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