Down syndrome is the most commoncause of mental retardation affecting 1 in 1,500 pregnancies with 1 in 400 babies been born every year worldwide with the incidence rates being increasingly high due to socioeconomic reasons. At the same time, as prospective parents are being informed on technological advances the demand for a safe, prenatal testing continuously increases.
The proposed study stems out from frontier research performed during the ERC Advanced Grant (322953) and ERC PoC (693555). For both projects fetal specific DNA and RNA differentially methylated regions (DMRs) were confirmed using novel methylation (DNA and RNA) immunoprecipitation protocols from low input of material (i.e plasma of pregnant women). The objective of this proposal is to extend the results of the two previous ERC funded Grants by utilizing the most robust biomarkers on a high precision ddPCR platform for the development and performance validation of a methylation-based assay for NIPT for Trisomy 21.
Specifically, following cell-free DNA (cfDNA) and cell-free RNA (cfRNA) isolation, samples were subjected to DNA and RNA methylation enrichment using Methylated DNA Immunoprecipitation (MeDIP) and Methylated RNA immunoprecipitation (MeRIP) respectively. Subsequently the enriched methylated RNA samples were subjected to RT-PCR, a conventional technique for the conversion of RNA to cDNA. cDNA and methylated DNA fractions were subjected to multiplex ddPCR using primers specifically designed for the enriched fetal specific regions. During the development phase, all work was completed according to Annex I however due to biological and technical limitations the RNA DMRs did not perform as expected. As such, we proceeded to the final stage (assay validation) using only DNA DMRs. Following the completion of the assay validation, we were able to achieve 91.4% (82.4-100%, 95% CI) specificity, and 100% sensitivity.
Overall, the following have been achieved towards the project’s objectives:
1. Selection of the most robust DNA and RNA DMRs for the development of the assay using specific criteria
2. Development a multiplex methyl-ddPCR assay
3. Performance validation of a fast, cost-efficient NIPT test for Trisomy 21 using DNA DMRs
Currently, the gold standard for NIPT is utilization of high throughput, high accuracy technologies such as Next Generation Sequencing. Although the sequencing cost has been dramatically decreased, NIPT is still inaccessible to many prospective parents especially in developing countries. Furthermore, albeit more accurate than traditional first trimester screening using ultrasound and biochemical markers, NIPT has not been established as a first-line screening by the majority of Health care systems because of its higher cost. Our assay provides the first step towards the provision of a fast, accurate, cost-efficient NIPT for Trisomy 21, overcoming the limitations of other NIPTs and still fulfilling the needs of healthcare providers and most importantly the needs of prospective parents as it will be accessible to all pregnant women independent from social and economic status.
