Extracellular chromosome 21 – derived microRNAs in maternal circulation: evaluation of their diagnostic potential for screening of Down syndrome

Czech version

Authors: I. Hromadníková ¹; K. Kotlabová ¹; J. Doucha ²; D. Chudoba ³; P. Calda ⁴ ; K. Dlouhá ⁵
Authors‘ workplace: Oddělení molekulární biologie a patologie buňky, Gynekologicko-porodnická klinika, 3. LF UK, Praha, přednosta kliniky doc. MUDr. E. Kučera, CSc. ¹; Gynekologicko-porodnická klinika, FN Motol a 2. LF UK, přednosta kliniky prof. MUDr. L. Rob, CSc. ²; Oddělení lékařské cytogenetiky, Ústav biologie a lékařské genetiky, FN Motol a 2. LF UK, přednosta ústavu prof. MUDr. M. Macek jr., DrSc. ³; Gynekologicko-porodnická klinika, VFN a 1. LF UK, Praha, přednosta kliniky prof. MUDr. A. Martan, DrSc. ⁴; Ústav pro péči o matku a dítě, Praha, ředitel ústavu doc. MUDr. J. Feyereisl, CSc. ⁵
Published in: Ceska Gynekol 2012; 77(5): 395-402

Overview

Objective:
Initially, we focused on the detection of extracellular microRNAs in maternal circulation, whose genes are located on human chromosome 21 (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802). Subsequently, we studied if plasmatic concentrations and/or expression profile of extracellular chromosome 21-derived microRNAs would distinguish between pregnancies bearing euploid foetuses and those affected with Down syndrome.

Design:
Pilot study.

Setting:
Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague.

Methods:
12 women with normal course of gestation (mean 16.4 weeks, median 16.0 weeks), 12 pregnancies bearing Down syndrome foetus (mean 18.2 weeks, median 18.5 weeks) and 6 non-pregnant individuals were involved in the retrospective study. RNA enriched for small RNAs (including microRNAs) was isolated from 1ml of plasma sample. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay.

Results:
Commercial systems enabled reliable detection of 4 out of 5 extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155). Expression profile of extracellular miR-99a, miR-125b-2 and miR-155 was significantly higher in the cohort of pregnant women than in non-pregnant individuals. Also plasmatic levels of miR-99a and miR-125b-2 were significantly increased in pregnant women. Unfortunately, the concentrations and gene expression of extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome.

Conclusion:
Analysis of extracellular chromosome 21-derived microRNAs does not distinguish between pregnancies with euploid and aneuploid foetuses and has no benefit for screening programmes.

Key words:
Down syndrome, gene expression, microRNA, placenta, real-time PCR.

Sources

1. Calin, GA., Dumitru, CD., Shimizu, M., et al. Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA, 2002, 99 (24), p. 15524–15529.

2. Ehrich, M., Deciu, C., Zwiefelhofer, T., et al. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting. Am J Obstet Gynecol, 2011, 204, (3), 205.e1–11.

3. Fan, HC., Blumenfeld, YJ., Chitkara, U., et al. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A, 2008, 105 (42), p. 16266–16271.

4. Grishok, A., Pasquinelli, AE., Conte, D., et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell, 2001, 106, p. 23–34.

5. Hammond, SM., Bernstein, E., Beach, D., et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000, 404, p. 293–296.

6. Hromadníková, I., Kotlabová, K., Jirásek, JE., et al. Detekce placentárně specifických mikroRNA v mateřské cirkulaci. Čes Gynek, 2010, 75, s. 252–256.

7. Hromadnikova, I., Kotlabova, K., Doucha, J., et al. Absolute and relative quantification of placenta-specific microRNAs in maternal circulation with placental insufficiency-related complications. J Mol Diagn, 2012, Jan 14, [Epub ahead of print].

8. Hromadnikova, I., Houbova, B., Hridelova, D., et al. Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies. BMC Pregnancy Childbirth, 2002, 28, 2 (1), p. 4.

9. Hromadníková, I. Současné možnosti neinvazivní prenatální diagnostiky na bázi extracelulárních nukleových kyselin v mateřské cirkulaci. Gynekolog, 2010, 2, s. 57–62.

10. Hutvágner, G., Zamore, PD. A microRNA in a multiple-turnover RNA enzyme complex. Science, 2002, 297, p. 2056–2060.

11. Chen, CZ., Li, L., Lodish, HF., et al. MicroRNAs modulate hematopoietic lineage differentiation. Science, 2004, 303 (5654), p. 83–86.

12. Ketting, RF., Fischer, SE., Bernstein, E., et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev, 2001, 15, p. 2654–2659.

13. Kotlabova, K., Doucha, J., Hromadnikova, I. Placental-specific microRNA in maternal circulation-identification of appropriate pregnancy-associated microRNAs with diagnostic potential. J Reprod Immunol, 2011, 89, p. 185–191.

14. Krichevsky, AM., King, KS., Donahue, CP., et al. A microRNA arrays reveals extensive regulation of microRNAs during brain development. RNA, 2003, 9 (10), p. 1274–1281.

15. Kuhn, DE., Nuovo, GJ., Martin, MM., et al. Human chromosome 21-derived miRNAs are overexpressed in down syndrome brains and hearts. Biochem Biophys Res Commun, 2008, 370 (3), p. 473–477.

16. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., Tuschl, T. Identification of novel genes coding for small expressed RNAs. Science, 2001, 294 (5543), p. 853–858.

17. Lau, NC., Lim, LP., Weinstein, EG., Bartel, DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science, 2001, 294, p. 858–862.

18. Lee, PL., Glasner, ME., Yekta, S., et al. Vertebrate microRNA genes. Science, 2003, 299 (5612), p. 1540.

19. Lewis, BP., Shih, IH., Jones-Rhoades, MW., et al. Prediction of mammalian microRNA targets. Cell, 2003, 115 (7), p. 787–798.

20. Livak, KJ., Schmittgen, TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25, p. 402–408.

21. Llave, C., Xie, Z., Kasschau, KD., Carrington JC. Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science, 2002, 297, p. 2053–2056.

22. Martinez, J., Patkaniowska, A., Urlaub, H., et al. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell, 2002, 110, p. 563–574.

23. Michael, MZ., O’Connor, SM., van Holst Pellekaan, NG., et al. Reduced accumulation of specific microRNAs in colorectal neoplasia. Mol Cancer Res., 2003, 1 (12), p. 882–891.

24. Ohashi, Y., Miharu, N., Honda, H., et al. Quantitation of fetal DNA in maternal serum in normal and aneuploid prenancies. Hum Genet, 2001, 108 (2), p. 123–127.

25. Vandesomple, J., De Preter, K., Pattyn, F., et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genom Biol, 2002, 3, RESEARCH0034.

26. Zamore, PD., Tuschl, T., Sharp, PA., Bartel, DP. RNAi : double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 2000, 101 (1), p. 25–33.