The objective of this study was to assess the frequency of
Nowadays, it is well recognized that the neonatal immune system relies largely on the function of innate immunity. Monocytes, granulocytes, mast cells, natural killer cells, and other innate immune system mediators, including complement factors and acute phase proteins, are critically important to prevent infections [
MBL is an acute phase protein that plays a key role in the first line immune responses as component of neonate innate immunity, since the adaptive immunity arms are not sufficiently developed. MBL belongs to the collectin family of proteins, which also includes lung surfactant protein-A (SP-A) and SP-D [
Although MBL serum concentrations show important changes with age [
MBL deficiency, characterized by low MBL levels and/or the low expression genotype (XA/O or O/O) [
One hundred and thirty-four neonates (93 term and 51 preterm) admitted to the NICU and 150 healthy neonates from the Department of Neonatology of the General Hospital of Nikea “Agios Panteleimon,” Athens, Greece, were enrolled in the study, after obtained informed consent from their parents. Blood was obtained from the neonates (and never from the umbilical cord) usually 2-3 h, and always within 12 h, after birth. In cases of a suspected infection, a comprehensive laboratory examination was performed, including blood and urine cultures, leukocyte counts, and C-reactive protein (CRP) levels. The diagnosis of all aspects of respiratory morbidity, including respiratory distress syndrome (RDS), was based on standard definition criteria [
Genomic DNA was extracted from whole blood using QIAmp DNA Blood Mini Kit (Qiagen, UK) according to manufacturer’s instructions. The detection of
PCR-RFLP protocol for the detection of
PCR primers and conditions
Region | Position* | Sequence# | Conditions of both reactions | PCR product |
---|---|---|---|---|
Promoter | 4421-4446 |
5′-GAAAATGCTTACCCAG(G) |
94°C for 5 min, followed by 30 cycles (94°C for 30 s, 64°C for 30 s, and 72°C for 30 s) and a final elongation at 72°C for 5 min | 134 bp |
Exon 1 | 5191-5218 |
5′-CATCAACGGCTTCCCAGG(C) |
410 bp |
Expected fragments of RFLP genotyping for
Polymorphic sites | Enzyme | RFLP conditions | Wild-type homozygotes (bp) | Mutant homozygotes (bp) | Wild-type & mutant heterozygotes (bp) |
---|---|---|---|---|---|
-550G>C (rs11003125) |
|
4 h at 37°C | 410 (H allele) | 388 + 22 (L allele) | 410 + 388 + 22 (H & L) |
-221G>C (rs7096206) |
|
4 h at 37°C | 410 (X allele) | 352 + 58 (Y allele) | 410 + 352 + 58 (X & Y) |
Arg52Cys (rs5030737) |
|
12 h at 60°C | 109 + 25 | 134 | 134 + 109 + 25 |
Gly54Asp |
|
4 h at 37°C | 99 + 35 | 134 | 134 + 99 + 35 |
Gly57Glu |
|
4 h at 37°C | 134 | 78 + 56 | 134 + 78 + 56 |
Genotypes | Respective haplotypes∧ | 410 bp | 388 bp | 352 bp | 330 bp |
---|---|---|---|---|---|
|
✓ | ||||
HY/HY | HYA/HYA, HYA/HYD, HYD/HYD | ✓ | |||
HY/LY | HYA/LYA, HYA/LYB, HYA/LYC, HYD/LYA, HYD/LYB, HYD/LYC | ✓ | ✓ | ||
HY/LX | HYA/LXA, HYD/LXA | ✓ | ✓ | ||
LX/LX | LXA/LXA | ✓ | |||
LX/LY | LXA/LYA, LXA/LYB, LXA/LYC | ✓ | ✓ | ||
LY/LY | LYA/LYA, LYA/LYB, LYA/LYC, LYB/LYB, LYB/LYC, LYC/LYC | ✓ |
#In forward primers of both reactions, the nucleotides in parentheses were modified and changed to the underlined ones.
∧The common haplotypes of
For the confirmation of results, about half of all PCR products were also purified by Qiagen PCR Purification System (Qiagen) and directly sequenced using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) and a Big Dye Terminator DNA sequencing kit (Applied Biosystems).
The part of blood sample for the measurement of MBL levels was collected in a covered test tube without anticoagulant. Fifteen to thirty minutes after collection, the blood samples were centrifuged at 2,000 ×g for 10 min at room temperature and serum samples were stored at −80°C until analysis. The oligomerized MBL levels were measured using an immunoassay (MBL Oligomer ELISA, Antibody Shop, Copenhagen, Denmark), according to manufacturer’s instructions. The linear range was 0–40
Categorical variables were analyzed with Fisher’s exact test. Normality of continuous variables was assessed with Kolmogorov-Smirnov test. Normally distributed data were analyzed with Student’s
Healthy and NICU neonates, as well as the NICU subgroups (term and preterm), were classified on the basis of the combined genotypes of promoter and exon 1 polymorphisms, in six groups (YA/YA, YA/XA, XA/XA, YA/O, XA/O, and O/O). Subsequently, a further classification in three groups was performed according to MBL expression levels, namely, high (YA/YA, YA/XA), medium (XA/XA, YA/O), and low (XA/O, O/O) [
The detected polymorphisms of
The observed genotype frequencies of
Overview of MBL concentrations and
Healthy | NICU (ne | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
(number 150) | Total (number 134) | Term (number 83) | Preterm (number 51) | |||||||
MBL (ng/mL) | MBL (ng/mL) | MBL (ng/mL) | MBL (ng/mL) | |||||||
|
Median (range) |
|
Median (range) |
|
|
Median (range) |
|
Median (range) |
|
|
| ||||||||||
YA/Y |
50 (33.3) | 1725 |
42 (31.3) | 1515 |
0.798 |
23 (27.7) | 1610 |
19 (37.3) | 1190 |
0.407 |
YA/ΧA | 26 (17.3) | 1290 |
21 (15.7) | 1230 |
0.750 |
13 (15.7) | 1350 |
8 (15.7) | 1055 |
0.998 |
ΧA/ΧA | 6 (4.0) | 885 |
9 (6.7) | 600 |
0.333 |
7 (8.4) | 600 |
2 (3.9) | 1065 |
0.341 |
YA/O | 42 (28.0) | 235 |
33 (26.7) | 180 |
0.623 |
24 (28.9) | 210 |
9 (17.6) | 105 |
0.247 |
XA/O | 16 (10.7) | 45 |
17 (12.7) | 30 |
0.637 |
11 (13.3) | 10 |
6 (11.8) | 10 |
0.825 |
O/O | 10 (6.7) | 10 |
12 (9.0) | 10 |
0.505 |
5 (6.0) | 10 |
7 (13.7) | 10 |
0.169 |
|
||||||||||
| ||||||||||
High |
76 (50.7) | 1600 |
63 (47.0) | 1350 |
0.719 |
36 (43.4) | 1600 |
27 (52.9) | 1170 |
0.521 |
Medium |
48 (32.0) | 260 |
42 (31.3) | 265 |
0.932 |
31 (37.3) | 330 |
11 (21.6) | 180 |
0.160 |
Low |
26 (17.3) | 25 |
29 (21.6) | 10 |
0.451 |
16 (19.3) | 10 |
13 (25.5) | 10 |
0.499 |
|
||||||||||
MBL functional deficiency |
|
|
||||||||
|
||||||||||
MBL levels |
30 (10.0) | 42 (31.3) | 0.091 | 24 (28.9) | 18 (35.3) | 0.578 | ||||
MBL levels |
61 (40.7) | 62 (46.3) | 0.550 | 35 (42.2) | 27 (52.9) | 0.465 | ||||
MBL levels |
68 (45.3) | 72 (53.7) | 0.411 | 44 (53.0) | 28 (54.9) | 0.907 |
Overview of
NICU (neonatal intensive care) | ||||||
---|---|---|---|---|---|---|
Healthy |
Total |
Term |
Preterm |
|||
|
|
|
|
|
|
|
| ||||||
YA/YA | ||||||
HYA/HYA | 13 (8.7) | 10 (7.5) | 0.732 | 6 (7.2) | 4 (7.8) | 0.903 |
LYA/LYA | 7 (4.7) | 4 (3.0) | 0.480 | 1 (1.2) | 3 (5.9) | 0.136 |
HYA/LYA | 31 (20.7) | 28 (20.9) | 0.969 | 16 (19.3) | 12 (23.5) | 0.636 |
|
||||||
Y |
||||||
LYA/LXA | 9 (6.0) | 10 (7.5) | 0.645 | 4 (4.8) | 6 (11.8) | 0.171 |
HYA/LXA | 16 (10.7) | 11 (8.2) | 0.521 | 9 (10.8) | 2 (3.9) | 0.188 |
|
||||||
Χ |
||||||
LXA/LXA | 6 (4.0) | 9 (6.7) | 0.333 | 7 (8.4) | 2 (3.9) | 0.341 |
|
||||||
Y |
||||||
HYA/HYD | 4 (2.7) | 2 (1.5) | 0.501 | 2 (2.4) | 0 (0) | 0.270 |
HYA/LYB | 26 (17.3) | 20 (14.9) | 0.640 | 15 (18.1) | 5 (9.8) | 0.257 |
HYA/LYC | 0 (0) | 1 (0.7) | 0.291 | 1 (1.2) | 0 (0) | 0.434 |
LYA/HYD | 4 (2.7) | 6 (4.5) | 0.425 | 3 (3.6) | 3 (5.9) | 0.557 |
LYA/LYB | 8 (5.3) | 4 (3.0) | 0.346 | 3 (3.6) | 1 (2.0) | 0.595 |
|
||||||
XA/O | ||||||
LXA/LYB | 9 (6.0) | 14 (10.4) | 0.206 | 9 (10.8) | 5 (9.8) | 0.863 |
LXA/HYD | 7 (4.7) | 2 (1.5) | 0.139 | 1 (1.2) | 1 (2.0) | 0.730 |
LXA/LYC | 0 (0) | 1 (0.7) | 0.291 | 1 (1.2) | 0 (0) | 0.434 |
|
||||||
O/O | ||||||
LYB/LYB | 5 (3.3) | 7 (5.2) | 0.449 | 4 (4.8) | 3 (5.9) | 0.799 |
LYB/HYD | 5 (3.3) | 3 (2.2) | 0.588 | 1 (1.2) | 2 (3.9) | 0.314 |
HYD/HYD | 0 (0) | 2 (1.5) | 0.136 | 0 (0) | 2 (3.9) | 0.075 |
|
||||||
| ||||||
HYA | 103 (34.3) | 82 (30.6) | 0.498 | 55 (33.1) | 27 (26.5) | 0.399 |
LYA | 66 (22.0) | 56 (20.9) | 0.797 | 28 (18.9) | 28 (27.5) | 0.097 |
LXA | 53 (17.7) | 56 (20.9) | 0.422 | 38 (22.9) | 18 (17.6) | 0.404 |
LYB | 58 (19.3) | 55 (20.5) | 0.772 | 36 (21.7) | 19 (18.6) | 0.624 |
HYD | 20 (6.7) | 17 (6.3) | 0.884 | 7 (4.2) | 10 (9.8) | 0.089 |
LYC | 0 | 2 (0.7) | 0.135 | 2 (1.2) | 0 (0) | 0.269 |
The most common
The presence of genotypic MBL deficiency was significantly associated with an increased probability of respiratory morbidity (
As presented in Table
(a) Scatterplot of MBL serum levels in the neonates of the study, according to the high, medium, and low
Considering that there is no consensus on the definition of the functional MBL deficiency, we initially used three different cut-off values of MBL concentration (150 ng/mL, 400 ng/mL, and 700 ng/mL) (Table
In order to further identify the best possible discrimination between the low and high value (cut-off) of MBL levels for the prediction of the development of respiratory morbidity, we performed ROC curve analysis. Interestingly, using MBL serum levels as a single variable, their prognostic value for the development of respiratory morbidity was poor (AUC < 0.7). Taking into account that the development of respiratory morbidity was also significantly affected by the male sex (
Multivariate logistic regression model indicated the probability of respiratory morbidity according to the MBL serum levels, corrected for prematurity and sex.
Bearing in mind both the fact that MBL serum levels are fundamentally affected by
Moreover, we demonstrated that the development of RDS was weakly associated with MBL serum levels (OR: 0.928, 95% CI: 0.864–0.996,
Considering other parameters of neonatal morbidity, a univariate logistic regression analysis indicated that the development of jaundice was also significantly associated with MBL serum levels (
Our results suggest that the presence of genotypic MBL deficiency is significantly associated with respiratory complications in NICU, while any increase of MBL serum levels seems to result in a decrease of the risk of respiratory morbidity and RDS and might have important therapeutic implications.
Previous studies have shown that serum MBL levels are lower in newborns, depending also on the gestational age, and increase during the first weeks after birth [
Neonatal respiratory morbidity, including RDS, TTN, perinatal asphyxia due to fetal distress, congenital pneumonia, air leaks, and persistent pulmonary hypertension, has been attributed to abnormal levels and/or composition of surfactants [
Interestingly, the contribution of MBL deficiency in the pathophysiology of several respiratory diseases has extensively been analyzed in recent studies [
The mechanism by which MBL protects the lungs is unclear, but several mechanisms could be suggested, involving both anti-infectious and anti-inflammatory processes [
In conclusion, the results of this study indicate that low MBL levels and/or genotypic MBL deficiency predispose to respiratory complications in neonates, especially TTN and RDS. Considering that the substitution therapy with MBL is now available and safe, the confirmation of the abovementioned association might provide the rationale for a controlled trial to evaluate the efficacy of early administration of MBL in the management of respiratory complications of NICU neonates with low MBL serum levels and/or genotypic MBL deficiency.
The authors declare no competing financial interests.
This work was supported by grants of the Research Committee of the University of Thessaly, Thessaly, Greece.