Genetic Contributions to the Development of Complications in Preterm Newborns.

AIM: We aimed to identify specific polymorphisms of genes encoding for vascular endothelial growth factor A (VEGFA), endothelial nitric oxide synthase (eNOS), renin-angiotensin system (angiotensinogen gene [AGT], angiotensinogen type 1 receptor [AGTR1], angiotensin-converting enzyme [ACE]), and heme oxygenase-1 (HMOX-1) in a cohort of preterm infants and correlate their presence with the development of respiratory distress syndrome (RDS) requiring mechanical ventilation (MV), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH) and retinopathy of prematurity (ROP). STUDY
DESIGN: We carried out a retrospective study to evaluate the allele frequency and genotype distribution of polymorphisms of VEGFA, eNOS, AGT, AGTR1, ACE, and HMOX-1 in a population of preterm neonates (n=342) with a gestational age ≤28 weeks according to the presence or absence of RDS requiring MV, BPD, IVH, or ROP. Moreover, we evaluated through the haplotype reconstruction analysis whether combinations of the selected polymorphisms are related to the occurrence of RDS, BPD, IVH and ROP.
RESULTS: In our population 157 infants developed RDS requiring MV, 71 BPD, 70 IVH, and 43 ROP. We found that TC+CC rs2070744 eNOS (41.7 vs. 25.4%, p=0.01) and GT+TT rs1799983 eNOS (51.8 vs. 35.2%, p=0.01) polymorphisms are independent risk factors for BPD. Haplotype reconstruction showed that haplotypes in VEGF and eNOS are significantly associated with different effects on RDS, BPD, IVH, and ROP in our population.
CONCLUSIONS: We found that TC+CC rs2070744 eNOS and GT+TT rs1799983 eNOS polymorphisms are independent predictors of an increased risk of developing BPD. Haplotypes of VEGFA and eNOS may be independent protective or risk markers for prematurity complications.

Introduction

There is increasing evidence that some genepolymorphismsare implicated in the development of severe complications of preterm birth, including respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), and retinopathy of prematurity (ROP).

Vascular endothelial growth factor A (VEGFA) is important for the physiological growth of retinal vessels, and experimental and clinical data suggest that VEGF may play a causative role in the development of ROP [1]. Similarly, VEGFA plays a central role in vascular lung repair and its absence results in impaired fetal lung microvascular development [2]. Accordingly, clinical studies suggest a relevant association between VEGFA activity and BPD development in preterm infants [3,4].

Nitric oxide (NO) has several vascular actions which may contribute to the growth and protection of vessels in preterm infants [5]. This seems to be confirmed by the reported association between endothelial NO synthase (eNOS) gene activity and the risk of developing RDS, BPD, and IVH in this population [6].

The renin-angiotensin system (RAS) is important for regulating systemic blood pressure and volume, and its genetic modifications could affect the outcome of preterm infants. Previous studies have reported the possible association between angiotensin-converting enzyme (ACE) and angiotensin type 1 receptor (AGTR1) polymorphisms and the risk of developing BPD [7-10] and ROP [11-12], but results are still inconclusive.

Heme oxygenase-1 (HMOX-1) is an enzyme degrading heme to iron ions, carbon monoxide, and biliverdin. Products of HO-1 activity perform important physiological functions in the vascular system, which are ultimately linked to the protection of endothelium through cytoprotective, promitogenic, and anti-inflammatory action [13]. The efficacy of this enzyme has been found to be affected by repeat polymorphisms in the promoter of HMOX-1 gene [13]. Although ample clinical data indicate its influence on cardiovascular complications in adult patients, the possible correlation between HMOX-1 gene transcription and the outcome in preterm infants has never been investigated.

On the basis of these considerations, we hypothesized that the occurrence of severe complications in preterm infants—RDS requiring mechanical ventilation (MV), BPD, IVH and ROP—might be affected by polymorphisms in genes coding the VEGFA, eNOS enzyme, RAS system (Angiotensinogen gene [AGT], AGTR1,ACE), and HMOX-1 enzyme. To assess this hypothesis we aimed to genotype specific polymorphisms of these genes in a cohort of preterm infants and correlate their presence to the development of RDS requiring MV, BPD, IVH, and ROP.

Materials and Methods

We performed this study following approval of the local medical ethics committee of Careggi University Hospital. Parental consent was not obtained because patient records/information were rendered anonymous and de-identified prior to analysis.

Study design

We carried out a retrospective study to evaluate the possible relationship between twelve polymorphisms in genes encoding for VEGFA, eNOS, RAS system, and HMOX-1 and the occurrence of RDS requiring MV, BPD, IVH and ROP in a population of preterm newborns. By using haplotype reconstruction analysis we evaluated whether combinations of the selected polymorphisms are related to the occurrence of the aforementioned prematurity complications.

Patient population

We studied 342 preterm neonates with a gestational age ≤28 weeks, admitted consecutively to the Neonatal Intensive Care Unit of the Careggi University Hospital of Florence, from January 2004 to December 2012. Exclusion criteria were the diagnosis of major congenital malformations, inherited errors of metabolism, and any other congenital syndrome. All subjects investigated were of Caucasian origin in order to guarantee a homogenous ethnic background.

The DNA analysis was performed on dried blood spots collected from infants at 48 hours of life for the local screening program [14].

Clinical Features and Outcome

Birth weight, gestational age, type of delivery, antenatal steroid treatment, gender, Apgar Score at 5 min, RDS occurrence, need of MV and surfactant, type and duration of respiratory assistance [nasal continuous positive airways pressure (NCPAP), patient-triggered ventilation (PTV),high frequency oscillatory ventilation (HFOV)] were recorded for each newborn infant.

The RDS diagnosis was made as previously reported [15]. Infants were given MV when the pH was <7.20, pO2 was <50 mmHg with FiO2>0.50, and pCO2 was >65 mmHg.

We reported the occurrence of patent ductus arteriosus (PDA), sepsis, IVH, periventricular leukomalacia (PVL),BPD, necrotizing enterocolitis (NEC), ROP, and mortality. Diagnosis of BPD was based on the need for supplemental oxygen to maintain adequate oxygenation at36 weeks of post-conceptional age [16]. Intraventricular hemorrhage, PVL, and NEC were diagnosed following Papile’ [17], de Vries’ [18], and Bell’s criteria [19], respectively. ROP was diagnosed following the International Classification criteria [20].

All clinical data were reported on data sheets designed for this study.

Studied polymorphisms

The criteria for selection of candidate genes in the present study were their potential involvement in the pathogenesis of the aforementioned prematurity complications. The DNA analysis was performed on dried blood spots collected from infants at 48 hours of life for the local screening program using the QIAamp DNA Micro Kit according to the manufacturer’s instructions (QiagenGmbh, Hilden, Germany).

We studied 12 single nucleotide polymorphisms (SNPs) in the 6 candidate genes (ACE, AGTR1, AGT, eNOS, VEGFA, HMOX-1) according to their demonstrated or putative function based on literature data, localization in the functional regions, as extracted from the dbSNP database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp&cmd=search&term=) (Table 1). SNP information was assessed in dbSNP NCBI and ENSEMBL (http://www.ensembl.org/index.html) databases.

Table 1

Description of the 12 studied single nucleotide polimorphisms (SNPs) in ACE, AGTR1, AGT, eNOS, VEGF and HMOX1 genes.

Gene Symbol Chr. Position	Reference Sequence Number	db SNP ID number(Taqman assay number)	Common SNP name and nucleotide substitution	Region	Relative Distance
ACE17q23.3	NG_011648.1	rs4291(C__11942507_10)	-240A>T	5’ near gene(promotor)	0
		rs1799752 Custom TaqMan SNP Genotyping Assays	Alu I/D (287 bp)	Intron 16	11696
AGTR13q24	NG_008468.1	rs5186(C___3187716_10)	1166 A>C	3’-UTR	0
AGT1q42.2	NG_008836.1	rs699(C___1985481_20)	803 A>GMet 235 Thr	Exon 2	0
eNOS	NG_011992.1	rs2070744 (C__15903863_10)	-786 T>C	5’ near gene	0
7q36
		rs61722009	eNOS 4a4b	Intron 4	4147
		rs1799983(C___3219460_20)	894 G>TGlu298Asp	Exon 7	5866
VEGFA	NG_008732.1	rs1547651(C___8311590_10)	A>T	5’ near gene	0
6p12
		rs833058(C___1647396_20)	C>T	5’ near gene	1210
		rs833061(C___1647381_10)	-460 T>C	5’ near gene	6842
		rs3025039(C__16198794_10)	936C>T	3’-UTR	21892
HMOX122q13.1		rs3074372	GT repeat	5’ near gene	0

Genotyping of ACE gene Insertion/Deletion (I/D) polymorphisms was performed by TaqMan designed assay, according to Koch et al. [21]. The eNOS 4a4b polymorphism was evaluated by PCR method, as previously described [22].

The other polymorphisms [ACE rs4291; AGTMrs699; AGTR1 rs5186; eNOSrs2070744 and rs1799983; VEGFA rs1547651,rs833058, rs833061 and rs3025039] were analyzed by using TaqMan SNP genotyping assays through the real-time PCR (7900HT Fast Real-Time PCR System, Life Technologies Italia, Monza, Italy).

The (GT)n repeat polymorphism in the promoter of HMOX-1 was detected according to Vazquez-Armenta G et al. [23].

Data analysis

Statistical analysis was performed using the SPSS package v18.0. Continuous variables were expressed as mean and standard deviation, and categorical variables were expressed as frequencies and percentages. Tests for conformity with Hardy—Weinberg equilibrium were performed using a standard χ2 test. Genotype distributions were compared between groups by χ2 analysis. We assessed the association between each polymorphism and clinical adverse outcomes (RDS, BPD, IVH, ROP) by using a dominant model of inheritance, which compares individuals with one or two rare alleles (heterozygotes+homozygotes) to a baseline group of homozygous wild type subjects.

The association of the studied polymorphisms with gestational age and birth weight was also evaluated. Comparisons between median values of gestational age and birth weight among genotypes were performed using the non-parametric Mann-Whitney U test. Logistic regression analysis was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). The independent association of polymorphisms with the different clinical endpoints was evaluated by logistic regression analysis adjusted for gestational age and weight at birth. A value of p = 0.05 was chosen as the cut-off level for statistical significance.

Haplotype analysis

In order to perform haplotype reconstruction analysis (to infer the most probable set of polymorphisms of each studied gene that are inherited together from one parent) in our population, data files were processed in R environment (http://www.r-project.org). Haplotype reconstruction and frequency estimation were independently performed using the PHASE v2.1 software [24], and R package haplo.stats by expectation-maximization strategy (EM algorithm). The haplo.stats package was also used to identify statistically significant associations between haplotypes and clinical endpoints by means of generalized linear models: positive or negative coefficient values indicate the risk or the protection conferred by the haplotypes, respectively, and the absolute values of the coefficient indicate the magnitude of the associations. In order to reduce the type I error, we applied the false discovery rate (FDR) multiple testing correction in generalized linear model analysis [25].

Results

We found that 157 infants developed RDS requiring MV, 71 BPD, 70 IVH, and 43 ROP in our study population (S1 Appendix). Table 2 shows clinical and demographic data of the enrolled preterm infants. Genotype distributions and allele frequencies of twelve polymorphisms investigated in our population are shown in Table 3.

Table 2

Demographic and clinical characteristics of enrolled infants.

Mean±SD and rate and (%).

Characteristics	n = 342
Birth weight (g)	915 ± 249
Gestational age (wks)	27.1 ± 1.8
Cesarean delivery, n (%)	227 (67)
Antenatal steroids, n (%)	240 (71)
Male, n (%)	160 (47)
Apgar score at 5’ th min	7.9 ± 0.86
RDS	160 (47)
Mechanical Ventilation	157 (46)
Surfactant	299 (87)
PDA	279 (82)
Sepsis	86 (25)
IVH	70 (21)
PVL	12 (4)
BPD	71 (21)
NEC	9 (3)
ROP	43 (13)
Death	23 (15)

Table 3

Genotype distribution and allele frequency of the studied polymorphisms.

Gene Symbol	dbSNP	Genotypes	n (%)	F	MAF
ACE	rs1799752	II	58 (16.9)
		ID	166 (48.5)
		DD	118 (34.6)
		D		0.59	0.51°
ACE Prom	rs4291	AA	119 (34.8)
		AT	171 (50.0)
		TT	52 (15.2)
		T		0.40	0.39°
AGTR1	rs5186	AA	211 (61.7)
		AC	108 (31.9)
		CC	22 (6.4)
		C		0.22	0.29°
AGT	rs699	MM	85 (24.9)
		MT	166 (49.1)
		TT	89 (26.0)
		T		0.51	0.43°
eNOS	rs2070744	TT	131 (38.3)
		TC	147 (43.0)
		CC	64 (18.7)
		C		0.40	0.41°
	rs1799983	GG	166 (48.5)
		GT	145 (42.4)
		TT	31 (9.1)
		T		0.30	0.36°
	rs61722009	bb	247 (72.2)
		ab	85 (24.9)
		aa	10 (2.9)
		a		0.15	0.17°
VEGFA	rs3025039	CC	240 (70.2)
		CT	88 (25.7)
		TT	14 (4.1)
		T		0.17	0.15*
	rs1547651	AA	255 (74.6)
		AT	77 (22.5)
		TT	10 (2.9)
		T		0.14	0.13*
	rs833058	CC	109 (31.9)
		CT	160 (46.8)
		TT	73 (21.3)
		T		0.45	0.39*
	rs833061	TT	99 (28.9)
		TC	170 (49.7)
		CC	73 (21.3)
		C		0.46	0.38*
HMOX1	rs3074372	LL	150 (43.8)
		SL	162 (47.4)
		SS	30 (8.8)
		S		0.31	-

There was a higher prevalence of TC+CC rs2070744 eNOS (74.6 vs 58.3%, p = 0.01) and GT+TT rs1799983 eNOS (64.8 vs 48.0%, p = 0.01) polymorphisms in infants with BPD compared to those without BPD (Table 4). Multivariate analysis indicates that these associations were statistically significant [TC+CC rs2070744: OR 1.89 (1.03–3.46), p = 0.04; GT+TT rs1799983: OR1.92 (1.09–3.39), p = 0.02] after adjustment for gestational age and birth weight (Table 5). The contemporary presence of TC or CC rs2070744 and GT or TT rs1799983 eNOS genotypes was associated with an increased OR for BPD (OR 2.26 (1.30–3.95), p = 0.004).

Table 4

Genotype distribution of the 12 polymorphisms in infants with or without RDS requiring MV and BPD.

			RDS			BPD
Gene Symbol	db SNP	Genotypes	Non = 182 (%)	Yesn = 160 (%)	p	Non = 271 (%)	Yesn = 71 (%)	p
ACE	rs1799752	II	26 (14.3)	32 (20.0)		48 (17.7)	10 (14.1)
		ID+DD	156 (85.7)	128 (80)	0.2	223 (82.3)	61 (85.9)	0.5
ACE Prom	rs4291	AA	66 (36.3)	53 (33.1)		101 (37.3)	18 (25.4)
		AT+TT	116 (63.7)	107 (66.9)	0.5	170 (62.7)	53 (74.6)	0.06
AGTR1	rs5186	AA	117 (64.3)	94 (58.8)		164 (60.5)	47 (66.2)
		AC+CC	65 (35.7)	66 (41.3)	0.3	107 (39.5)	24 (33.8)	0.4
AGT	rs699	MM	48 (26.4)	37 (23.1)		73 (26.9)	12 (16.9)
		MT+TT	134 (73.6)	123 (77.9)	0.5	198 (73.1)	59 (83.1)	0.08
eNOS	rs2070744	TT	71 (39.0)	60 (37.5)		113 (41.7)	18 (25.4)
		TC+CC	111 (61.0)	100 (62.5)	0.8	158 (58.3)	53 (74.6)	0.01
	rs1799983	GG	90 (49.5)	76 (47.5)		141 (52.0)	25 (35.2)
		GT+TT	92 (50.5)	84 (52.5)	0.7	130 (48.0)	46 (64.8)	0.01
	rs61722009	bb	133 (73.1)	114 (71.2)		197 (72.7)	50 (70.4)
		ab+aa	49 (26.9)	46 (28.8)	0.7	74 (27.3)	21 (29.6)	0.7
VEGFA	rs3025039	CC	126(69.2)	114(71.2)		184(67.9)	56(78.9)
		CT+TT	56(30.8)	46(28.8)	0.6	87(32.1)	15(21.1)	0.07
VEGFA	rs1547651	AA	143(78.6)	112(70.0)		202(74.5)	53(74.6)
		GG+GA	39(21.4)	48(30.0)	0.06	69(25.5)	18(25.4)	0.9
VEGFA	rs833058	CC	52(28.6)	57(35.6)		89(32.8)	20(28.2)
		CT+TT	130(71.4)	103(64.4)	0.1	182(77.2)	51(72.8)	0.4
VEGFA	rs833061	TT	54(29.7)	46(28.8)		76(28.0)	24(33.8)
		CT+CC	128(70.3)	114(71.2)	0.8	195(72.0)	47(66.2)	0.3
HMOX1	rs3074372	LL	79 (43.4)	71 (44.4)		115 (42.4)	35 (49.3)
		SL+SS	103 (56.6)	89 (55.6)	0.8	156 (57.6)	36 (50.7)	0.3

Table 5

Univariate and multivariate analysis for VEGF, ACT, AGT, eNOS polymorphisms.

RDS
	Univariate Analysis		Multivariate Analysis*
	OR (95%CI)	p	OR (95%CI)	p
rs 1547651 VEGFA T	1.57 (0.96–2.56)	0.07	1.47 (0.82–2.63)	0.2
BPD
	Univariate Analysis		Multivariate Analysis*
	OR (95%CI)	p	OR (95%CI)	p
rs 4291 ACE Prom -240T	1.75 (0.97–3.15)	0.06	1.61 (0.87–2.97)	0.13
rs699 AGT 235T	1.82 (0.93–3.58)	0.08	1.87 (0.92–3.84)	0.08
rs2070744 eNOS -786C	2.11 (1.17–3.79)	0.01	1.89 (1.03–3.46)	0.04
rs1799983 eNOS 894T	1.98 (1.15–3.41)	0.01	1.92 (1.09–3.39)	0.02
rs3025039 VEGF 936T	0.57 (0.30–1.06)	0.07	0.59 (0.31–1.12)	0.1
ROP
	Univariate Analysis		Multivariate Analysis*
	OR (95%CI)	p	OR (95%CI)	p
rs1799983 eNOS 894T	1.90 (1.00–3.71)	0.05	1.73 (0.83–3.64)	0.1
rs833061 VEGF -460C	0.66 (0.34–1.29)	0.2	0.74 (0.35–1.58)	0.4
rs833058 VEGFA T	1.63 (0.77–3.45)	0.2	1.28 (0.57–2.90)	0.5

We observed a trend toward a different prevalence of AT+TT rs4291 ACE polymorphism of the promoter region, MT+TT rs699 AGT, and CT+TT rs3025039 VEGFA polymorphisms in infants with BPD compared to those without BPD (Table 4), and of GT+TT rs1799983 eNOS in infants with ROP compared to those without ROP (Table 6). However, these associations disappeared after adjusting for gestational age and birth weight (Table 5).

Table 6

Genotype distribution of the 12 polymorphisms in infants with or without retinopathy of prematurity (ROP) and intraventricular hemorrhage (IVH).

			ROP			IVH
Gene Symbol	db SNP	Genotypes	Non = 299 (%)	Yesn = 43 (%)	p	Non = 272 (%)	Yesn = 70 (%)	p
ACE	rs1799752	II	47 (15.7)	11 (25.6)		46 (16.9)	12 (17.1)
		ID+DD	252 (84.3)	32 (74.4)	0.1	226 (83.1)	58 (82.9)	0.9
ACE Prom	rs4291	AA	103 (34.4)	16 (37.2)		92 (33.8)	27 (38.6)
		AT+TT	196 (65.6)	27 (62.8)	0.7	180 (66.2)	43 (61.4)	0.4
AGTR1	rs5186	AA	188 (62.9)	23 (53.5)		164 (60.3)	47 (67.1)
		AC+CC	111 (37.1)	20 (46.5)	0.2	108 (39.7)	23 (32.1)	0.3
AGT	rs699	MM	74 (24.7)	11 (25.6)		70 (25.7)	15 (21.4)
		MT+TT	225 (75.3)	32 (74.4)	0.9	202 (74.3)	55 (78.6)	0.4
eNOS	rs2070744	TT	116 (38.8)	15 (34.9)		105 (38.6)	26 (37.1)
		TC+CC	183 (61.2)	28 (65.1)	0.6	167 (61.4)	44 (62.9)	0.8
	rs1799983	GG	151 (50.5)	15 (34.9)		135 (49.6)	31 (44.3)
		GT+TT	148 (49.5)	28 (65.1)	0.05	137 (50.4)	39 (55.7)	0.4
	rs61722009	bb	214 (71.6)	33 (76.7)		197 (72.4)	50 (71.4)
		ab+aa	85 (28.4)	10 (23.3)	0.5	75 (27.6)	20 (28.6)	0.9
VEGFA	rs3025039	CC	210(70.2)	30(69.8)		193(71.0)	47(67.1)
		CT+TT	89(29.8)	13(30.2)		79(29.0)	33(32.9)	0.5
					0.9
VEGFA	rs1547651	AA	221(73.9)	34(79.1)		203(74.6)	52(74.3)
		GG+GA	78(26.1)	9(20.9)	0.5	69(25.4)	18(25.7)	0.9
VEGFA	rs833058	CC	99(33.1)	10(23.3)		87(32.0)	22(31.4)
		CT+TT	200(66.9)	33(76.7)	0.1	185(68.0)	48(68.6)	0.9
VEGFA	rs833061	TT	84(28.1)	16(37.2)		76(27.9)	24(34.3)
		CT+CC	215(72.9)	27(62.8)	0.2	196(72.1)	46(65.7)	0.3
HMOX1	rs3074372	LL	132 (44.1)	18 (41.9)		121 (44.5)	29 (41.4)
		SL+SS	167 (55.9)	25 (58.1)	0.8	151 (55.5)	41 (58.6)	0.6

Gestational age and birth weight were not associated with any of the analyzed polymorphisms (Table 7).

Table 7

Gestational age and birth weight at according to genotype distribution of studied polymorphisms.

			Gestational Age at birth			Weight at birth
Gene Symbol	db SNP	Genotypes	n	Median weeks (IQR Range)	p	n	Median grams (IQR range)	p
ACE	rs1799752	II	58	26.7 (25.3–28.3)		58	900 (730–1120)
		ID+DD	284	27.6 (25.8–28.5)	0.2	284	872 (725–1077)	0.5
ACE Prom	rs4291	AA	119	27.6 (25.8–28.9)		119	910 (730–1120)
		AT+TT	223	27.4 (25.7–28.4)	0.5	223	865 (730–1070)	0.3
AGTR1	rs5186	AA	211	27.4 (25.4–28.7)		211	870 (707.5–1082.5)
		AC+CC	131	27.6 (26.0–28.4)	0.9	131	897.5 (747.5–1100)	0.4
AGT	rs699	MM	85	27.2 (25.7–28.7)		85	910 (760–1125)
		MT+TT	257	27.6 (25.5–28.4)	0.6	257	871 (711.2–1080)	0.3
eNOS	rs2070744	TT	131	27.4 (26.3–28.5)		131	940 (754–1110)
		TC+CC	211	27.6 (25.4–28.6)	0.6	211	865 (720–1075)	0.2
	rs1799983	GG	166	27.7 (25.9–28.7)		166	900 (735–1120)
		GT+TT	176	27.4 (25.4–28.4)	0.2	176	871 (720–1060)	0.2
	rs61722009	Bb	247	27.6 (25.9–28.6)		247	890 (750–1100)
		ab+aa	95	27.1 (25.3–28.3)	0.3	95	870 (700–1053.7)	0.3
VEGFA	rs3025039	CC	240	27.6 (25.6–28.6)		240	867.5 (720–1097.5)
		CT+TT	102	27.3 (25.8–28.4)	0.9	102	910 (760–1082.5)	0.4
VEGFA	rs1547651	AA	255	27.6 (25.7–28.7)		255	880 (730–1100)
		AT+TT	87	27.2 (25.6–28.3)	0.1	87	860 (720–1050)	0.4
VEGFA	rs833058	CC	109	27.7 (26.1–28.6)		109	890 (755–1100)
		CT+TT	233	27.4 (25.4–28.6)	0.5	233	880 (720–1080)	0.3
VEGFA	rs833061	TT	100	27.2 (25.4–28.3)		100	872.5 (742.5–1047.5)
		CT+CC	242	27.6 (26.0–28.6)	0.2	242	890 (720–1110)	0.6
HMOX1	rs3074372	LL	150	27.6 (25.7–28.6)		150	915 (745–1105)
		SL+SS	192	27.6 (25.8–28.6)	0.9	192	865 (720–1085)	0.3

According to the multivariate analysis, adjusted for gestational age and birth weight, we found that:

1) VEGFA haplotypes increase (ACCC, ACTC, ACTT, ATCC, TCCT) or decrease (ACCT, TCCC) birth weight (Table 8); 2) the eNOS haplotype (TaG) decreases gestational age (Table 8). We also found that the: 1) VEGFA haplotype (ACTT) decreases the risk of RDS and increases the risk of BPD (Table 9); 2) the VEGFA haplotype (TCCT) decreases the risk of ROP and increases the risk of IVH (Tble 9); 3) the rare (CaT and TaT) and CbT eNOS haplotypes were significantly associated with different effects (risk for RDS and BPD and protection from IVH and ROP) on clinical prematurity complications (Table 9).

Table 8

Haplotypes of VEGF and eNOS genes significantly associated with weight and gestational age at birth.

Gene Symbol	Haplotype	Frequency	Coefficient	Standard Error	p-value
		Weight at birth
VEGFA
	ACCC	0.232	37.2	5.58	<0.0001
	ACCT	0.065	-15.2	1.23	<0.0001
	ACTC	0.103	22.2	2.08	<0.0001
	ACTT	0.012	35.2	0.19	<0.0001
	ATCC	0.022	40.6	0.39	<0.0001
	TCCC	0.101	-47.9	1.91	<0.0001
	TCCT	0.029	73.2	0.46	<0.0001
SNP1 = rs1547651, SNP2 = rs833058, SNP3 = rs833061, SNP4 = rs3025039
		Gestational age at birth
VEGFA
	ACCC	0.232	0.33	0.19	0.08
eNOS
	TaG	0.021	-1.33	0.53	0.01
SNP1 = rs2070744, SNP2 = rs61722009, SNP3 = rs1799983

Table 9

Haplotypes of the VEGF and eNOS genes significantly associated with the adverse outcomes.

Gene Symbol	Outcome	Haplotype	Frequency in subjects without adverse outcome	Frequency in subjects with adverse outcome	Coefficient	Standard Error	p
VEGF
	BPD	ACTT	0.0009	0.038	0.8	0.01	<0.0001
	RDS	ACTT	0.013	0.008	-0.08	0.01	<0.0001
	ROP	TCCT	0.036	NA	-0.1	0.07	0.03
	IVH	TCCT	0.019	0.064	0.3	0.1	0.02
SNP1 = rs1547651, SNP2 = rs833058, SNP3 = rs833061, SNP4 = rs3025039
eNOS
	BPD	CbT	0.197	0.294	0.09	0.04	0.02
SNP1 = rs2070744, SNP2 = rs61722009, SNP3 = rs1799983

NA: not applicable.

Discussion

In this study we evaluated the possible association between VEGFA, eNOS, AGT, AGTR1, ACE, and HMOX-1 gene polymorphisms and the development of the most frequent prematurity complications, namely RDS requiring MV, BPD, IVH and ROP in a large population of preterm infants. Our data demonstrate for the first time that the T allele of the rs1799983 eNOS polymorphism and confirm that C allele of the rs2070744 eNOS polymorphism are independent predictors of an increased risk of BPD in preterm infants. We also discovered that haplotypes of VEGFA and eNOS genes may independently affect birth weight and gestational age.

Although further associations emerged from the univariate analysis, these correlations disappeared after data adjustment for gestational age and birth weight, with the exception of TC+CC rs2070744 eNOS and GT+TT rs1799983 eNOS genotypes which were independent risk factors for BPD. We speculated that the lack of correlations of the different polymorphisms “per se” was due to the multifactorial pathogenesis of studied prematurity complications which were mainly governed by gestational age [26-30].

The role of the rs2070744 eNOS polymorphism in the pathogenesis of RDS and BPD has been previously investigated in a cohort (n = 124) of preterm infants by Vannemreddy et al. [6]. They found that the status of rs2070744 allele C carrier(TC+CC) was significantly more frequent in infants with RDS, BPD, and IVH compared to control patients (15.3 vs. 7.3%, p = 0.03) and represents a significant risk factor for the development of these pathologies [6] Our data are in partial agreement with these findings because we did not observe any relationship between the polymorphism and the risk of severe RDS and IVH, but we could confirm that TC+CC rs2070744 eNOS genotypes are a risk factor for BPD. Previous reports have demonstrated that TC heterozygotes and CC homozygotes for the rs2070744 eNOS polymorphism increase the risk of cardiovascular diseases in adults and decreases NO levels [6,31]. Considering the crucial role of NO in promoting lung angiogenesis during the fetal/neonatal period [2,32,33], we can reasonably speculate that the C allele of the polymorphism increases the risk of BPD through the decrease of endothelial NO synthesis that impairs lung vessel growth and in turn alveologenesis.

Similarly, the role of the rs1799983 eNOS polymorphism has been demonstrated in the pathogenesis of cardiovascular diseases in adults [34,35], but has never been investigated in the pathogenesis of prematurity complications. We found that GT+TT rs1799983 eNOS genotypes increase the risk of BPD. It has been demonstrated that the T allele carrier status is associated with reduced function of the enzyme and decreased NO production by endothelial cells compared to GG homozygous subjects [36,37]. Therefore, these polymorphisms might also favor BPD development through a negative effect on lung vessels and alveolar growth.

These considerations seem to be confirmed by the results of Ballard’s large randomized controlled study demonstrating that prolonged inhaled NO treatment increases survival without BPD in high- risk preterm infants, although its routine use can not be recommended [38].

Interestingly, the multivariate analysis (adjusted for gestational age and birth weight) of the polymorphism combinations by haplotype analysis of the polymorphisms localized in the same gene showed that haplotypes of VEGFA increase or decrease birth weight and one haplotype of eNOS decreases gestational age. Moreover, the VEGFA haplotype increases the risk of IVH and BPD or decreases the risk of RDS and ROP, while the eNOS haplotype increases the risk of RDS and BPD or decreases the risk of IVH and ROP. Although it is difficult to explain these different effects, if we consider the crucial role of VEGFA [1,2] and NO [5,6] on fetal and neonatal vessel growth and protection, we may speculate that protective haplotypes might enhance their beneficial effects, and promote adequate tissue vascularization and perfusion, while unfavorable combinations might attenuate them and induce inadequate vessel development and blood flow.

Haplotype analysis allow us to identify alleles in which polymorphisms per se associated with the diseases are included, and helps to better identify subjects who are either at risk or not (e.g. VEGFA haplotypes for RDS, BPD, ROP, and IVH). Thus, our findings suggest that different SNPs in VEGFA and eNOS genes of low frequency might change the patients’ susceptibility to prematurity complications by altering either their function or their expression.

The limitations of our study include the relatively small size of the study which could affect the demonstration of correlation between studied complications and polymorphisms; the lack of measurement of infants’ VEGFA levels and NOS activity which precludes the possibility of relating specific polymorphisms and haplotype combinations with their expression in our patients. Thus, our findings do not allow us to demonstrate definitively whether the aforementioned SNPs and haplotypes are themselves responsible for their beneficial or detrimental effects in our population or whether they reflect linkage disequilibrium with variation in the remainder of the genes or perhaps in a nearby flanking gene. However, given the homogeneity of our population, it is unlikely that the observed associations are a statistical artefact resulting from a population substructure.

Conclusions

We confirm that the C allele of rs2070744 eNOS polymorphism is an independent predictor of an increased risk of BPD in preterm infants and demonstrate for the first time the same thing for the T allele of the rs1799983 eNOS polymorphism. We also find that haplotypes of VEGFA and eNO genes may independently affect birth weight and gestational age, and act as protecting or risk markers for prematurity complications. These results could be useful in identifying patients at high risk of developing prematurity complications in whom prevention strategies would be particularly indicated.

Database of clinical characteristics and polymorphisms of studied infants.

(XLS)

Click here for additional data file.