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

TLDR

This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test. The study aimed to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell‑free DNA from 11 pregnant women was isolated and subjected to massively parallel sequencing with ~10^9 25‑mer tags mapped to hg19, then counted and normalized to 1 Mb or 100 kb bins to identify copy‑number changes. All seven nonmosaic microdeletion, duplication, translocation, and trisomy 20 cases were detected blindly, including a 300 kb deletion, and MPS identified translocation breakpoints and chromosomal origins of unknown material in two cases, whereas mosaic abnormalities were not detected, showing that in nonmosaic pregnancies, maternal plasma cfDNA MPS yields a fetal molecular karyotype equivalent to microarray and sometimes superior to metaphase karyotype.

Abstract

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10(9) tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.