Amniotic fluid and serum biomarkers from women with neural tube defect–affected pregnancies: a case study for myelomeningocele and anencephaly

Clinical article

Restricted access

Object

The authors sought to identify novel biomarkers for early detection of neural tube defects (NTDs) in human fetuses.

Methods

Amniotic fluid and serum were drawn from women in the second trimester of pregnancy. The study group included 2 women pregnant with normal fetuses and 4 with fetuses displaying myelomeningocele (n = 1), anencephaly (n = 1), holoprosencephaly (n = 1), or encephalocele (n = 1). Amniotic fluid stem cells (AFSCs) were isolated and cultured. The cells were immunostained for the stem cell markers Oct4, CD133, and Sox2; the epigenetic biomarkers H3K4me2, H3K4me3, H3K27me2, H3K27me3, H3K9Ac, and H3K18Ac; and the histone modifiers KDM6B (a histone H3K27 demethylase) and Gcn5 (a histone acetyltransferase). The levels of 2 markers for neural tube development, bone morphogenetic protein–4 (BMP4) and sonic hedgehog (Shh), were measured in amniotic fluid and serum using an enzyme-linked immunosorbent assay.

Results

The AFSCs from the woman pregnant with a fetus affected by myelomeningocele had higher levels of H3K4me2, H3K4me3, H3K27me2, and H3K27me3 and lower levels of KDM6B than the AFSCs from the women with healthy fetuses. The levels of H3K9ac, H3K18ac, and Gcn5 were also decreased in the woman with the fetus exhibiting myelomeningocele. In AFSCs from the woman carrying an anencephalic fetus, levels of H3K27me3, along with those of H3K9Ac, H3K18ac, and Gcn5, were increased, while that of KDM6B was decreased. Compared with the normal controls, the levels of BMP4 in amniotic fluid and serum from the woman with a fetus with myelomeningocele were increased, whereas levels of Shh were increased in the woman pregnant with a fetus displaying anencephaly.

Conclusions

The levels of epigenetic marks, such as H3K4me, H3K27me3, H3K9Ac, and H3K18A, in cultured AFSCs in combination with levels of key developmental proteins, such as BMP4 and Shh, are potential biomarkers for early detection and identification of NTDs in amniotic fluid and maternal serum.

Abbreviations used in this paper:AFSC = amniotic fluid stem cell; BMP4 = bone morphogenetic protein–4; ELISA = enzymelinked immunosorbent assay; NTD = neural tube defect; Shh = sonic hedgehog.

Article Information

* Drs. Tsurubuchi, Ichi, and Shim contributed equally to this work.

Address correspondence to: C. Shekhar Mayanil, Ph.D., M/C 204, Ann and Robert H. Lurie Children's Hospital of Chicago Research Center, 2430 N. Halsted St., Chicago, IL 60614. email: smayanil@northwestern.edu.

Please include this information when citing this paper: published online August 23, 2013; DOI: 10.3171/2013.7.PEDS12636.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Amniotic fluid stem cells express stem cell markers. Neurospheres were grown from total AFSCs as described in Methods. Maternal cells do not survive in neurosphere culture medium (Neurobasal Media with basic fibroblast growth factor and epidermal growth factor). Embryonic stem cells grew in Neurobasal Media with these 2 growth factors and formed neurosphere colonies and represented AFSCs. Colonies were immunostained for Sox2, Oct4, CD133, and nestin. The DAPI stain was used to stain cell nuclei. The Sox2, Oct4, CD133, and nestin markers were present in AFSCs from tissues recovered from normal (A), myelomeningocele (MM)- (B), and anencephaly- (C) affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

  • View in gallery

    Differentiated amniotic fluid stem cells express different markers for cell differentiation. Neurospheres were grown in Neurobasal Media without growth factors for 7 days and were immunostained for astrocyte (GFAP), neuron (TuJ1), and oligodendrocyte (O4) markers. The DAPI stain was used to stain the cell nuclei. The levels of GFAP and TuJ1 were similar in AFSCs obtained from normal (A), myelomeningocele (MM)- (B), and anencephaly- (C) affected pregnancies. The O4 staining intensity was MM > anencephaly > normal. Each experiment was performed at least 4 times and in 3 technical replicates.

  • View in gallery

    Methylation of H3K4 assessed through immunostaining of cultured AFSCs from normal and from MM- and anencephaly-affected pregnancies. A: Staining for H3K4me2 and Oct4. B: Staining for H3K4me3 and Oct4. All cells were counterstained with DAPI nuclear stain. Presence of Oct4, a stem cell marker, demonstrated that stained cells were of embryonic and not of maternal origin. Compared with the cells from normal pregnancies, both H3K4me2 and H3K4me3 immunostaining were increased in cells from MM- but not in cells from anencephaly-affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

  • View in gallery

    Methylation of H3K27 and KDM6B assessed through immunostaining of cultured AFSCs from normal, myelomeningocele (MM)-, and anencephaly-affected pregnancies. A: Staining for H3K27me2 and Oct4. B: Staining for H3K27me3 and Oct4. C: Staining for KDM6B and Oct4. All cells were counterstained with DAPI nuclear stain. The immunostaining of H3K27me2 and H3K4me3 was increased in the MM-affected pregnancy, whereas in the anencephaly-affected pregnancy, only H3K27me3 staining was increased. The immunostaining of KDM6B was decreased in both MM- and anencephaly-affected pregnancies. Each experiment was performed at least 4 times and in 3 technical replicates.

  • View in gallery

    Acetylation of H3K9, H3K18, and Gcn5 assessed through immunostaining of AFSCs from normal and MM- and anencephaly-affected pregnancies. A: Staining for H3K9Ac and Gcn5. B: Staining for H3K18Ac and Gcn5. All cells were counterstained with DAPI nuclear stain. In the AFSCs cultured from the woman pregnant with an anencephalic fetus, the staining intensity of H3K9Ac and H3K18Ac was increased. Each experiment was performed at least 4 times and in 3 technical replicates.

  • View in gallery

    Summary of the densitometry results for the epigenetic markers. A minimum of 6 different fields of immunostained cells were measured by densitometry, and arbitrary densitometry units with that for Oct4 set to 1 are shown. Compared with immunostaining intensities in cells from women with normal pregnancies, the staining intensities for H3K4me2, H3K4me3, H3K27me2, and H3K27me3 were high in the cells from the woman with the myelomeningocele-affected pregnancy. Staining intensities for H3K27me3, H3K9Ac, H3K18Ac, and Gcn5 were high in AFSCs derived from anencephaly-affected pregnancy, and KDM6B levels were low in both myelomeningocele and anencephalic samples relative to its levels in the normal sample. The error bars indicate the SEM.

  • View in gallery

    Amniotic fluid and serum levels of BMP4 and Shh in normal and NTD-affected pregnancies. Serum and amniotic fluid showed similar trends in these levels. A: The levels of BMP4 in amniotic fluid were higher in myelomeningocele-affected and lower in anencephaly- and encephalocele-affected pregnancies. In the holoprosencephaly-affected pregnancy, BMP4 levels were similar to those in the normal pregnancies. B: The levels of serum BMP4 were high in the myelomeningocele-affected pregnancy but nondetectable in the anencephaly-affected pregnancy. C: The Shh levels in amniotic fluid were higher in the anencephaly-affected pregnancy. D: Serum Shh levels were highest in the anencephaly-affected pregnancy and lowest in the myelomeningocele-affected pregnancy. Each experiment was done in triplicate.

References

1

Akizu NEstarás CGuerrero LMartí EMartínez-Balbás MA: H3K27me3 regulates BMP activity in developing spinal cord. Development 137:291529252010

2

Bernstein BEMikkelsen TSXie XKamal MHuebert DJCuff J: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125:3153262006

3

Blanco-Muñoz JLacasaña MBorja-Aburto VH: Maternal miscarriage history and risk of anencephaly. Paediatr Perinat Epidemiol 20:2102182006

4

Bol KACollins JSKirby RS: Survival of infants with neural tube defects in the presence of folic acid fortification. Pediatrics 117:8038132006

5

Bowman RMMcLone DGGrant JATomita TIto JA: Spina bifida outcome: a 25-year prospective. Pediatr Neurosurg 34:1141202001

6

Boyd PADevigan CKhoshnood BLoane MGarne EDolk H: Survey of prenatal screening policies in Europe for structural malformations and chromosome anomalies, and their impact on detection and termination rates for neural tube defects and Down's syndrome. BJOG 115:6896962008

7

Bredaki FEPoon LCBirdir CEscalante DNicolaides KH: First-trimester screening for neural tube defects using alphafetoprotein. Fetal Diagn Ther 31:1091142012

8

Cho AKo HWEggenschwiler JT: FKBP8 cell-autonomously controls neural tube patterning through a Gli2- and Kif3a-dependent mechanism. Dev Biol 321:27392008

9

Cuckle HS: Recurrence risk of neural tube defects following a miscarriage. Prenat Diagn 3:2872891983

10

Doherty DShurtleff DB: Pediatric perspective on prenatal counseling for myelomeningocele. Birth Defects Res A Clin Mol Teratol 76:6456532006

11

Elicha Gussin HAElias S: Culture of fetal cells from maternal blood for prenatal diagnosis. Hum Reprod Update 8:5235272002

12

Felder BStegmann KSchultealbert AGeller FStrehl EErmert A: Evaluation of BMP4 and its specific inhibitor NOG as candidates in human neural tube defects (NTDs). Eur J Hum Genet 10:7537562002

13

Fuccillo MJoyner ALFishell G: Morphogen to mitogen: the multiple roles of hedgehog signalling in vertebrate neural development. Nat Rev Neurosci 7:7727832006

14

Greene NDStanier PMoore GE: The emerging role of epigenetic mechanisms in the etiology of neural tube defects. Epigenetics 6:8758832011

15

Ichi SCosta FFBischof JMNakazaki HShen YWBoshnjaku V: Folic acid remodels chromatin on Hes1 and Neurog2 promoters during caudal neural tube development. J Biol Chem 285:36922369322010

16

Jansen MWKorver-Hakkennes Kvan Leenen DBrandenburg HWildschut HIWladimiroff JW: How useful is the in vitro expansion of fetal CD34+ progenitor cells from maternal blood samples for diagnostic purposes?. Prenat Diagn 20:7257312000

17

Johnson CYHonein MAFlanders WDHowards PPOakley GP JrRasmussen SA: Pregnancy termination following prenatal diagnosis of anencephaly or spina bifida: a systematic review of the literature. Birth Defects Res A Clin Mol Teratol 94:8578632012

18

Kaur GSrivastav JKaur AHuria AGoel PKaur R: Maternal serum second trimester screening for chromosomal disorders and neural tube defects in a government hospital of North India. Prenat Diagn 32:119211962012

19

Kavalakis ISouka APPilalis APapastefanou IKassanos D: Assessment of the posterior brain at 11–14 weeks for the prediction of open neural tube defects. Prenat Diagn 32:114311462012

20

Keenen Bde la Serna IL: Chromatin remodeling in embryonic stem cells: regulating the balance between pluripotency and differentiation. J Cell Physiol 219:172009

21

Lan FBayliss PERinn JLWhetstine JRWang JKChen S: A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 449:6896942007

22

McMahon JATakada SZimmerman LBFan CMHarland RMMcMahon AP: Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite. Genes Dev 12:143814521998

23

Mikkelsen TSKu MJaffe DBIssac BLieberman EGiannoukos G: Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448:5535602007

24

Mitchell LE: Epidemiology of neural tube defects. Am J Med Genet C Semin Med Genet 135C:88942005

25

Monsoro-Burq AHDuprez DWatanabe YBontoux MVincent CBrickell P: The role of bone morphogenetic proteins in vertebral development. Development 122:360736161996

26

Murdoch JNCopp AJ: The relationship between sonic Hedgehog signaling, cilia, and neural tube defects. Birth Defects Res A Clin Mol Teratol 88:6336522010

27

Nakazaki HReddy ACMania-Farnell BLShen YWIchi SMcCabe C: Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development. Dev Biol 316:5105232008

28

Nicolaides KH: Turning the pyramid of prenatal care. Fetal Diagn Ther 29:1831962011

29

Nuss SBrebaum DGrond-Ginsbach C: Maternal cell contamination in amniotic fluid samples as a consequence of the sampling technique. Hum Genet 93:1211241994

30

Pan GTian SNie JYang CRuotti VWei H: Wholegenome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell Stem Cell 1:2993122007

31

Perin LSedrakyan SDa Sacco SDe Filippo R: Characterization of human amniotic fluid stem cells and their pluripotential capability. Methods Cell Biol 86:85992008

32

Rice JCAllis CD: Histone methylation versus histone acetylation: new insights into epigenetic regulation. Curr Opin Cell Biol 13:2632732001

33

Shukla VVaissière THerceg Z: Histone acetylation and chromatin signature in stem cell identity and cancer. Mutat Res 637:1152008

34

Siddiqi SMills JMatushansky I: Epigenetic remodeling of chromatin architecture: exploring tumor differentiation therapies in mesenchymal stem cells and sarcomas. Curr Stem Cell Res Ther 5:63732010

35

Siegel NValli AFuchs CRosner MHengstschläger M: Induction of mesenchymal/epithelial marker expression in human amniotic fluid stem cells. Reprod Biomed Online 19:8388462009

36

Stottmann RWBerrong MMatta KChoi MKlingensmith J: The BMP antagonist Noggin promotes cranial and spinal neurulation by distinct mechanisms. Dev Biol 295:6476632006

37

Zhao XDHan XChew JLLiu JChiu KPChoo A: Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell Stem Cell 1:2862982007

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 61 61 13
Full Text Views 66 66 7
PDF Downloads 43 43 12
EPUB Downloads 0 0 0

PubMed

Google Scholar