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

Abstract

Background: Non-invasive prenatal testing (NIPT) has been widely adopted for the detection of fetal aneuploidies and microdeletion syndromes, nevertheless, limited clinical utilization has been reported for the non-invasive prenatal screening of monogenic diseases. In this study, we present the development and validation of a single comprehensive NIPT for prenatal screening of chromosomal aneuploidies, microdeletions and 50 autosomal recessive disorders associated with severe or moderate clinical phenotype. Results: We employed a targeted capture enrichment technology powered by custom TArget Capture Sequences (TACS) and multi-engine bioinformatics analysis pipeline to develop and validate a novel NIPT test. This test was validated using 2033 cell-fee DNA (cfDNA) samples from maternal plasma of pregnant women referred for NIPT and paternal genomic DNA. Additionally, 200 amniotic fluid and CVS samples were used for validation purposes. All NIPT samples were correctly classified exhibiting 100% sensitivity (CI 89.7-100%) and 100% specificity (CI 99.8-100%) for chromosomal aneuploidies and microdeletions. Furthermore, 613 targeted causative mutations, of which 87 were unique, corresponding to 21 monogenic diseases, were identified. For the validation of the assay for prenatal diagnosis purposes, all aneuploidies, microdeletions and point mutations were correctly detected in all 200 amniotic fluid and CVS samples. Conclusions: We present a NIPT for aneuploidies, microdeletions, and monogenic disorders. To our knowledge this is the first time that such a comprehensive NIPT is available for clinical implementation.

A Novel Graphic-Aided Algorithm (gNIPT) Improves the Accuracy of Noninvasive Prenatal Testing

“…Koumbaris et al validated their NIPT using 2033 cell-free DNA samples in 2019 and found 27 positive cases for T13/T18/T21. The overall positive rate of their test is 1.32% (27/2033) [21]. Compared with this value, the positive rate of 0.39% in this study seems quite low.…”

Simultaneous detection of fetal aneuploidy, de novoFGFR3 mutations and paternally derived β‐thalassemia by a novel method of noninvasive prenatal testing

“…Recently, three studies reported targeted capture NIPT to screen for chromosome aneuploidy, copy number variation, and monogenic disease. [32][33][34] such as early pregnancies or certain aneuploidies. 2,,35 A recent study has demonstrated the importance of prenatal screening for genetically dominant disorders caused by de novo mutations.…”

“…, 31 However, these methods require experiments that cannot cope with conventional NIPT method for chromosome analysis. Recently, three studies reported targeted capture NIPT to screen for chromosome aneuploidy, copy number variation, and monogenic disease 32–34 . However, their methods were time‐consuming due to 16–72 hours of hybridization capture after the preparation of cfDNA sequencing library.…”

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…”

“…According to the traditional cytogenetic testing and analysis, the chromosome number abnormalities in genetic mutations include the polyploid and aneuploid. Chromosomal aneuploidy is the most common chromosomal disease, accounting for 80–90% of all chromosomal diseases, 4 and it is the main cause of birth defects. Different types of fetal chromosomal aneuploidy have different prognoses.…”