Development and Validation of NIPD Trisomy 21 Test

Down syndrome (trisomy 21) is the most common cause of mental impairment with an incidence of 1:700 births.  The early detection of trisomy 21 is critical for prospective parents and is an integral component of many national healthcare programs.  Current methods for the prenatal screening and diagnosis of trisomy 21 have limitations.  Prenatal screening tests using sonography and/or measurements of compounds in maternal serum have relatively low sensitivities (~70-90%), very low specificities with high false positive rates and do not provide the most effective prenatal diagnosis.  Even though invasive prenatal diagnostic tests by chorionic villus sampling (CVS), amniocentesis and chordocentesis are accurate and specific, they have other limitations such as a considerable risk for miscarriage (~1-2%) and fetal loss.  Thus, invasive prenatal diagnostic tests can not be applied to all pregnancies and can not provide the most effective prenatal diagnosis.


There is a vital need to develop a Non-Invasive Prenatal Diagnostic (NIPD) test that can be offered to all pregnancies, does not put the fetus at risk for miscarriage and provides a more effective prenatal diagnosis for Down syndrome and other genetic disorders.  The development of such a test is widely anticipated by prospective parents, health care providers and the scientific community.  Several groups have been working towards this goal for the past fifteen years.  However, the development of a NIPD test has been a great challenge due to the limited amount of free fetal DNA (ffDNA) (3-5%) in maternal circulation.

NIPD Genetics has been successful in developing a NIPD test for the diagnosis of trisomy 21 during the early stages of pregnancy (as early as the 10th week of gestation). The results of this study were published  last year in the prestigious journal, Nature Medicine (details are available in our Publications section). Our Non-Invasive Prenatal Diagnostic test for trisomy 21 is based on the identification of differentially methylated fetal-specific markers and the use of those Differentially Methylated Regions (DMRs) for the discrimination between a trisomy 21 fetus and a normal fetus by real-time quantitative PCR (see graph for example). By assaying multiple DMRs across chromosome 21 we demonstrated a sensitivity and specificity of 100% from testing and correctly classifying 34 trisomy 21 and 46 normal cases.


Last year, the validity of this approach was further demonstrated in a second, larger blind validation study which was published in October, 2012 in Prenatal Diagnosis (refer to Publications section for details). In the study, the team at NIPD Genetics improved the identification and characterization of 8 DMRs on chromosome 21 and developed a new diagnostic formula using 7 of these DMRs to better classify trisomy 21 pregnancies and normal pregnancies. To test the improved method, a blind, larger validation study using maternal peripheral blood from 175 pregnant women between 11 to 14 weeks gestation was performed. The study found that all 50 trisomy 21 cases and 124 of 125 normal cases were correctly classified (one normal case was found as false positive), thus showing the refined test has a sensitivity of 100% and a specificity of 99.2%.


Recently, the results of a study which investigated whether certain factors may affect test result determination (i.e. proper classification of a normal or affected fetus) have been published (details are available in our Publications section). It has been known that the amount of fetal DNA available for testing can affect other molecular trisomy 21 tests, so NIPD Genetics evaluated whether the amount of free fetal DNA (ffDNA), total DNA and ‘fetal fraction’ found in 83 maternal plasma samples affected trisomy 21 classification. The study found that variability of ffDNA and total DNA from maternal peripheral blood samples did not affect the enrichment ratios of the DMRs used in the method and, more importantly, trisomy 21 classification using MeDIP-RTqPCR methodology.

The application of this novel test is sensitive, specific, safe, simple, affordable and does not require specialized or complex laboratory equipment, software or know-how. Results can be obtained within 3 days and the cost is lower than that of current invasive methods. With so many advantages, the test is suitable for routine laboratory procedures and can be readily introduced by any genetic diagnostic laboratory.



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