OBJECTIVE: To examine whether selected genetic polymorphisms in the infant are associated with spontaneous preterm birth (less than 37 weeks) among children with or without later-diagnosed cerebral palsy.
METHODS: Exploratory case–control study investigating the relationship of gestational age at delivery to 31 single nucleotide polymorphisms measured in newborn screening bloodspots. Among all 443 children with later-diagnosed cerebral palsy born to white women in South Australia in 1986–1999, 234 were born after spontaneous onset of labor, and 108 of these were preterm (gestational age less than 37 weeks). The comparison group was 549 infants born after spontaneous onset of labor, of whom 147 were preterm. Distributions of genotypic frequencies were examined in preterm compared with term infants with and without cerebral palsy. Genotyping was performed using a Taqman assay.
RESULTS: In children without cerebral palsy, preterm birth after spontaneous onset of labor was more frequent in association with a variant of the β2 adrenergic receptor gene (ADRB2 Q27E, P=.003), inducible nitric oxide synthase (iNOS or NOS2A, P=.042), or thrombomodulin (G127A, P=.006). Among children with cerebral palsy, preterm birth was associated with polymorphisms in genes for endothelial nitric oxide synthase (eNOS -922, P=.012), plasminogen activator inhibitor-2 (P=.015 and .019), and alpha adducin (ADD1, P=.047).
CONCLUSION: We confirm previous observations that variants of the β adrenergic receptor and of nitric oxide synthase are associated with prematurity, and suggest that genetic variants of the placental antifibrinolytic plasminogen activator inhibitor-2, and thrombomodulin and alpha adducin may be contributors to risk of spontaneous preterm birth.
LEVEL OF EVIDENCE: II
In infant blood, genetic variants of the &#x03B2; adrenergic receptor, nitric oxide synthase, and the placental plasminogen activator inhibitor are associated with spontaneous preterm birth.
From the 1School of Paediatrics and Reproductive Health, University of Adelaide, Adelaide, Australia; 2Department of Microbiology and Infectious Diseases, Women’s and Children’s Hospital, Adelaide, Australia; 3National Institute of Neurological Diseases and Stroke, Bethesda, Maryland; and 4Laboratory of Molecular Technology, SAIC-Frederick, Inc., National Cancer Institute, Frederick, Maryland.
Funded by the Australian National Health and Medical Research Council, The Channel Seven Children’s Research Foundation, and the South Australian Government Captive Insurance Corporation, and by funds from the National Cancer Institute and National Institute of Neurological Disorders and Stroke, the former under contract NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
The authors thank the other contributing members of the South Australian Cerebral palsy Research Group for their assistance with the clinical data linkage system: A. Chan, E.A. Haan, K. Priest, E. Ranieri, H. Scott, and P. Sharpe.
Corresponding author: Karin B. Nelson, MD, National Institutes of Health, Building 31, room 8A03, Bethesda, MD 20892-2540; e-mail: email@example.com.
The rate of preterm birth is increasing in many countries, and in the United States is 12.5% and rising.1 Premature birth, delivery before 37 weeks gestation, is a major risk factor for perinatal mortality or long-term neurologic disability, especially cerebral palsy. There are no effective means as yet for prevention of preterm birth.2
The risk of preterm birth is increased in women who have had a previous preterm birth and increases with the number of previous preterm deliveries.3 Both environmental4 and genetic factors4,5 are relevant to risk of premature birth. An Australian twin study estimated the heritability of delivery 2 or more weeks before term to be about 27%,6 and a Swedish twin study estimated the heritability of gestational age at birth to be 25–40%.7 A number of genes have been examined for association with preterm birth, including the adrenergic β receptor,8–10 genes related to thrombophilias,11–13 and those related to immune processes.4,14–17 Recent reviews have summarized findings to date.18,19
Despite some progress, evidence on genetic polymorphisms and risk of preterm birth remains scarce, and only a narrow range of possible contributing factors has been explored. In an effort to develop information for the design of further studies, we examined in an exploratory study the association of 31 candidate single nucleotide polymorphisms (SNPs) with risk of delivery before 37 weeks of gestation in white children born in South Australia. This sample, which is population-based for children with cerebral palsy, was assembled for a study of genotype and cerebral palsy, the subject of a recently published20 and future reports. The examined SNPs were selected because of their association with early stroke or cardiovascular disease or with cerebral palsy in very preterm infants in a previous study.21
The study population consisted of all children with cerebral palsy born 1986–1999 in South Australia (SA) to white mothers (N=443), ascertained by the South Australia Cerebral Palsy Register from notifications by pediatricians, hospitals, and child treatment centers. A total of 883 infants without cerebral palsy born to white mothers between the years 1986 and 1999 were selected for comparison. Newborn screening cards were identified by the South Australia Newborn Screening Program for each case. Potential controls were the four infants who survived at least to age 1 year, whose dates of birth were within a few days of each child with cerebral palsy, whose hospital where blood was sampled was of the same category (metropolitan teaching, metropolitan private, or country), and whose blood was sampled on approximately the same day of life as the cases. The control population included a higher proportion of preterm infants than the general population, because many of the cases of cerebral palsy were born preterm and had been referred to metropolitan teaching hospitals. Linkage was attempted for all cases and potential controls to the South Australia Perinatal Data Collection of births, with its large number of sociodemographic and clinical variables. This was successful for all cases and 1,691 controls. A total of 268 (15.8%) of these 1,691 controls were excluded because they were children of nonwhite mothers (n=102), children who had a birth defect as identified from the SA Birth Defects Register (n=161), or children who died in the first year of life (n=37) to ensure that they were not potentially cases of cerebral palsy. Some controls were excluded for more than one reason. Two controls were then selected from the remaining controls in each group of four, using random numbers, to form the control population of 886. Because three controls had inadvertently been selected more than once, the final number of controls was 883. The data from all cases and controls were deidentified before testing for polymorphisms and statistical analysis. We limited the study to infants whose delivery followed spontaneous onset of labor, yielding 227 case children and 538 control children. All testing for polymorphisms was performed blind to cerebral palsy or control status. Indecisive calls were excluded, leading to slightly different numbers for different SNPs. This research was approved by the Research Ethics Committee of the Women’s and Children’s Hospital, Adelaide, Australia.
The 5′ nuclease assay was used, with oligonucleotide probes labeled with two fluorescent dyes, FAM and VIC, to distinguish between the two alleles of each biallelic SNP. All assays were designed and developed either using Assay-by-Design (Applied Biosystem Inc, Foster City, CA) or Primer Express 2.0 software (Applied Biosystem). All oligo primers and probes were synthesized by Applied Biosystem, Inc. Assays were validated and optimized using in-house samples of DNA collected for European subjects, with a no-template control and control DNAs of known genotype run on each assay plate for quality control. Assays were set up in 384-well plates by using 2.5 μL of 2X the Taqman Universal Master Mix (No AmpErase UNG; Applied Biosystem), which contains all four deoxynucleotides, Taq polymerase, and Taqman buffer, 0.125 μL of 40X Assay Mix including forward and reverse primers and FAM and VIC labeled probes, and 1–5 ng of genomic DNA diluted in distilled H2O in a final volume of 5 μL reactions. The thermal cycling conditions for the ABI 7900HT Sequence Detector were an initial denaturation step of 95°C for 5 minutes followed by 40 cycles each of 92°C for 15 seconds and 60°C for 1 minute.
Data output was processed and downloaded electronically into analysis programs. The SDS 2.1 was used to determine the genotypes of the samples. Some data points did not cluster well; these were eliminated in the analysis.
This study is exploratory and the usual statistical caveats for association studies of complex traits with genotypes prevail. Descriptive statistics for gestational age, birth weight, and gender by categories of prematurity are provided in Table 1. Associations of genotypic distribution by three levels of prematurity were assessed separately for children with and without cerebral palsy using the exact Fisher-Freeman-Halton test statistics22 and Monte Carlo confidence intervals for P values were obtained using 10,000 samples as implemented in Stat Exact 6.23 Adjustments for multiple comparisons were not employed, and SNPs with P values less than .10 are indicated in Table 2.
The SNPs tested, and their abbreviations, gene locations, and identifying numbers, are indicated in Table 3.
Among the 538 children delivered after spontaneous onset of labor who were without later-diagnosed cerebral palsy, 402 were born at 37 weeks gestational age or later, and 147 were preterm. Of the 234 children with cerebral palsy, 126 were born at term, 102 before 37 weeks (Table 1). Mean gestational age, birth weight, gender distribution, and the variances of these were comparable, within gestational age groups and overall, in children with and without later-diagnosed cerebral palsy. However, consistent with the known association of cerebral palsy risk with prematurity and the method of control selection in this study, fewer children with than without cerebral palsy were born at term (P<.001). The association of genotype with prematurity among children born after spontaneous onset of labor was examined separately for children with and without cerebral palsy.
Among children without cerebral palsy who were born after spontaneous onset of labor, those delivered preterm were more frequently homozygous for the minor allele of the β2 adrenergic receptor gene (ADRB2 Q7E), and less frequently heterozygous, than those born at term (Table 3, P=.003). The THBD A allele was observed more frequently in preterm than in term infants (P=.006). Preterm infants were more often heterozygous for inducible nitric oxide synthase (iNOS NOS2A, P=.042). For all of these SNPs, patterns were similar in male and female infants and in the total.
In children with later-diagnosed cerebral palsy, homozygosity for the minor allele of all three SNPs examined for plasminogen activator inhibitor-2 (PAI-2; Serp B) (P=.015 and .019) was associated with preterm birth, significantly for two. The same five children with cerebral palsy were homozygous for all three of these PAI-2 SNPs.
Heterozygosity for ADD1 was associated with preterm birth (P=.047), more strikingly in boys. Premature infants with later-diagnosed cerebral palsy were less frequently heterozygous for eNOS (P=.050). Heterozygosity for factor V Leiden was negatively associated with preterm birth in boys (P=.049).
This study included all children with cerebral palsy born to white women in South Australia over a 13-year period, and a comparison group of white children without cerebral palsy, born in hospitals of the same characteristics over the same time period. The relative proportion of children with and without cerebral palsy was not the same as in general population, and considering these groups together would not approximate the general population. Therefore, associations with the studied genetic factors were evaluated separately in children with and without cerebral palsy, comparing term with preterm children with cerebral palsy, and term with preterm children without cerebral palsy. Consistent with the increase in risk of cerebral palsy with prematurity, children with cerebral palsy were more often preterm than was the comparison group; it is not obvious that this disparity should lead to bias in distribution of genotypes within groups.
We investigated only infant genotypes. The relationship of one or both variant alleles in fetal blood with preterm birth might be related at least in part to the maternal genotype, or be due to gene expression in the placenta or fetus of genes inherited from the father. Examination of maternal as well as infant genotypes will be important in future investigations.
Preterm birth is initiated by the onset of preterm labor, by premature rupture of membranes, or by iatrogenic intervention. The biology of preterm labor is still incompletely understood but includes roles for inflammation and for intrinsic mechanisms that influence uterine quiescence compared with contractility and affect cervical ripening.22–26
We observed in children with and in those without cerebral palsy that genotypes for genes related to nitric oxide synthase were differently distributed in term and preterm infants born after spontaneous onset of labor. Nitric oxide is important in maintenance of uterine quiescence during pregnancy27 and in cervical ripening associated with labor28 as well as control of blood flow in uterus, placenta, and brain. Nitric oxide also functions in inflammation and in nitrative injury.29 The mRNAs for iNOS, eNOS, and neuronal NOS are present in the human cervix, in levels higher in preterm as compared with term labor.30 Hao et al31 found NOS2A associated with preterm delivery in white subjects. Endothelial NOS SNPs were related to cerebral palsy risk in preterm infants in a previous study.21
In children without cerebral palsy, we observed an association of a β-2 adrenergic receptor variant in infant blood with preterm birth. Three previous reports, with varying exclusions and in differing ethnic groups, have examined selected samples for a possible association of an ADRB2 SNP with birth before 37 weeks, finding associations of arg16gly or gly27glu with preterm birth.8–10 These variants are in linkage disequilibrium, and their frequency differs by ethnic group.32 Adrenergic activation is inhibitory on smooth muscle in many tissues including the uterus, decreasing uterine contractility and altering vascular regulation.33 β-adrenergic receptor function also influences interactions between neuroendocrine and immune systems.34,35
Three variants of PAI-2 were observed with greater frequency in preterm birth among children with cerebral palsy; the same five prematurely born boys with cerebral palsy in this study were homozygous for the minor allele of these three SNPs. Impairment of plasminogen activator inhibitory activity may contribute to premature termination of pregnancy.36 The fibrinolytic system, tissue plasminogen activator, and its inhibitors PAI-1 and PAI-2 (Serpin 1 and SerpinB 2) are involved in degradation of intravascular clots and in tissue remodeling and fetal development.37 The plasminogen activators and their inhibitors influence matrix metalloproteinases,38 involved in cervical ripening. The PAI-2 protein, presumably of placental origin, is detectable in adult plasma only in pregnant women. Levels of PAI-1 and PAI-2 increase during pregnancy and are at their highest at term and in the puerperium.
Thrombomodulin binds thrombin, a potent stimulator of contractility,38 and may be part of the mechanism relating intrauterine bleeding conditions with preterm labor. Thrombin–antithrombin complexes precede and predict preterm birth.39,40 The rarity of the minor allele complicates interpretation of the observed association of the thrombomodulin SNP with preterm birth in this study. Our findings suggest that variants of PAI-2, the placental antifibrinolytic, may contribute to preterm birth in children with cerebral palsy. Adducin, related to hypertension and to myocardial and cerebrovascular infarction,41 has apparently not previously been explored for association with preterm birth.
The findings of this study in blood of white infants support previous observations indicating an association of genetic variants of nitric oxide synthase and ADRB 2 with premature birth. It was unanticipated that some of the genetic variants associated with preterm birth were observed to differ in children with compared with those without cerebral palsy, an observation consistent with the possibility that pathways to preterm birth may differ in the degree to which they predispose to brain injury.
1. Preterm birth: crisis and opportunity. Lancet 2006;368:339.
2. Goldenberg RL, Rouse DJ. Prevention of premature birth. N Engl J Med 1998;339:313–20.
3. Bloom SL, Yost NP, McIntire DD, Leveno KJ. Recurrence of preterm birth in singleton and twin pregnancies. Obstet Gynecol 2001;98:379–85.
4. Adams KM, Eschenbach DA. The genetic contribution towards preterm delivery. Semin Fetal Neonatal Med 2004;9 445–52.
5. Ward K. Genetic factors in preterm birth. BJOG 2003;110 suppl:117.
6. Treloar SA, Macones GA, Mitchell LE, Martin NG. Genetic influences on premature parturition in an Australian twin sample. Twin Res 2000;3:80–2.
7. Clausson B, Lichtenstein P, Cnattingius S. Genetic influence on birthweight and gestational length determined by studies in offspring of twins. BJOG 2000;107:375–81.
8. Landau R, Xie HG, Dishy V, Stein CM, Wood AJ, Emala CW, et al. beta2-Adrenergic receptor genotype and preterm delivery. Am J Obstet Gynecol 2002;187:1294–8.
9. Ozkur M, Dogulu F, Ozkur A, Gokmen B, Inaloz SS, Aynacioglu AS. Association of the Gln27Glu polymorphism of the beta-2-adrenergic receptor with preterm labor. Int J Gynecol Obstet 2002;77:209–15.
10. Doh K, Sziller I, Vardhana S, Papp Z, Witkin SS. Beta2-adrenergic receptor gene polymorphisms and pregnancy outcome. J Perinat Med 2004;32:413–7.
11. Resch B, Gallistl S, Kutschera J, Mannhalter C, Muntean W, Mueller WD. Thrombophilic polymorphisms—factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations—and preterm birth. Wien Klin Wochenschr 2004;116:622–6.
12. Hartel C, von Otte S, Koch J, Ahrens P, Kattner E, Segerer H, et al. Polymorphisms of haemostasis genes as risk factors for preterm delivery. Thromb Haemost 2005;94:88–92.
13. Valdez LL, Quintero A, Garcia E, Olivares N, Celis A, Rivas F Jr, et al. Thrombophilic polymorphisms in preterm delivery. Blood Cells Mol Dis 2004;33:51–6.
14. Annells MF, Hart PH, Mullighan CG, Heatley SL, Robinson JS, Bardy P, et al. Interleukins-1, -4, -6, -10, tumor necrosis factor, transforming growth factor-beta. FAS, and mannose-binding protein C gene polymorphisms in Australian women: risk of preterm birth. Am J Obstet Gynecol 2004;191:2056–67.
15. Hao K, Wang X, Niu T, Xu X, Li A, Chang W, et al. A candidate gene association study on preterm delivery: application of high-throughput genotyping technology and advanced statistical methods. Hum Mol Genet 2004;13:683–91.
16. Hartel Ch, Finas D, Ahrens P, Kattner E, Schaible T, Muller D, et al. Polymorphisms of genes involved in innate immunity: association with preterm delivery. Mol Hum Reprod 2004;10:911–5.
17. Engel SA, Erichsen HC, Savitz DA, Thorp J, Chanock SJ, Olshan AF. Risk of spontaneous preterm birth is associated with common proinflammatory cytokine polymorphisms. Epidemiology 2005;16:469–77.
18. Crider KS, Whitehead N, Buus RM. Genetic variation associated with preterm birth: A HuGE review. Genet Med 2005;7:593–604.
19. Giarratano G. Genetic influences on preterm birth. MCN Am J Matern Child Nurs 2006;31:169–75.
20. Gibson CS, MacLennan AH, Hague WM, Haan EA, Priest K, Chan A, et al. Associations between inherited thrombophilias, gestational age, and cerebral palsy. Am J Obstet Gynecol 2005;193:1437.
21. Nelson KB, Dambrosia JM, Iovannisci DM, Cheng S, Grether JK, Lammer E. Genetic polymorphisms and cerebral palsy in very preterm infants. Pediatr Res 2005;57:494–9.
22. Freeman GH, Halton JH. Note on an exact treatment of contingency, goodness of fit and other problems of significance. Biometrika 1951;38:141–9.
24. Terzidou V, Bennett PR. Preterm labour. Current Opin Obstet Gynecol 2002;14:105–13.
25. Buxton IL. Regulation of uterine function: a biochemical conundrum in the regulation of smooth muscle relaxation. Mol Pharmacol 2004;65:1051–9.
26. Hertelendy F, Zakar T. Regulation of myometrial smooth muscle functions. Curr Pharm Des 2004;10:2499–517.
27. Wetzka B, Schafer WR, Stehmans A, Zahradnik HP. Effects of nitric oxide donors on the contractility and prostaglandin synthesis of myometrial strips from pregnant and non-pregnant women. Gynecol Endocrinol 2001;15:34–42.
28. Facchinnetti F, Venturini P, Blasi I, Giannella L. Changes in the cervical competence in preterm labour. BJOG 2005;112 suppl:23–7.
29. Hooper WC. The relationship between inflammation and the anticoagulant pathway: the emerging role of endothelial nitric oxide synthase (eNOS). Curr Pharm Des 2004;10:923–7.
30. Tornblom SA, Maul H, Klimaviciute A, Garfield RE, Bystrom B, Malmstrom A, et al. mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour. Reprod Biol Endocrinol 2005;3:33–42.
31. Hao K, Wang X, Niu T, Xu X, Li A, Chang W, et al. A candidate gene association study on preterm delivery: application of high-throughput genotyping technology and advanced statistical methods. Hum Mol Genet 2004;13:683–91.
32. Xie HG, Stein CM, Kim RB, Xiao ZS, He N, Zhou HH, et al. Frequency of functionally important beta-2 adrenoceptor polymorphisms varies markedly among African-American, Caucasian and Chinese individuals. Pharmacogenetics 1999;9:511–516.
33. Turki J, Pak J, Green SA, Martin RJ, Liggett SB. Genetic polymorphisms of the beta 2-adrenergic receptor in nocturnal and nonnocturnal asthma. Evidence that Gly16 correlates with the nocturnal phenotype. J Clin Invest 1995;95:1635–41.
34. Wahle M, Krause A, Pierer M, Hantzschel H, Baerwald CG. Immunopathogenesis of rheumatic diseases in the context of neuroendocrine interactions. Ann N Y Acad Sci 2002;966:355–64.
35. Podojil JR, Sanders VM. CD86 and beta2-adrenergic receptor stimulation regulate B-cell activity cooperatively. Trends Immunol 2005;26:180–5.
36. Glueck CJ, Phillips H, Cameron D, Wang P, Fontaine RN, Moore SK, et al. The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type 1 gene: an independent risk factor for serious pregnancy complications. Metabolism 2000;49:845–52.
37. Kruithof EK, Baker MS, Bunn CL. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood 1995;86:4007–24.
38. Nicholl SM, Roztocil E, Davies MG. Plasminogen activator system and vascular disease. Curr Vasc Pharmacol 2006;4:101–16.
39. Elovitz MA, Baron J, Phillippe M. The role of thrombin in preterm parturition. Am J Obstet Gynecol 2001;185:1059–63.
40. O’Sullivan CJ, Allen NM, O’Loughlin AJ, Friel AM, Morrison JJ. Thrombin and PAR1-activating peptide: effects on human uterine contractility in vitro. Am J Obstet Gynecol 2004;190:1098–105.
41. Morrison AC, Doris PA, Folsom AR, Nieto FJ, Boerwinkle E. G-protein beta3 subunit and alpha-adducin polymorphisms and risk of subclinical and clinical stroke. Stroke 2001;32:822–9.
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