Noninvasive prenatal diagnosis by genome-wide haplotyping of cell-free plasma DNA.

PURPOSE: Whereas noninvasive prenatal screening for aneuploidies is widely implemented, there is an increasing need for universal approaches for noninvasive prenatal screening for monogenic diseases. Here, we present a cost-effective, generic cell-free fetal DNA (cffDNA) haplotyping approach to scan the fetal genome for the presence of inherited monogenic diseases. METHODS: Families participating in the preimplantation genetic testing for monogenic disorders (PGT-M) program were recruited for this study. Two hundred fifty thousand single-nucleotide polymorphisms (SNPs) captured from maternal plasma DNA along with genomic DNA from family members were massively parallel sequenced. Parental genotypes were phased via an available genotype from a close relative, and the fetal genome-wide haplotype and copy number were determined using cffDNA haplotyping analysis based on estimation and segmentation of fetal allele presence in the maternal plasma. RESULTS: In all families tested, mutational profiles from cffDNA haplotyping are consistent with embryo biopsy profiles. Genome-wide fetal haplotypes are on average 97% concordant with the newborn haplotypes and embryo haplotypes. CONCLUSION: We demonstrate that genome-wide targeted capture and sequencing of polymorphic SNPs from maternal plasma cell-free DNA (cfDNA) allows haplotyping and copy-number profiling of the fetal genome during pregnancy. The method enables the accurate reconstruction of the fetal haplotypes and can be easily implemented in clinical practice.

Breast Cancer Detection and Treatment Monitoring Using a Noninvasive Prenatal Testing Platform: Utility in Pregnant and Nonpregnant Populations.

BACKGROUND: Numerous publications have reported the incidental detection of occult malignancies upon routine noninvasive prenatal testing (NIPT). However, these studies were not designed to evaluate the NIPT performance for cancer detection. METHODS: We investigated the sensitivity of a genome-wide NIPT pipeline, called GIPSeq, for detecting cancer-specific copy number alterations (CNAs) in plasma tumor DNA (ctDNA) of patients with breast cancer. To assess whether a pregnancy itself, with fetal cell-free DNA (cfDNA) in the maternal circulation, might influence the detection of ctDNA, results were compared in pregnant (n = 25) and nonpregnant (n = 25) cancer patients. Furthermore, the ability of GIPSeq to monitor treatment response was assessed. RESULTS: Overall GIPSeq sensitivity for detecting cancer-specific CNAs in plasma cfDNA was 26%. Fifteen percent of detected cases were asymptomatic at the time of blood sampling. GIPSeq sensitivity mainly depended on the tumor stage. Also, triple negative breast cancers (TNBC) were more frequently identified compared to hormone-positive or HER2-enriched tumors. This might be due to the presence of high-level gains and losses of cfDNA or high ctDNA loads in plasma of TNBC. Although higher GIPSeq sensitivity was noted in pregnant (36%) than in nonpregnant women (16%), the limited sample size prohibits a definite conclusion. Finally, GIPSeq profiling of cfDNA during therapy allowed monitoring of early treatment response. CONCLUSIONS: The results underscore the potential of NIPT-based tests, analyzing CNAs in plasma cfDNA in a genome-wide and unbiased fashion for breast cancer detection, cancer subtyping and treatment monitoring in a pregnant and nonpregnant target population.

Predicting fetoplacental mosaicism during cfDNA-based NIPT.

PURPOSE OF REVIEW: Cell-free DNA-based noninvasive prenatal testing (cfDNA-based NIPT) using maternal blood is highly sensitive for detecting fetal trisomies. However, false-positive and false-negative results can occur, which prevents NIPT from being a diagnostic test. Fetoplacental mosaicism is one of the main reasons for discordant test results. It is therefore important to understand this phenomenon to enable more comprehensive and appropriate genetic counselling. The present review aims to summarize the current knowledge of fetoplacental mosaicism ascertained during cfDNA-based NIPT and refers to the development of recent analytical pipelines for its detection during pregnancy. RECENT FINDINGS: Publications are emerging demonstrating that genome-wide approaches to analyzing cfDNA can detect chromosomal aneuploidy other than the common trisomies. Despite the high accuracy of current cfDNA-based NIPT, a substantial number of false-positive and false-negative test results remain. Biological causes, such as fetal or (confined) placental mosaicism have been identified using advanced bioinformatics algorithms. Fetoplacental mosaicism can occur as part of normal pregnancy development, hence clinical practice standards recommend confirmation of positive NIPT results with a diagnostic karyotype or microarray study. SUMMARY: cfDNA-based NIPT for fetal chromosomal aneuploidies is not diagnostic because of false-positive and false-negative test results. Recently, novel algorithms have been described that identify pregnancies with an increased risk of fetoplacental mosaicism. Reporting the presence of fetoplacental mosaicism during pregnancy can influence risk estimation and improve genetic counseling.

Maternal liver transplant: Another cause of discordant fetal sex determination using cell-free DNA.

Noninvasive prenatal testing (NIPT) can very accurately determine fetal sex during pregnancy. We present an exceptional case where NIPT contradicts the ultrasound-based sex determination. The pregnant woman was recipient of a liver transplant from a male donor. Graft-derived cell-free DNA released into the maternal circulation clouded the NIPT-based sex determination. Hence, NIPT is not advisable when the pregnant mother underwent an organ transplant.

Predicting fetoplacental chromosomal mosaicism during non-invasive prenatal testing.

OBJECTIVE: Non-invasive prenatal detection of aneuploidies can be achieved with high accuracy through sequencing of cell-free maternal plasma DNA in the maternal blood plasma. However, false positive and negative non-invasive prenatal testing (NIPT) results remain. Fetoplacental mosaicism is the main cause for false positive and false negative NIPT. We set out to develop a method to detect placental chromosomal mosaicism via genome-wide circulating cell-free maternal plasma DNA screening. METHOD: Aneuploidy detection was combined with fetal fraction determination to enable the detection of placental mosaicism. This pipeline was applied to whole genome sequencing data derived from 19 735 plasma samples. Following an abnormal NIPT, test results were validated by conventional invasive prenatal or postnatal genetic testing. RESULTS: Respectively 3.2% (5/154), 12.8% (5/39), and 13.3% (2/15) of trisomies 21, 18, and 13 were predicted and confirmed to be mosaic. The incidence of other, rare autosomal trisomies was ~0.3% (58/19,735), 45 of which were predicted to be mosaic. Twin pregnancies with discordant fetal genotypes were predicted and confirmed. CONCLUSION: This approach permits the non-invasive detection of fetal autosomal aneuploidies and identifies pregnancies with a high risk of fetoplacental mosaicism. Knowledge about the presence of chromosomal mosaicism in the placenta influences risk estimation, genetic counseling, and improves prenatal management.

Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

BACKGROUND: There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS: We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS: Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS: The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions.

DNA methylation is an epigenetic marker that has been shown to vary significantly across different tissues. Taking advantage of the methylation differences between placenta-derived cell-free DNA and maternal blood, several groups employed different approaches for the discovery of fetal-specific biomarkers. The aim of this study was to analyse whole-genome fetal and maternal methylomes in order to identify and confirm the presence of differentially methylated regions (DMRs). We have initially utilized methylated DNA immunoprecipitation (MeDIP) and next-generation sequencing (NGS) to identify genome-wide DMRs between chorionic villus sampling (CVS) and female non-pregnant plasma (PL) and peripheral blood (WBF) samples. Next, using specific criteria, 331 fetal-specific DMRs were selected and confirmed in eight CVS, eight WBF and eight PL samples by combining MeDIP and in-solution targeted enrichment followed by NGS. Results showed higher enrichment in CVS samples as compared to both WBF and PL samples, confirming the distinct methylation levels between fetal and maternal DNA for the selected DMRs. We have successfully implemented a novel approach for the discovery and confirmation of a significant number of fetal-specific DMRs by combining for the first time MeDIP and in-solution targeted enrichment followed by NGS. The implementation of this double-enrichment approach is highly efficient and enables the detailed analysis of multiple DMRs by targeted NGS. Also, this is, to our knowledge, the first reported application of MeDIP on plasma samples, which leverages the implementation of our enrichment methodology in the detection of fetal abnormalities in maternal plasma.

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.

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.

Targeted capture enrichment assay for non-invasive prenatal testing of large and small size sub-chromosomal deletions and duplications.

Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers.