Cumulative evidence of the genetic association between SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome

Abstract Objective: Surfactant protein SP-B, an important protein in pulmonary surfactant, is required for the stabilization of surfactant films in the lung and maintenance of postnatal lung function. Although the association between SP-B polymorphisms and the risk of neonatal respiratory distress syndrome (RDS) has been evaluated, the results have been inconsistent. We investigated the association between SP-B polymorphisms and the risk of neonatal RDS. Methods: Relevant studies were systematically searched in PubMed, EMBASE, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) electronic databases until June 2022. Data were collected independently by two reviewers and converted to odds ratios (ORs) with 95% confidence intervals (CIs). Meta-analysis, subgroup analysis, sensitivity analysis, and publication bias assessment were performed using Stata 12.1 software and Review Manager 5.3. Results: Fourteen studies were included. SP-B C1580T polymorphism was significantly associated with neonatal RDS in five genetic models (T vs. C: OR = 0.70, 95% CI 0.57–0.86, I2 = 78%; TT vs. CC: OR = 0.63, 95% CI 0.53–0.86, I2 = 39%; CT vs. CC: OR = 0.65, 95% CI 0.50–0.84, I2 = 54%; TT + CT vs. CC: OR = 0.62, 95% CI 0.49–0.78, I2 = 59%; TT vs. CC + CT: OR = 0.78, 95% CI 0.67–0.91, I2 = 43%). The CT and TT genotypes may decrease the risk of RDS in neonates. Subgroup analyses revealed that the association of SP-B C1580T polymorphism with neonatal RDS was stable, independent of preterm birth and Hardy–Weinberg equilibrium. In addition, the Han Chinese were more likely to be affected by SP-B C1580T polymorphisms than Caucasians and Finnish. Conclusions: Our findings suggest that SP-B C1580T polymorphism may be a protective factor against neonatal RDS.


Introduction
Neonatal respiratory distress syndrome (RDS, also known as hyaline membrane disease) is a clinical sign of early neonatal respiratory distress with consistent radiological features and the need for supplemental oxygen to maintain more than 85% saturation in the first 24 h of life [1].RDS is a major cause of neonatal morbidity and mortality [2].Although the incidence of RDS is only 1% of all births, it increases with decreasing gestational age.The incidence is 50% at 30 weeks of gestation and up to 90% at 28 weeks of gestation [3].Premature infants are at increased risk of RDS, mainly because of a lack of pulmonary surfactants, which mature during the perinatal period, and underdeveloped lung anatomy [4].Both preterm birth and the genes encoding pulmonary surfactants are critical for the production of pulmonary surfactants and the development and maintenance of healthy lungs [5].
Pulmonary surfactants, synthesized and secreted by alveolar type II cells in the alveoli, are complex mixtures of phospholipids and specific associated proteins that reduce the surface tension at the air-liquid interface of the distal conduction airways and alveoli [6,7].Pulmonary surfactants consist of approximately 90% phospholipids and 10% specific associated proteins, known as surfactant proteins (e.g.SP-A, SP-B, SP-C, and SP-D) [7].Although the hydrophobic SP-B accounts for only 1-1.5% of the total surfactant weight, it plays a key role in the formation and stabilization of surfactant films in the lung [8].Moreover, SP-B promotes the adsorption of phospholipid molecules into the expanding surfactant films and enhances their stability during the compression and expansion processes that occur during the respiratory cycle [9].
SP-B is an essential protein associated with pulmonary surfactant, which plays a pivotal role in preserving postnatal lung function and ensuring survival.A complete lack of SP-B in mice and humans can cause fatal respiratory distress [10].Abnormal production of SP-B is mainly associated with mutations and polymorphisms in its encoding gene, such as the 121 ins2 frameshift mutation and the C1580T polymorphism [11].This single nucleotide polymorphism at position 1580 in exon 4 of SP-B results in a 131 amino acidvariant that has threonine (Thr) substituted by isoleucine (Ile) in the recognition sequence, which affects N-linked glycosylation [12].The Thr allele is associated with several adult pulmonary diseases, such as idiopathic pulmonary fibrosis in smokers [13].However, the link between SP-B C1580T polymorphism and neonatal RDS remains controversial.Therefore, we conducted a meta-analysis to explore this link.

Information sources and search strategies
This study was conducted in accordance with the Metaanalyses of Observational Studies in Epidemiology (MOOSE) guidelines and registered in PROSPERO (CRD42022340427).All eligible studies on the association between the SP-B C1580T polymorphism and the risk of neonatal RDS were systematically searched in the electronic databases of PubMed, EMBASE, Web of Science, and Chinese National Knowledge Infrastructure (CNKI).The search was performed from 1990 to 2022.A structured search strategy was designed in accordance with Medical Subject Headings (MeSH) search terms: ("Surfactant Protein SP-B" OR "Pulmonary Surfactant-Associated Protein B" OR "SPB" OR "SP-B") AND ("Polymorphism" OR "Mutation" OR "Variant") AND ("Infantile Respiratory Distress Syndrome" OR "Neonatal Respiratory Distress Syndrome" OR "Respiratory Distress Syndrome, Infant" OR "Respiratory Distress Syndrome, Newborn").The search strategy was adapted for other electronic databases used.

Study eligibility criteria
The included studies had to meet the following criteria.
(1) The studies evaluated the association between the SP-B C1580T polymorphism and the risk of neonatal RDS.(2) The studies had sufficient data on allele or genotype frequencies to calculate the odds ratio (OR) with a 95% confidence interval (CI).(3) The neonates in the studies were clinically diagnosed as having RDS.The diagnosis of RDS was substantiated by clinical manifestations, encompassing grunting, retraction, flaring, sustained reliance on supplemental oxygen for a duration exceeding 48 h, or necessitating the administration of exogenous surfactant therapy.In addition, radiographic criteria, notably the presence of a diffuse reticulogranular pattern and air bronchograms, were employed to corroborate the diagnosis of RDS [14].Studies that deviated from the Hardy-Weinberg equilibrium (HWE) in the genotype distribution of neonates in the control group were also included to avoid selection bias.Moreover, there were no restrictions in terms of language, publication date, publication status, ethnicity, or geographical region.Narrative reviews, animal studies, case reports, comments, and editorials were excluded.One reviewer performed the database search and screened for duplicates.After removing duplicates, two reviewers (TR and WL) screened the titles and abstracts of all the records and assessed the full text of the eligible articles.

Data collection
Data collection included the following items: name of the author, year of publication, place of study, ethnicity of the study population, sample size, genotyping method, study design, and genotype frequency of cases and controls.Data extraction was conducted by two independent reviewers (TR and WL).

Assessment of risk of bias
The Newcastle-Ottawa scale (NOS) was used to evaluate the risk of bias in the studies included in the meta-analysis [15].The NOS includes the following domains: selection of study groups, comparability of groups, and ascertainment of outcomes.The selection domain included four sub-items, each item for one star and up to four stars in the domain.The comparability domain included only one sub-item with up to two stars in the domain.The outcome domain included three sub-items and scored up to three stars.Two reviewers (TR and WL) independently assessed the NOS scores for each included study.Study quality was classified into three grades based on quality: low (0-3), moderate (4-6), and high (7-9).

Statistical analysis
Data from the selected studies were processed using Stata 12 software (StataCorp, College Station, TX, USA) and Review Manager 5.3 (Cochrane Collaboration, Oxford, UK).We calculated the total sample size of the included studies by summing the individual sample sizes.The HWE among the control neonates in each study was evaluated using the Pearson v 2 test.We used models of five genetic patterns, allelic genetic model (T vs. C), homozygous genetic model (TT vs. CC), heterozygous genetic model (CT vs. CC), dominant genetic model (CT þ TT vs. CC), and recessive genetic model (TT vs. CC þ CT), to evaluate the association between SP-B C1580T polymorphism and the risk of neonatal RDS by pooling the ORs and their corresponding 95% CIs.
The I 2 statistic and the Q test were used to evaluate the heterogeneity of each study.If the heterogeneity was significant and I 2 was >50%, a random-effects model was used; otherwise, a fixed-effects model was used.A sensitivity analysis was used to assess the robustness of the association by omitting one study at a time.Publication bias was assessed using funnel plots and Begg's tests.Subgroup analyses were conducted by preterm birth, ethnicity/race, and HWE to further validate the association between SP-B C1580T polymorphism and the risk of neonatal RDS.

Study selection and data
The study selection process is outlined in the flowchart shown in Figure 1.In total, 606 studies matched the search terms.After removing 134 duplicates, titles and abstracts of 472 articles were screened for eligibility.Among them, 61 articles were included in the full-text review.23 studies were excluded because they failed to include data on 1580 C/T polymorphisms in SP-B.Nine studies were excluded as they did not provide sufficient data.Nine studies were excluded because they were either conference abstracts (three) or review articles (six).In addition, six studies, conducted in the same center, were excluded because of overlapping participants.Finally, 14 studies were included in this meta-analysis [14,[16][17][18][19][20][21][22][23][24][25][26][27][28].
The main characteristics of the included studies are summarized in Table 1.The 14 studies included 1857 cases and 2402 controls.The participants were recruited from China, Finland, Greece, and Brazil.The clinical characteristics of neonates extracted from the included studies were shown in Table S1.All included studies utilized polymerase chain reaction (PCR) to detect the genotype of SP-B C1580T polymorphisms.The genotype distribution of the control group in all but three studies was consistent with the HWE.Among the 14 studies, 10 showed high methodological quality [18][19][20][21][22][24][25][26][27][28], whereas 4 had moderate methodological quality according to their NOS scores [14,16,17,23] (Table S2).

Association between SP-B C1580T polymorphism and RDS risk
We used five types of genetic models (allelic, homozygous, heterozygous, dominant, and recessive) to evaluate the association between SP-B C1580T polymorphism and the risk of neonatal RDS.The pooled ORs of the five genetic models showed that SP-B C1580T polymorphism had a significant association with the risk of RDS: T vs. C(allelic genetic model): OR ¼ 0.70, 95% CI 0.57-0.86,I 2 ¼ 78%, Figure 2; TT vs. CC(homozygous genetic model): OR ¼   2).Next, we evaluated the possibility of a publication bias using the Begg's test and funnel plot.The funnel plot resembled a symmetrical distribution of the five types of genetic models (Figure S1).The results of the Begg's test (p > .05, in all genetic models) revealed no evidence of publication bias in the overall analysis (Table 2).Moreover, a sensitivity analysis was conducted to assess the robustness of the results, which showed that the ORs of all genetic models were still robust after removing a single study each time (Figure S2).

Subgroup analysis of association between SP-B C1580T polymorphism and RDS risk
Stratified subgroup analyses were conducted based on different factors, including preterm birth, ethnicity, and HWE (Table 2).First, the SP-B C1580T polymorphism was significantly associated with the risk of RDS in preterm neonates in four genetic models, except for the recessive genetic model (Table 2, Figure S3).The Begg's test in all genetic models showed no publication bias in the preterm subgroup (Table 2).Second, as for ethnicities, SP-B C1580T polymorphism showed a significant association with the risk of RDS in Han Chinese neonates (Table 2, Figure S4).However, the Begg's test for all genetic models showed publication bias.However, there were no missing studies identified by the trimming estimator,  suggesting that the potential publication bias did not influence the pooled result of the association between SP-B C1580T polymorphism and RDS in Han Chinese neonates.Moreover, no significant association was found in Caucasian neonates (T vs. C: OR ¼  S4).Finally, subgroup analysis stratified by HWE revealed that SP-B C1580T polymorphism was related to reduced risk in all genetic models.
(Figure S5, Table 2) The Begg's test for all genetic models showed no publication bias in the HWE subgroup analysis.

Discussion
Neonatal RDS, often due to a deficiency in pulmonary surfactant, is one of the leading causes of neonatal mortality [2,3].SP-B is important for enhancing the rate of adsorption and surface spreading of phospholipids in pulmonary surfactants [29].The frameshift mutation in exon 4 (121 ins2) of SP-B is the most common cause of SP-B deficiency [30].However, it remains unclear whether the SP-B C1580T polymorphism is linked to SP-B abnormalities that lead to neonatal RDS.Therefore, this meta-analysis was conducted to provide clarity in this regard.Fourteen studies involving 1857 cases and 2402 controls were included in the meta-analysis.Overall, we found a significant association between SP-B C1580T polymorphism and neonatal RDS in all five genetic models.Our results indicate that the CT and TT genotypes may decrease the risk of RDS in neonates.Heterogeneity was observed in the overall analysis.Therefore, subgroup analyses were performed stratified by several covariates influencing the association between SP-B C1580T polymorphism and RDS in neonates, including preterm birth, ethnicity, and HWE.
As preterm birth has a considerably elevated risk for RDS in neonates, we only included preterm infants in the subgroup analysis.SP-B C1580T polymorphism was found significantly associated with the risk of RDS in preterm births in four genetic models, except for the recessive genetic model (TT vs. CT þ CC).This showed that the CT and TT genotypes reduce the risk of RDS in preterm births.No significant result for preterm births in the recessive genetic models (TT vs. CT þ CC) was observed, probably because the CT genotype also reduced the RDS risk.The prevalence and incidence of neonatal RDS vary widely according to race, which may be due to both nongenetic and genetic factors [31].Therefore, we performed a subgroup analysis based on ethnicity and found that the risk of RDS in Han Chinese neonates was affected by SP-B C1580T polymorphism.However, the Caucasian and Finnish neonate groups showed no significant correlation with this polymorphism.A limited number of the included studies examined the association between the SP-B C1580T polymorphism and the risk of RDS in three ethnicities, including Han Chinese neonates, Caucasian neonates, and Finnish neonates.The remaining studies did not specifically investigate the impact of ethnicities on this association.Therefore, SP-B C1580T-racial background correlation requires further investigation.Deviations from HWE in genetic association studies were mainly used in the control group as representative of the study quality [32].Deviations from the HWE in the control group are related to the design and conduct of genetic association studies, particularly due to problems caused by population stratification, genotyping errors, or selection bias [32].Stratification analysis based on HWE suggested an association between the SP-B C1580T polymorphism and RDS in the five genetic models, regardless of whether the study was consistent with the HWE.This finding strongly suggests that the T allele is a potential risk factor for the risk of neonatal RDS.
The interaction of SP-B with lipid vesicles results in the formation of phospholipids and enhanced stability of the phospholipids at the air-liquid interface [33].Wang et al. found that the C allele in the SP-B C1580T polymorphism leads to SP-B containing two functional N-linked glycosylation sites at residues Asn 129 and Asn 311 , whereas the T allele presents only one N-linked glycosylation site at Asn 311 [34].This posttranslational modification change may result in alteration of SP-B function.Moreover, the SP-B C1580T polymorphism was significantly associated with bronchopulmonary dysplasia in the Han Chinese population [35].Sumita et al. found that the T/T genotype of the SP-B C1580T polymorphism could reduce the risk of interstitial lung disease [36].This result is similar to our finding in neonatal RDS.
This meta-analysis has several advantages.First, the included studies were searched from four main databases to reduce inclusion loss.Second, the genotype information of the patient was mainly obtained from PCR analysis of the patient's tissue DNA.Furthermore, pertinent neonatal clinical data were obtained from electronic medical records in the primary studies.These data collection methods significantly reduced the recall bias to a large extent.Third, all eligible studies were qualified as having no high risk of bias, indicating the relatively high quality of the findings.Nevertheless, this study has several limitations.First, we did not include ongoing or unpublished studies.Second, the number of original studies on Caucasians (one study) and Finnish (two studies) was relatively limited.The included studies didn't involve multiple centers with large sample sizes.In order to validate and strengthen these findings, further studies are warranted, encompassing diverse ethnic groups and larger sample sizes.
In conclusion, the present meta-analysis showed that the SP-B C1580T polymorphism is significantly associated with RDS in neonates.The CT and TT genotypes of the SP-B C1580T polymorphism reduced the risk of RDS in neonates, especially in the Han Chinese.These findings will contribute to the identification of the genetic risk factors for RDS in neonates.

Figure 1 .
Figure 1.Flow chart of the study selection procedure.

Figure 2 .
Figure 2. Forest plots of the overall association of SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome in Allelic genetic model: T vs. C.

Figure 3 .
Figure 3. Forest plots of the overall association of SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome in Homozygous genetic model: TT vs. CC.

Figure 4 .
Figure 4. Forest plots of the overall association of SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome in Heterozygous genetic model: CT vs. CC.

Figure 6 .
Figure 6.Forest plots of the overall association of SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome in Recessive genetic model: TT vs. CC þ CT.

Figure 5 .
Figure 5. Forest plots of the overall association of SP-B C1580T polymorphisms and risk of neonatal respiratory distress syndrome in Dominant genetic model: TT þ CT vs. CC.

Table 1 .
Characteristics of included studies.