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A Novel Non-Invasive Prenatal Diagnosis for Genetic Disorders

Final Report Summary - NIPD (A Novel Non-Invasive Prenatal Diagnosis for Genetic Disorders)

NIPD Genetics has successfully developed and validated a novel non-invasive prenatal test for the detection of fetal syndromes caused by aneuploidies of chromosomes 13, 18 and 21. We discovered hundreds of fetal specific differentially methylated regions between fetal and maternal DNA using a newly developed method. We also reconstructed the fetal and maternal methylome at base pair resolution using whole genome bisulfide sequencing.
Correlation between MeDIP-NGS and bisulfide sequencing results resulted in the discovery of hundreds of fetal specific DMRs. A subset of these were pre-validated using ddPCR, while all DMRs were confirmed and characterized using MeDIP-Targeted NGS, a double enrichment approach developed for the purposes of this project. Two rounds of biomarker discovery were performed to ensure the selection of the most robust DMRs and to increase the sensitivity and specificity of the test. Multiple fetal and maternal samples were then investigated based on their inter-individual methylation variability. Results showed that the extend of variability is not large enough to mask the levels of differential methylation between fetal and maternal DNA.
Towards the development of the a NIPT assay, we applied MeDIP-Targeted NGS on normal and abnormal spike-in samples at different percentages representing different fetal fraction (5-20%). Using three different mathematical models we successfully classified all normal and abnormal samples. Following the assessment of the NIPT assay on spiked-in samples, we tested its performance in a proof-of-concept study for the detection of fetal aneuploidies (trisomy 13, 18 and 21) in 44 real pregnancy plasma samples including 4 trisomy 21, one trisomy 13 and one trisomy 18 sample with fetal fraction ranging from 5-16%. Bioinformatics pipelines and mathematical models were developed for read depth normalization and GC bias correction. All normal samples were correctly classified.

The final objective of the project was the blind validation of 288 plasma pregnancy samples. All samples were subjected to MeDIP-Targeted NGS resulting in the detection of 6 out of 6 trisomy 21 samples and 281 out of 282 normal samples with 100% [54%-100%] sensitivity and 99.6% [98%-99.9%] specificity. All samples were found to be normal for trisomy 13 and trisomy 18 with 100% [98.7%-100%] specificity.
Conclusively, we have successfully completed all the objectives. We discovered hundreds of fetal specific DMRs. A subset of robust and well-characterized DMRs was subsequently used for the development and validation of a novel and accurate double enrichment NIPT assay for the detection of chromosomes 13, 18 and 21.