Previous studies have begun to address the role of genetic factors influencing common neonatal morbidities. A retrospective, multicenter study of 450 twin pairs found a genetic contribution for several morbidities of the preterm infant such as bronchopulmonary dysplasia, necrotizing enterocolitis, and intraventricular hemorrhage (16
). Retinopathy of prematurity has also been found to have a strong genetic predisposition (17
). As mentioned earlier, only one paper to date has focused on genetic polymorphisms contributing to the PDA in preterm infants (9
Our approach was to evaluate sequence polymorphisms within candidate genes for association with isolated (non-syndromic) PDA in high-risk preterm infants. We identified seven SNPs associated with the development of a PDA with a p-value of less than 0.01. Another sixteen markers were found to have a p-value between 0.01 and 0.05. We chose a p-value of 0.01 to provide a first level cutoff for markers that might be of interest and warranted replication of results in the second sample population. Replication with a smaller number of SNPs tested does not require the extreme rigor of the Bonferroni adjustment as the initial phase of this study, which used a large number of SNPs and a modest number of cases. How to appropriately analyze large numbers of samples and hypotheses from a statistical standpoint represents a significant challenge that is now arising in large genome-wide association studies where hundreds of thousands of markers are tested on thousands of cases (18
Sequence variations in one SNP (rs987237) in TFAP2B
(transcription factor AP-2B), the gene mutated in Char syndrome, was found to be associated with both PDA and failure of the PDA to close with indomethacin in our population of normal preterm infants. This gene is expressed in neural crest derivatives and is generally involved with development, cell-cycle control, and apoptosis (19
). Embryologic studies using the chick model system have identified cardiac neural crest cells in the wall of the ductus arteriosus, further supporting a role for a gene controlling differentiation of neural crest derivatives in the persistent patency of the ductus arteriosus (21
). Recently, TFAP2B
were both found to be expressed in the smooth muscle of the mouse DA, playing key roles in ductal closure in this animal model by participating in a transcriptional network that regulates ductal smooth muscle development. (Ronald Clyman, personal communication). The specific TFAP2B
marker of interest (rs987237) is located in a highly conserved region between exons three and four containing a number of putative transcription factor binding sites. The SNP rs987237 is present in a haplotype block that, in Caucasians, extends from the intronic region between exons one and two to beyond the end of the gene. This block encompasses exons two, four, and five, where several mutations reported to cause Char syndrome are located (7
). The actual genetic variation(s) responsible for PDA in preterm infants could lie anywhere within this relatively large haplotype block. More detailed mapping of this region and confirmation of the finding in other preterm populations is necessary to further define the actual etiologic polymorphism.
A second SNP (rs1056567), in a haplotype block adjacent to the TRAF1
gene (tumor necrosis factor receptor-associated factor 1), was also found to be associated with PDA in our population. This protein mediates the activity of NF-kappaB and plays a role in modulating both the inflammatory and apoptotic pathways (22
). Both apoptosis and inflammation are known to play an important role in the second (more permanent) phase of ductal closure, fibrosis following muscular contraction and hypoxia. The SNP rs1056567 is also in a haplotype block with a second gene, PHF19
(PHD finger protein 19). PHF19
, which is a human homologue of the Drosophila polycomb-like gene, encodes a protein that binds to DNA and is postulated to play a role in transcriptional regulation (24
Haplotype analysis determines whether combinations of neighboring polymorphisms, rather than individual sequence variations alone, are positively associated with a phenotype. In addition to a two-SNP window in the TRAF1
gene, a second two-SNP combination was found to be associated with PDA in the PTGIS
(prostaglandin I2 synthase) gene. This gene is responsible for the production of prostaglandin I2 (prostacyclin), a potent vasodilator which plays a critical role in fetal patency of the ductus arteriosus (reviewed in (25
)). It is possible that alterations in the regulation of this gene could lead to abnormal postnatal closure of the DA in a preterm infant, since prostacyclin levels are elevated in preterm infants and the concentration of a prostacyclin metabolite (6-ketoprostaglandin F1-alpha) has been shown to correlate with ductal diameter (26
None of the genetic polymorphisms that were positively associated with persistent patency of the DA were located in coding regions; therefore they would not be expected to alter protein structure. They could, however, alter gene expression by changing transcriptional regulatory regions or methylation patterns. Recent reports suggest that haplotype structure may be more important in determining the effect of a variant on function rather that the individual SNP (27
). While none of the genetic variants that we identified is likely to be a causative factor for PDA, sequence variations located within the identified haplotype blocks they represent may be etiologic. Our approach has identified a region of interest for further fine mapping for potential genetic contributors to PDA.
One limitation of a hypothesis-generating (i.e., data-mining), candidate gene approach such as ours is the large number of observations that are produced by the analysis. Because of these multiple observations, it becomes difficult to delineate what p-value represents an appropriate level of significance. We recognize the importance of this issue in the present study. Our use of p=0.01 as a first-phase criteria was a compromise to limit false positives, but may have also led to us to miss some significant genes where our power to detect an affect was limited by sample size. In fact, the second phase of our study demonstrated that five of the seven original positive signals were likely false positives rather than truly associated with PDA. Employing the traditional Bonferroni correction for multiple comparisons in the initial phase of the study would have led to a very stringent level for statistical significance (0.00013), eliminating all candidate genes in the study. This degree of stringency is likely not appropriate when investigating common complex human diseases where an individual gene, along with environmental forces, may play a variable, but important, role in the development of a disease.
PDAs are found with high frequency in preterm infants. It is possible then, that the significant allelic variation described in this report may be associated with preterm birth and are positive simply because PDA is a common morbidity of the preterm infant. In order to eliminate preterm birth as a possible confounding factor in the analysis for predisposition to PDA, we also performed an analysis for association with preterm delivery with the same data set of infants less than 32 weeks gestation. None of the polymorphisms that were positively associated with PDA were found to be associated with preterm birth overall.
Gene-environment interactions almost certainly play a role in PDA. It is very likely that extreme prematurity is the environmental condition upon which a genetic predisposition to PDA becomes manifest, a hypothesis supported by the very low incidence of PDA in term infants. The cardiovascular environment of a preterm neonate is very different from that of a term infant in a number of ways (e.g
., lower PaO2
, lower systemic blood pressure, need for positive pressure ventilation, etc.) that may promote persistent patency of the DA. Additional conditions, such as birth via cesarean section and lower hematocrit at birth, have been associated with failure of the PDA to close with indomethacin treatment (28
). It has also been demonstrated that preterm infants of mothers who received indomethacin tocolysis have an increased incidence of symptomatic PDA (29
). Prematurity and the abnormal anatomy and physiology that goes along with it, coupled with a genetic predisposition, may lead to the development of a PDA. Infants born at term, even with a genetic risk, may not have a PDA because they are simply not exposed to the inciting environment.
There are practical benefits in delineating genetic factors associated with PDA. It is common practice to attempt closure with non-steroidal anti-inflammatory medications such as indomethacin or ibuprofen. The number of such attempts before surgical closure is undertaken varies by institution. Medical treatment of the PDA, however, is not without potential risks and side effects. Recent studies have shown that adverse effects from the early use of indomethacin may offset the benefits of early DA closure. While early closure of the PDA has been shown to reduce incidence of IVH, no reduction in BPD has been seen (30
). If an infant’s genotype is associated with failure of medical closure and could reliably predict the need for surgical ligation, we may one day incorporate genotype data into whether to subject the infant to medical therapy before definitive closure of the PDA surgically. In this study we did see a borderline significant association of alleles in the TFAP2B
genes with the subsequent need for surgical ligation that, if replicated, might afford an opportunity for such early predictions. Finally, identifying genes and pathways that play a role in PDA closure can advance our understanding of developmental cardiovascular physiology in humans.