ATT AGA-3′
5′-CCC TTT CTC TCC CAG GTT CT-3′
59.1°C, 40 cycl.
TaiI

rs10519230
5′-AAT GTG ACC GCA TAT TGG TG-3′
5′-CAT TTT GGG AAC TGG GGT AA-3′
56.5°C, 35 cycl.
TaqI

FGF-18
rs4559013
5′-GAG GCA CAG AAT GAG GAA GG-3′
5′-TAC CTG GGC TCT CAA CAT CC-3′
56.5°C, 35 cycl.
Alw26I

SIRPA
rs3828016
5′-TCA AAC CCA GGC TTT CTG AC-3′
5′-AAC CAA GGC TGG CAC ATA AG-3′
63°C, 40 cycl.
NlaIII

rs4814734
5′-CTG TGT ACA TCC TCT TAA ATG GAC-3′
5′-TGC TGT GTG CCA GAG TAT GC-3′
59.1°C, 40 cycl.
HinfI

TTF-1
rs11628131
5′-TGC AGC CTC ATT CAG CTT TA-3′
5′-GGT TGG AGA GGA GGA AAG GA-3′
56.5°C, 40 cycl.
TaqI

rs999460
5′-ACC TGG GCG AGA AGT GAG TA-3′
5′-AGA GAG GGG AGA AGG AAT GG-3′
56.5°C, 40 cycl.
SspI

2.3. Sequencing

Accuracy of the RFLP was confirmed by sequencing via dideoxy chain termination method [31], respectively, three controls (homozygous wildtype, heterozygous, and homozygous mutation) for each polymorphism using the Big Dye Terminator cycle sequencing kit on an ABI 310 sequencer (Applied Biosystems). The RFLP analyses included these control references.

2.4. Statistical Analysis

Genotyping data of our case-control populations were analysed by using Armitage’s trend test (ATT) for possible association with BPD and RDS as specified previously [28]. Moreover ATT was used to calculate Hardy Weinberg equilibrium (HWE) for each polymorphism.

2.5. Approval

The collection of blood/buccal swabs and the experimental procedures were approved by the Ethical Committee of the University of Freiburg. Parents were given written and verbal information about the study and a statement of informed consent was signed by the parents of all enrolled children.

3. Results

The results of the 27 studied polymorphisms (Table 1) for association with bronchopulmonary dysplasia and neonatal respiratory distress are specified in Table 3 (BPD) and Table 4 (RDS).

Table 3: Results for BPD. Genotype distribution as numbers of individuals for each genotype. values are given in accordance with Hardy-Weinberg equilibrium (HWE) and for association with BPD by Armitage’s trend test.

Gene
Polymorphism
Genotypes

BPD
HWE

BPD
Genotypes

controls
HWE

controls
values for association

MMP-1
rs1799750
16, 25, 14
0,590
34, 44, 39
0.009
0.532

rs7125062
41, 15, 3
0,364
74, 43, 11
0.241
0.126

MMP-2
rs7201
21, 28, 9
1,000
43, 64, 21
0.860
0.747

rs243865
35, 18, 6
0,170
80, 35, 12
0.013
0.675

rs17301608
27, 25, 11
0,280
51, 63, 21
0.857
0.769

MMP-9
rs20544
17, 19, 17
0,053
27, 60, 37
0.856
0.508

rs3918242
35, 12, 4
0,077
76, 36, 4
1.000
0.894

rs17576
24, 18, 12
0,042
57, 46, 22
0.032
0.639

MMP-12
rs2276109
44, 13, 2
0,328
86, 38, 2
0.522
0.574

rs652438
52, 8, 0
1,000
10, 19, 1
0.587
0.615

MMP-16
rs2664352
12, 31, 19
1,000
40, 57, 35
0.120
0.185

FGFR-2
rs2981579
21, 32, 10
0,799
51, 58, 25
0.281
0.858

rs1219648
19, 33, 12
0,804
52, 61, 20
0.854
0.215

FGFR-4
rs376618
35, 23, 5
0,744
84, 40, 10
0.132
0.432

rs1966265
47, 15, 1
0,473
79, 45, 9
1.000
0.023

FGF-2
rs11938826
48, 14, 1
1,000
96, 38, 1
0.305
0.557

FGF-3
rs11263591
25, 22, 15
0,037
45, 61, 29
0.382
0.710

rs10796856
17, 26, 22
0,135
37, 59, 40
0.125
0.632

FGF-4
rs3740639
33, 23, 7
0,365
74, 46, 15
0.086
0.815

FGF-7
rs16962440
17, 35, 12
0,614
42, 70, 25
0.730
0.656

FGF-7, C15 or f33
rs4316697
28, 30, 4
0,377
65, 55, 15
0.550
0.868

FGF-7, C15 or f33
rs10519230
37, 23, 3
1,000
73, 50, 11
0.517
0.421

FGF-18
rs4559013
22, 23, 17
0,045
45, 62, 22
1.000
0.387

SIRPA
rs3828016
20, 31, 12
1,000
48, 59, 27
0.288
0.788

rs4814734
16, 37, 10
0,206
46, 62, 25
0.598
0.553

TTF-1
rs11628131
20, 27, 16
0,311
54, 50, 29
0.012
0.287

rs999460
34, 19, 11
0,019
60, 51, 24
0.040
0.414

Table 4: Results for RDS. Genotype distribution as numbers of individuals for each genotype. values are given for Hardy-Weinberg equilibrium (HWE) and for association with RDS.

Gene
Polymorphism
Genotypes RDS
HWE

RDS
Genotypes controls
HWE controls
value for association

MMP-1
rs1799750
25, 44, 20
1,000
24, 29, 33
0.004
0.163

rs7125062
51, 36, 10
0.003
66, 21, 9
0.330
0.076

MMP-2
rs7201
30, 49, 16
0.678
34, 43, 16
0.067
0.648

rs243865
58, 26, 11
0.012
58, 27, 7
0.139
0.540

rs17301608
42, 47, 18
0,422
37, 43, 14
0.827
0.838

MMP-9
rs20544
29, 34, 24
0,828
14, 44, 30
0.052
0.033

rs3918242
55, 23, 6
0,178
57, 25, 2
1.000
0.421

rs17576
38, 30, 21
0,005
45, 34, 13
0.153
0.164

MMP-12
rs2276109
67, 27, 2
1,000
64, 23, 1
1.000
0.595

rs652438
89, 10, 0
1,000
73, 17, 1
1.000
0.047

MMP-16
rs2664352
26, 46, 27
0,546
26, 43, 27
0.312
0.998

FGFR-2
rs2981579
33, 40, 20
0,840
40, 52, 15
0.289
0.351

rs1219648
42, 49, 16
0,837
30, 47, 16
0.833
0.347

FGFR-4
rs376618
71, 27, 9
0,018
51, 36, 6
1.000
0.286

rs1966265
76, 28, 2
1,000
50, 35, 8
0.605
0.003

FGF-2
rs11938826
80, 26, 1
0,690
67, 26, 1
0.684
0.586

FGF-3
rs11263591
31, 48, 25
0,438
40, 37, 19
0.085
0.127

rs10796856
33, 50, 26
0,442
21, 38, 36
0.092
0.036

FGF-4
rs3740639
57, 41, 7
1,000
50, 30, 16
0.009
0.209

FGF-7
rs16962440
30, 57, 21
0,566
30, 49, 17
0.836
0.588

FGF-7 C15 or f33
rs4316697
56, 43, 7
1,000
38, 44, 12
1.000
0.044

FGF-7, C15 or f33
rs10519230
61, 41, 5
0,790
51, 33, 9
0.301
0.418

FGF-18
rs4559013
32, 45, 26
0,236
35, 43, 13
1.000
0.079

SIRPA
rs3828016
34, 53, 21
1,000
37, 38, 19
0.134
0.487

rs4814734
31, 58, 18
0,334
32, 43, 17
0.674
0.671

TTF-1
rs11628131
36, 47, 23
0,326
41, 30, 22
0.002
0.453

rs999460
56, 37, 16
0,027
38, 36, 19
0.082
0.121

3.1. Matrix Metalloproteinases

Among the 11 genotyped polymorphisms in different MMP genes (see Table 1) there was no BPD-associated polymorphism (Table 3) but two polymorphisms associated () with RDS (rs20544 in MMP-9: ; rs652438 in MMP-12: , see Table 4). Both SNPs show no significant deviation from Hardy-Weinberg equilibrium, neither in the control nor in the case population. Analysis of rs20544 (C/T) identifies the T allele as protective against respiratory distress. For the genotyping results of the amino acid substitution rs652438 (A/G, Asn357Ser) the complete absence of the G/G homozygous genotype in the respiratory distress case population must be taken in account. The other MMP-SNPs showed no association, inclusively rs2664352 in MMP16, that had been associated with protection from BPD [14].

3.2. Fibroblast Growth Factor Receptors

The FGFR-4 SNP rs1966265, located in the exon region and causing an amino acid substitution of Isoleucine (Ile) for Valine (Val) is associated with both BPD () and RDS (). Here the A/A genotype (Ile) could be identified as protective allele variant against our studied lung diseases. The association results from significant differences in allele frequencies: in both BPD and RDS analysis the G allele is more frequent in the disease populations (see Tables 3 and 4). The other SNPs in the FGFR genes showed no association with neither BPD nor respiratory distress.

3.3. Fibroblast Growth Factors

Whereas no association could be detected between the eight FGF-SNPs and BPD, rs10796856 in FGF-3 and rs4316697 in FGF-7 showed associations with RDS. Correspondent values are (rs10796856) and (rs4316697), and no deviations from Hardy-Weinberg equilibrium were detected (see Table 4).

3.4. SIRPA and TTF-1

The four SNPs in SIRPA and TTF-1 showed no association with neither BPD nor RDS. Analysis of TTF-1 rs999460 unfolds deviation from Hardy-Weinberg equilibrium in both case and one control populations in our Caucasian population (see Tables 3 and 4).

4. Discussion

Bronchopulmonary dysplasia and respiratory distress syndrome of preterm infants have complex pathogenic mechanisms. The aim of this study has been to identify genetic risk factors in an ethnically homogenous Caucasian population.

Genetic contribution to BPD is suggested on the basis of twin studies demonstrating that at least half of the susceptibility is hereditary [8, 9, 23]. Additionally, Lavoie et al. [9] could differentiate in their study that mild BPD (according to the National Institute of Child Health and Human Development consensus definition [6]) had been mainly attributable to shared environmental factors whereas moderate or severe BPD had been attributable to genetic influence. Following these findings, we defined our control population as neonates with no BPD or mild BPD, whereas our BPD population included neonates with moderate or severe BPD. Furthermore, we recruited only preterm neonates ≤28 weeks of gestational age for the BPD population to avoid false associations based on the fact that BPD hardly develops in newborn older than 30 weeks of gestational age.

In contrast to BPD, the results of twin studies on RDS susceptibility showed mostly contradictory results [24, 32–35]. A twin study by Levit et al. with 332 twin pairs of a heterogeneous population has been the first one to include and assess the influence of several independent covariates, revealing that 50% of the variance to RDS susceptibility is hereditary [24].

Given these lines of evidence for genetic contribution, we have chosen the candidate-gene approach for our association study based on the hypothesis that genes fundamental in lung organogenesis and alveolar remodelling, that is, MMP and FGF, determine susceptibility to BPD and RDS.

Known genetic risk factors for RDS are mostly allelic polymorphisms of the genes encoding surfactant proteins SP-A1, SP-A2, and SP-B [36]. Anyhow, other determinants than components of the surfactant system might also affect the liability to RDS. Genes encoding for growth factors or enzymes that account for alveolarization through proper secondary septation and extracellular remodeling might affect the gas-exchange and therefore aggravate respiratory distress at birth.

Supposed genetic risk factors for BPD are mostly genes encoding components of innate immunity and antigen-presentation, cytokines, antioxidant defences, and angiogenic growth factors such as: mannose-binding lectin (MBL2) [37], tumor necrosis factor-alpha (TNF-) [28, 38], human leucocyte antigen (HLA)-A, -B, and -C alleles [39], glutathione-S-transferase-P1 [40], and vascular endothelial growth factor (VEGF) [28]. Some years ago, two MMP-16 gene polymorphisms were demonstrated to protect from BPD and moreover to be associated with lower tracheal MMP-2 and -16 levels [14].

Matrix metalloproteinases are a family of zinc-dependent endopeptidases [11], and they degrade extracellular components and play a crucial role in lung development, especially during alveolarization. Particularly MMP-2 and -9 (so-called gelatinases A and B) seem to be relevant in extracellular remodeling and even pulmonary host defense. They degrade type IV collagen, fibronectin, elastin, and denatured collagen (gelatin). MMP-2 deficient mice show an abnormal saccular development with larger and simplified alveoles [12]. In line with this finding, newborns developing BPD showed low MMP-2 tracheal levels at birth [41, 42]. Recently, MMP-9 could be identified as a pathogenic key mediator in a murine model of BPD [13]. On the other hand, increased tracheal levels of MMP-9 early after birth have been associated with resolving RDS, suggesting that increase in MMP-9-activity is a physiologic repair response [43]. Dik et al. demonstrated that increased MMP-9 activity in neonatal lungs early after birth correlated with resolving respiratory distress syndrome, demonstrating a likely role of MMP-9 in pulmonary host defense [43].

In our study we identified an SNP (rs20544) in the MMP-9 gene to be associated () with RDS, but not BPD. Respiratory distress syndrome has been defined as need of surfactant (see Supplementary Material). On one hand, ethnically homogenous populations like our Caucasian population are favourable to detect possible pathogenetic determinants, but one must bear in mind that the size of our RDS population is limited and the total numbers of neonates studied for each polymorphism vary slightly according to the recruiting time point. Furthermore, association studies on RDS are prone to confounding factors. Other pulmonary conditions such as a transient tachypnea provoked by wet lung syndrome or pulmonary infection might mimic respiratory distress syndrome caused by surfactant deficiency and thereby hamper the results of our study.

In our study, we included MMP-16 polymorphisms that had been associated with BPD in a French population (rs2664352) [14]. In our population rs2664352 did not show any association.

The FGF-3 polymorphism rs10796856 showed association with RDS (). Up to now, the role of FGF3 has been mainly studied in cancer diseases, that is, lung cancer [44], but its exact role in pulmogenesis remains elusive. FGF-3 is encoded by the fgf3/int2 gene. There is evidence for FGF-3 upregulation to be associated with alveolar type 2 cell hyperplasia [45] and downregulation to be associated with an excessive recruitment of free alveolar macrophages [45] which might lead to symptoms of respiratory distress. Furthermore it has been shown that FGF-3 stimulates the secretion of MMP-2 and -9 propeptides in vitro [46].

FGFR-4 polymorphism rs1966265 showed association with both respiratory distress () and bronchopulmonary dysplasia (). The A/A genotype (encoding for Isoleucin instead of Valine) has been protective in our association study. The exact-test showed no deviation from Hardy Weinberg equilibrium for this SNP in both case and control populations, suggesting that the association does not result from population admixture or genotyping errors.

FGFR-1 to FGFR-4 are expressed in the lung and FGFR-3 and -4 signalling, in particular, appears to be fundamental in alveolar formation. Weinstein et al. demonstrated that mice deficient in both FGFR-3 and -4 show a completely blocked alveolarization and fail to show any formation of secondary septae, whereas solely FGFR-4(−/−) animals exhibit no significant abnormalities, revealing a cooperative effect of FGFR-3 and -4 in lung development [22]. Hyperoxia-exposed (FiO2 0.85) mice show a BPD-like lung pattern of enlarged airspaces and furthermore a reduced expression of FGFR-3 and -4, suggesting a pathogenic role in arrested lung development [47]. Srisuma et al. [48] replicated these results in FGFR-3 and -4 deficient mice and demonstrated in addition that FGFR-3/-4 signaling contributes to excessive elastin production and its alveolar accumulation, which is another typical feature of BPD. But these abnormalities have not been due to fibroblast defects but due to increased expression of paracrine factors of alveolar type 2 cell (AT2) [48].

If a reduction in FGFR-3 and -4 expression affects distal lung development, a functionally significant polymorphism within the correspondent gene possibly alters the susceptibility to alveolar disease such as BPD and RDS. Powell et al. showed that there is a peak of FGFR-4 expression at the day of birth, when respiratory distress syndrome occurs [21]. This supports the conclusion that defective FGFR-4 signalling possibly results in neonatal lung diseases.

Our associations do not justify general interpretation. False-positive results can only be excluded by replications in other study populations.

In conclusion, we describe five SNPs in MMP-9, MMP-12, FGFR-4, FGF-3, and FGF-7 that are associated () in our Caucasian population with respiratory distress syndrome of the newborn, defined as surfactant application within the first 24 hours after birth. Among these polymorphisms one polymorphism in FGFR-4 (rs1966265) is additionally associated with bronchopulmonary dysplasia, demonstrating its possible role in the pathogenesis of newborn lung diseases on grounds of pulmonal immaturity.

Consent

The experiments with genetic material used from humans for this paper were undertaken with the understanding and written consent of each subject. The study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964).

References

W. H. Northway Jr., R. C. Rosan, and D. Y. Porter, “Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia,” The New England Journal of Medicine, vol. 276, no. 7, pp. 357–368, 1967.

M. C. Walsh, Q. Yao, P. Gettner et al., “Impact of a physiologic definition on bronchopulmonary dysplasia rates,” Pediatrics, vol. 114, no. 5, pp. 1305–1311, 2004.

J. K. Chye and P. H. Gray, “Rehospitalization and growth of infants with bronchopulmonary dysplasia: a matched control study,” Journal of Paediatrics and Child Health, vol. 31, no. 2, pp. 105–111, 1995.

V. C. Smith, J. A. F. Zupancic, M. C. McCormick et al., “Rehospitalization in the first year of life among infants with bronchopulmonary dysplasia,” Journal of Pediatrics, vol. 144, no. 6, pp. 799–803, 2004.

B. Schmidt, E. V. Asztalos, R. S. Roberts et al., “Impact of bronchopulmonary dysplasia, brain injury, and severe retinopathy on the outcome of extremely low-birth-weight infants at 18 months: results from the trial of indomethacin prophylaxis in preterms,” The Journal of the American Medical Association, vol. 289, no. 9, pp. 1124–1129, 2003.

A. H. Jobe and E. Bancalari, “Bronchopulmonary dysplasia,” The American Journal of Respiratory and Critical Care Medicine, vol. 163, no. 7, pp. 1723–1729, 2001.

J. P. Kinsella, A. Greenough, and S. H. Abman, “Bronchopulmonary dysplasia,” The Lancet, vol. 367, no. 9520, pp. 1421–1431, 2006.

V. Bhandari, M. J. Bizzarro, A. Shetty et al., “Familial and genetic susceptibility to major neonatal morbidities in preterm twins,” Pediatrics, vol. 117, no. 6, pp. 1901–1906, 2006.

P. M. Lavoie, C. Pham, and K. L. Jang, “Heritability of bronchopulmonary dysplasia, defined according to the consensus statement of the national institutes of health,” Pediatrics, vol. 122, no. 3, pp. 479–485, 2008.

J. J. Coalson, “Pathology of new bronchopulmonary dysplasia,” Seminars in Neonatology, vol. 8, no. 1, pp. 73–81, 2003.

K. J. Greenlee, Z. Werb, and F. Kheradmand, “Matrix metalloproteinases in lung: multiple, multifarious, and multifaceted,” Physiological Reviews, vol. 87, no. 1, pp. 69–98, 2007.

F. Kheradmand, K. Rishi, and Z. Werb, “Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP2,” Journal of Cell Science, vol. 115, no. 4, pp. 839–848, 2002.

A. Harijith, R. Choo-Wing, S. Cataltepe et al., “A role for matrix metalloproteinase 9 in IFNγ-mediated injury in developing lungs: relevance to bronchopulmonary dysplasia,” The American Journal of Respiratory Cell and Molecular Biology, vol. 44, no. 5, pp. 621–630, 2011.

A. Hadchouel, F. Decobert, M. Franco-Montoya et al., “Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: identification of MMP16 as a new player in lung development,” PLoS ONE, vol. 3, no. 9, Article ID e3188, 2008.

P. R. Brauer, “MMPs—role in cardiovascular development and disease,” Frontiers in Bioscience, vol. 11, no. 1, pp. 447–478, 2006.

J. M. Shannon and B. A. Hyatt, “Epithelial-mesenchymal interactions in the developing lung,” Annual Review of Physiology, vol. 66, pp. 625–645, 2004.

K. Sekine, H. Ohuchi, M. Fujiwara et al., “Fgf10 is essential for limb and lung formation,” Nature Genetics, vol. 21, no. 1, pp. 138–141, 1999.

C. A. Dionne, G. Crumley, F. Bellot et al., “Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors,” EMBO Journal, vol. 9, no. 9, pp. 2685–2692, 1990.

K. Keegan, D. E. Johnson, L. T. Williams, and M. J. Hayman, “Isolation of an additional member of the fibroblast growth factor receptor family, FGFR-3,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 4, pp. 1095–1099, 1991.

J. Partanen, T. P. Mäkelä, E. Eerola et al., “FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern,” EMBO Journal, vol. 10, no. 6, pp. 1347–1354, 1991.

P. P. Powell, C. Wang, H. Horinouchi et al., “Differential expression of fibroblast growth factor receptors 1 to 4 and ligand genes in late fetal and early postnatal rat lung,” The American Journal of Respiratory Cell and Molecular Biology, vol. 19, no. 4, pp. 563–572, 1998.

M. Weinstein, X. Xu, K. Ohyama, and C. X. Deng, “FGFR-3 and FGFR-4 function cooperatively to direct alveogenesis in the murine lung,” Development, vol. 125, no. 18, pp. 3615–3623, 1998.

R. A. Parker, D. P. Lindstrom, and R. B. Cotton, “Evidence from twin study implies possible genetic susceptibility to bronchopulmonary dysplasia,” Seminars in Perinatology, vol. 20, no. 3, pp. 206–209, 1996.

O. Levit, Y. Jiang, M. J. Bizzarro et al., “The genetic susceptibility to respiratory distress syndrome,” Pediatric Research, vol. 66, no. 6, pp. 693–697, 2009.

S. J. Gardai, Y. Xiao, M. Dickinson et al., “By binding SIRPα or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation,” Cell, vol. 115, no. 1, pp. 13–23, 2003.

K. Takenaka, T. K. Prasolava, J. C. Y. Wang et al., “Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells,” Nature Immunology, vol. 8, no. 12, pp. 1313–1323, 2007.

V. Boggaram, “Thyroid transcription factor-I (TTF-I/Nkx2.I/TITFI) gene regulation in the lung,” Clinical Science, vol. 116, no. 1, pp. 27–35, 2009.

B. Mailaparambil, M. Krueger, U. Heizmann, K. Schlegel, J. Heinze, and A. Heinzmann, “Genetic and epidemiological risk factors in the development of bronchopulmonary dysplasia,” Disease Markers, vol. 29, no. 1, pp. 1–9, 2010.

Y. Zhu, M. R. Spitz, L. Lei, G. B. Mills, and X. Wu, “A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances lung cancer susceptibility,” Cancer Research, vol. 61, no. 21, pp. 7825–7829, 2001.

L. Joos, J. Q. He, M. B. Shepherdson et al., “The role of matrix metalloproteinase polymorphisms in the rate of decline in lung function,” Human Molecular Genetics, vol. 11, no. 5, pp. 569–576, 2002.

F. Sanger, S. Nicklen, and A. R. Coulson, “DNA sequencing with chain-terminating inhibitors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 74, no. 12, pp. 5463–5467, 1977.

R. Marttila, R. Haataja, M. Rämet, J. Löfgren, and M. Hallman, “Surfactant protein B polymorphism and respiratory distress syndrome in premature twins,” Human Genetics, vol. 112, no. 1, pp. 18–23, 2003.

L. van Sonderen, E. F. W. Halsema, E. J. H. Spiering, and J. G. Koppe, “Genetic influences in respiratory distress syndrome: a twin study,” Seminars in Perinatology, vol. 26, no. 6, pp. 447–449, 2002.

N. C. Myrianthopoulos, J. A. Churchill, and A. J. Baszynski, “Respiratory distress syndrome in twins,” Acta Geneticae Medicae et Gemellologiae, vol. 20, no. 2, pp. 199–204, 1971.

R. Marttila, J. Kaprio, and M. Hallman, “Respiratory distress syndrome in twin infants compared with singletons,” The American Journal of Obstetrics and Gynecology, vol. 191, no. 1, pp. 271–276, 2004.

M. Hallman and R. Haataja, “Genetic basis of respiratory distress syndrome,” Frontiers in Bioscience, vol. 12, no. 7, pp. 2670–2682, 2007.

A. Hilgendorff, K. Heidinger, A. Pfeiffer et al., “Association of polymorphisms in the mannose-binding lectin gene and pulmonary morbidity in preterm infants,” Genes and Immunity, vol. 8, no. 8, pp. 671–677, 2007.

S. N. J. Kazzi, U. O. Kim, M. W. Quasney, and I. Buhimschi, “Polymorphism of tumor necrosis factor-alpha and risk and severity of bronchopulmonary dysplasia among very low birth weight infants,” Pediatrics, vol. 114, no. 2, pp. e243–e248, 2004.

G. Rocha, E. Proença, A. Areias et al., “HLA and bronchopulmonary dysplasia susceptibility: a pilot study,” Disease Markers, vol. 31, no. 4, pp. 199–203, 2011.

M. H. Manar, M. R. Brown, T. W. Gauthier, and L. A. S. Brown, “Association of glutathione-S-transferase-P1 (GST-P1) polymorphisms with bronchopulmonary dysplasia,” Journal of Perinatology, vol. 24, no. 1, pp. 30–35, 2004.

C. Danan, P. Jarreau, M. Franco et al., “Gelatinase activities in the airways of premature infants and development of bronchopulmonary dysplasia,” The American Journal of Physiology—Lung Cellular and Molecular Physiology, vol. 283, no. 5, pp. L1086–L1093, 2002.

C. G. Schulz, G. Sawicki, R. P. Lemke, B. M. Roeten, R. Schulz, and P. Cheung, “MMP-2 and MMP-9 and their tissue inhibitors in the plasma of preterm and term neonates,” Pediatric Research, vol. 55, no. 5, pp. 794–801, 2004.

W. A. Dik, A. H. L. C. van Kaam, T. Dekker et al., “Early increased levels of matrix metalloproteinase-9 in neonates recovering from respiratory distress syndrome,” Biology of the Neonate, vol. 89, no. 1, pp. 6–14, 2006.

A. L. S. Tai, J. S. T. Sham, D. Xie et al., “Co-overexpression of fibroblast growth factor 3 and epidermal growth factor receptor is correlated with the development of nonsmall cell lung carcinoma,” Cancer, vol. 106, no. 1, pp. 146–155, 2006.

B. Zhao, S. S. Chua, M. M. Burcin et al., “Phenotypic consequences of lung-specific inducible expression of FGF-3,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 10, pp. 5898–5903, 2001.

A. Hajitou, E. N. Baramova, K. Bajou et al., “FGF-3 and FGF-4 elicit distinct oncogenic properties in mouse mammary myoepithelial cells,” Oncogene, vol. 17, no. 16, pp. 2059–2071, 1998.

M. S. Park, E. Rieger-Fackeldey, B. L. Schanbacher et al., “Altered expressions of fibroblast growth factor receptors and alveolarization in neonatal mice exposed to 85% oxygen,” Pediatric Research, vol. 62, no. 6, pp. 652–657, 2007.

S. Srisuma, S. Bhattacharya, D. M. Simon et al., “Fibroblast growth factor receptors control epithelial-mesenchymal interactions necessary for alveolar elastogenesis,” The American Journal of Respiratory and Critical Care Medicine, vol. 181, no. 8, pp. 838–850, 2010.