Background: Respiratory distress syndrome (RDS) is a major acute postnatal pulmonary disease that influences mainly preterm neonates. Recently, polymorphism of immunomodulatory genes has been suggested to be associated with RDS development. Here, we aimed at investigating the association of CNR2 gene Q63R polymorphism with the development of RDS. Materials and Methods: In this multicenter case series study, we enrolled three hundred preterm newborns . RDS diagnosed based on clinical and radiographic findings. Polymerase Chain Reaction with sequence-specific primer method was used for genotyping.Results: One hundred and forty neonates out of 300 were diagnosed with RDS.
The overall frequency of the QQ, QR and RR genotypes of rs35761398 were 23.7%, 50.7% and 25.6%, respectively. In the present study the difference in birth weight, birth Height, gestational age (GA) and severity of respiratory distress were statistically significant between the two groups. We found no statistically significant differences in either allele (P=0.624) or genotype (P=0.461) distributions between the healthy group and RDS patients.Conclusions: Gestational age <28-weeks has the highest impact on predisposition to RDS. No association was found betweenrs35761398 and RDS in a group of Iranian preterm infants.
Keywords: Respiratory distress syndrome, RDS, SNP, CNR2, cannabinoids Receptors type 2
Respiratory distress syndrome (RDS) is a major concern of pulmonary disease management in preterm newborns. In spite of extensive efforts and improvement in therapeutic strategies, it has remained an important cause of neonatal mortality and morbidity. In order to multifactorial etiology of RDS, genetic factors have been addressed as a molecular contributor of RDS pathogenesis. Since surfactant deficiency has been thought as the primary cause of RDS, most of studies assessing the association of genetic factors with RDS development have focused on surfactant gene polymorphisms and valuable results and insights have been obtained.
There is growing evidence which shows that some aspects of fetal lung maturation were mediated by inflammatory cells. Bry et al. reported increased expression of SP-A and SP-B mRNA after injection of Interleukin-1a (IL-1a), a proinflammatory cytokine, to the amniotic fluid sacs in preterm labor, associated with lung maturation improvement. They also concluded that IL-1 in amniotic fluid could be part of a host-defense mechanism.
Reportedly IL-10 levels increased in the RDS affected preterm infants, in this way not only proinflammatory cytokines expression suppressed, but also inflammatory process controlled. Also, the association of IL-10-1082 polymorphism with RDS development has been shown in two different studies in Italy and Iran. Taking together, the balance between proinflammatory and anti-inflammatory cytokines have been proposed as another important factor in acute RDS.
A novel family of immunological mediators (Endocannabinoids) controls immune functions and play a role in immune homeostasis. The ability of cannabinioid recepetor 2 (CB2) to decrease the inflammatory response has been shown in different studies. It is well-known that a missense polymorphism at CB2 gene (CNR2) position 63(Q63R) can result in two fold decrease in CB2 receptor function. The association of this polymorphism with several immunity-associated disorders including celiac disease, childhood immune thrombocytopenic purpura and also risk of hospitalization in children with acute respiratory tract infection (ARTI) has been reported previously.
Despite of considerable tendency to role of the endocannabinoid system in human diseases, their effects on pulmonary disease is still unclear. In this study, we aimed whether CNR2 gene Q63R polymorphism (rs35761398) associated with the development and severity of RDS in a group of Iranian preterm neonates.
Materials and Methods
The Human Subjects Committees (HSC) at the Maternal, Fetal and Neonatal Research Center (MFNRC) and the Tehran University of Medical Sciences (TUMS) approved this study (Code No. 94-01-91-28579), and all parents provided informed consent and signed HSC-approved consent forms. This study was conducted according to the Declaration of Helsinki. Population StudyThree hundred preterm Caucasian infants born before 34 weeks GA who were admitted to the neonatal intensive care unit (NICU) at three TUMS hospitals in Tehran- Iran, from May 2013 to April 2015 were recruited into the study.
Those with known genetic disorder(s) or congenital anomaly (ies) were excluded from the study. In this study, neonatologists filled out a designed questionnaire (as previously described) that comprised RDS development factors ,. Based on the standard RDS diagnosis protocol, our newborns divided in two groups, RDS group and Control. The RDS group involved those infants that showed RDS in the first 6 hours of life. Also, our physician’s team ruled out other causes of RDS according to clinical presentations and radiographic outcomes.Downes’ score made our criteria for respiratory distress severity. Dawnes’ score < 8 was considered as “mild” and Dawnes’ score ≥ 8 was regarded as “severe”. Infants were classified as extremely preterm, very preterm and moderate to late preterm, respectively, based on GA <28 w, 28-31 w + 6 d and 32-34 w.
Genotyping of CNR2 Q63R polymorphismPeripheral blood were collected from the study participants into EDTA-containing tubes and genomic DNA was extracted from whole blood samples by modified salting out extraction and stored at -20°C until use. Genotyping of rs35761398 was performed by PCR with sequence-specific primer (SSP-PCR) assay. Briefly, in two PCR tube either the AA-specific primer 5’-TATCTGATCCTGTCCTCCCACCAA-3’ and GG-Specific primer 5’-TATCTGATCCTGTCCTCCCACCGG-3’ in combination with reverse primer 5’-TAGTCACGCTGCCAATC-3’ were used to amplify a 178 bp product which were correspond to Glutamine (Q) and Arginine (R) alleles, respectively. PCR products were electrophoresed in 2% agarose gel (PCR condition is available on request).
For statistical analysis we used a Statistical Package for Social Sciences (SPSS13.5). Also, the power of this study was 80%. Using Chi-square and Fisher’s exact test, deviations from Hardy–Weinberg equilibrium (HWE) and genotype and allele frequencies of rs35761398 were assessed. Continuous variables such as birth weight and gestational age were compared with the Student t test for. Logistic regression analysis was used to assess the independent relationship between RDS development and other variables. All statistical tests were two-sided and a value of P<0.05 was considered significant.
The participants included 300 preterm infants of both sexes 152 (50.7%) male and 148 (49.3%) female. A total of 140 (46.6%) was diagnosed with RDS and the remaining 160 (53.4%) were not. We observed GA ranged from 23 w+ 5 d to 33 w+ 6 d (mean: 30 w + 6 d) and birth weight ranged from 0.5kg to 3.1kg (mean: 1573 g).The participants included 93 (30.66%) twin or multiple pregnancies that showed no association with RDS. Moreover, there was no significant difference in monozygous twins distribution between two groups (P=0.579).We could find no significant difference in preterm birth risk factors, including preeclampsia, maternal diabetes and preterm premature rupture of membranes (PROM) between the two groups. Also the difference between group case and control is not significant due to maternal medical history, antenatal glucocorticoid treatment, mode of delivery, birth order and gender.
The mortality rate was significantly higher in RDS affected infants than in control participants (14.3% vs. 2.5%, respectively) (P=0.0001).Also, a significant difference (P=0.0001) was seen in RDS rate associated with of preterm severity in our RDS participants (extremely preterm: 85.7%, very preterm: 40% and moderate to late preterm: 34.3%).
The observed frequency in the distribution of QQ, QR and RR genotypes in the cases, compared with the controls showed no significant difference(24.3%, 47.1% and 28.6% vs. 23.1%, 53.8% and 23.1%, respectively) (P=0.461). The total frequency of the QQ, QR and RR genotypes of rs35761398 were 23.7%, 50.7% and 25.6%, respectively. Also, the frequency of Q or R alleles did not significantly differ between RDS cases and controls (49.7% and 52.1% vs. 50.0% and 50.0%).We observed no association between severity of respiratory distress and either allele or genotype distribution of rs35761398 in our RDS preterm infants (data not shown here). Similarly, there was no significant association between response to therapy and neither genotype nor allele distribution of rs35761398 (data not shown).
Here, we determined CNR2 gene rs35761398 polymorphism and investigated its potential association with the occurrence of RDS in a group of premature neonates. As the evidence suggests, there is a difference between extremely preterm and near-term infants in genetic susceptibility factors, so in this study only preterm infants were enrolled. Matched appropriately the cases and controls with respect to RDS risk factors such as gender, maternal diseases history and antenatal glucocorticoid therapy, decrease confounding effect which is a common problem in association studies.
In the present study, the RDS infants showed significantly lower birth weight and gestational age compared to those without RDS. The rate of RDS was in reverse correlation with GA so that GA<28 weeks was the most important factor in RDS development. The RDS rate in this group was 85.7%. This result is in accordance with most of the previous reports. Few studies suggested some gene polymorphisms to be associated with preterm birth, but these findings were not confirmed in the other studies [6, 20]. According to our results, no association was observed between CNR2 rs35761398 and risk of extremely premature birth. Due to the need for definitive etiology, RDS pathomechanism has been remained a certain challenge.
It is now evident that prematurity alone does not determine the risk of developing RDS and an association of SP-A, SP-B, SP-C and SP-D gene polymorphisms with RDS have been shown in different populations. In the Iranian preterm infants, we also reported association of SFTPB gene 9306 A/G polymorphism (rs7316) and SFTPC gene codon 186 G/A polymorphism (rs1124) with RDS development.
In addition to the genes related to the surfactant structure and function, polymorphisms of genes contributed to inflammatory response has been proposed as the other candidate genes for the development of RDS. CNR2 is a G-protein- coupled cannabinoid receptor that is mainly expressed by immune cells. Inducible expression of this receptor in inflammatory conditions suggests CB2-dependency of anti-inflammatory and immunomodulatory action of cannabinoids. The polymorphism at CNR2 gene position 63(Q63R) that substitute glutamine by argenine can result in two fold decrease in CB2 receptor function and results in a reduced immune modulation function when activated by cannabinoids.
Moreover, association of CNR2 RR genotype with immune mediated conditions like celiac disease and childhood immune thrombocytopenic purpura has been shown in different studies. Also, the association of CB2 Q63R variation with increased risk of hospitalization in children with acute respiratory tract infection (ARTI) has been shown recently. Tahmatn et al reported that children carrying the QQ genotype were more prone to developing severe ARTI. They also reported that the risk of developing severe ARTI following RSV infection increased more than two-fold in children carrying the Q allele.
Taken together the rational of this study is based on the assessed association between the CNR2 Q63R polymorphism and several immunity-associated diseases as well as on the well-known anti-inflammatory and immunomodulatory effects of CB2 signaling. According to our results, no significant differences were observed among either CNR2 Q63R polymorphism allele or genotype distributions in patients and controls (table 2). This finding may suggest that the implication of CB2 in the development of RDS is less likely. This result might be warranted by knowing that the action of CB2 receptor is cannabinoid-dependent.
Previous studies showed that CB2 receptor was localized only to placental macrophages and the placenta may form a barrier preventing maternal-fetal transfer of endogenous cannabinoids. This may consequently result in lack of stimuli for CB2 during pregnancy period. This idea worth to be investigated in future studies.This study suffers from relatively small sample size. In a multifactorial trait such as RDS, most of the genetic polymorphisms will have minor effects that are not detectable in association studies with small sample size.
Since this is the first study that assessed the potential association of CNR2 Q63R polymorphism with RDS development, knowing whether this polymorphism is associated with RDS development remains to be elucidated in future studies. Conclusion:According to our results gestational age<28 weeks was the major risk factor for RDS. Although no significant association between CNR2 Q63R polymorphism and risk of RDS was found, further studies are needed to verify this point.
This study was supported by a grant from Tehran University of Medical Sciences (Grant no. 94-01-91-28579).