Noninvasive detection of fetal subchromosome abnormalities via deep sequencing of maternal plasma

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.

Figure 1

Family 1313 z_7j 1 Mb Bin Results for Chr 7 The data show the 0% (solid circles) and 10% (empty circles) mixtures of the affected son’s DNA mixed with the mother’s DNA. The red circle highlights bin 98. The figure shows a 20 Mb deletion on Chr 7 in the DNA mixture, covering the region between 38 Mb and 58 Mb of the chromosome. Additionally, a potential maternal copy number increase, not shared by the son, is seen at 98 Mb.

Figure 2

Family 2877 z_ij 1 Mb Bin Results The data show results for Chr 11 (A) and Chr 15 (B) with 0% (solid circles) and 10% (empty circles) mixture of the affected son’s DNA mixed with the mother’s DNA. (A) An 8 Mb deletion for Chr 11 in both mother-only and the mixed DNAs, covering the region from 41 Mb to 49 Mb of Chr 11; this deletion is shared by mother and son. (B) The son-specific CNV in Chr 15 from 27 Mb to 66 Mb.

Figure 3

Maternal Plasma Sample BE3096 z_ij 1 Mb Bin Results with a Fetal Karyotype with a Duplication in Chromosome 6 Expanded regions show z_6j 1 Mb bin results. The figure shows a 38 Mb duplication, covering the region between 64 Mb and 102 Mb of Chr 6.

Figure 4

Maternal Plasma Sample BF3404 z_ij 1 Mb Bin Results across the Genome with a Fetal Karyotype with a Deletion in Chromosome 7 This clinical sample has a karyotype with a small deletion in chromosome 7 (blue circle). Another small deletion is detected in chromosome 8 (red circle). Expanded regions show z_7j and z_8j 1 Mb and 100 kb bin data. The figure shows a 1 Mb deletion at bin number 150 Mb on Chr 7 (A). At higher resolution (B), this deletion is found to be 300 kb long, in the region from 150.3 Mb to 150.6 Mb of Chr 7. Note: the putative copy-number gain seen in Chr 7 at bin number 156 in the 1 Mb data is not seen in the same region in the 100 kb data. The figure also shows a 2 Mb deletion on Chr 8 (A) covering bins 46 Mb and 47 Mb. At higher resolution (B), this resolves into a 900 kb deletion, covering the region from 46.9 Mb to 47.7 Mb of Chr 8.

Figure 5

Maternal Plasma Sample BE3236 z_ij 1 Mb Bin Results across the Genome for Clinical Sample with a Fetal Karyotype with an Unbalanced Translocation Involving Chromosome 15 Expanded region shows z_15j 1 Mb bin data. The figure shows a 17 Mb deletion in the region between 22 Mb and 39 Mb of Chr 15.

Figure 6

Maternal Plasma Sample AF1019 z_ij 1 Mb Bin Results across the Genome with Additional Unspecified Material in the Fetal Karyotype Expanded regions show z_10j and z_17j 1 Mb bin data. The figures show a 19 Mb duplication of the region from 62 Mb to 81 Mb on Chr 17 and a 2 Mb deletion on Chr 10 from 134 Mb to 135 Mb.