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a Departments of Pediatrics
b Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut
c Department of Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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OBJECTIVES. Our goal was to assess the familial and genetic susceptibility to intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia.
METHODS. Mixed-effects logistic-regression and latent variable probit model analysis were used to assess the contribution of several covariates in a multicenter retrospective study of 450 twin pairs born at 
32 weeks of gestation. To determine the genetic contribution, concordance rates in a subset of 252 monozygotic and dizygotic twin pairs were compared.
RESULTS. The study population had a mean gestational age of 29 weeks and birth weight of 1286 g. After controlling for effects of covariates, the twin data showed that 41.3%, 51.9%, and 65.2%, respectively, of the variances in liability for intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia could be accounted for by genetic and shared environmental factors. Among the 63 monozygotic twin pairs, the observed concordance for bronchopulmonary dysplasia was significantly higher than the expected concordance; 12 of 18 monozygotic twin pairs with 
1 affected member had both members affected versus 3.69 expected. After controlling for covariates, genetic factors accounted for 53% of the variance in liability for bronchopulmonary dysplasia.
CONCLUSIONS. Twin analyses show that intraventricular hemorrhage, necrotizing enterocolitis, and bronchopulmonary dysplasia are familial in origin. These data demonstrate, for the first time, the significant genetic susceptibility for bronchopulmonary dysplasia in preterm infants.
Key Words: premature newborn • intraventricular hemorrhage • necrotizing enterocolitis • bronchopulmonary dysplasia • logistic regression • zygosity
Abbreviations: IVH—intraventricular hemorrhage • NEC—necrotizing enterocolitis • BPD—bronchopulmonary dysplasia • BW—birth weight • GA—gestational age • RDS—respiratory distress syndrome • INST—treating institution • OR—odds ratio • CI—confidence interval • TNF—tumor necrosis factor • IL—interleukin
Despite significant clinical advances in neonatal care over the last 2 decades, intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), and bronchopulmonary dysplasia (BPD) continue to account for most of the morbidity and mortality in premature newborns. The National Institute of Child Health and Human Development Neonatal Research Network1 reported a decrease in mortality in infants of birth weight (BW) 500 to 1500 g from 1987–1994. Between 1994 and 2000, however, mortality and the incidence of these conditions plateaued. Thus, the persistence of these major morbidities remains a serious concern.1
The primary factor common to IVH, NEC, and BPD is immaturity of the target organs, brain, gastrointestinal tract, and lungs. Other contributing factors include cerebral blood flow fluctuations,2 changes in mesenteric blood flow, feeding practices,3 ventilator-induced trauma, and hyperoxia.4 We hypothesized that, in addition to these factors, there is a genetic susceptibility for the development of these gestational age (GA)-dependent disorders. Our goal was to conduct a multicenter retrospective study of a large cohort of twins to determine the familial and genetic contributions to IVH, NEC, and BPD.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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32 weeks of gestation between 1994 and 2004 were collected from 4 centers: Yale University, Brown University (Providence, RI), University of Kentucky (Lexington, KY), and the University of Connecticut (Farmington, CT). Zygosity data were available for analyses from 2 centers: Yale and the University of Connecticut. Only infants who survived beyond 36 weeks' postmenstrual age were included. The institutional review boards at each participating center approved this study.
Definitions
Respiratory distress syndrome (RDS) was defined as the presence of respiratory distress with an oxygen requirement in the first 6 hours of life, accompanied by a characteristic chest radiograph. IVH was defined as blood in the germinal matrix or ventricular system with or without ventricular dilatation and/or periventricular hemorrhagic infarction.5 BPD was defined as the need for oxygen supplementation at 36 weeks' postmenstrual age in association with characteristic radiologic changes.6 NEC was defined as stage 2 or more as per modified Bells' criteria.7 Zygosity was determined by histopathological examination of the placenta at Yale and the University of Connecticut with an additional confirmation of the gender.
Statistical Analyses
For the mixed-effect logistic-regression analyses, IVH, NEC, or BPD was the primary outcome variable. The covariates were male gender, GA, BW, treating institution (INST), and RDS. The status of the outcomes from twin pairs was treated as a correlated event in the mixed-effects models. INST was evaluated as an overall variable, as well as individual institutions (1, 2, or 3) compared with a reference institution (4) chosen at random. Differences in demographic variables between groups were compared by using the Student's t or 
2 tests, as appropriate. To estimate the variances in liability for IVH, BPD, and NEC, we used latent variable probit models for twin data.8,9
Mixed-effects logistic-regression models were fitted to produce Table 2 to assess the relationship between the covariates listed in Table 2 with the respective outcome, incorporating the correlation within twin pairs. Mixed-effects probit models were fitted to estimate the genetic contribution to the respective outcome, adjusting for the significant covariates. In this model, a liability variable is assumed underlying the respective outcome. This liability variable is assumed to follow a normal distribution. The mean of the normal distribution depend on the covariates, and the variance can be partitioned into a genetic component, a shared nongenetic component, and a random component. The sum of the first 2 components constitutes the overall sharing between twins and can be estimated from the correlated twins (both monozygotic and dizygotic), and Table 3 reports the results. The genetic component is estimated from the correlation between monozygotic twins beyond that of dizygotic twins.
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of .05, the power (1 – ß) was in excess of 99% for all of the comparisons. Even if the OR were as low as 2.2, the power would be in excess of 80%.
For the 2 analyses of the zygosity data, the IVH, NEC, or BPD rate was calculated as the number of twin pairs with 1 or both affected with the outcome condition divided by the total number of twin pairs. The expected concordance was the probability of both twins in a pair having IVH, NEC, or BPD and was calculated as [(rate)2]. The number of expected concordant twin pairs was calculated as [n(rate)2] where n refers to the total number of twin pairs in the monozygotic and dizygotic groups. The observed concordance was the actual IVH, NEC, or BPD concordance between the twins in a pair. Observed versus expected concordance was compared by using 2 analysis.
To calculate the heritability of susceptibility to BPD, we used the formula 2(CMZ – CDZ) where CMZ is the concordance rate for monozygotic twins and CDZ is the concordance rate for dizygotic twins.11 We also performed mixed-effects logistic-regression analyses on the zygosity data in the same way as we did for the entire data. In addition, we used the latent variable probit model to estimate the genetic variance in liability for BPD.
Anonymized clinical data, formatted in Microsoft (Redmond, WA) Excel spreadsheets, were forwarded from each institution to the statistical cores at Yale and the University of Alabama. Statistical analyses were performed using SAS 9.1 (PROC GLIMMIX and PROC NLMIXED; SAS Institute, Inc, Cary, NC), SPSS 11.5 for Windows and Macintosh (SPSS, Inc, Chicago, IL), and GraphPad Prism 3.0 (GraphPad Software, Inc, San Diego, CA). P < .05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Despite the significant effects of those risk factors, after controlling for their effects, genetic modeling of twin data indicated that 41.3% (P = .02; 95% CI: 7.0–75.6%), 51.9% (P < .001; 95% CI: 33.2–70.6%), and 65.2% (P < .001; 95% CI: 52.6–78.9%), respectively, of the variance in liability for IVH, NEC, and BPD could be accounted for by shared genetic and environmental factors (Table 3).
We analyzed 63 monozygotic and 189 dizygotic twin pairs. Although there were more dizygotic twins (378) than monozygotic twins (126), demographic data for BW, GA, Apgar score at 5 minutes, gender, RDS, IVH, NEC, duration of ventilation, and duration of supplemental oxygen were comparable (Table 4). Monozygotic twins, however, had more BPD (P = .04) and a longer length of stay (P = .03; Table 4).
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BPD occurred in either 1 or both of the twins in 18 (29%) of 63 pairs of monozygotic twins and in 43 (23%) of 189 pairs of dizygotic twins. The ratio between the observed concordance to the expected concordance was significantly higher (P < .0001) in the monozygotic versus the dizygotic group (Table 5). Without adjusting for covariates, BPD had a heritability of 63.6% in the subset of subjects with zygosity data. After controlling for covariates, genetic factors accounted for 53% (P = .004; 95% CI: 16–89%) of the variance in liability for BPD.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In addition to mixed-effects logistic regression, latent variable probit models of our twin pairs also support familial factors contributing to susceptibility for IVH. Published reports of family data and IVH are sparse, but an early retrospective study (in the presurfactant era) examined the incidence of IVH in preterm twins.13 Among 70 newborns with BW <1500 g, of whom 20 had IVH, the only significant association with IVH was RDS. A multiple logistic-regression analysis of these newborns categorized by IVH status demonstrated significant effects of twinship itself and of birth order within a given twinship.13 In another study of 41 twins (mean BW: 929 g), however, there was no difference in the incidence of IVH between monozygotic (n = 17) and dizygotic (n = 24) twins.14
A number of small studies have examined specific mutations that might predispose a preterm newborn to IVH. A point mutation in the factor V Leiden gene, Gln506-FV, was more common in newborns with IVH than in the general population,15 but the carrier state of a factor V Leiden or prothrombin G20210A mutation was predictive for a low rate of IVH grades 2 to 4.16 In contrast, prothrombin G20210A was found to be more prevalent in a cohort of newborns with IVH (12%) than in those without (2%), although the difference was not statistically significant.17 In another study, newborns with the tumor necrosis factor (TNF)- –308 A allele had increased risk of IVH.18
Our data show for the first time that familial factors contribute to susceptibility for NEC. A published review of a large population of multiple-gestation pregnancies showed prematurity to be the only consistent risk factor for NEC with no familial tendency.19 Despite this, candidate genes that contribute to NEC have been sought without conclusive findings. A recent study of TNF- promoter gene variants reported no differences between NEC and control subjects.20 Using the same cohort, these investigators found that newborns with NEC carried the mutant variant of interleukin (IL)-4ra less frequently than controls, even after adjustment for risk factors of NEC.21 No significant differences, however, were found in the allelic frequencies of IL-1ß, IL-6, and IL-10 genes.21
Consistent with published studies, our analyses show that RDS is an important predictor of BPD.22,23 Recently, specific alleles of surfactant apoproteins were shown to have protective and deleterious effects on the pathogenesis of RDS.24–26 Although we used RDS as an independent predictor of BPD, they may share common genetic influences.
Genetic Factors
Zygosity analyses show that a large proportion of the variance in liability for BPD is attributable to genetic factors. The observed concordance among monozygotic twin pairs for BPD is significantly higher than the expected concordance. Because monozygotic twins share 100% of their genome, whereas on average dizygotic twins share 50%, these data strongly support a significant genetic component. Adjusting for the major covariates, such as male gender, RDS, BW, and INST, 53% of the variance in liability is attributable to genetic factors, 63.6% without adjusting for covariates.
This is the first demonstration of a significant genetic contribution to BPD. An earlier published study without zygosity data showed a familial tendency.27 After correcting for potentially significant risk factors, BPD status of the first twin was found to be a significant predictor of BPD in the second twin.27 Others have sought to associate gene mutations with BPD. In a small sample, there was a significantly increased frequency of the SP-A1 polymorphism 6A6 in newborns with BPD.28 In another study, after controlling for race and gender, BPD cases were less likely to be homozygous for the more efficient Val/Val allele of glutathione S-transferase P1 and more likely to have the less efficient Ile isoform.29 Other polymorphisms, TNF- –308, MCP-1 –2518, and TGF-ß1 +915, were not associated with BPD.18
As presented in Table 2, there are significant differences among the INSTs for the 3 diseases; therein lies the advantage of multicenter studies in making the data more generalizable. The statistical significant differences among the GA, BW, RDS, IVH, and BPD are an effect of the large sample size and are not clinically significantly different between the entire cohort and the zygosity subset (Table 1). The limitations of this study include the retrospective nature of the data collection and the variability of the 3 major morbidities among the sites. Prospective data collection and a larger number of sites would address these issues. Thus, whereas we have shown that familial factors contribute to susceptibility for IVH and NEC, a larger sample size will be needed to define the genetic contribution. In addition, the retrospective nature precluded DNA confirmation of zygosity. Placental histopathology and gender were used to determine zygosity status. A monochorionic placenta was regarded as representing monozygotic.30 Approximately 9% of same gender dichorionic placentas are, in fact, monozygotic.31 The reverse may also be true; in rare circumstances, dizygotic twins may present with a monochorionic placenta.30 When adjustments were made for these potential misclassifications using worst-case scenarios, we observed no significant impact on our findings.
These data herald new paradigms for the pathophysiology and treatment of the common diseases of preterm newborns. Traditionally, IVH, NEC, and BPD have been considered to result from interrupted development exacerbated by life-sustaining but detrimental effects of invasive neonatal practice. Certainly this model has been validated by the strides in mortality and morbidity evident from 1970 through 1993, largely because of the advent of surfactant, total parenteral nutrition, promulgation of gentle noninvasive ventilation, and restraint in overly aggressive fluid resuscitation. But since 1994, outcome measures have flattened, perhaps signaling optimization in care and revealing a fixed threshold below which additional and costly treatment measures would yield marginal improvements. Our data suggest that, for BPD, this threshold may be composed of unknown genetic factors, which, if identified by additional studies, could be addressed by specific novel therapies.
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ACKNOWLEDGMENTS |
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Centrs and participants of the Neonatal Genetics Study Group include the following: Yale University School of Medicine: Department of Pediatrics (V. Bhandari, M.J. Bizzarro, A. Shetty, R.A. Ehrenkranz, I. Gross, L.R. Ment, J.R. Gruen), Department of Pathology (L.M. Ernst), Department of Epidemiology and Public Health (X. Zhong, R. Feng, H. Zhang); Brown University School of Medicine: Department of Pediatrics (B.R. Vohr); University of Kentucky School of Medicine: Department of Pediatrics (N. Desai, H.S. Bada); University of Connecticut School of Medicine: Department of Pediatrics (N. Hussain); and University of Alabama at Birmingham: Department of Biostatistics (G.P. Page).
We thank Trupti Akella for help with the compilation of the data.
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FOOTNOTES |
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Address correspondence to Laura Ment, MD, Yale University School of Medicine, Division of Neurology, Department of Pediatrics, Children's Hospital LMP 3089, PO Box 208064, New Haven, CT 06520-8064. E-mail: laura.ment{at}yale.edu
Drs Bizzarro and Shetty contributed equally to this work.
The authors have indicated they have no financial relationships relevant to this article to disclose.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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