MicroRNAs in maternal plasma for the non-invasive prenatal diagnosis of Down syndrome (Trisomy 21)
Julian Kamhieh-Milz1,2, Reham Fadl Hassan Moftah3, Omid Khorramshahi2, Martin
Burow2, Gundula Thiel4, Annegret Stuke-Sontheimer4, Rabih Chaoui5, Sundrela
Kamhieh-Milz1 and Abdulgabar Salama1
1 Institute for Transfusion Medicine, Charité University Medicine, Augustenburger Platz 1, 13353, Berlin,
Germany, 2 Research Center for Immune Science (RCIS), Charité University Medicine, Hessische Str. 3-4,
10115 Berlin, Germany, 3 Institute for Human Genetics, Charité University Medicine, Augustenburger
Platz 1, 13353, Berlin, Germany, 4 Practice for Human Genetics, Friederichstraße 147, 10117, Berlin,
Germany, 5 Prenatal Diagnosis and Human Genetics Centre, Friederichstraße 147, 10117, Berlin,
Germany.
Although differentially expression patterns of miRNAs between
Down syndrome pregnancies compared to euploid
pregnancies can be observed, the exact transport
mechanisms of extracellular, cell-free miRNAs into the maternal
circulation are currently unknown. Here, we present some
hypothetical mechanisms.
A Connection of the mother and foetus via the placenta. B
Chorionic villi are responsible for sustaining the placenta with
nutrients and oxygen. The intervillus space is filled with maternal
blood. C Cellular release mechanisms and extracellular
transportation systems of miRNAs (10). In the cytoplasm, miRNAs
can be incorporated into small vesicles, exosomes, which stem
from the endosome, and are released from cells when
multivesicular bodies coalesce with the plasma membrane.
MiRNAs are also found in circulation in a microparticle-free
form (associated with Ago-2 or HDL).
DISCUSSION
A wide range of organ systems are affected in Down syndrome individuals,
with some congenital whereas others are progressive, and include cardiac
malformations, increased frequency of childhood leukaemia, varying
degrees of intellectual disability and central nervous system abnormalities
(7). MiRNAs are considered to play an active role in the regulation of
developmental processes. With the assumption that ~50% of all genes are
miRNA-controlled (8), we were able to identify Down syndrome-specific
miRNA profiles in maternal plasma. These miRNA pattern have potential to
be used for non-invasive diagnostic purposes. When using a subset of 10 or
20 miRNAs, a clear identification of Down syndrome is possible. The exact
transport mechanisms of extracellular, cell-free miRNAs into the maternal
circulation are currently unknown. Trisomy 21 may leads to a dysregulation
of gene expression including miRNAs. These miRNAs may then enhance the
dysregulation of genes in a genome-wide fashion, resulting in mild or severe
symptoms, depending on the dysregulation. It has been previously
demonstrated that cells can select some miRNAs for cellular release while
others are retained (9). Their exocytosis may represent a protective
mechanism in which harmful miRNAs are actively enveloped and secreted
out of the cells via exosomes. The Down syndrome-specific miRNA profile
may therefore reflect the severity of symptoms e.g. in respect to mental
retardation and cardiomyopathy, thus classifying miRNAs as the first
prognostic biomarkers in the NIPD of Down syndrome. Furthermore, miRNA
may also allow for antagomiR strategies to cure or at least partially reduce
serious Down syndrome symptoms during prenatal and postnatal
development of Down syndrome patients in future.
ABSTRACT
Most developmental processes are under the control of small regulatory
RNAs called microRNAs (miRNAs). We hypothesise that different foetal
developmental processes might be reflected by extracellular miRNAs in
maternal plasma and may be utilised as biomarkers for the non-invasive
prenatal diagnosis of chromosomal aneuploidies. In this proof-of concept
study, we report on the identification of extracellular miRNAs in maternal
plasma of Down syndrome pregnancies. Using high throughput-quantitative
PCR (HT-qPCR), 1043 miRNAs were investigated in maternal plasma via
comparison of seven Down syndrome pregnancies with age and foetal sex
matched controls. Six hundred and ninety-five miRNAs were identified. Thirty-
six significantly differentially expressed mature miRNAs were identified as
potential biomarkers. Hierarchical cluster analysis of these miRNAs resulted in
the clear discrimination of Down syndrome from euploid pregnancies. Gene
targets of the differentially expressed miRNAs were enriched in signalling
pathways such as mucin type-O-glycans, ECM-receptor interactions, TGF-
beta and endocytosis, which have been previously associated with Down
syndrome. MiRNAs are promising and stable biomarkers for a broad range of
diseases and may allow a reliable, cost-efficient diagnostic tool for the non-
invasive prenatal diagnosis of Down syndrome. Furthermore, they exhibit the
potential of being the first prognostic marker to characterise the severity of
Down syndrome disabilities and may allow for antagoMir strategies to tread
common Down syndrome features in the future.
MATERIALS AND METHODS
Blood samples collected in EDTA were obtained from pregnant women for
whom invasive genetic testing was recommended due to increased
maternal age or suspicious ultrasound findings. Invasive diagnostics were
performed in accordance to routine procedures of the Practice for Human
Genetics Friedrichstrasse by conventional chromosome analysis
(karyotyping). In this proof-of-concept study, plasma from seven Trisomy 21
pregnancies and seven appropriately matched controls (similar maternal
age, identical gestational week and foetal gender) were selected. For HT-
qPCR, miRNAs were isolated from 200 µL plasma with the miRNeasy Mini Kit
(Qiagen, Hilden, Germany). High-throughput quantitative PCR (HT-qPCR) was
performed on individual samples using the SmartChip Human miRNA Panel
V3.0 (WaferGen, CA, USA) in accordance to the manufacturer’s instructions.
HT-qPCR analysis was performed using both qBase Software (Biogazelle,
Belgium) and BioConductor (HT-qPCR Package). The WaferGen qPCR
Software report generated which provides a short overview on raw data (replicates) Cq´s, distance between sample and NTC, and normalized
relative quantities (NRQ). These data were also used as a template for
downstream analysis using R and Bioconductor. In order to identify predicted
miRNA targets, the Diana mirPath tool V2 was used.
Hypothesised mechanisms on the entrance of foetal / placental miRNAs in the maternal circulation.
RESULTS: Column plot and hierarchical cluster analysis
Left: Column plots of selected miRNAs representing an expression profile between Down syndrome
versus euploid pregnancies. Although a particular trend of these miRNAs can be observed, a
single miRNA that discriminated Down syndrome from euploid pregnancies does not exist. Right:
When using 20 miRNAs which were found to be most differentially expressed, a clear identification
of Down Syndrome is possible.
RESULTS: Predictive miRNA targets and Down syndrome severity
References
Take-home messages: Down syndrome-specific miRNA profiles can be found in maternal plasma.
miRNAs may be of foetal and not of placental origin.
miRNAs may represent the first predictive marker for the characterisation
of the severity of Down syndrome symptoms.
miRNA may allow for antagoMir strategies to ‘treat’ Down syndrome
symptoms in future.
1. Chandrasekaran EV, Xue J, Xia J, Locke RD, Patil SA, Neelamegham S, Matta KL. Mammalian sialyltransferase
st3gal-ii: Its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated
glycoproteins and specific gangliosides. Biochemistry 2011;50:9475-87. 2. Birken S. Specific measurement of o-
linked core 2 sugar-containing isoforms of hyperglycosylated human chorionic gonadotropin by antibody b152.
Tumour Biol 2005;26:131-41. 3. Grossman TR, Gamliel A, Wessells RJ, Taghli-Lamallem O, Jepsen K, Ocorr K, et al.
Over-expression of dscam and col6a2 cooperatively generates congenital heart defects. PLoS Genet
2011;7:e1002344. 4. Wang LL, Zhang Z, Li Q, Yang R, Pei X, Xu Y, et al. Ethanol exposure induces differential
microrna and target gene expression and teratogenic effects which can be suppressed by folic acid
supplementation. Hum Reprod 2009;24:562-79.
5. van der Wal EA, Gomez-Pinilla F, Cotman CW. Transforming growth factor-beta 1 is in plaques in alzheimer
and down pathologies. Neuroreport 1993;4:69-72. 6. Bromage SJ, Lang AK, Atkinson I, Searle RF. Abnormal
tgfbeta levels in the amniotic fluid of down syndrome pregnancies. Am J Reprod Immunol 2000;44:205-10. 7.
Haydar TF, Reeves RH. Trisomy 21 and early brain development. Trends Neurosci 2012;35:81-91. 8. Krol J, Loedige
I, Filipowicz W. The widespread regulation of microrna biogenesis, function and decay. Nat Rev Genet
2010;11:597-610. 9. Pigati L, Yaddanapudi SC, Iyengar R, Kim DJ, Hearn SA, Danforth D, et al. Selective release of
microrna species from normal and malignant mammary epithelial cells. PLoS One 2010;5:e13515. 10. Creemers
EE, Tijsen AJ, Pinto YM. Circulating micrornas: Novel biomarkers and extracellular communicators in
cardiovascular disease? Circ Res 2012;110:483-95.
Mucin type O-Glycan: Human chorionic gonadotropin (HCG) is a glycoprotein
hormone produced by placental trophoblasts and trophoblastic tumours (1). The
antibody B152, which mainly recognizes the core-2 O-glycans at Ser-132, has been
demonstrated to be useful in the prediction of Down syndrome pregnancies (2).
ECM receptor interaction: Grossman and colleagues studied the effect of the co-
expression of DSCAM and COL6A2, interaction partners that are overexpressed in Down
syndrome patients and are associated with cardiomyopathy (3). In their study, the gene
expression profile of co-overexpression versus normal expression also revealed the KEGG
gene ontology terms ECM-receptor interaction as the most significant pathway.
Glycosaminoglycans: GAGs are associated with mental retardation and multiple organ
failure, and already serve as biomarkers in prenatal diagnostics. This pathway was
identified due to the strong linkage of HSA-MIR-362 and HSA-MIR-124. Interestingly, HSA-
MIR-362 has been associated with foetal teratogenesis and mental retardation in mice
(4).
TGF-ß: Abnormal TGF-ß has been associated with plaque formation in the brains of
Alzheimer’s disease (AD) and Down syndrome patients (5). Moreover, abnormal TGF-ß
levels have been identified in the amniotic fluid of Down syndrome pregnancies (6).
These findings provide evidence that the differentially expressed
miRNAs in this study are not randomly identified, but point towards its
strong linkage to many well-known Down syndrome
pathomechanisms. Thus, miRNA exhibit the potential to be the first
prognostic factors to explore the severity of Down syndrome
symptoms by NIPD.
University Medicine