EP1259824A1

EP1259824A1 - Trisomy 21 cytotrophoblast cultures, and uses thereof for obtaining trisomy 21 markers

Info

Publication number: EP1259824A1
Application number: EP01913925A
Authority: EP
Inventors: Danièle EVAIN-BRION; Jean-Louis Frendo
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2000-03-03
Filing date: 2001-03-02
Publication date: 2002-11-27
Also published as: US20030138435A1; JP2003525056A; WO2001065264A1; FR2805824A1; CA2401603A1; FR2805824B1

Abstract

The invention concerns trisomy 21 cytotrophoblast cultures. Proteins differentially secreted by said cultures and by normal cytotrophoblast cultures are in particular useful as trisomy 21 markers.

Description

CROPS OF TRISOMY 21 CYTOTROPHOBLASTS AND THEIR USE FOR OBTAINING TRISOMY 21 MARKERS

The invention relates to cultures of trisomy 21 cytotrophoblasts, and to their uses. Trisomy 21, responsible for Down syndrome more commonly known as mongolism, is the most common autosomal trisomy since it is observed on nearly 1 in 800 newborns during childbirth [FERGUSON-SMITH et al., Prenat. Diagn. , 4, 5-44, 1984)]. It is the major genetic cause of mental retardation [CUNNINGHAM et al., Williams Obstetrics, ed. 19, 919-938, (1993)].

The prenatal diagnosis of trisomy 21 is essentially based on the establishment of the fetal karyotype, an examination practiced most often on fetal cells of the amniotic fluid collected by amniocentesis, more rarely on the trophoblastic cells taken by biopsy of the choπal villi.

However, establishment of the fetal karyotype cannot be practiced systematically, and has long been reserved for women aged 38 years and over and / or those with signs of ultrasound call. Certain ultrasound warning signs, and in particular the thickening of the neck of the fetus in the first trimester of pregnancy, make it possible to detect 80% of cases of trisomy 21 [NICOLAIDES et al., BMJ, 304, 867-869, (1992);

NICOLAIDES et al., Br. J. Obstet. Gynecol. , 101, 782-786,

(1994); SNIJDERS et al., Lancet, 352, 343-346, (1998)].

However, the effectiveness of this screening method depends directly on the operator's expertise. On the other hand, although the risk of Down's syndrome increases with age, nearly 70% of children with Down's syndrome 21 are born to mothers under the age of 35 [WALD et al., Bπt. Med. J., 297, 883-887, (1988)].

Under these conditions, it has been proposed to complete these examinations by assaying maternal seπques markers, with the aim in particular of carrying out the prior detection of cases in which the establishment of the fetal karyotype would be recommended. In 1987, BOGART et al. [Prenat. Diagn., 7, 623-630] described in two thirds of pregnancies with a fetus with Down's syndrome a decrease in α-fetoprotein associated with an increase in hCG in maternal serum in the second trimester. Many studies have since confirmed the link between the risk of Down's syndrome and high levels of hCG, and in particular of hCGβ in the mother [BROCK et al., Prenat. Diagn., 10, 245-251, (1990); CHARD et al., The embryo. Spπnger Verlag, London, 209-226, (1991); HADDOW et al., N. Engl. J. Med., 338, 955-961, (1998)]. In addition, there are also low levels of unconjugated estriol in maternal blood.

[HADDOW et al., N. Engl. J. Med., 327, 588-593, (1992);

HADDOW et al., N. Engl. J. Med., 330, 1114-1118, (1994);

VAN-LITH, Prenat. Diagn., 12, 495-504, (1992)]. As these three markers are not interdependent, it is possible to calculate the risk of fetal trisomy 21 by combining their determination; this screening method is called "triple test".

It should be noted, however, that the use of unconjugated estriol as an additional marker in the "triple test" is very controversial [cf. for example SPENCER, Screening for Down's syndrome, Cambridge University Press, 141-162, (1994)] and, for reasons of public health economics, its determination is not systematically carried out.

These tests make it possible to detect a little more than 60% of trisomies 21; there are also about 5% false positives whose fetal karyotype will prove, in fact, normal. It therefore appears desirable to have available other markers of trisomy 21, which would make it possible to improve the sensitivity and / or the specificity of the screening.

Furthermore, the origin of the abnormal maternal hormonal profile, observed in trisomy 21, remains completely unknown.

For example, it has been hypothesized that the increase in circulating levels of hCG in the mother in the event of trisomy 21, could result from an increased production by the trophoblast at a given gestational age, or a decrease in the clearance of the molecule in connection with a post-translational modification. Thus, ELDAR-GEVA et al. [J. Clin. Endocπnol. Metab., 80, 3528-3531, (1996)], report a very significant increase in the expression and secretion of hCG in trophoblastic cells of trisomy 21 collected in the second trimester of pregnancy, and propose it as one of the possible causes of increased maternal hCG. Other authors [BRIZOT et al., Mol. Hmm. Reprod., 10, 2506-2509, (1995)] do not observe any difference in expression of 1 hCG between normal placentas and trisomy 21 placentas in the first trimester of gestation. COLE et al. [Prenatal. Diagnosis, 18, 926-933,

(1998)], highlighted the presence in the maternal urine of cases of trisomy 21, of a hyperglycosylated form of hCG similar to that produced in case of choriocarcmome; these authors propose the sequential or uπnal assay of this hyperglycosylated form for the detection of trisomy 21. It therefore appears that few studies have been carried out on the molecular anomalies observed in the trisomy 21 placenta, and that the results are contradictory. .

The present invention aims to provide new means of studying cellular and molecular dysfunctions occurring during placental development in trisomy 21, as well as new markers of trisomy 21. For this purpose, the inventors have put in culture cytotrophoblastic cells from normal placentas and placentas with fetal trisomy 21. The cytotrophoblastic cell is the key cell of the human placenta. It is differentiated m vi tro and m if you at the placental level as a syncytiotrophoblast. The syncytiotrophoblast is the endocrine tissue of the human placenta. It produces steroid and polypeptide hormones at very high rates (several grams per day).

In vi tro, the cytotrophoblasts isolated and purified from the normal placenta aggregate then merge to form the syncytiotrophoblast. This highly polarized cell secretes m vi tro into the culture medium the hormones which it normally pours into the maternal circulation. The inventors have isolated, purified and cultured cytotrophoblasts from trisomy 21 placentas to all terms of pregnancy, and have compared their development, as well as their ability to express and secrete different peptide hormones, with that of normal cytotrophoblasts. They thus observed that cultures of cytotrophoblasts originating from trisomy 21 placentas, did not form a syncytiotrophoblast, or formed a defective syncytiotrophoblast, and that this formation occurred much later than in cultures of cytotrophoblasts originating from normal placentas collected at the same stage. of gestation. They also measured the expression and secretion in the culture medium, of certain

(hCGα, hCGβ, hPL, PGH, and leptm) peptide hormones normally produced by the trophoblast, and found that in cultures of cytotrophoblasts from trisomy 21 placentas, abnormalities in syncytiotrophoblast formation were accompanied by significant decrease in these hormones. These results being in contradiction with those previously reported by ELDAR-GEVA et al. (publication cited above), the Inventors have, in order to verify the absence of artifact, measured the expression of the peptide hormones mentioned above in samples of placental tissues. They thus observed in the tissues of trisomy 21 placentas, a level of expression much lower than the average level of expression of the same hormones in tissues derived from normal placentas.

In addition, they found that transformed cytotrophoblasts, overexpressing copper and zinc-dependent superoxide dismutase (Cu / Zn SOD), exhibited the same abnormalities in syncytiotrophoblast formation, and decreased production of hCGα, the hCGβ, hPL, and PGH. In humans, the Cu / Zn SOD gene is carried by the long arm of chromosome 21, and this enzyme is one of those that are overexpressed in subjects with Down's syndrome.

The subject of the present invention is a culture of cytotrophoblasts, characterized in that said cytotrophoblasts are trisomal for human chromosome 21, or for a region thereof comprising at least the gene coding for Cu / Zn superoxide dismutase, and in that '' they express after 3 days of culture at least one peptide hormone chosen from human placental lactogen (hPL), the human choπonic gonadotroph

(hCG), the human choπonic gonadotrophm β (hCGβ), human placental growth hormone (PGH), and leptm, in an amount less than that expressed on average by normal cytotrophoblasts from placentas at the same stage of gestation, and grown under the same conditions.

The term “normal cytotrophoblasts” is understood here to mean cytotrophoblasts having a normal karyotype, and which are capable of forming a syncytiotrophoblast when cultivated in vitro. Cytotrophoblast cultures according to the invention can be obtained from trisomy 21 placentas, by enzymatic digestion in several stages, followed by purification on a Ficoll gradient.

The cells thus isolated can then be cultured in a conventional manner, according to the techniques usually used for cultures of normal cytotrophoblasts.

Cytotrophoblast cultures according to the invention can be used as an in vitro model to study the anomalies of differentiation of the syncytiotrophoblast in trisomy 21, for example cell fusion anomalies. In particular, as the trophoblastic cells secrete in the culture medium the same soluble proteins as those which they secrete in vi vo in the maternal blood or in the fetal compartment, the cultures of cytotrophoblasts in accordance with the invention can be used to identify and characterize biological molecules, in particular proteins, released into the blood maternal by placentas of fetuses with Down's syndrome, and quantitatively and / or qualitatively different from the biological molecules released into maternal blood by placentas of normal fetuses, and thus being able to constitute markers of Down's syndrome.

One can in particular analyze the differential expression of genes and proteins, between normal trophoblastic cell cultures and cell cultures according to the invention, during the various stages of formation of the syncytiotrophoblast, including the development of the differentiated syncytiotrophoblast.

For example, cultures of cytotrophoblasts in accordance with the invention can be used for obtaining differential expression libraries of nucleic acids between normal cytotrophoblasts and trisomal cytotrophoblasts for chromosome 21, and / or between trisomal cytotrophoblasts for chromosome 21 at different stages of culture.

The present invention thus encompasses differential expression libraries of nucleic acids obtained using at least one culture of cytotrophoblasts according to the invention. Such libraries can consist, for example: of a population of total or partial cDNA sequences of genes expressing themselves in the cytotrophoblast cultures according to the invention, and not expressing themselves in the cytotrophoblast cultures normal to same stage of syncytiotrophoblast formation; a population of total or partial cDNA sequences of genes not expressing themselves in the cultures of cytotrophoblasts in accordance with the invention, and expressing themselves in cultures of normal cytotrophoblasts at the same stage of formation of the syncytiotrophoblast; of a population of total or partial sequences of cDNAs of genes whose expression is greater in the cultures of cytotrophoblasts according to the invention than in the cultures of normal cytotrophoblasts at the same stage of formation of the syncytiotrophoblast; a population of total or partial sequences of cDNAs of genes whose expression is less important in the cultures of cytotrophoblasts in accordance with the invention than in the cultures of normal cytotrophoblasts at the same stage of formation of the syncytiotrophoblast; - A population of total or partial cDNA sequences of genes whose expression varies between different stages of formation of the syncytiotrophoblast in cell cultures according to the invention; a population of total or partial sequences of cDNAs of genes whose expression in cell cultures in accordance with the invention varies according to environmental conditions, or in response to the application of a physical or chemical agent

(for example at 2 different temperatures, in the presence or absence of oxygen, etc.). Differential expression libraries of nucleic acids according to the invention can be obtained by methods known in themselves to those skilled in the art. By way of nonlimiting examples, mention will be made, for example, of methods such as DDRT-PCR [LIANG and PARDEE, Science, 257, 967-971, (1992)], subtractive hybridization [JIANG and FISHER, Mol. Cell. Different., 1, 285-299, (1993)], RAP-PCR [MCCLELLAND and WELSH, PCR Methods & Applications, 4, S66-81, (1994)], SAGE analysis [VELCULESCU et al., Science , 270, 484-487, 1995], and their different variants. They can then be used in particular to identify, characterize and clone genes involved in abnormalities in the differentiation of the syncytiotrophoblast, and / or to study the regulatory mechanisms of these genes.

It is also possible, to study the genes involved in the differentiation of the syncytiotrophoblast, and the transcription factors regulating these genes, using nuclear extracts prepared from cultures of cytotrophoblasts in accordance with the invention or of syncytiotrophoblasts formed by these cultures. Cytotrophoblast cultures in accordance with the invention can also be used to study the expression and / or differential secretion of proteins during the various stages of syncytiotrophoblast formation, by comparing their expression and / or secretion profiles. proteins with those of normal cytotrophoblast cultures, or with those of other cytotrophoblast cultures according to the invention, at another stage of development or cultivated under different conditions. One can for example compare the quantity of proteins produced and / or secreted, as well as the qualitative differences between proteins, in particular post-translational modifications. The comparison can be carried out on total proteins, or on protein subpopulations, for example on intracellular proteins or on those secreted in the culture medium, on glycosylated proteins or on those which are not, etc. .

The methods of separation, detection, and quantification or qualitative analysis which allow this comparison to be made are known in themselves to those skilled in the art.

Mention will be made, for example, of two-dimensional electrophoresis, optionally coupled to a mass spectrometry, which makes it possible to quickly identify, by comparison of electrophoresis gels, the differences between two protein expression profiles. Likewise, the glycosylation or the absence of glycosylation of a protein can for example be detected by isoelectric focusing; according to their glycosylation (in particular their sialic acid content), the different glycoforms will migrate until they arrive in a pH zone corresponding to their isoelectric point; this isoelectric focusing profile can be compared with that obtained after treatment with an enzyme (for example neurammidase or mannosidase) which hydrolyzes the glycans. Glycosylated proteins can also be detected by affinity electrophoresis on a gel containing a lectme; the glycoforms are separated according to their affinity for lectme.

Proteins separated on gel can for example be detected by conventional staining or by immunodetection after transfer to nitrocellulose, and their quantity can be evaluated by densitometry. The proteins for which differential expression is observed can then be purified from the gels to complete their identification and characterization.

The proteins in the culture medium can also be analyzed by chromatography. For example, to detect and / or quantify glycosylated proteins, it is possible to perform affinity chromatography on lectme.

By way of example, the comparison of the expression of mRNAs and of the secretion of proteins in cultures of cytotrophoblasts in accordance with the invention and in cultures of normal cytotrophoblasts enabled the inventors to identify proteins which may constitute serum markers for Down's syndrome.

The inventors have in fact found that the synthesis of certain peptide hormones, which is very important in the cultures of normal cytotrophoblasts, at the time of the formation of the syncytiotrophoblast, was very weak in the cultures of cytotrophoblasts in accordance with the invention. These serum hormones should therefore be present in smaller quantities in the serum of women with a Down's syndrome fetus than in that of women with a normal fetus. However, hCG, which is one of the peptide hormones whose expression is reduced in Cytotrophoblast cultures according to the invention is, on the contrary, known to be present in a greater quantity in the serum of women carrying a trisomic fetus 21 than in that of women carrying a normal fetus.

The inventors have hypothesized that this apparent contradiction is explained by a longer half-life of hCG in serum, resulting from an abnormality in glycosylation in trisomal cytotrophoblasts for chromosome 21, such as that described by COLE et al. (1998, publication cited above).

In this case, the proteins which are produced by the cytotrophoblasts in non-glycosylated form will, if they are produced in less quantity, also present in smaller quantity in the serum of women carrying a fetus with Down's syndrome than in that of women carrying a normal fetus, whereas on the contrary the proteins produced in glycosylated form, like hCG, will be glycosylated abnormally in the cytotrophoblasts trisomiques 21, and even if they are produced in smaller quantity, will be found in more quantity important in maternal blood.

To verify this hypothesis, the inventors carried out the assay, in sera of women carrying a trisomic fetus 21 and in sera of women carrying a normal fetus, 1 hCG and another glycosylated hormone, l growth hormone, as well as that of the following hormones: human placental lactogen (hPL) and leptm, which are not produced by cytotrophoblasts in glycosylated form.

They thus found that in the sera of women carrying fetuses with Down's syndrome, 1 hPL and leptm were present in significantly lower quantity, whereas on the contrary, hCG and placental growth hormone were present in quantity more important.

These results make it possible to propose new detection methods and new markers of Down's syndrome. The subject of the present invention is a method for detecting a risk of trisomy 21 in a fetus, characterized in that it comprises the measurement, in a sample of a biological fluid obtained from the mother, of at least one protein chosen from:

- the non-glycosylated proteins secreted by a culture of cytotrophoblasts according to the invention in a smaller amount than by a culture of normal cytotrophoblasts; - the non-glycosylated proteins secreted by a culture of cytotrophoblasts in accordance with the invention in greater quantity than by a culture of normal cytotrophoblasts; glycosylated proteins, secreted by a culture of cytotrophoblasts according to the invention, with the exception of hCG.

These proteins therefore appear to be usable as markers of Down's syndrome, in accordance with the invention. Their assay can be carried out, depending on the protein concerned, either by detection and quantification of the whole protein, or, if it is a protein which is degraded in the organism, by detection of its degradation products ( including peptide fragments). Proteins "secreted by a culture of cytotrophoblasts according to the invention in a smaller amount than by a culture of normal cytotrophoblasts" also include proteins which are not secreted by cytotrophoblasts according to the invention but are secreted by normal cytotrophoblasts .

Likewise, proteins "secreted by a culture of cytotrophoblasts according to the invention in greater quantity than by a culture of normal cytotrophoblasts" also include proteins which are secreted by cytotrophoblasts according to the invention but are not by normal cytotrophoblasts. By "biological liquid" is meant in particular serum and urine. For the implementation of the process according to the invention, the assay of said non-glycosylated protein will preferably be carried out from a serum sample. Proteins that can be used can be easily identified by those skilled in the art, by comparing the protein secretion profiles of the 2 cultures, and / or by detecting glycosylation or the absence of glycosylation, as indicated above. A separate dose of one or more of said proteins different from the average dose encountered in women carrying a normal fetus indicates a risk of Down's syndrome in the fetus. In the case of a glycosylated protein, due to the greater half-life resulting from an abnormal glycosylation in the trisomal cytotrophoblasts 21, a dose of said protein greater than the average dose encountered in women with a normal fetus indicates a risk of Down's syndrome in the fetus. Using the CLONTECH Microarray device

"Atlas Human 1.2", the inventors thus found that the cytotrophoblastic cell expressed secreted proteins such as: IGF ₂ , Thymosme beta 10, Calgranulme B, "Macrophage colony stimulatmg factor", "C. reactive protem precursor ", EGF," FMLP-related receptor 1 ", small cytok e mductible A 5, IGF BPi," Hepatocyte growth factor like protem ", PDGF, and some of the IL-1 mterleukmes IL-18. They also found that cytotrophoblastic cells expressed the precursor of the amyloid peptide (APP). They also observed that this expression was much greater in the cultures of cytotrophoblasts according to the invention than in the cultures of normal cytotrophoblasts.

The precursor of the amyloid peptide (APP) is a transmembrane protein (SWISS-PROT access number:

P05067), the extracellular domain of which is released in soluble form after proteolytic cleavage by an α-secretase activity (release of a soluble protein comprising the amino acids 18-699 of APP) or by a β-secretase activity (release of a soluble protein comprising amino acids 18-671 of APP), (for review, see OCTAVE et al., Medicine / Sciences, 11, 1251-1259, 1995). The extracellular domain of the APP protein, or fragments thereof, therefore appears usable, in accordance with the invention, as maternal serum markers of trisomy 21.

The inventors have also identified hPL and leptm, among the non-glycosylated proteins secreted by normal and non-secreted cytotrophoblast cultures, or secreted in less quantity, by the cytotrophoblast cultures in accordance with the invention. They also identified 1 mterleukme 13, among the glycosylated proteins secreted by the cultures of cytotrophoblasts in accordance with the invention. This protein is not secreted by normal cytotrophoblasts.

According to a preferred embodiment, the method according to the invention comprises the assay, in a biological sample obtained from the mother, of at least one protein chosen from glycosylated proteins secreted by a culture of cytotrophoblasts in accordance with the invention, and at least one protein chosen from non-glycosylated proteins secreted by a culture of cytotrophoblasts according to the invention.

In the context of this embodiment, said glycosylated protein can be for example hCG, placental growth hormone, the extracellular domain of APP protein or any other glycosylated protein secreted by cultures of cytotrophoblasts in accordance with l 'invention. Glycosylated proteins which can be used can be easily identified by a person skilled in the art, from the protein secretion profile of a cytotrophoblast culture according to the invention, by detecting glycosylation or the absence of glycosylation, as indicated above.

The dosage of the selected proteins is carried out according to the usual methods, known in themselves. same to those skilled in the art. In particular, immunoassay methods can be used, using antibodies directed against the protein concerned. In the case of proteins which are known per se as hPL, placental growth hormone, leptm, hCG, or APP, suitable antibodies are available commercially, or can readily be obtained by conventional techniques, from purified preparations of these proteins. In the case of proteins identified only from protein secretion profiles of cytotrophoblast cultures, which were not previously known and for which no specific antibody is available, suitable antibodies can be obtained, for example, from proteins eluted from two-dimensional gels. These antibodies can then be used to purify larger quantities of the protein concerned from cytotrophoblast cultures.

The detection method according to the invention can be implemented in addition to another method of detecting trisomy 21, for example in addition to an assay method using other serum markers, or a method of detection by ultrasound.

For example, to confirm or deny an ultrasound diagnosis, one can perform the assay of hPL, which constitutes a very discriminating marker from the 19 ^th week of gestation.

The present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples of obtaining and characterizing cultures of cytotrophoblasts according to the invention, and of the use of hormones peptides produced by these cultures as markers of Down's syndrome.

EXAMPLE 1: OBTAINING TRISOMY CYTOTROPHOBLAST CULTURES 21 Collection of placental tissues:

Placental tissue samples were taken during abortion for abnormalities severe fetal, between 12 and 35 weeks of gestation, either in cases of trisomy 21 detected by karyotype, or as a control, in cases of fetal malformation without karyotype anomalies. The samples were taken in accordance with the provisions of French law for the consent of the patients concerned.

The samples from the two groups were matched to correspond to identical gestation durations. It was verified, for the samples taken in the case of trisomy 21, that the placental tissue was also trisomic for chromosome 21, and for the control samples, that the karyotype was normal. Isolation and culture of cytotrophoblasts

The placental villi were stripped of membranes and vessels, rinsed and minced in a balanced solution of HANK (HBSS) devoid of calcium and magnesium.

Cytotrophoblast cells were isolated by trypsin-DNAse digestion, using the protocol described by KLIMAN et al. [Endocrmology, 118, 1567-1582, (1986)], modified as follows:

The digestion is carried out using an enzymatic preparation prepared extemporaneously, and comprising 0.5% (w / v) of trypsm powder (DIFCO), 5 IU / ml of DNAsel (SIGMA, 375000 U, 2600 Kunitz u / mg solid), 25 mM HEPES, 4.2 mM MgS0 ₄ and 1% (w / v) penicillin / streptomycin, in HBSS.

The minced villi are covered with this preparation; after 30 mm. digestion at 37 ° C, the liquid is discarded and replaced by a fresh enzyme preparation. A further 25 min incubation is carried out, at the end of which the liquid is discarded and again replaced by a fresh enzymatic preparation. 5 digests are then carried out successively, each time using a fresh enzymatic preparation: 1 digestion of 20 min., 1 digestion of 15 mm. and 3 digests of 10 mm. The liquids from the last 5 digests are stored and collected, then fractionated on a discontinuous Percoll gradient of 10 to 70%.

A homogeneous population of mononuclear cells is thus obtained. After centrifugation, the cell pellet is taken up in 4 ml of DMEM medium. These 4 ml are deposited on the gradient comprising more than 90% of viable cytotrophoblastic cells [ALSAT et al. , J. Clin. Encodocrmol. Metab., 73, 288-294, (1991)]. The cells are cultured in dishes 60 mm in diameter (3 × 10 ⁶ cells per dish, in 3 ml of DMEM medium, supplemented with 25 mM of HEPES, 2 mM of glutam, 20% of fetal calf serum inactivated by heat, and in the presence of antibiotics (100 IU per ml of penicillin and 100 μg per ml of streptomycin). The cultures are maintained at 37 ° C. in a humid atmosphere: 95% air

(19% 0 ₂ , and 76% NH ₂ ); 5% C0 ₂ . on DMEM medium (Dulbecco's

Modified Eagle Medium), in the presence of 25mM HEPES, 2mM glutam, 20% heat-inactivated fetal calf serum, and in an atmosphere consisting of 95% air and 5% C0 ₂ .

Comparison of the properties of cytotrophoblasts obtained from trisomy 21 placentas and cytotrophoblasts obtained from control placentas

The cytotrophoblasts isolated from the control placentas aggregate, then after 24 to 48 hours, fuse and differentiate into syncytiotrophoblasts.

Cytotrophoblasts isolated from trisomy 21 placentas remain aggregated in culture dishes. After 3 to 4 days of culture, only very few syncytiotrophoblasts are observed.

Desmoplaqume, and cadherme-E which disappear normally during the formation of syncytium, have been sought in cultures of control cytotrophoblasts, and in cultures of trisomy 21 cytotrophoblasts, using anti-desmoplaquma monoclonal antibodies or anti-E-cadherme. For the detection of desmoplaqume or E-cadherme, the cells in culture were rinsed with PBS, fixed, and permeabilized by immersion in methanol at -20 ° C for 20 mm. An anti-desmoplaqum or anti-E-cadherm monoclonal antibody, diluted to l / 400 ^th (SIGMA) is then applied to the treated cells, and the antigen-antibody complex is revealed by anti-mouse goat immunoglobulins, fluorescently labeled. (SIGMA).

After 3 days of culture, desmoplaqume and E-cadherme are absent from normal syncytiotrophoblasts, but present at mtercellular contacts in cultures of trisomy 21 cytotrophoblasts.

These results clearly show that there is, in the cultures of cytotrophoblasts of trisomy 21, a delay and / or a defect in differentiation of the cytotrophoblast into syncytiotrophoblast.

EXAMPLE 2: CHARACTERIZATION OF THE EXPRESSION AND SECRETION OF PROTEINS IN CULTURES OF TRISOMY CYTOTROPHOBLASTS Study of transcription

Total RNA is extracted from cytotrophoblast cultures after 24, 48 or 72 hours using _t QIAGEN, following the manufacturer's protocol.

Human placental lactogen (hPL), human choonic gonadotrophm α (hCGα), human chorionic gonadotrophm β (hCGβ), human placental growth hormone (PGH), and leptm mRNAs are quantified as described below, from total RNA in solution, previously transcribed inversely into cDNA. The cDNA is amplified by RT-PCR in real time using the TaqMan ^ method (PE Applied Biosystems). This method is based on the use of the 5 '3' exonuclear activity of the Taq polymerase in order to digest a labeled probe hybridized to a target sequence, during the extension phase of a PCR amplification. The labeling of the probe is designed so that its digestion generates a fluorescent signal. For each reaction, the parameter C _τ (cycle threshold) is defined as the number of cycles for which the fluorescence generated by the digestion of the probe exceeds a threshold fixed beforehand; this number is lower the higher the initial quantity of the target molecule. The quantity of each target sequence in samples of unknown composition is thus determined by measuring C _τ and using a calibration curve to determine the initial quantity of the target sequence.

The primers used for reverse transcription, as well as the amplification primers and the detection probes are chosen from the nucleic sequences of hPL), of hCGα, of hCGβ, of PGH and of leptum. available on the databases, using OLIGO 4.0 (NATURAL BIOSCIENCES) and Primer express (PERKIN-ELMER BIOSYSTEMS) software. Their specificity, and the absence of polymorphism was verified by research using BLASTN

[ALTSCHUL et al. Nucleic Acids Res. 25: 3389-3402, (1997)]. on the bases dbEST and nr. The PCR amplifications were carried out in an ABI PRISM 7700 apparatus (PERKIN-ELMER BIOSYSTEMS).

The standard curve was established using series of 10 in 10 dilutions of cDNA obtained by reverse transcription from 1 μg of total RNA extracted from placenta collected during the 1 ^st trimester of pregnancy. To correct possible biases caused by an imprecise evaluation of the quantity of total RNA used in each reaction, as well as its quality (i.e. the absence of significant degradation), an internal control was carried out by quantification of transcripts of the PPIA gene (coding for human peptidyl prolyl isomerase A), and each sample was normalized on the basis of its PPIA content. For each sample, the quantity of target RNA to be quantified and that of the control PPIA RNA was determined from the standard curve. Then, the amount of target RNA was divided by the amount of PPIA RNA to obtain the normalized value. Secretion study

Measurement of hormones produced by cytotrophoblasts

The concentration of hCG in the culture media was determined by an ELISA test (VITAS system, BIOMERIEUX) following the protocol indicated by the manufacturer. The detection sensitivity of this test is 2 mU / ml.

The concentration of secreted hPL was determined using the IRMA AMERLEX hPL kit, (AMERSHAM) in concentrated medium 4 times. The sensitivity of the test is 0.5 μg / ml). HPL has also been detected from cell lysates of cytotrophoblasts. The solubilized proteins (5 μg) were revealed after electrophoresis and transfer to nitrocellulose using a polyclonal rabbit antibody directed against 1 hPL and diluted to 250 ^erae (DACO, FRANCE). The band corresponding to 1 hPL is revealed by chemiolummescence (PIERCE SUPERSIGNAL, INTERKIM, FRANCE) after incubation with an anti-lapm antibody coupled to peroxidase.

The leptm was measured in concentrated medium 4 times using the RIA kit “Sensitive Human Leptm RIA Kit” (LINCO, ST LOUIS, USA). The sensitivity of the assay is 0.5 ng / ml.

For each assay, three independent measurements were made. Immunoblotinσ

The results of these experiments are illustrated in Figures 1 to 3:

FIGS. 1A and 1B show the expression of the mRNAs of 1 hCGα and 1 hCGβ during the differentiation of cytotrophoblasts isolated from control placentas (N) and trisomy 21 placentas (T21) and cultured for 24 h, 48 h or 72 h. Figure 1C illustrates the secretion of hCG by control cells (N) or T21 cells after 24 h, 48 h or 72 h of culture. In normal placental cell cultures, the level of transcripts of the hCG β subunit increases by more than 10 ³ times between cytotrophoblast and differentiated syncytiotrophoblast m vi tro. Likewise, the secretion of βhCG gradually increases in the culture medium. In cultures of T21 placenta cells, the level of transcripts and hCG secretion are decreased. Figures 2A to 2C illustrate the expression of

MRNA, secretion into the culture medium, and intracellular production of hPL during the differentiation of cytotrophoblasts isolated from control (N) or trisomal (T21) placentas. Figure 2A illustrates the level of transcription, expressed as a normalized amount of mRNA. Figure 2B illustrates the secretion of hPL in the culture medium, expressed in μg / ml. Figure 2C shows the detection of intracellular hPL after immunoelectrophoretic transfer. In T21 cells, we do not detect after

3 days a very small amount of hPL transcripts, and 1 hPL is not detectable in cells.

Figure 3 illustrates the expression of leptm (Figure 3A) and PGH (Figure 3C) mRNAs, as well as leptm secretion (Figure 3B) during differentiation of cytotrophoblasts isolated from control placentas (N) or of T21 placentas grown for 3 days.

In normal placenta cultures, the level of mRNA is increased by more than 20 times (leptm) and 140 times (placental GH) between cytotrophoblasts and syncytiotrophoblasts. In the case of trisomy 21, there is a defect in transcription and in the secretion of these two hormones in the culture media.

These results show that, in the cells obtained from control placentas, after 72 h, which corresponds to the formation of the syncytiotrophoblast, a significant increase in the mRNAs of hCGα, hCGβ, hPL, leptum and PGH is observed. Simultaneously, an increase in hCG, hPL and leptm secretion is observed in the culture media.

In contrast, in cells obtained from trisomy 21 placentas, defective syncytiotrophoblast formation is associated with a decrease important of hCGα, hCGβ, hPL, leptm and PGH mRNAs.

At the same time, there is a significant decrease in the secretion of hCG in the culture medium, and after 3 days no secretion of hPL or of leptm can be detected in the culture medium. Likewise, hPL is not detectable in trisomy 21 cells, whereas it is present in normal cells.

The secretion of PGH in the culture medium has not been measured since it is known that this secretion is inhibited by glucose, which is present in this medium.

These results indicate that the defects and / or the delay in the morphological differentiation into syncytiotrophoblasts of cytotrophoblasts isolated from trisomy 21 placentas are associated with a simultaneous decrease in the expression and secretion of the hormones specifically synthesized by the syncytiotrophoblast.

At the same time, the transcription of the hCGα, hCGβ, PGH, hPL, and leptm genes was measured during placental ontogenesis, using the protocol described above, in samples of normal placentas and T21. the results obtained indicate that throughout pregnancy, the transcription of these genes is much lower in the placentas of trisomy 21 than in normal placentas.

EXAMPLE 3 USE OF PROTEINS SECRETED BY CULTURES OF CYTOTROPHOBLASTS OF TRISOMY 21 AS MARKERS OF TRISOMY 21.

The following proteins were assayed on a sample of 86 sera taken from women with Down's syndrome fetuses, and 247 sera taken from women with normal fetuses.

Non-glycosylated proteins

1. hPL: The assay is carried out on serum. The test portion is adapted according to the stage of pregnancy (at the start of pregnancy 200 μl, at the end of pregnancy 20 μl). The Assay is carried out using the AMERLEX-hPL-IRMA IRMA Kit (Ortho Clmical Diagnostics, United Kingdom).

Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 5.9% to 10% coefficient of variation. The concentration range is

0.5 to 16.5 μg / ml. The detection limit of the test is

0.06 μg / ml.

The results are illustrated in Figure 4, which shows that in the case of T21, the levels of HPL are abnormally low and that from 19-20 weeks, they become a very discriminating marker. Maternal serum hPL levels are therefore a direct reflection of the anomaly in the formation of the syncytiotrophoblast in T21. 2. Leptme:

The assay is carried out on serum. The test portion is 100 μl. The assay is performed using the RIA Human Leptm RIA KIT assay (Linco, USA).

Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 3.3% to 8.9% of coefficients of variation. The concentration range is from 0.5 to 100 ng / ml. The detection limit is 0.1 ng / ml

There is a decrease in maternal serum leptm levels in the case of fetal trisomy 21. Glycosylated protein

GH placental

The assay is carried out on serum. The test portion is 100 μl. The assay is carried out using the hPGH IRMA kit (BC1017) BIOCODE. Each serum is dosed in duplicate with a precision on the mter-series measurement ranging from 5.5% to 7.9% of coefficients of variation. The concentration range is from 1 to 180 ng / ml. The detection limit is 0.2 ng / ml.

The results are illustrated in Figure 5, which confirms that the placental GH is higher in the maternal blood in the case of fetal trisomy 21 from 16 to 17 weeks.

Claims

CLAIMS 1) Culture of cytotrophoblasts characterized in that said cytotrophoblasts are trisomic for human chromosome 21, or for a region thereof comprising at least the gene coding for Cu / Zn superoxide dismutase, and in that they express after 3 days of culture at least one peptide hormone chosen from human placental lactogen (hPL), human choπonic gonadotrophm α (hCGα), human choπonic gonadotrophm β (hCGβ), human placental growth hormone (PGH), and leptm, in an amount lower than that expressed on average by normal cytotrophoblasts from placentas at the same stage of gestation, and cultivated under the same conditions. 2) Use of a cytotrophoblast culture according to claim 1 to identify and characterize markers of trisomy 21.

3) Differential expression library of nucleic acids obtained using at least one culture of cytotrophoblasts according to claim 1.

4) Method for detecting a risk of Down's syndrome in a fetus, characterized in that it comprises the determination, in a biological sample obtained from the mother, of at least one protein chosen from: glycosylated secreted by a culture of cytotrophoblasts according to claim 1 in a lower amount than by a culture of normal cytotrophoblasts;

- the non-glycosylated proteins secreted by a culture of cytotrophoblasts according to claim 1 in a larger amount than by a culture of normal cytotrophoblasts; glycosylated proteins secreted by a culture of cytotrophoblasts according to claim 1, with the exception of hCG.

5) Method according to claim 4, characterized in that one carries out the assay of at least one non-glycosylated protein selected from 1 hPL and leptm. 6) Method according to claim 4, characterized in that one carries out the assay of at least one glycosylated protein from the placental growth hormone, 1 mterleukme 13, and the extracellular domain of the precursor of the amyloid protein.

7) Method according to any one of claims 4 or 5, characterized in that it further comprises the determination of at least one protein chosen from glycosylated proteins secreted by a culture of cytotrophoblasts according to claim 1, and at least a protein chosen from non-glycosylated proteins secreted by a culture of cytotrophoblasts according to claim 1.

8) Method according to claim 7, characterized in that said glycosylated protein is chosen from hCG, placental growth hormone, 1 mterleukme 13, and the extracellular domain of the precursor of the amyloid protein.

9) Use of at least one protein as defined in claim 4 as a marker for the in vitro diagnosis of trisomy 21.