John M. Dagle

Evaluation of fetal and maternal genetic variation in the progesterone receptor gene for contributions to preterm birth

Progesterone plays a critical role in the maintenance of pregnancy and has been effectively used to prevent recurrences of preterm labor. We investigated the role of genetic variation in the progesterone receptor (PGR) gene in modulating risks for preterm labor by examining both maternal and fetal effects. Cases were infants delivered prematurely at the University of Iowa. DNA was collected from the mother, infant, and father. Seventeen single nucleotide polymorphisms (SNP) and an insertion deletion variant in PGR were studied in 415 families. Results were then analyzed using transmission disequilibrium tests and log-linear-model–based analysis. DNA sequencing of the PGR gene was also carried out in 92 mothers of preterm infants. We identified significant associations between SNP in the PGR for both mother and preterm infant. No etiologic sequence variants were found in the coding sequence of the PGR gene. This study suggests that genetic variation in the PGR gene of either the mother or the fetus may trigger preterm labor.

Chromosome Targeting at Short Polypurine Sites by Cationic Triplex-forming Oligonucleotides

Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15–30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine orN,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine >N,N-dimethylaminopropylamine ≫ phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly,N,N-diethylethylenediamine andN,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively chargedN,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.

Role of polymorphic variants as genetic modulators of infection in neonatal sepsis.

This study is a retrospective, case control study involving 535 preterm infants examining the roles of sequence polymorphisms in genes that mediate host immune responses to bacterial infection in newborn infants. A total of 49 single nucleotide polymorphisms (SNPs) in 19 candidate genes including inflammatory cytokines (IL6, IL10, IL1B, and TNF), cytokine receptors (IL1RN), toll-like receptors (TLR2, TLR4, and TLR5), and cell surface receptors (CD14) were genotyped. Subjects were stratified into three groups (sepsis, suspected sepsis, and control). The data were analyzed using a family-based transmission disequilibrium test. We found that birth weight, gestational age, duration of rupture of membranes, and presence of clinical chorioamnionitis were strongly associated with sepsis. Polymorphisms in TLR2 (rs3804099), TLR5 (rs5744105), IL10 (rs1800896), and PLA2G2A (rs1891320) genes were associated with sepsis. Allelic variants in PLA2G2A and TLR2 were associated with Gram-positive infections, whereas IL10 was associated with Gram-negative infections (p < 0.05). We conclude allelic variations in PLA2G2A, TLR2, TLR5, and IL10 may moderate the predisposition to sepsis in preterm infants.

Maternal and fetal variation in genes of cholesterol metabolism is associated with preterm delivery

Objective:To examine the contribution of variants in fetal and maternal cholesterol metabolism genes in preterm delivery (PTD).Study Design:A total of 40 single-nucleotide polymorphisms (SNPs) in 16 genes related to cholesterol metabolism were examined for 414 preterm infants (gestational ages 22 to 36 weeks; comprising 305 singletons and 109 twins) and at least 1 parent. Fetal effects were assessed using the transmission disequilibrium test (TDT) for each SNP, followed by a log linear model-based approach to utilize families with missing parental genotypes for those SNPs showing significance under TDT. Genetic variant effects were examined for a role in PTD, gestational age and birth weight. Maternal effects were estimated using a log linear model-based approach.Result:Among singleton gestations, suggestive association (P<0.01 without adjusting for multiple comparisons) was found between birth weight and fetal DHCR7 gene/SNP combinations (rs1630498, P=0.002 and rs2002064, P=0.003). Among all gestations, suggestive associations were found between PTD and fetal HMGCR (rs2303152, P=0.002) and APOA1 (rs 5070, P=0.004). The result for HMGCR was further supported by the log linear model-based test in the single births (P=0.007) and in all births (P=0.006). New associations (APOE and ABCA1) were observed when birth weight was normalized for gestational age suggesting independent effects of variants on birth weight separate from effects on PTD. Testing for maternally mediated genetic effects has identified suggestive association between ABCA1 (rs4149313, P=0.004) and decreased gestational age.Conclusion:Variants in maternal and fetal genes for cholesterol metabolism were associated with PTD and decreased birth weight or gestational age in this study. Genetic markers may serve as one mechanism to identify high-risk mothers and fetuses for targeted nutritional treatment and/or prevention of low birth weight or PTD.

Genetic Contributions to the Development of Retinopathy of Prematurity

There is growing support for the role of genetic factors in the development of retinopathy of prematurity (ROP), a serious visual morbidity resulting from preterm birth. We used both candidate gene and data-mining approaches to investigate the role of genetic polymorphisms in the development of ROP. Our study population consisted of 330 infants, less than 32 wk gestation, and their parents. We initially studied 24 single nucleotide polymorphisms (SNPs) in 11 candidate genes. Using a family-based analysis strategy, we found an association between SNPs in the EPAS1 gene and the development of ROP (p = 0.007). Logistic regression analysis showed three SNPs associated with development of ROP, two in the CFH gene (p = 0.01) and one in the EPAS1 gene (p = 0.001). Extending this analysis to include genotyping data from a larger genetic study of prematurity (455 SNPs in 153 genes), we found SNPs in five genes associated with the development of ROP: IHH (p = 0.003), AGTR1 (p = 0.005), TBX5 (p = 0.003), CETP (p = 0.004), and GP1BA (p = 0.005). Our data suggest that genetic risk factors contribute to the development of ROP.

Individual and Center-Level Factors Affecting Mortality Among Extremely Low Birth Weight Infants

OBJECTIVE: To examine factors affecting center differences in mortality for extremely low birth weight (ELBW) infants. METHODS: We analyzed data for 5418 ELBW infants born at 16 Neonatal Research Network centers during 2006–2009. The primary outcomes of early mortality (≤12 hours after birth) and in-hospital mortality were assessed by using multilevel hierarchical models. Models were developed to investigate associations of center rates of selected interventions with mortality while adjusting for patient-level risk factors. These analyses were performed for all gestational ages (GAs) and separately for GAs <25 weeks and ≥25 weeks. RESULTS: Early and in-hospital mortality rates among centers were 5% to 36% and 11% to 53% for all GAs, 13% to 73% and 28% to 90% for GAs <25 weeks, and 1% to 11% and 7% to 26% for GAs ≥25 weeks, respectively. Center intervention rates significantly predicted both early and in-hospital mortality for infants <25 weeks. For infants ≥25 weeks, intervention rates did not predict mortality. The variance in mortality among centers was significant for all GAs and outcomes. Center use of interventions and patient risk factors explained some but not all of the center variation in mortality rates. CONCLUSIONS: Center intervention rates explain a portion of the center variation in mortality, especially for infants born at <25 weeks’ GA. This finding suggests that deaths may be prevented by standardizing care for very early GA infants. However, differences in patient characteristics and center intervention rates do not account for all of the observed variability in mortality; and for infants with GA ≥25 weeks these differences account for only a small part of the variation in mortality.

Improving survival of extremely preterm infants born between 22 and 25 weeks of gestation.

OBJECTIVE: To estimate observed compared with predicted survival rates of extremely premature infants born during 2000–2009, to identify contemporary predictors of survival, and to determine if improved survival rates occurred during the decade. METHODS: We conducted a retrospective cohort analysis of 237 inborn neonates without major congenital anomalies born from 2000 to 2009 after 22 to 25 completed weeks of gestation. Observed survival rates at each gestational age were compared with predicted survival rates based on gestational age, birth weight, sex, singleton or multiple gestation, and antenatal corticosteroid administration estimated by a Web-based calculator that was derived from 1998 to 2003 outcomes of a large national cohort. Multivariable logistic regression analysis was used to identify significant predictors of survival of the study cohort, including year of birth. RESULTS: Survival rates for the decade by gestational age (compared with predicted rates) were: 22 weeks, 33% (compared with 19%); 23 weeks, 58% (compared with 38%); 24 weeks, 87% (compared with 58%); and 25 weeks, 85% (compared with 70%). Antenatal corticosteroids were administered in 96% of pregnancies. Variables that significantly predicted survival and their odds ratios (OR) with 95% confidence intervals (CI) are: antenatal corticosteroid administration (OR 5.27, CI 1.26–22.08); female sex (OR 3.21, CI 1.42–7.26); gestational age (OR 1.89, CI 1.27–2.81); 1-minute Apgar score (OR 1.39, CI 1.15–1.69); and birth year (OR 1.17, CI 1.02–1.34). The number needed to treat with any antenatal corticosteroid therapy to prevent one death was 2.4. CONCLUSION: In this single-institution cohort treated aggressively (antenatal corticosteroid administration [even if less than 24 weeks], tocolysis until steroid course complete, cesarean for fetal distress) by perinatologists and neonatologists, survival rates at 22–25 weeks of gestation age for inborn infants during the 2000s exceeded predicted rates, with increasing odds of survival during the decade. Antenatal corticosteroid administration had a significant effect on survival. LEVEL OF EVIDENCE: II

Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis.

The experimental manipulation of early embryologic events, resulting in the misexpression of the homeobox transcription factor pitx2, is associated with subsequent defects of laterality in a number of vertebrate systems. To clarify the role of one pitx2 isoform, pitx2c, in determining the left-right axis of amphibian embryos, we examined the heart and gut morphology of Xenopus laevis embryos after attenuating pitx2c mRNA levels using chemically modified antisense oligonucleotides. We demonstrate that the partial depletion of pitx2c mRNA in these embryos results in alteration of both cardiac morphology and intestinal coiling. The most common cardiac abnormality seen was a failure of rightward migration of the outflow tract, while the most common intestinal laterality phenotype seen was a full reversal in the direction of coiling, each present in 23% of embryos injected with the pitx2c antisense oligonucleotide. An abnormality in either the heart or gut further predisposed to a malformation in the other. In addition, a number of other cardiac anomalies were observed after pitx2c mRNA attenuation, including abnormalities of atrial septation, extracellular matrix restriction, relative atrial-ventricular chamber positioning, and restriction of ventricular development. Many of these findings correlate with cardiac defects previously reported in pitx2 null and hypomorphic mice, but can now be assigned specifically to attenuation of the pitx2c isoform in Xenopus.

Genetic Associations of Surfactant Protein D and Angiotensin-Converting Enzyme with Lung Disease in Preterm Neonates

Objective:To replicate genetic associations with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in genes related to surfactant deficiency, inflammation and infection, and the renin–angiotensin system.Study Design:We examined eight candidate genes for associations with RDS and BPD in 433 preterm birth (PTB—<37 weeks) infants (251 with RDS and 134 with BPD). Both case–control and family-based analyses were performed in preterm (<37 weeks) and very preterm birth (VPTB—<32 weeks) infants.Result:We replicated a previous finding that rs1923537, a marker downstream of surfactant protein D (SFTPD) is associated with RDS in VPTB infants in that the T allele was overtransmitted from parents to offspring with RDS (P=8.4 × 10−3). We also observed the A allele of rs4351 in the angiotensin-converting enzyme (ACE) gene was overtransmitted from parents to VPTB offspring with BPD (P=9.8 × 10−3).Conclusion:These results give further insight into the genetic risk factors for complex neonatal respiratory diseases and provide more evidence of the importance of SFTPD and ACE in the etiology of RDS and BPD, respectively.

TBP paralogs accommodate metazoan- and vertebrate-specific developmental gene regulation

In addition to TATA‐binding protein (TBP), a key factor for transcription initiation, the metazoan‐specific TBP‐like factor TLF/TRF2 and the vertebrate‐specific factor TBP2/TRF3 are known to be required for transcription of specific subsets of genes. We have combined an antisense‐knockdown approach with transcriptome profiling to determine the significance and biological role of TBP‐independent transcription in early gastrula‐stage Xenopus laevis embryos. Here, we report that, although each of the TBP family members is essential for embryonic development, relatively few genes depend on TBP in the embryo. Most of the transcripts that depend on TBP in the embryo are also expressed maternally and in adult stages, and show no functional specialization. In contrast, TLF is linked to preferential expression in embryos and shows functional specialization in catabolism. A requirement for TBP2 is linked to vertebrate‐specific embryonic genes and ventral‐specific expression. Therefore TBP paralogs are essential for the gene‐regulatory repertoire that is directly linked to early embryogenesis.