Single nucleotide polymorphisms in surfactant protein A1 are not associated with a lack of responsiveness to antenatal steroid therapy in a pregnant sheep model

Abstract

Treatment with antenatal steroids (ANS) is standard practice for reducing the risk of respiratory distress in the preterm infant. Despite clear overall benefits when appropriately administered, many fetuses fail to derive benefit from ANS therapies. In standardized experiments using a pregnant sheep model, we have demonstrated that around 40% of ANS-exposed lambs did not have functional lung maturation significantly different from that of saline-treated controls. Surfactant protein A is known to play an important role in lung function. In this genotyping study, we investigated the potential correlation between polymorphisms in SFTPA1, messenger RNA and protein levels, and ventilation outcomes in animals treated with ANS. 45 preterm lambs were delivered 48 h after initial ANS therapy and 44 lambs were delivered 8 days after initial ANS therapy. The lambs were ventilated for 30 min after delivery. SFTPA1 mRNA expression in lung tissue was not correlated with arterial blood PaCO₂ values at 30 min of ventilation in lambs delivered 48 h after treatment. SFTPA1 protein in lung tissue was significantly correlated with PaCO₂ at 30 min of ventilation in lambs ventilated both 48 h and 8 days after ANS treatment. Six different single nucleotide polymorphisms (SNPs) in the Ovis aries SFTPA1 sequence were detected by Sanger Sequencing. No individual SNPs or SNP haplotypes correlated with alterations in PaCO₂ at 30 min of ventilation or SFTPA1 protein levels in the lung. For the subset of animals analyzed in the present study, variable lung maturation responses to ANS therapy were not associated with mutations in SFTPA1.

Keywords: antenatal corticosteroids; lung maturation; preterm birth; sheep; single nucleotide polymorphisms.

FIGURE 1

Map of primers. (a, b) Ovis SFTPA1 consists of five exons with 5′ and 3′ UTRs. Primer 1 and primer 7 were set as forward and reverse primers respectively to include all SFTPA1 sequences. Then Primer 1–6 were used for Sanger Sequencing. (c) PCR product using primer 1 and 7 was at 4.4 kbp.

FIGURE 2

Correlations between SFTPA1 mRNA and lung function (48 h study only). The graph shows the correlation between SFTPA1 mRNA expression and PaCO₂ in two‐day study animals. There was no significant correlation between these values. (p = 0.147).

FIGURE 3

Correlation between SFTPA1 protein and lung function. (a–c) The graphs show correlations between SFTPA1 protein fold change against saline control group and PaCO₂ in 48 h study, 8 days study, and all animals from 48 h and 8 days studies, respectively. Each graph shows a significant correlation between them (p < 0.001). (d–f) The graphs show a correlation between SFTPA1 protein fold change against a saline control group and lung compliance at 30 min ventilation in 48 h study, 8 days study, and all animals from 48 h and 8 days studies, respectively. While animals in 48 h study (d) and all animals (f) showed a significant correlation between them (p < 0.001), animals in 8 days study (e) did not (p = 0.116).

FIGURE 4

SNPs and PaCO₂. The graphs show PaCO₂ at 30 min of ventilation in different SNPs and alleles. There was no significant difference between alleles in each group.

FIGURE 5

SNPs and SFTPA1. Graphs show SFTPA1 protein fold changes for different SNPs and alleles. There was no significant difference between alleles in each group.

FIGURE 6

Haplotypes or diplotypes and lung maturation. (a, b) Graphs show PaCO₂ or SFTPA1 protein fold changes for different haplotypes. There were no significant differences between haplotypes. (c, d) The graphs show PaCO₂ or SFTPA1 protein fold changes in different diplotypes including haplotype C. There were no significant differences between diplotypes.