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

Date Published: February 3, 2017

Publisher: Public Library of Science

Author(s): Maria C. Neofytou, Kyriakos Tsangaras, Elena Kypri, Charalambos Loizides, Marios Ioannides, Achilleas Achilleos, Petros Mina, Anna Keravnou, Carolina Sismani, George Koumbaris, Philippos C. Patsalis, Noam Shomron.


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.

Partial Text

Large and small size sub-chromosomal deletions and duplications are associated with genetic disorders and syndromes [1]. This group of clinically recognizable disorders is characterized by diverse phenotypes including intellectual disability (ID), autism and other neurodevelopmental disorders (NDD) [2]. Chromosomal abnormalities can result from genomic structural changes, such as copy number changes, leading to abnormal gene dosage with a dramatic impact on gene expression and phenotype [1].

Non-invasive prenatal testing has focused mainly on whole chromosome aneuploidies and has advanced rapidly in clinical practice. Recent microarray studies showed that clinically relevant deletions or duplications are found in 1.7% of pregnancies with clinical indications for prenatal diagnosis [6]. Whole genome sequencing and targeted sequencing of cffDNA found in maternal circulation has enabled the detection of fetal sub-chromosomal events avoiding the risk of miscarriage [28, 29, 32].

We have developed and assessed an accurate and cost-effective assay for the NIPT of sub-chromosomal small and large size deletions and duplications using targeted capture enrichment technology. The use of TACS on selected syndromic regions offers high multiplex capabilities reducing the sequencing cost per base for each sample. In addition this approach requires less time for bioinformatics analysis since it is limited to a specific number of genomic regions. This assay exhibited very high accuracy, resulting from the inherently very high read depth of the targeted microdeletion/microduplication genomic regions. Due to its targeted nature, the assay avoids the detection of CNVs of unknown clinical significance and enables the detection of deletions or duplications as small as 0.5Mb in size. The analytical performance of the assay was successfully evaluated using cfDNA samples from normal pregnancies and serial dilutions of abnormal DNA in plasma. These results indicate that detection of small and large size deletions and duplications is feasible. Additional validations with prospective and retrospective studies using affected plasma pregnancies should be performed to obtain diagnostic sensitivity and specificity rates. Early stage noninvasive identification of microdeletion and microduplication syndromes is clinically important as it empowers prospective parents to make informed decisions and enables health professionals to offer optimal pregnancy management, and postnatal interventions with long term improvements in the health of the newborn.




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