Genetic and Biochemical Predictors of Neonatal Bronchopulmonary Dysplasia

Abstract

Bronchopulmonary dysplasia (BPD) is a common complication of prematurity with a multifactorial etiology, influenced by both genetic susceptibility and environmental factors on the immature lung. Fibroblast growth factor receptor-3 and -4 (FGFR-3 and FGFR-4) are abundantly expressed in both the epithelium and mesenchyme in the developing mammalian lung. FGFR-4 may play a role in developing BPD as it is associated with airway inflammation and remodeling; studies showed a link between BPD and a polymorphism in the FGFR-4 gene. The aim of this study was to study the significance of FGFR-4 in developing BPD and to investigate the correlation between its serum level and its genetic polymorphism in relation to development of BPD in preterms. This case–control study was performed on 80 preterm neonates (<32 weeks) divided into two groups: group I included 50 preterms with respiratory distress syndrome (RDS) who developed BPD and group II included 30 preterms with RDS only. The mean serum level of FGFR-4 was significantly lower in group I than in group II ( p -value < 0.05). There was no significant correlation between the serum levels of FGFR-4 and the degree of severity of BPD. Allele variation in the FGFR-4 gene was similar in both groups. The serum level of FGFR-4 was significantly lower in preterms with BPD, although the gene polymorphism was not significantly different in the studied groups.

Introduction

Bronchopulmonary dysplasia (BPD) is a chronic inflammatory lung disease of premature infants characterized by lung damage, triggered by mechanical ventilation and hyperoxia. ¹

According to the definition proposed by National Institute of Child Health and Human Development (NICHD), infants depending on supplemental oxygen for a minimum of 28 days suffer from BPD. ²

It is the most common neonatal chronic lung disease, affecting around 25 to 35% of very low birth weight <1,500 g neonates, and is associated with increased risk of rehospitalization, cognitive delay, and neurosensory deficits. The underlying etiology is multifactorial, influenced by both genetic susceptibility and environmental factors on the immature lung; the pathophysiology is characterized by inflammation, abnormal microvascularization, and impaired alveolarization. ³

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Investigations have revealed that fibroblast growth factors (FGFs), secreted glycoproteins, are involved in regulating cell migration and proliferation in embryonic development. Their signaling depends on membrane-located receptors (FGFRs) with a tyrosine kinase domain, encoded by four different genes ( FGFR 1–4 ). They are suggested to play major roles in modifying distal lung patterns during alveolarization. ^{3 4}

Due to their functional importance, FGFRs have been considered as promising drug targets for the therapy of various cancers. Multiple small molecule inhibitors targeting this family of kinases have been developed, and some of them are in clinical trials. ⁵

In the study by Park et al in 2007 which tested the hypothesis that “exposure of new born mice to sublethal hyperoxia would alter lung development and expressions of fibroblast growth factor receptors FGFRs-3 and FGFR-4,” they concluded that the temporally and spatially specific alterations in the expressions of FGFR-3, FGFR-4, and FGF-7 in these mice may contribute to aberrant lung development. ⁶ This aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders and cancer. ⁷

Regarding FGFR-4 in humans, one study by Rezvani et al in 2013 found that five single nucleotide polymorphisms in genes encoding matrix metalloproteinases 9 and 12, FGFs 3 and 7, and FGFR-4 were associated with respiratory distress syndrome (RDS) of the newborn in their Caucasian population; among these polymorphisms one polymorphism in FGFR-4 (rs1966265) was additionally associated with BPD. ³

Based on previous studies that highlighted the significance of FGFR-4 in the pathogenesis of RDS and subsequently BPD, we designed this study, aiming at investigating the relation of FGFR-4 SNPs and the possible genetic risk of developing BPD in premature neonates with RDS, in addition to assessing the correlation to its serum level.

Subjects and Methods

Compliance with Ethical Standards

The study protocol was approved by the local ethics committee of the Faculty of Medicine, Cairo University, as to be in accordance with Helsinki Declaration II, Finland. Written informed consent was obtained from the legal guardians of all participating subjects.

Subjects

This is a case–control study recruiting a total of 80 neonates admitted to the Neonatal Intensive Care Unit (NICU) of Abo-Elreesh Children Hospital and Kasr El Aini hospital, Cairo University, during the period from October 2016 to January 2018. They were divided into two groups: Group I included 50 preterm neonates with gestational age <32 weeks admitted to the NICU presenting with RDS and developed BPD and group II included 30 preterm neonates with gestational age <32 weeks admitted to the NICU presenting with RDS and did not develop BPD. Neonates with congenital malformations, chromosomal aberrations and those, whose mothers suffered from preeclampsia, prenatal infections and diabetes (conditions affecting lung maturation), were excluded. Data was collected including, maternal history of gestational diabetes, placental insufficiency, use of antenatal steroids, mode of delivery normal vaginal delivery or cesarean section, Apgar scoring, sex, gestational age (new Ballard score), birth weight, diagnosis of RDS clinically with increased work of breathing, retractions and increased oxygen requirements to maintain normal saturation, use of surfactant therapy, mode and duration of oxygen support, associated morbidities in the form of Patent Ductus arteriosus (PDA), pulmonary hypertension, septicemia, and outcome of babies (mortality, morbidity). Degree of BPD was assessed for infants born at <32 weeks' gestation who received supplemental oxygen for their first 28 days. The National Institutes of Health defined BPD at 36 weeks' postmenstrual age as mild: no supplemental O ₂ requirement, moderate: supplemental O ₂ requirement <30%, and severe: supplemental O ₂ requirement ≥ 30% and/or continuous positive airway pressure or ventilator support. ⁸

Chest radiograph was done for all neonates for the diagnosis of RDS and BPD. RDS appeared in the form of low lung volumes, homogeneous microatelectasis that has the appearance of ground glass, and air bronchogram highlighted by the surrounding micro-atelectasis. Initially, in BPD, there appeared diffuse haziness and normal-to-low lung volumes. However, in more severe disease, chronic changes included homogenous regions of opacification and hypertranslucency with superimposed hyperinflation.

Samples Collection and Preparation for Biochemical and Genetic Analysis

Whole blood and serum samples were collected and the entire biochemical and genetic workout was performed in the Molecular and Genetic Studies Unit, Chemical Pathology Department, Kasr al Ainy Hospitals, Cairo University. Peripheral blood samples were collected from all the neonates on day 28 of life through a venipuncture. Collected samples were divided into two tubes. One sample was placed in a plain tube, serum-coagulated at room temperature for 10 to 20 minutes and centrifuged at the speed of 2000 to 3000 rpm for 20 minutes. The supernatant was removed for serum separation and kept at –20°C until used for estimation of serum FGFR-4. The other sample was placed in a tube containing ethylenediaminetetraacetic acid and kept at 4°C until assayed.

Biochemical Analysis

Detection of serum levels of FGFR-4 using enzyme-linked immunosorbent assay Kit (Glory Science Co., Ltd; www.glorybios.com ) was done to the manufacturer's instructions.

Genetic Analysis

Deoxyribonucleic acid (DNA) extraction was done using Thermo Scientific GeneJET Whole Blood Genomic DNA Purification Mini kit # K0781 in accordance with the manufacturer's instructions.

DNA amplification was performed by polymerase chain reaction (PCR) technique. Genotyping of FGFR-4 single nucleotide variation (SNV) (rs1966265, G > A, Val10Ile) was done using PCR-RFLP technique using conditions previously referenced by Rezvani et al. ³

The used primer sequence was forward 5′-CAAAGGTGCACGTGTAGC AG-3′, reverse 5′-TCCCACCTCAGAAGCCATAC-3′. PCR reaction components consisted of, in a total volume of 25 μL, 12.5μL ready-to-use PCR Master Mix (lot: MTRX-515109), 5.5 μL nuclease-free water, 1 μL of each of forward and reverse Primers (25 pmole), and 5 μL of extracted genomic DNA. The reaction cells were vortexed and spinned. PCR cycle conditions were adjusted to initial denaturation step at 95°C for 3 minutes, 40 cycles at 95°C for 20 seconds, 56.6°C for 30 seconds, 72°C for 30 seconds, and for 72°C for 1 minute. PCR reaction finally yielded a PCR amplicon of 212 bp. The presence of the PCR amplicon was checked by agarose gel electrophoresis and ethidium bromide staining. Lastly, genotyping was done using restriction fragment length polymorphism (RFLP) using restriction enzyme: Bam HI. In case of the G allele a fragment of 212bp was obtained, while in case of the A allele two fragments of 126bp + 86bp were obtained ( Fig. 1 ).

Fig. 1.

Representative genotyping of fibroblast growth factor receptor 4 gene using polymerase chain reaction-restriction fragment length polymorphism analysis by gel electrophoresis.

Statistical Analysis

Data was coded and entered using the statistical package SPSS version 25. Quantitative parametric variables were summarized using mean (standard deviation [SD]). Categorical variables were expressed as frequencies (percentages). For comparing categorical variables, χ ² was performed. Exact test was used instead when the expected frequency is less than 5. Correlations between quantitative variables were done using Spearman correlation coefficient. P -values less than 0.05 were considered as statistically significant.

Results

This case–control study included 80 preterm neonates (< 32 weeks of gestation) diagnosed with RDS. Out of these, 50 preterms (group I) developed BPD and 30 preterms (group II) did not develop BPD. Demographic data of the studied population are shown in Table 1 .

Table 1. Demographic data of the studied population.

Variable	Group I ( n  = 50)	Group II ( n  = 30)	p -Value
Gestational age (weeks) a	29.74 (1.19)	30.13 (1.2)	0.078
Male	31	13	0.104
Female	19	17
Birth weight
ELBW	7	2	0.714
VLBW	34	22
LBW	9	6
Mode of delivery NVD	18	9	0.583
CS	32	21
Mortality	13	1	0.01
Surfactant therapy	28	9	0.024
Mode of O 2 support			<0.001
Noninvasive	1	22
Invasive	49	8
Days of O 2 therapy a	34.76(8.32)	12.10(4.26)	< 0.001

Abbreviations: CS, cesarean section; ELBW, extremely low birth weight; NVD, normal vaginal delivery; LBW, low birth weight; VLBW very low birth weight; SD, standard deviation.

^aData are displayed as mean (SD).

There was a statistically significant difference between the two groups regarding mode of O ₂ therapy administered. The mean duration of O ₂ therapy in group I was 34.76 days versus group II 10.5 days, which was statistically significant ( p -value <0.001). The mortality rate was higher in group I than in group II (26 vs. 3.3%) with a p -value of 0.01.

Among the encountered comorbidities in the studied population, pulmonary hypertension, PDA, pneumothorax, and sepsis were significantly more frequent in group I (82, 82, 22, and 88%) than in group II (20, 43, 0, and 66.7%), respectively, as shown in Table 2 .

Table 2. Comparison between the comorbidities of the studied groups.

Variables	Group I ( n  = 50)	Group II ( n  = 30)	p- Value
Pulmonary hypertension
Mild (25–40 mm Hg)	33 (66)	6 (20)	≤ 0.001
Moderate (40–55 mm Hg)	8 (16)	0 (0)
Patent ductus arteriosus
Small (<1.5 mm)	8 (16)	3 (10)	≤ 0.001
Moderate (1.5–3 mm)	23 (46)	10 (33.3)
Large (>3 mm)	10 (20)	0 (0)
Pneumothorax	22 (44)	0 (0)	<0.001
Intraventricular hemorrhage	9 (18)	3 (10)	0.520
Retinopathy of prematurity	4 (8)	2 (6.7)	0.09
Sepsis	44 (88)	20 (66.7)	0.021
Necrotizing enterocolitis	3 (6)	4 (13.3)	0.416
Jaundice	22 (44)	23 (76.7)	0.004
Systemic hypertension	2 (4)	5 (16.7)	0.096

Note: Data are displayed as frequency (%)

The serum level of FGFR-4 in group I ranged from 21 to 4,121 pg/mL with a mean (±SD) of 791.8 (±977) pg/mL and a median of 468.75 pg/mL. Higher levels of FGFR-4 with a mean (±SD) of 1250.38 (±1881) pg/mL ranging from 0.00 to 7855 pg/mL and a median of 1250.38 pg/mL were estimated in group II ( p  = 0.041).

The results of the genetic analysis of the FGFR-4 gene, where we studied the FGFR-4 SNP rs1966265, located in the exonic region and causing an amino acid substitution of isoleucine (Ile) for valine (Val), showed that the genotype (GG) had a higher frequency (90%) in both studied groups. In group I, 90% of the cases showed genotype GG, 8% genotype GA and 2% (one patient) genotype AA and in group II, 90% showed genotype GG, 3.3% (one patient) genotype GA and 6.7% (two patients) genotype AA. Accordingly, there was no statistically significant difference ( p  = 0.574) between the allele frequencies in both groups: allele G in 94% of the cases in group I and 91.7% in group II, whereas allele A in 6% of the cases in group I and 8.3% in group II. There was no significant difference between the genotypes and the serum levels of FGFR-4 in the BPD (group I) versus non-BPD (group II) with ( p  < 0.05) ( Table 3 ).

Table 3. Comparison between serum FGFR-4 and the genotypes in the studied population.

		BPD genotype group I				RDS genotype group II
		AA (no = 1)	GA (no = 4)	GG (no = 45)	p- Value	AA (no = 2)	GA (no = 1)	GG (no = 27)	p- Value
S.FGFR-4 (pg/mL)	Mean	4121.00	583.13	736.37	0.234	646.75	7855.00	1050.48	0.207
	Standard Deviation	–	555.43	887.34		23.69	–	1483.20

Abbreviations: FGFR-4, fibroblast growth factor receptor 4; RDS, respiratory distress syndrome.

Preterms with BPD were classified according to the disease severity into two groups: mild to moderate (35 cases) and severe (15 cases). The level of serum FGFR-4 was lower in severe cases (median of 452 pg/mL, mean ± SD of 632.93 ± 552.5 pg/mL) compared with the mild-to-moderate group (median of 481.5 pg/mL, mean ± SD of 859.89 ± 1113.4 pg/mL), with no significant difference ( p  = 0.966).

There was no significant correlation between FGFR-4 level and the gestational age of the studied cases (correlation coefficient = 0.140, p  = 0.215) nor with the duration of oxygen exposure (correlation coefficient =–0.141, p  = 0.213).

Receiver operating characteristic (ROC) curve was used as a tool to find out the sensitivity and specificity of serum FGFR-4 that were 75 and 60%, respectively. The cutoff value for serum FGFR-4 was found to be at (558.75 pg/mL) with area under the curve = 0.639, as illustrated in Fig. 2 .

Fig. 2.

Receiver operating characteristic (ROC) curve for cutoff value of serum fibroblast growth factor receptor 4 level in bronchopulmonary dysplasia group.

Discussion

The pathogenesis of BPD involves an interaction between environmental factors such as hyperoxia and ventilation induced lung injury, intrauterine and postnatal inflammation, and host factors including gene regulatory pathways involved in alveolar and vascular development. ⁹

FGFs are mitogenic signaling molecules that play important roles in the control of cell proliferation, differentiation, and survival in many tissues. ¹⁰ Studies have reported that FGF signaling through receptors Fgfr3 and Fgfr4 is crucial for alveologenesis. ¹¹

To our knowledge, there are no documented studies, measuring the level of FGFR-4 in premature neonates. In the current study, we aimed at comparing the serum level and genotypes of FGFR-4 between the preterms with RDS and BPD (group I) and those with RDS only (group II). The level of FGFR-4 in group I was significantly lower than that of group II. However, the results of the ROC curve in this study did not point out serum levels of FGFR-4 to be a reliable marker for the development of BPD in preterms with RDS.

Research in this matter is still deficient. Among the few studies done on preterm neonates, Rezvani et al in 2013 found that reduction in FGFR-4 expression affects alveolar formation and that a functionally significant polymorphism within the correspondent gene possibly alters the susceptibility to alveolar disease such as BPD and RDS. ³

Stages of lung development are comparable among mammalian species, including humans and rodents; accordingly, rodents are widely used to study lung disease. Studies done by Park et al and Srisuma et al found altered expression of FGFR-3 and FGFR-4 messenger ribonucleic acid in the lungs of hyperoxic mice, suggesting mechanisms for arrested lung development. They reported hyperoxia-induced enhancements of rates of receptor protein degradation, which may contribute to the lower levels of FGFR-3 and FGFR-4 proteins. Increased production of reactive oxygen species in response to hyperoxia could stimulate degradation and turnover of FGFRs. In addition, increased expression of FGFRs 3 and 4 coincides with alveolar formation and reduced expression of FGFR3 and FGFR4 is associated with reduced alveolar formation in the lung of neonates after hyperoxic injury. ^{6 12}

In this work, the result of genetic analysis of the FGFR-4 gene showed that the genotype (GG) had a higher frequency in both studied groups. These results are in agreement with Rezvani et al in 2013. ³ Accordingly, the AA genotype may be considered as a protective allele variant against lung diseases, while the G allele is a risk factor for developing the disease. Moreover, there was an insignificant relationship between the genotypes of FGFR-4 and the degree of BPD in our population.

Several comorbidities including pulmonary hypertension, PDA, and pneumothorax were found to be significantly higher in the BPD group in agreement with many studies. ^{13 14 15 16}

The duration of oxygen exposure and invasive ventilation was among the clinical factors associated with increased risk of BPD in our work consistent with other studies. ¹⁷ The mortality rate in the current study was significantly higher in group I than in group II. Cokyaman and Kavuncuoglu stated an increase in mortality rate with increase in the incidence of BPD. ¹⁶

Among our limitations were the relatively small number of studied cases and the absence of previous research related to this topic.

Conclusion

Serum FGFR-4 level may play a significant role in airway inflammation, alveolarization, and airway remodeling being lower in the preterm neonates with BPD. The distribution of FGFR-4 polymorphisms was the same in both studied groups, with G allele predominance, suggesting it to be a risk factor for developing lung disease. Further large-scale studies are encouraged to improve our knowledge about FGFRs and their role in BPD.

Funding Statement

Funding None.