Objective
We previously demonstrated that maternal and fetal genotypes are associated independently with neonatal respiratory distress syndrome. The objective of the current study was to determine the impact of maternal and fetal single-nucleotide polymorphisms (SNPs) in key betamethasone pathways on respiratory outcomes that serve as markers for severity of disease.
Study Design
DNA was obtained from women who were given betamethasone and from their infants. Samples were genotyped for 73 exploratory drug metabolism and glucocorticoid pathway SNPs. Clinical variables and neonatal outcomes were obtained. Logistic regression analysis that controlled for relevant clinical variables to determine SNP impact on bronchopulmonary dysplasia (BPD), the need for respiratory support, and surfactant therapy use was performed.
Results
Data from 109 women who delivered 117 infants were analyzed: 14.5% of the infants experienced BPD; 70.8% of the infants needed some respiratory support after birth, and 27.5% of the infants needed surfactant therapy. In a multivariable regression analysis, gestational age at delivery was associated with most neonatal respiratory outcomes ( P ≤ .01), and chorioamnionitis was associated with BPD ( P < .03). The following genotypes were associated with respiratory severity outcomes: BPD–fetal Importin 13 gene ( IPO13 ; rs4448553; odds ratio [OR], 0.01; 95% confidence interval [CI], 0.00–0.92); surfactant use–maternal IPO13 (rs2428953 and 2486014; OR, 13.8; 95% CI, 1.80–105.5; and OR, 35.5; 95% CI, 1.71–736.6, respectively).
Conclusion
Several discrete maternal and fetal SNPs in the IPO13 family may be associated with neonatal respiratory outcomes after maternal antenatal corticosteroid treatment for anticipated preterm birth.
Antenatal corticosteroids are one of the most significant interventions to improve neonatal morbidity and mortality rates. Although the corticosteroids are administered to essentially all women with threatened early preterm delivery, it has been documented that not all neonates experience the same benefits. Differences in neonatal respiratory outcomes are seen in different ethnic groups, independent of gestational age, weight, and other sociodemographic factors. Additionally, respiratory distress syndrome (RDS)–related death has a racial disparity that cannot be explained by demographic characteristics.
Pharmacogenetics is one potential explanation for some of the differences that have been seen in therapeutic response. Genetic polymorphisms in drug-metabolizing enzymes (such as the cytochrome P450 [CYP] families), transporters, and receptors have been able to explain differences in drug response and side-effects for several drugs. We previously described genetic polymorphisms in both maternal and fetal genotypes for drug-metabolizing enzymes such as CYP3A5 and CYP3A7 and for steroid pathway genes such as adenylate cyclase 9 that were associated with the development of RDS in infants who were born to mothers after the administration of betamethasone to enhance fetal maturity in situations of anticipated preterm delivery. These associations were present after being controlled for several key maternal and delivery variables that included maternal race, infant sex, and gestational age at delivery. We are unaware of any study on the pharmacogenetic impact of single-nucleotide polymorphism (SNP) variants in other markers of the severity of respiratory disease in newborn infants after the administration of betamethasone.
The objective of this project was to determine the impact of genetic polymorphisms in the drug metabolism and glucocorticoid pathways of betamethasone on neonatal respiratory outcomes that serve as markers for severity of disease. Additionally, because pregnancy is a unique environment in which both the maternal and fetal/placental compartments play a role in drug metabolism, we sought to determine the impact on these outcomes by analyzing both the maternal and fetal genotypes.
Materials and Methods
Subjects and samples
This was a planned secondary analysis of our betamethasone pharmacogenetics cohort study. The full details of study recruitment, sample acquisition and processing, and analysis plans are contained in the original report. Briefly, women who were admitted to the hospital with threatened preterm delivery who received at least 1 dose of betamethasone were recruited to the study. Informed consent was obtained for all women who were enrolled; the governing institutional review board approved the study. Participants had to be at least 18 years old and at least 23 weeks’ gestation. Exclusion criteria included known fetal anomaly or inability to provide consent. Standard clinical care at the providers’ discretion was provided to the woman, and standard neonatal resuscitation and care practices were provided by the pediatric/neonatal services. Clinical care was not guided by this observational cohort study. The outcome of need for any respiratory support included oxygen use more than “blow-by” oxygen at initial resuscitation and any ventilator support or respiratory support that included continuous positive airway pressure or nasal cannula. Surfactant use was extracted from the neonatal chart. Surfactant therapy was administered per general clinical guidelines by the neonatal care providers. For babies who were not breathing during the resuscitation, surfactant therapy usually is given soon after delivery. For those babies who are breathing but are getting respiratory support, if the work of breathing is moderate or very high or if the fraction of inspired oxygen is >30-40% on continuous positive airway pressure, then surfactant therapy is given to the neonate. The diagnosis of bronchopulmonary dysplasia (BPD) was made by the pediatricians following standard the National Institute of Child Health and Human Development Neonatal Research Network criteria.
Maternal DNA was obtained from whole blood or from a salivary sample if we were unable to obtain blood. Neonatal DNA was obtained from umbilical cord blood or from buccal swabs if we were unable to obtain umbilical cord blood at the time of delivery. Salivary or buccal samples were collected and processed with the use of the Oragene saliva kit (DNA Genotek Inc, Kanata, Ontario, Canada). DNA isolation was done according to manufacturer instructions. Samples were frozen at −80°C until quantification. DNA was extracted from blood samples with the QIAamp DNA mini kits (Qiagen Inc, Valencia, CA). Manufacturer spin protocol instructions were followed for all kits. When manufacturer protocols listed steps for highly concentrated DNA, those steps were followed. Isolated DNA was transferred into cryovials, and all samples were stored at −80°C until quantification.
Genotyping
Seventy-three SNPs were genotyped with a combination of methods that are specific to the desired SNP. Genotypic designations that were assigned from assayed SNPs in CYP3A4, CYP3A5, CYP3A7 , sulfotransferase, multidrug resistance gene-1 ( ABCB1 ), glucocorticoid receptor, and associated pathway gene assays were listed fully in the previous report. The SNPs were selected on the basis of known metabolism pathways of glucocorticoids and previous work on relevant SNPs for glucocorticoid response in asthma. The individuals were genotyped for most SNPs with the use of the OpenArray TaqMan genotyping platform (Applied Biosystems, Foster City, CA). A high throughput genotyping 32-SNP chip was used for some of the SNPs. For SNPs that did not have valid OpenArray assays, predesigned and commercially available Taqman or fragment analysis real-time polymerase chain reaction assays were used according to the manufacturer’s published methods (Applied Biosystems). For other SNPs that did not have predesigned assays (namely CYP3A7 and glucocorticoid receptor), Sanger dideoxy-DNA bidirectional sequencing with high throughput capillary sequencing instrumentation was performed ( http://polymorphicdna.com/reeqvardisc.html ). Sulfotransferase 1A1 copy number variation was determined with semiquantitative polymerase chain reaction followed by fragment analysis. For each SNP, once a platform was chosen, all samples were genotyped with the use of the same platform.
Analysis
Clinical variables of maternal age, maternal race, diabetes mellitus, estimated gestational age at delivery, birthweight, infant gender, cesarean (vs vaginal) delivery, estimated gestational age at first dose of betamethasone, and the presence of chorioamnionitis were recorded as potential cofactors. Estimated gestational age and the woman’s due date were determined by last menstrual period that was confirmed by ultrasound measurements of the fetus as per routine clinical care. Neonatal outcomes of any respiratory or oxygen support after birth, surfactant use, and BPD were all recorded as outcomes. The outcomes of “any respiratory support after birth” did not include short amounts of oxygen or pressure support immediately after delivery. These infants were excluded from this outcome to keep the outcome representative of more significant respiratory difficulty after birth and the initial neonatal resuscitation. A single SNP association analysis was performed initially to assay for any 1-on-1 SNP to outcome associations. Because many clinical variables may influence the development and severity of neonatal respiratory outcomes, we rigorously tested our genetic hypothesis through multivariate analysis. Demographic and delivery information listed earlier were analyzed as covariate variables in a multivariate analysis. Odds ratios (ORs) and 95% confidence interval (CI) were calculated for each SNP. Adjustment for multiple comparisons was not performed because of the small sample size.
Genotype was analyzed in the model in different ways. Genotypic models assess each of the 3 possible allele combinations separately. The dominant model assumes that 1 or 2 copies of the risk allele (variant) lead to increased risk. The recessive model assumes that the 2 copies of risk variant develop risk of the neonatal respiratory outcome. The trend model (or additive model) looked at an “allele-dose” effect in which the more variant alleles that were present were tested for associations with risk of disease. For the analyses that were reported, the dominant model is reported because it consistently performed best in this analysis and our previous analysis. This means that, when a listed SNP is given, the presence of at least 1 minor allele (Aa or aa) is compared with the homozygous wild type (AA). The full results of the recessive and additive models are not displayed because of space considerations and are available from the authors on request. A CYP3A5 genotype score was calculated on the basis of the individual SNPs to categorize the mothers as either expressing or not expressing CYP3A5 . Women who possess two *3 allele do not express CYP3A5 , where as those who are CYP3A5*1/*1,*1/*3 *1/*6 , or *1/*7 do demonstrate some CYP3A5 activity.
Our initial sample size was calculated based on the incidence of RDS. Because this is a secondary analysis, a sample size calculation was not performed for the outcomes that are reported here.
Results
Full details regarding the overall subject population that was recruited can be found in the earlier report. After excluding subjects with missing DNA data, the final cohort that was analyzed included 109 women and 117 neonates with adequate DNA and outcome data. The mean maternal age was 26.5 ± 5.7 years. The median gravidity was 3, and the median parity was 1. There were 4 sets of twins included. Nineteen women (16.4%) were Hispanic. Racial distribution was white (n = 58; 54%), black (36; 33%), other (n = 11; 10%), mixed (n = 2; 2%), and Indian (n = 1; 1%). Paternal ethnicity was Hispanic for 17 (16%) of subjects. Paternal race was white (n = 46; 41%), unknown (n = 29; 26%), black (n = 27; 24%), other (n = 9; 8%), and Indian (n = 1; 1%). Thirteen women (11%) had gestational diabetes mellitus. The gestational age at receipt of the first dose of betamethasone was 28.8 ± 3.3 weeks gestation; the mean gestational age at delivery was 32.2 ± 3.9 weeks gestation. The admission diagnoses were preterm labor (37%), preterm premature ruptured membranes (35%), preeclampsia (15%), other (10%), and placenta previa (3%). Tocolytic medications were given to 35% of the women; nifedipine and magnesium sulfate were the most commonly used (45% and 42%, respectively, of those who received tocolytics). “Rescue” doses of betamethasone were used in 8 women. The mean number of days from first dose of betamethasone to delivery was 23.4 ± 22.2. All but 4 subjects received 2 doses of betamethasone. Sixty-four women (55%) delivered babies vaginally.
The mean birthweight of the babies was 1913 ± 785 g. One-half of the babies were male (51%). Median 1- and 5-minute Apgar scores were 7 and 8, respectively. Sixty-four babies (49%) were diagnosed with RDS; subsequently, 19 babies (14.5%) were diagnosed with BPD. Necrotizing enterocolitis developed in 9 babies (7%); intraventricular hemorrhage developed in 10 babies (7.6%), and neonatal death occurred in 6 babies (4.6%). The gestational age at birth of the babies who died ranged from 24–29 weeks. Neonatal sepsis occurred in 23 babies (17.6%). Thirty-nine babies (30%) were intubated; 70 babies (53.4%) were placed on continuous positive airway pressure; 34 babies (26%) were placed on high-flow nasal cannula, and 38 babies (29%) were treated with nasal cannula room air (fraction of inspired oxygen, 0.21) for respiratory support. Thus, there were 80 infants who required some form of respiratory support beyond the initial newborn resuscitation (70.8%). Surfactant therapy was given to 36 babies (27.5%). Mean total hospital stay was 40 ± 39 days, and mean total days of respiratory support in hospital was 19 ± 33.
All SNPs that were assayed were in Hardy-Weinberg equilibrium ( P ≥ .00083) after correction for multiple comparisons. The overall SNP frequencies can be found in the original report. Maternal carriers of at least 1 copy of the minor allele in CYP3A7 and CRHR1 were associated with BPD ( CYP3A7: rs113874418 [ P = .006]; novel CRHR1 sequence: Chr 17- 43895531 [ P = .006]; CYP3A7*1E : rs28451617 [OR, 4.97; 95% CI, 1.12–22.05; P = .02]). Fetal SNP in the glucocorticoid receptor was associated with BPD ( NR3C1 : rs41423247 [OR, 2.56; 95% CI, 1.11–5.95; P = .02]). No maternal single SNPs were associated with the outcome of any respiratory support, although several had probability values just above .05. The exon 12 SNP in the ABCB1 gene was associated with the need for respiratory support in the newborn infants (rs1128503 [OR, 2.23; 95% CI, 1.05–4.74; P = .03]). A fetal SNP in oxidized low density lipoprotein receptor 1 ( OLR1 ) was associated with surfactant replacement therapy (rs3736233 [OR, 0.35; 95% CI, 0.17–0.71; P = .003]).
The Table displays significant genetic analysis results from the multivariable logistic regression analysis for the 3 outcomes that mark severity of neonatal respiratory disease. In a multivariable regression analysis, gestational age at delivery was associated with most neonatal respiratory outcomes ( P ≤ .01); chorioamnionitis was associated with BPD ( P < .03). The following genotypes were associated with non-RDS respiratory outcomes: surfactant use: maternal importin 13 ( IPO13 ; rs2428953 and 2486014 [OR, 13.8; 95% CI, 1.80–105.5; and OR, 35.5; 95% CI, 1.71–736.6, respectively]); BPD fetal IPO13 (rs4448553 [OR, 0.01; 95% CI, 0.00–0.92]). Trends were seen for independent associations of maternal ABCB1 exon 26 with the need for respiratory support ( P = .05), fetal OLR1 with surfactant use ( P = .07), and maternal CYP3A7*1E and trafficking protein particle complex 5/Fc fragment of IgE, low affinity II receptor ( TRAPPC5/FCER2 ) with BPD ( P = .07 for both).
