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Indian Pediatr 2015;52:
391-394 |
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Human Surfactant Proteins A2 (SP-A2)
and B (SP-B) Genes as Determinants of Respiratory
Distress Syndrome
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Walaa A Abuelhamed, Nancy Zeidan, Walaa A Shahin,
*Hoda I Rizk and
#Walaa A Rabie
From Departments of Pediatrics, *Public Health and
Community Medicine, and #Clinical and Chemical Pathology; Faculty of
Medicine, Cairo University, Cairo, Egypt.
Correspondence to: Dr W Rabie, Lecturer, Department
of Clinical and Chemical Pathology, Faculty of Medicine, Cairo
University, Cairo, Egypt.
Email: [email protected]
Received: August 20, 2014;
Initial review: November
18, 2014;
Accepted: February 02, 2015.
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Objective: To study the relationship between SP-A2
and SP-B gene polymorphisms and respiratory distress syndrome in
preterm neonates.
Design: Cross-sectional.
Setting: Neonatal intensive care
unit and the Molecular Biology unit of the Chemical Pathology
Department, Kasr Alainy hospital, Cairo University.
Participants: Sixty-five preterm
infants with respiratory distress syndrome and 50 controls. The genomic
DNA was isolated using DNA extraction kits. SYBR Green-based real-time
PCR was used to determine the variant genotypes of SP-A2 c.751
G>A and SP-B c.8714 G>C single nucleotide polymorphisms.
Results: Homozygosity of SP-A
(OR 46, 95% CI 14-151) and SP-B (OR
5.2, 95% CI 2.3-11.4) alleles increased the risk of respiratory distress
syndrome. The logistic regression model showed that genotypes SP-A2
(OR 164) and SP-B (OR 18) were directly related to the occurrence
of respiratory distress syndrome, whereas gestational age (OR 0.57) and
5-minute Apgar score (OR 0.19) were inversely related to its occurrence.
Conclusions: There is a possible
involvement of SP-A2 and SP-B genes polymorphisms in the
genetic predisposition to respiratory distress syndrome.
Keywords: Neonate; Polymorphisms; Respiratory
distress syndrome; Surfactant protein.
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According to Egypt Demographic and Health Survey
2008 [1], neonatal mortality was 16 per 1000 live births. Identifying
the causes of neonatal morbidity and mortality is essential for planning
to its reduction, as one of the Millennium Development Goals [2]. The
inability of premature neonates to produce surfactant and immaturity of
the lung constitute the primary etiologies of respiratory distress
syndrome (RDS). The surfactant protein (SP) genes have
been used as candidate genes. So far, five human proteins have been
identified: SP-A1, SP-A2, SP-B, SP-C, and SP-D [3]. The SP-A gene
is located in chromosome 10q21 with two 99% homologous genes– SP-A1
and SP-A2 — in which a large number of single-nucleotide
polymorphisms (SNPs) exist [4-6]. SP-B is secreted by type II
cells in the lung and is essential for its normal function. Its absence
results in respiratory failure and death shortly after birth [7-11].
Several studies have evaluated the association of SP-A and
SP-B SNPs with RDS [12-14], but the functional consequences of their
allelic variations are not well understood. The study of their
polymorphisms aids in understanding the susceptibility of various
individuals to RDS. The aim of this study was to investigate the
association between SP-A2 and SP-B genes polymorphisms and
the risk of RDS in preterm neonates.
Methods
We conducted this cross-sectional study at Children’s
Hospital, Faculty of Medicine, Cairo University, during the period
between May 2013 and January 2014. Sixty-five preterm infants with RDS
and 50 without RDS as controls were recruited from the neonatal
intensive care unit (NICU). The study protocol was approved by the
Ethics committee of Faculty of Medicine, Cairo University. Written
informed consent was obtained from the legal guardians.
The criteria for the diagnosis of RDS included the
presence of respiratory rate >60/min, dyspnea, grunting, cyanosis,
respiratory acidosis in addition to the presence of typical chest X-ray
findings [15-17]. Bronchopulmonary dysplasia (BPD) was diagnosed
according to the National Institute of Health (US) criteria [18].
Neonates with genetic syndromes, congenital malformations, and other
associated pathologies were excluded.
We collected 3 mL of blood in EDTA vacutainers, kept
at 4ºC until DNA was extracted. Genomic DNA was extracted using High
Pure PCR Template Preparation Kit (Roche Applied Science, USA).
Genotype analysis was carried out at the Molecular
Biology unit of the Chemical Pathology Department, Faculty of Medicine,
Cairo University. The c.751 G>A of SP-A2 and c.8714 G>C in the
3’UTR of SP-B genes were genotyped using SYBR Green-based
polymerase chain reaction (PCR) and consequent melting curve analysis
was performed using LightCycler 2.0 Instrument (Roche Applied Science,
Germany). PCR amplification was done using the Primers sequences as
previously reported for SP-B [19, 20] and SP-A2 [21]
genes. The 20 µL reaction mixture included 15 µL of master mix with the
following components: 9µL water, PCR grade; 1 µL forward Primers,
10×conc.; 1 µL reverse Primers, 10× conc.; 4µL 5×conc,
LightCyclerFastStart DNA Master PLUS
SYBR Green I (Roche Applied Science, Germany); and 5 µL of 50 ng genomic
DNA, under the following conditions: initial denaturation at 95ºC for 10
min, followed by 45 cycles of denaturation at 95 ºC for 10 s, annealing
at 59ºC for 20s, and extension at 72ºC for 25s. After amplification,
melting curve analysis was performed by heating the reaction mixture
from 65 to 95 ºC at a rate of 0.1ºC/s. LightCycler 2.0 PCR Systems
automatically calculated the negative derivative of the change in
fluorescence and generated a melting curve for each sample
Web
Fig. 1.
Statistical analysis: Data analysis was done
using SPSS Version 15, and Epicalc version 2000. Pearson Chi-Square,
Fisher’s Exact, and z tests were used; the predictors for RDS were
tested using logistic regression analysis. P<0.05 was considered
significant.
Results
Mean (SD) gestational age, birth weight, and Apgar
score were significantly (P<0.001) lower in the RDS group
compared to controls. Otherwise, there was no significant difference
between the two groups regarding the remaining demographic and clinical
data (Table I).
TABLE I Demographic and Clinical Data of The Studied Groups
Variables |
RDS |
Controls |
P- |
|
(n = 65) |
(n=50) |
value |
Gestational age (wks)* |
29.4 (2.42) |
32.5 (2.02) |
<0.001 |
Birth weight (kg)* |
1.2 (0.33) |
1.6 (0.32) |
<0.001 |
Apgar at 5 min* |
4.9 (1.07) |
6.7 (1.32) |
<0.001 |
#Males |
37 (57) |
28 (56) |
0.92 |
#Caesarian delivery |
47 (72) |
35 (70) |
0.79 |
#Antenatal steroid usage |
22 (33.8) |
18 (36) |
0.81 |
#Multiple pregnancies |
21(32.3) |
12 (24) |
0.22 |
#Premature rupture of membranes |
8 (12.3) |
3 (6) |
0.25 |
#Maternal hypertension |
20 (30.8) |
17 (34) |
0.71 |
#Maternal diabetes mellitus |
3 (4.6) |
7 (14) |
0.07 |
Values in * mean (SD) or # No. (%). |
TABLE II Genotype and Allele Frequencies in the Studied Groups
Variables |
RDS |
Controls |
P value |
OR |
|
(n=65) |
(n=50) |
|
(95% CI) |
Genotype frequencies |
|
|
|
|
SP-A2 |
|
|
|
|
GG |
13 (20) |
46 (92) |
<0.001 |
|
GA |
1 (1.5) |
2 (4) |
0.82 |
|
AA |
51(78.5) |
2 (4) |
<0.001 |
|
SP-B |
|
|
|
|
GG |
19 (29.2) |
34 (68) |
<0.001 |
|
GC |
5 (7.7) |
16 (32) |
0.001 |
|
CC |
41(63.1) |
0 (0) |
<0.001 |
|
Risky and Protective genotypes |
|
|
|
|
SP-A2* |
|
|
|
|
GA+ AA |
52 (80) |
4 (8) |
<0.001 |
46 (14-151) |
GG |
13 (20) |
46 (92) |
|
|
SP-B* |
|
|
|
|
GC+ CC |
46 (70.8) |
16 (32) |
<0.001 |
5.2 (2.3-11.4) |
GG |
19 (29.2) |
34 (68) |
|
|
Alleles |
|
|
|
|
SP-A2* |
|
|
|
|
G |
25 (19.2) |
94 (94) |
<0.001 |
65 (26 -167) |
A |
105 (80.8) |
6 (6.0) |
|
|
SP-B* |
|
|
|
|
G |
44 (33.8) |
84 (84) |
<0.001 |
10.3 (5.4-19.6) |
C |
86 (66.2) |
16 (16) |
|
|
Data expressed as number and percent. |
The amplification of the genomic DNA segments with
subsequent genotyping analysis was successful in 115 samples. There were
statistically significant differences in genotypes between the RDS and
control groups (Table II). Preterm neonates with SP-A2
(AA and GA) genotypes and SP-B (CC and GC) genotypes were more
prone to have RDS compared to neonates with the GG genotype. A and C
alleles of SP-A2 and SP-B genes, respectively, were
significantly higher (P<0.001) in the RDS group. There was no
significant association between variant genotypes of both SP-A2
and SP-B and severity of RDS or BPD among our patients. These
variant genotypes also did not differ between the infants who died
versus those who survived (Table III).
TABLE III Relation Between SP-A2 and SP-B Genotypes and RDS Grade, BPD and Outcome
Variables |
SP A2 genotypes |
|
P value |
SP B genotypes |
|
P value |
RDS grade |
CA+ AA |
CC |
|
GC+CC |
GG |
|
I and II |
15 (88.2) |
2 (11.8) |
0.49 |
12 (70.6) |
5 (29.4) |
0.99 |
III and IV |
37 (77.1) |
11 (22.9) |
|
34 (70.8) |
14 (29.2) |
|
BPD |
10 (76.9) |
3 (23.1) |
0.76 |
9 (19.6) |
4 (21.1) |
0.89 |
Death |
29 (55.8) |
9 (69.2) |
0.38 |
28 (60.9) |
10 (52.6) |
0.54 |
*Value in No. (%). |
In our study, the distribution of the combined
genotypes of SP-A2 and SP-B between RDS and the control as
(AA and CC) represents 49.2% of cases and 0% of the control, while (GG
and GG) represents 60% of the control and only 6.2% of cases (P<0.001).
For the regression analysis, the variables included
were: maternal hypertension, maternal diabetes mellitus, multiple
pregnancies, maternal use of antenatal steroids, premature rupture of
membrane, mode of delivery, gestational age, birth weight, sex, Apgar
score at 5 min, and SP-A2 and SP-B genotypes. There were
four significant predictors for RDS; two were directly related to the
occurrence of RDS (the SP-A2 genotype with OR =164; P<0.001;
and SP-B genotype with OR=18; P=0.008), while gestational
age (OR=0.57; P=0.01) and 5-minute Apgar score (OR=0.19; P=<0.001)
were inversely related.
Discussion
Factors affecting the development of RDS include
specific SNPs of SPs which affect the protein structure and function
[22], degree of prematurity, sex, and ethnicity [23]. In this study, we
found that preterm neonates with SP-A2 (AA) or SP-B (CC)
genotypes had higher odds of RDS compared to neonates with GG genotype.
However, Lyra, et al. [24] reported that there was no
statistically significant difference in the distribution of the
genotypes of the G/C polymorphism at nucleotide 8714 in patients with
and without RDS. Our results are in concordance with an earlier study
showing that the homozygous genotype for SPA was over represented
in RDS [23].
Studying the distribution of the combined genotypes
of SP-A2 and SP-B between RDS cases and the control showed
that combined (AA & CC) were present in higher number of cases than
controls, while combined (GG and GG) represented more of the control
than cases. This indicates that homozygous wild genes are essential for
surfactant function. Meanwhile, there was no statistically significant
synergistic effect of either SP-A2 or SP-B on the severity
of RDS, BPD, or neonatal mortality.
The limitations of present study include small sample
size and no matching of cases and controls for gestational age and birth
weight.
In conclusion, this study identified that the
SP-A2 [AA] and SP-B [CC] genotypes are risk factors of RDS. Further
studies on a larger group of patients in different populations are
required to confirm these findings.
Contributors: AWA: conceived and designed the
study, and revised the manuscript for important intellectual content.
She will act as guarantor of the study; ZN, SW: collected data, and
drafted the paper; RW: laboratory tests and interpretation, data
analysis and manuscript writing; RH: data analysis. The final manuscript
was approved by all authors.
Funding: None; Competing interests: None
stated.
What is Already Known?
• Low gestational age, maternal diabetes and
perinatal asphyxia are risk factors for respiratory distress
syndrome.
What this Study Adds?
• SP-B and SP-A polymorphisms are
associated with increased risk of respiratory distress syndrome.
|
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