Abstract
Intraventricular hemorrhage is a disorder of complex etiology. We analyzed genotypes for 7 genes from 224 inborn preterm neonates treated with antenatal steroids and Grade 3-4 intraventricular hemorrhage and 389 matched controls. Only methylenetetrahydrofolate reductase was more prevalent in cases of intraventricular hemorrhage, emphasizing the need for more comprehensive genetic strategies.
Converging data suggest that intraventricular hemorrhage (IVH) of the preterm neonate is a disorder of complex etiology. IVH has been attributed to changes in cerebral blood flow to the immature germinal matrix microvasculature,1 and the more severe grades are characterized by acute distension of the ventricular system (Grade 3) and parenchymal venous infarction (Grade 4). Nationally, ~15% of all very low birth weight infants have grade (Gr) 3–4 IVH,2 and one-half to three-quarters of survivors develop cognitive impairment and/or cerebral palsy.3-5 Despite advances in neonatal intensive care, the incidence of Gr 3-4 IVH has changed little over the past two decades.2 Recent studies have tested the hypothesis that IVH may be secondary to variability in risk genes, and candidate gene studies have implicated the coagulation, inflammatory and vascular pathways.6-8 However, few have been replicated in independent populations. Factors contributing to these findings include the wide range of ancestries of infants studied, small sample sizes and lack of appropriate controls.
If a major focus of perinatal care is to prevent brain injury and abnormal development,9 then physicians and scientists must better understand those factors that contribute to severe IVH. The objective of this report is to interrogate previously published genetic risk factors for Gr 3-4 IVH in a cohort of inborn appropriate for gestational age (AGA) preterm neonates.
METHODS
Inborn infants with birth weights (BW) 500-1250 g and Gr 3–4 IVH and neonates with normal cranial ultrasounds were enrolled prospectively at 24 universities; additional samples were provided from ELGAN,10 Iowa Prematurity6 and Oulu University11 cohorts. The protocol was approved by the institutional review board of each institution.
Candidate genes
PubMed searches were performed of original and review articles published prior to 1 January 2013 that reported significant associations between specific genotypes and Gr 3-4 IVH. PubMed terms included: candidate, gene, genetic, grades 3–4, infant, intraventricular hemorrhage, IVH, neonate, polymorphism, premature, preterm, and risk factor.
Eleven polymorphic genetic variants in 9 genes were identified. dbSNP ID numbers follow: COL4A1(rs1139941148); ESR1(rs223469312); F2(rs17999637,13); F5(rs60256,7,13); IL1B(rs11436276, rs169446,14); IL6(rs180079515); MTHFR(rs18011337; 18011317); TNF(rs180062916); and TNFB2(rs13843566917).
Description of cases and controls
Cases had Gr 3–4 IVH based upon blinded cUS review and met the following criteria: inborn; ANS exposure; BW 500–1250 g; AGA; no congenital malformations, infections or chromosomal disorder; no family history of coagulopathy; and not a sibling of an enrolled subject. Controls met these criteria and had normal ultrasounds. Analysis was performed on neonates of European ancestry to reduce confounding from racial admixture. Data were entered into a secure online database at Yale University.
Genotyping
Genomic DNA was isolated from buccal swabs, umbilical cords and blood, and genotyping was performed using Illumina 1 Million Quad and HumanOmniExpress-12 v1.0 arrays as previously described.18 For SNPs not on the arrays, proxy SNPs selected secondary to strong linkage disequilibrium with the candidate were employed. The D’ between the candidate and proxy19 was determined based on the 1000 genomes European data.20
dbSNP ID numbers for the variants follow: COL4A1(rs3825481); ESR1 (rs1643821); F2(rs5898); F5 (rs6015); IL1B(rs16944, rs1143627); IL6(rs1800797); MTHFR(rs1801133; rs1801131); TNF(rs1800629). D’ values for the best proxies for ESR1 and TNFB2 were 0.496 and 0.732, and thus these were not evaluated, permitting evaluation of 9 variants in 7 genes.
Statistical Analyses
Distributions for the 9 variants in cases and controls were examined for significant deviation (p< 0.01) from Hardy-Weinberg equilibrium. Each was pre-specified based on published reports in the primary analysis, and the frequencies of risk-associated variants and non-risk-associated variants were compared in cases and controls (Table I). In the secondary analysis, the distribution of all three genotypes was compared at each locus (Table II). Fisher exact test was applied to both.
Table 1.
Validation of predefined risk genotype comparisons in cases vs controls
| Gene | Varianta | Genotype comparison | SNP | Proxy (D’ value with original SNP)b | Odds Ratio (95% CI) | P value |
|---|---|---|---|---|---|---|
| COL4A1 | p.Gly1580Arg8 | CG vs GG | rs113994114 | rs3825481 (nac) | 0.89 (0.52-1.49) | 0.712 |
| F2 | c.97G>A7,13 | AG vs GG | rs1799963 | rs5898 (1.000) | 1.40 (0.88-2.22) | 0.139 |
| F5 | c.1601G>A6,7,13 | AG vs GG | rs6025 | rs6015 (1.000) | 1.07 (0.66-1.72) | 0.817 |
| IL1B | c.-87-511T>C6,14 | T vs C | rs16944 | - | 1.02 (0.79-1.30) | 0.902 |
| c.-87-31C>T14 | C vs T | rs1143627 | - | 1.03 (0.80-1.31) | 0.853 | |
| IL6 | c.-116-121C>G15 | CC vs CG or GG | rs1800795 | rs1800797 (1.000) | 1.08 (0.76-1.56) | 0.662 |
| MTHFR | c.677C>T7 | TT or CT vs CC | rs1801133 | - | 0.95 (0.67-1.34) | 0.799 |
| c.1298A>C7 | CC or CA vs AA | rs1801131 | - | 1.56 (1.10-2.20) | 0.009 | |
| TNF | c.-169-319G>A,16 | AA or AG vs GG | rs1800629 | - | 0.90 (0.62-1.31) | 0.587 |
c – cDNA; p - protein
D’ = D/Dmax where D = the deviation of the observed frequency from the expected; and Dmax = the theoretical maximum for the observed allele frequencies
Allele frequency 0 for the original SNP from1000 genomes database
COL4A1 – collagen 4A1; F2 – prothrombin; F5 – factor V Leiden; IL – interleukin; MTHFR - Methylenetetrahydrofolate reductate; TNF – tumor necrosis factor α.
Table 2.
Genotype frequencies and p values in cases with Grade 3 – 4 IVH and controls
| Gene | Variant | Genotype | Number (%)
|
P value | |
|---|---|---|---|---|---|
| Controls | Cases | ||||
|
| |||||
| COL4A1 | p.Gly1580Arg8 rs3825481* | AA | 85.8 | 87.4 | 0.817 |
| AG | 13.9 | 12.6 | |||
| GG | 0.3 | 0.0 | |||
|
| |||||
| F2 | c.97G>A7,13 rs5898* | GG | 84.5 | 79.9 | 0.306 |
| GA | 14.5 | 19.2 | |||
| AA | 1.0 | 0.9 | |||
|
| |||||
| F5 | c.1601G>A6,7,13 rs6015* | CC | 84.3 | 83.5 | 0.919 |
| CT | 15.2 | 16.1 | |||
| TT | 0.5 | 0.5 | |||
|
| |||||
| IL1B | c.-87-511T>C6,14 rs16944 | GG | 41.4 | 37.5 | 0.206 |
| AG | 44.7 | 51.8 | |||
| AA | 13.9 | 10.7 | |||
|
| |||||
| c.-87-31C>T14 rs1143627 | TT | 41.4 | 37.1 | 0.184 | |
| CT | 44.7 | 52.2 | |||
| CC | 13.9 | 10.7 | |||
|
| |||||
| IL6 | c.-116-121C>G15 rs1800797* | GG | 35.7 | 33.9 | 0.873 |
| AG | 46.8 | 47.3 | |||
| AA | 17.5 | 18.8 | |||
|
| |||||
| MTHFR | c.677C>T7 rs1801133 | CC | 42.4 | 43.8 | 0.782 |
| CT | 44.5 | 45.1 | |||
| TT | 13.1 | 11.2 | |||
|
| |||||
| c.1298A>C7 rs1801131 | AA | 54.8 | 43.8 | 0.021 | |
| AC | 37.0 | 48.2 | |||
| CC | 8.2 | 8.0 | |||
|
| |||||
| TNF | c.-169-319G>A16 rs1800629 | GG | 68.4 | 70.5 | 0.843 |
| AG | 29.3 | 27.7 | |||
| AA | 2.3 | 1.8 | |||
Proxy SNP
Continuous variables were compared using Student t test and categorical variables using Fisher’s exact test. P-value <0.01 was considered statistically significant without adjustment for multiple comparisons. Analyses were performed with SAS (version 9.3).
RESULTS
Cases had lower BW and GA than controls (Table III). Case mothers had less preeclampsia and cesarean deliveries but more chorioamnionitis and multiple gestation pregnancies than control mothers. Similarly, there were more cases with five minute Apgar scores < 3 and more receiving intubation for resuscitation.
Table 3.
Study Subjects
| Gr 3 - 4 IVH | Controls | p value | |
|---|---|---|---|
|
| |||
| Number of subjects | 224 | 389 | |
|
| |||
| Birth weight grams (N, SD) | 810.5 (224, 181.5) | 864.3 (389, 174.0) | <0.0001 |
|
| |||
| GA wks (N, SD) | 25.6 (224, 1.5) | 26.5 (389, 1.5) | <0.0001 |
|
| |||
| Male subjects (%) | 128/224 (57.1%) | 231/389 (59.4%) | 0.61 |
|
| |||
| IVH status | |||
| Grade 3 | 95 | ||
| Grade 4 | 129 | ||
|
| |||
| Maternal variables | |||
|
| |||
| Maternal prenatal visit (%) | 216/222 (97.3%) | 384/389 (98.7%) | 0.2193 |
|
| |||
| Preeclampsia (%) | 23/222 (10.4%) | 91/388 (23.5%) | <0.0001 |
|
| |||
| Clinical chorioamnionitis (%) | 69/223 (30.9%) | 69/389 (17.7%) | 0.0003 |
|
| |||
| Any ANS w/in 7 d prior to delivery (%) | 195/223 (87.4%) | 336/388 (86.6%) | 0.8043 |
|
| |||
| Complete ANS within 7 d (%) | 118/224 (52.7%) | 260/389 (66.8%) | 0.0006 |
|
| |||
| Multiple gestation (%) | 81/224 (36.2%) | 90/386 (23.3%) | 0.0008 |
|
| |||
| Cesarean delivery | 133/224 (59.4%) | 281/388 (72.4%) | 0.0012 |
|
| |||
| Delivery room variables | |||
|
| |||
| Apgar 1 minute < 3 (%) | 70/222 (31.5%) | 89/388 (22.9%) | 0.0216 |
|
| |||
| Apgar 5 minutes < 3 (%) | 19/223 (8.5%) | 14/388 (3.6%) | 0.0145 |
|
| |||
| Intubation for resuscitation | 166/203 (81.8%) | 243/351 (69.2%) | 0.0013 |
|
| |||
| Epinephrine for resuscitation | 8/162 (4.9%) | 5/215 (2.3%) | 0.2535 |
The overall genotype call rate for the variants was 99.8% (range, 98.2-100%). To validate the quality of the genotypes from the markers, deviation from Hardy-Weinberg equilibrium was calculated. All showed insignificant deviation from expected by Hardy-Weinberg equilibrium (p>0.01).
None of the putative risk variants showed significant differences in frequency between cases and controls except the MTHFR 1298A>C polymorphism. To confirm these results, we analyzed the genotypes by 2×3 Fisher’s exact (Table I). The MTHFR 1298A>C polymorphism did not attain significance, but nearly so (Table II).
DISCUSSION
Although recent data have implicated genes contributing to the pathogenesis of IVH, we were unable to confirm as common risk factors for severe IVH 8 of the 9 published variants, and the 1298A>C variant for MTHFR gave equivocal results. Hartman reported that 4/16 preterm neonates with atypical Gr 4 IVH had this variant,7 and our cases were significantly more likely than controls to have the C allele. MTHFR catalyzes the reduction of 5,10-methylenetrahydrofolate to 5-methyltetrahydrofolate, which is necessary for the conversion of homocyteine to methionine. Hyperhomocysteinemia is associated with polymorphisms at -677 and -1298, especially the 677 TT variant, and results in endothelial cell injury and alterations in coagulation.21-23
We report a large reported cohort of inborn AGA preterm neonates of European ancestry with exposure to ANS, Gr 3–4 IVH and genotyping. Limitations include the small sample size, failure to date onset of hemorrhage and lack of homocysteine levels for study participants. This study was not able to evaluate rare variants, and, although we have previously reported a COL4A1 duplication in twins with severe IVH,24 the technology we employed was not designed to identify short insertions. The strengths include prospective collection of preterm neonates with tertiary neonatal care, centrally read ultrasounds and plans for state-of-the-art genetic analyses.
Table IV.
| Site | Cases | Controls | Total |
|---|---|---|---|
| Gene Targets for IVH | 162 | 215 | 377 |
| ELGAN | 24 | 90 | 114 |
| Iowa | 17 | 46 | 63 |
| Oulu | 21 | 38 | 59 |
| Total | 224 | 389 | 613 |
Central blinded ultrasound readings were performed for all subjects from the Gene Targets for IVH, ELGAN and Iowa cohorts (554/613, 90%). Ultrasounds for Oulu subjects were performed by blinded readers but were not centrally read.
Acknowledgments
We are indebted to our medical, nursing and research colleagues and the infants and their parents who agreed to take part in this study. We also are indebted to Deborah Hirtz, MD, for scientific expertise, Jill Maller-Kesselman, MS, for study management, and Karen C. Schneider, MPH, for editorial expertise and assistance.
Supported by the National Institutes of Health (NS053865 and R01 DA016750-09 [to H.P.]). W.C. is a board member of Mednax.
ABBREVIATIONS
- AGA
Appropriate for gestational age
- ANS
Antenatal steroid
- BW
Birth weight
- COL4A1
Collagen 4A1
- cUS
Cranial ultrasonography
- GA
Gestational age
- Gr
Grade
- IL
Interleukin
- IVH
Intraventricular hemorrhage
- MTHFR
Methylenetetrahydrofolate reductase
- TNF
Tumor necrosis factor α
Footnotes
Finally, we appreciate the contribution of our collaborators: Elizabeth Newton Alfred, MS (Department of Biostatistics, Harvard School of Public Health and Department of Neurology, Children’s Hospital Boston [ELGAN Study], Susan K. Berends, MA, Allison Momany, RN, John Daigle, MD, Nancy Weathers, RN (Iowa Prematurity, Department of Pediatrics, University of Iowa), and Johanna Huusko, MD (Oulu University, Department of Pediatrics).
The other authors declare no conflicts of interest.
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References
- 1.Volpe JJ. Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances. Lancet Neurology. 2009;8:110–24. doi: 10.1016/S1474-4422(08)70294-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Stoll BJ, Hansen NI, Bell EF, et al. Neonatal outcomes of extremely preterm infants from the NICHD Neonatal Research Network. Pediatrics. 2010;126:443–56. doi: 10.1542/peds.2009-2959. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Sherlock RL, Synnes AR, Grunau RE, et al. Long term outcome after neonatal intraparenchymal echodensities with porencephaly. Archives of disease in childhood Fetal and neonatal edition. 2008;93:F127–31. doi: 10.1136/adc.2006.110726. [DOI] [PubMed] [Google Scholar]
- 4.Luu TM, Ment L, Allan W, Schneider K, Vohr BR. Executive and Memory Function in Adolescents Born Very Preterm. Pediatrics. 2011;127:e639–46. doi: 10.1542/peds.2010-1421. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Beaino G, Khoshnood B, Kaminski M, et al. Predictors of cerebral palsy in very preterm infants: the EPIPAGE prospective population-based cohort study. Developmental medicine and child neurology. 2010;52:e119–25. doi: 10.1111/j.1469-8749.2010.03612.x. [DOI] [PubMed] [Google Scholar]
- 6.Ryckman KK, Dagle JM, Kelsey K, Momany AM, Murray JC. Replication of genetic associations in the inflammation, complement, and coagulation pathways with intraventricular hemorrhage in LBW preterm neonates. Pediatric research. 2011;70:90–5. doi: 10.1203/PDR.0b013e31821ceb63. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Harteman JC, Groenendaal F, van Haastert IC, et al. Atypical timing and presentation of periventricular haemorrhagic infarction in preterm infants: the role of thrombophilia. Developmental medicine and child neurology. 2012;54:140–7. doi: 10.1111/j.1469-8749.2011.04135.x. [DOI] [PubMed] [Google Scholar]
- 8.de Vries LS, Koopman C, Groenendaal F, et al. COL4A1 mutation in two preterm siblings with antenatal onset of parenchymal hemorrhage. Annals of neurology. 2009;65:12–8. doi: 10.1002/ana.21525. [DOI] [PubMed] [Google Scholar]
- 9.Bauer SC, Msall ME. Optimizing neurodevelopmental outcomes after prematurity: lessons in neuroprotection and early intervention. Minerva Pediatr. 2010;62:485–97. [PubMed] [Google Scholar]
- 10.Helderman JB, O’Shea TM, Kuban KC, et al. Antenatal antecedents of cognitive impairment at 24 months in extremely low gestational age newborns. Pediatrics. 2012;129:494–502. doi: 10.1542/peds.2011-1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Karjalainen MK, Haataja R, Hallman M. Haplotype analysis of ABCA3: association with respiratory distress in very premature infants. Annals of medicine. 2008;40:56–65. doi: 10.1080/07853890701611094. [DOI] [PubMed] [Google Scholar]
- 12.Derzbach L, Treszl A, Balogh A, Vasarhelyi B, Tulassay T, Rigo JJ. Gender dependent association between perinatal morbidity and estrogen receptor-alpha Pvull polymorphism. Journal of perinatal medicine. 2005;33:461–2. doi: 10.1515/JPM.2005.082. [DOI] [PubMed] [Google Scholar]
- 13.Gopel W, Gortner L, Kohlmann T, Schultz C, Moller J. Low prevalence of large intraventricular haemorrhage in very low birthweight infants carrying the factor V Leiden or prothrombin G20210A mutation. Acta paediatr. 2001;90:1021–4. doi: 10.1080/080352501316978101. [DOI] [PubMed] [Google Scholar]
- 14.Baier RJ. Genetics of perinatal brain injury in the preterm infant. Frontiers in bioscience : a journal and virtual library. 2006;11:1371–87. doi: 10.2741/1890. [DOI] [PubMed] [Google Scholar]
- 15.Harding DR, Dhamrait S, Whitelaw A, Humphries SE, Marlow N, Montgomery HE. Does interleukin-6 genotype influence cerebral injury or developmental progress after preterm birth? Pediatrics. 2004;114:941–7. doi: 10.1542/peds.2003-0494-F. [DOI] [PubMed] [Google Scholar]
- 16.Adcock K, Hedberg C, Loggins J, Kruger T, Baier R. The TNF-alpha - 308, MCP - 1 - 2518 and TGF - beta 1 + 915 polymorphisms are not associated with the development of chronic lung disease in very low birth weight infants. Genes Immun. 2003;4:420–6. doi: 10.1038/sj.gene.6363986. [DOI] [PubMed] [Google Scholar]
- 17.Heep A, Schueller AC, Kattner E, et al. Association of two tumour necrosis factor gene polymorphisms with the incidence of severe intraventricular haemorrhage in preterm infants. Journal of medical genetics. 2005;42:604–8. doi: 10.1136/jmg.2004.021378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Yasuno K, Bilguvar K, Bijlenga P, et al. Genome-wide association study of intracranial aneurysm identifies three new risk loci. Nature genetics. 2010;42:420–5. doi: 10.1038/ng.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lewontin RC, Kojima K. The evolutionary dynamics of complex polymorphisms. Evolution. 1960;14:458–72. [Google Scholar]
- 20.Altshuler DM, Gibbs RA, Peltonen L, et al. Integrating common and rare genetic variation in diverse human populations. Nature. 2010;467:52–8. doi: 10.1038/nature09298. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Stuart S, Cox HC, Lea RA, Griffiths LR. The role of the MTHFR gene in migraine. Headache. 2012;52:515–20. doi: 10.1111/j.1526-4610.2012.02106.x. [DOI] [PubMed] [Google Scholar]
- 22.Faraci FM, Lentz SR. Hyperhonnocysteinemia, oxidative stress and cerebral vascular dysfunction. Stroke; a journal of cerebral circulation. 2004;35:345–7. doi: 10.1161/01.STR.0000115161.10646.67. [DOI] [PubMed] [Google Scholar]
- 23.Lievers KJ, Boers GH, Verhoef P, et al. A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk. J Mol Med (Berl) 2001;79:522–8. doi: 10.1007/s001090100253. [DOI] [PubMed] [Google Scholar]
- 24.Bilguvar K, DiLuna ML, Bizzarro MJ, et al. COL4A1 Mutation in preterm intraventricular hemorrhage. The Journal of pediatrics. 2009;155:743–5. doi: 10.1016/j.jpeds.2009.04.014. [DOI] [PMC free article] [PubMed] [Google Scholar]