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

Abstract

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.

Analysis of Prenatal Diagnosis Results and Pregnancy Outcome of Non-invasive Prenatal Screening High-risk Fetuses

“…Some previous studies reported that the PPV range of trisomy 21 was 65-94%, trisomy 18 was 47-85%, and trisomy 13 was 12-62% [9][10][11]. There is no difference between this study and the literature reports.…”

Non-invasive prenatal testing by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions

“…On the other hand, the drawback is that some genes with clinical significance might be hypothetically missed, as some of the deletions have overhangs from the critical regions possibly encompassing genes too. Our study brings also new information that could be used in the further specification of the size as well as localization of the tested microdeletions as both these parameters were found to be between the four most critical ones 6,12 , since there are only very few pathologic detections that overlap the critical regions both from ISCA and DECIPHER databases (Supplementary Table 2).…”

Targeted capture enrichment followed by NGS: development and validation of a single comprehensive NIPT for chromosomal aneuploidies, microdeletion syndromes and monogenic diseases

“…Even though the cost of a screening test is more easily amortized, compared to the cost of treating an affected child [33,34], a lower test cost allows for greater uptake. Our assay's targeted design significantly reduces the number of required reads, resulting in higher efficiency and scalability [7,35]. By combining all these features we can provide a cost-effective, extended carrier screening and NIPT solution to prospective parents, thereby extending the scope of prenatal care.…”