Non Invasive Prenatal Diagnosis of Trisomy 21 by Genetic Analysis of Circulating Fetal Cells

Study Overview
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Brief Summary
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The objective of this project is to develop a non-invasive prenatal diagnostic test for trisomy 21 which is reliable, sensitive and cost-effective, and thus, offers an alternative to the currently employed invasive diagnostic tests amniocentesis and chorionic villus sampling. Current prenatal screening methods (blood markers and ultrasound) for trisomy 21 (Down syndrome) detect about 90 % of cases and have a false positive rate of > 90 %. The results of these tests are expressed in risks for trisomy 21, the threshold being in France at 1/250. Women exhibiting a higher risk are offered to undergo invasive diagnostic testing, either by amniocentesis or chorionic villus sampling. However, these invasive diagnostic methods are associated with a considerable risk of miscarriage (1-3 %), and thus underline the importance to develop a safe and non-invasive prenatal diagnostic test for trisomy 21. The investigators have planned to assess the clinical impact of a non-invasive prenatal method to detect Trisomy 21 through genetic analysis of circulating trophoblastic cells.

Detailed Description
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The investigators have planned and developed the following approach: fetal cells are first enriched from blood of pregnant women, between 7 and 12 weeks gestation, employing the ISET (isolation by size of epithelial tumor cells) technique. Cells presumed to be of fetal origin are microdissected and subsequently genetically analyzed, using STR markers, to verify their fetal nature. The investigators then plan to test two strategies in order to assess the number of copies of chromosome 21. The first one involves the DNA of a single fetal cell to be analyzed with CGH (comparative genomic hybridization) array. In fact, our team has already developed an application of the metaphase CGH method to single cells isolated by ISET in which we were able to demonstrate the gain of chromosome 21 DNA in single fetal cells isolated from cord blood of a fetus with Down syndrome. The second strategy will be accomplished with the use of quantitative fluorescent PCR analysis of short tandem repeats (STRs), applied to single cells. At least 5-8 highly polymorphic STR markers specific for chromosome 21 will be tested to minimize the effects of a phenomenon called allele drop out, in which one allele fails to amplify, and to maximize the number of triallelic signals for an accurate diagnosis of disomy or trisomy 21. This survey is performed in collaboration with the Department of Gynaecology-Obstetrics - Reproductive Medicine of Antoine Béclère Hospital in Clamart. The women included in the survey will be taken a 20 ml blood sample and a cervical Pap smear before the invasive test (amniocentesis). The blood sample will be treated by the ISET method within 3 hours after collection and the filter will be stored at - 20°C. The cells obtained by Pap smear will be kept in an appropriate liquid and then treated by the ISET method in the Biochemistry Laboratory of Necker Hospital. The molecular analyses directed to the Trophoblastic cells for the NI-PND of Trisomy 21 will be performed in a blind study. The instigators have planned to use the ISET method in a blind study including 100 cases of trisomy 21 and 300 control cases with normal caryotype. This study will allow to obtain results with sensitivity higher than 97 % and specificity higher than 99 % (IC 95 % [70-100]). The validation will be obtained by the opening of the blind study and the comparison of results obtained by the invasive method (amniocentesis) and the non-invasive method.

Official Title
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Clinical Validation of the ISET Method for the Non Invasive Prenatal Diagnosis of Trisomy 21 by Genetic Analysis of Circulating Trophoblastic Cells

Conditions
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Trisomy 21

Intervention / Treatment
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* Other: sample blood


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Pregnant women older than 18 years old Pregnant women followed at a prenatal diagnostic centre Pregnant woman having a risk (> 1/250) of trisomy 21 based on the combined screening serological tests/nuchal ultrasonography  Sample of blood and cervical smear obtained between the 8th and the 10th WG Pregnant women accepting an invasive prenatal diagnosis Father of the child agreeing to participate in the clinical study (accepting to give a saliva sample) Pregnant women beneficiary of a national insurance program Pregnant women and fathers signing an informed consent Exclusion Criteria: Pregnant women with combined risk of trisomy 21 < 1/250 Pregnant women non accepting the invasive prenatal diagnosis Pregnant women participating another clinical study

Ages Eligible for Study
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Minimum Age: 18 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Diagnostic
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Pregnant women accepting an invasive prenatal diagnosis<br>Pregnant women accepting an invasive prenatal diagnosis and a sample blood (non invasive diagnosis) | Other: sample blood<br> <br> * Other names: Trisomy 21;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| non-invasive method of PND of Trisomy 21. | Clinical validation of a non-invasive method of PND of Trisomy 21. | 9 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Circulating Trophoblastic Cells, Non-Invasive Prenatal Diagnosis, Genetic analysis, Trisomy 21, Down's Syndrome