MeDIP combined with in-solution targeted enrichment followed by NGS: Inter-individual methylation variability of fetal-specific biomarkers and their implementation in a proof of concept study for NIPT

Abstract

DNA methylation is the most characterized epigenetic process exhibiting stochastic variation across different tissues and individuals. In non-invasive prenatal testing (NIPT) fetal specific methylated regions can potentially be used as biomarkers for the accurate detection of fetal aneuploidies. The aim of this study was the investigation of inter-individual methylation variability of previously reported fetal-specific markers and their implementation towards the development of a novel NIPT assay for the detection of trisomies 13, 18, and 21. Methylated DNA Immunoprecipitation (MeDIP) combined with in-solution targeted enrichment followed by NGS was performed in 29 CVS and 27 female plasma samples to assess inter-individual methylation variability of 331 fetal-specific differentially methylated regions (DMRs). The same approach was implemented for the NIPT of trisomies 13, 18 and 21 using spiked-in (n = 6) and pregnancy samples (n = 44), including one trisomy 13, one trisomy 18 and four trisomy 21. Despite the variability of DMRs, CVS samples showed statistically significant hypermethylation (p<2e-16) compared to plasma samples. Importantly, our assay correctly classified all euploid and aneuploid cases without any false positive results (n = 44). This work provides the starting point for the development of a NIPT assay based on a robust set of fetal specific biomarkers for the detection of fetal aneuploidies. Furthermore, the assay’s targeted nature significantly reduces the analysis cost per sample while providing high read depth at regions of interest increasing significantly its accuracy.

Prenatal testing for imprinting disorders: A laboratory perspective

“…The decision on prenatal testing has to weigh the benefits for the families and the methodological limitations. With the diagnostic implementation of high-throughput and deep-sequencing molecular approaches in methylation and prenatal testing 43,47,48 , further improvement of testing can be expected. However, the suitability of each test has to be monitored, and each test needs appropriate validation.…”

Amniocentesis and Next Generation Sequencing (NGS)-Based Noninvasive Prenatal DNA Testing (NIPT) for Prenatal Diagnosis of Fetal Chromosomal Disorders

“…Poor prognosis, organ abnormalities, mental retardation, and growth retardation are the common features. [3][4][5] Prenatal diagnosis is based on providing genetic counseling for highrisk pregnant females, with further applications of modern biology, biochemistry, immunogenetics, cytogenetics, and molecular genetics techniques to perform maternal or embryo/fetal testing to achieve the diagnosis of chromosomal abnormalities. Traditionally, among the procedures of prenatal diagnosis, the most commonly used and effective method of prenatal diagnosis for fetuses in mid-term pregnancy is amniocentesis and karyotyping of amniotic fluid…”

“…The NGS represented by NIPT, through the noninvasive method, amplifying and counting of the free fetal DNA fragments in the maternal plasma, is used for detection of the genetic material nucleotides to realize the bioinformatics interpretation of the DNA fragments. 2 , 3 , 6 NIPT is currently regarded as a breakthrough method for the detection of fetal chromosomal aneuploidy. A series of clinical verifications have confirmed that NIPT has high sensitivity and specificity for trisomy 21 and trisomy 18.…”

“…Results greater than or equal to 3.0 or less than or equal to −3.0 indicated a positive result. 3 …”

“…With the continuous improvement and comprehensive development of next generation sequencing (NGS), noninvasive prenatal DNA testing (NIPT) through the NGS of peripheral blood of pregnant females is becoming the new direction of prenatal screening for high-risk populations. 2 , 3 Owing to the short development duration of the NGS technology in the field of prenatal diagnosis, the impact of NIPT on the traditional prenatal diagnosis needs to be further confirmed. The present study aimed to detect the fetal free DNA in the peripheral blood of 2267 high-risk pregnant females with indications for the prenatal diagnosis, and perform comparative analysis of the gold standard of karyotyping by amniocentesis to provide a reference for the application of a new model of prenatal screening for fetal chromosomal disorders.…”