Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

Abstract

OBJECTIVES: Sequencing of cell-free fetal DNA from maternal plasma, also known as non-invasive prenatal testing (NIPT), has enabled accurate prenatal diagnosis of aneuploidy. This approach is gaining clinical acceptance, as it significantly reduces the necessity of invasive diagnostic procedures such as amniocentesis that carry a significant risk of fetal loss. Recent studies have demonstrated the potential for NIPT to detect subchromosomal abnormalities. We investigated whether NIPT using semiconductor sequencing could reliably detect subchromosomal deletions and duplications in a large population of women carrying high-risk fetuses. METHODS: We applied a sliding window method to reduce required sequencing depth. First, we demonstrated that increasing concentration of abnormal DNA as well as increasing number of sequencing reads improved detection of chromosomal abnormalities. We then analyzed plasma from 1456 pregnant women to develop a method to predict the fraction of fetal DNA based on the size distribution of DNA fragments (r = 0.818, p<2.2e À 16, linear regression model). Finally, we collected plasma from 938 of pregnant women having a fetus with structural abnormalities detected on ultrasound who also underwent amniocentesis, chorionic villous sampling, or umbilical cord blood sampling. We sequenced these samples using from 3M up to 15M reads to detect subchromosomal abnormalities, in parallel with array comparative genomic hybridization performed on each invasively-derived sample as a comparison. RESULTS: In total, 100% (57/57) instances of aneuploidy and 36/40 (90%) of samples with subchromosomal abnormalities greater than 5MB in size were detected using only 3 million (3 M) reads, while 5M reads was required to detect two more samples subchromosomal abnormalities greater than 5MB in size and 12/19 samples with chromosomal abnormalities between 1.5 and 5 MB. While increasing sequencing depth up to 15M reads increased accuracy for larger deletions/duplications, 88.1% (52/59) abnormalities larger than 1.5M could be detected using this method, while 5 abnormalities under 1.5MB were not reliably detected. CONCLUSIONS: This study demonstrates the viability of NIPT using semiconductor sequencing to accurately detect subchromosomal abnormalities greater than 1.5MB in a high-risk population.

Noninvasive prenatal screening for fetal aneuploidy, 2016 update: a position statement of the American College of Medical Genetics and Genomics

“…Expanding NIPS to include detection of specific conditions caused by a CNV (e.g., 22q11.2 deletion, 1p36 deletion, 15q11.2-13 deletion) is technically possible (analytical validity). [60][61][62][63] The phenotypes associated with these conditions can be severe; therefore, they may be appropriate conditions for prenatal screening. However, providers and patients must be aware that expanding the use of NIPS to include the detection of CNVs requires in-depth knowledge of the limitations of the technology, return of results, and follow-up.…”

“…Validation studies make the point that DR and SPEC depend on many variables (e.g., depth of read), 10,[60][61][62][63] which can change the false-positive and false-negative rate when NIPS is used for prenatal detection of CNVs. Pretest and posttest counseling is further confounded by variable expressivity and penetrance of the conditions being screened, size of the deletion being screened, specific genes within the critical region of the locus interrogated, and the number of genes within the critical region being screened.…”

Limited Clinical Utility of Non-invasive Prenatal Testing for Subchromosomal Abnormalities

“…Using one billion reads, Srinivasan et al detected fetal CNVs as small as 300 kb, 14 whereas Chen et al claimed that their pipeline can detect all fetal CNVs bigger than 10 Mb with just two to eight million reads. 13 Until now, NIPT of subchromosomal abnormalities has been reported only in a small number of cases of affected pregnancies, although a larger series using spiked samples has been reported.…”

“…10 However, in principle, sequencing of cfDNA can also be used for detecting other unbalanced chromosomal rearrangements prenatally, and a number of proof-of-concept studies using a variety of sequencing depths and bioinformatics approaches have detected a range of fetal subchromosomal abnormalities in maternal plasma. [11][12][13][14][15] Indeed, several commercial providers have expanded their NIPT platform to include a panel of syndromes characterized by recurrent microdeletions and microduplications.…”

Noninvasive Prenatal Testing: The Indian Perspective

“…The common microdeletions tested are 22q11.2 deletion/DiGeorge syndrome (may have cardiac defects detected on ultrasound), 1p36 deletion, Angelman syndrome, Prader-Willi syndrome, and Cri-du-chat syndrome. One company screens additionally for chromosome 16 and 22, and deletions of 4p (Wolf-Hirschhorn syndrome), 8q (Langer-Gideon syndrome), and 11q (Jacobsen syndrome) [28]. The 22q microdeletion is the commonest and has the greatest clinical importance.…”