MicroRNAs in maternal plasma for the non-invasive prenatal diagnosis of Down syndrome (Trisomy 21) Julian Kamhieh-Milz 1,2 , Reham Fadl Hassan Moftah 3 , Omid Khorramshahi 2 , Martin Burow 2 , Gundula Thiel 4 , Annegret Stuke-Sontheimer 4 , Rabih Chaoui 5 , Sundrela Kamhieh-Milz 1 and Abdulgabar Salama 1 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