Objective
The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP)–based noninvasive prenatal test for 5 microdeletion syndromes.
Study Design
Four hundred sixty-nine samples (358 plasma samples from pregnant women, 111 artificial plasma mixtures) were amplified with the use of a massively multiplexed polymerase chain reaction, sequenced, and analyzed with the use of the Next-generation Aneuploidy Test Using SNPs algorithm for the presence or absence of deletions of 22q11.2, 1p36, distal 5p, and the Prader-Willi/Angelman region.
Results
Detection rates were 97.8% for a 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1), and cri-du-chat syndromes (24/24). False-positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442), or 1p36 deletion syndromes (0/422).
Conclusion
SNP-based noninvasive prenatal microdeletion screening is highly accurate. Because clinically relevant microdeletions and duplications occur in >1% of pregnancies, regardless of maternal age, noninvasive screening for the general pregnant population should be considered.
The discovery in the maternal circulation of cell-free DNA (cfDNA) of fetal/placental origin has led to a revolution in prenatal screening. Common whole-chromosome fetal aneuploidies can now be detected with high sensitivity and specificity and have facilitated a significant reduction in the number of invasive diagnostic procedures that have been performed. In the United States, 2 noninvasive prenatal testing (NIPT) approaches have been commercialized: quantitative “counting” that uses massive or targeted parallel sequencing and a single-nucleotide polymorphism (SNP)–based approach that relies on the identification of maternal and fetal allele distributions. Both methods can detect pregnancies at high risk for trisomy 21 (Down syndrome), trisomy 18, trisomy 13, and sex chromosome abnormalities. The SNP-based approach is also able to detect triploidy.
Subchromosomal abnormalities (microdeletions and duplications) may result in physical and/or intellectual impairments that can be more severe than whole chromosome abnormalities. Unlike the risks of aneuploidy that is associated with nondisjunction, the incidence of subchromosomal copy number variations (CNVs) is independent of maternal age. Clinically relevant microdeletions and duplications occur in 1-1.7% of all structurally normal pregnancies. In younger women, the risk for a clinically significant deletion exceeds the risk for Down syndrome. Because some infants with subchromosomal abnormalities may benefit from early therapeutic intervention, prenatal detection is important for optimal management. In support of this, it is recommended that chromosome microarray analysis be offered to all women who undergo invasive diagnostic testing. However, with the introduction of NIPT for aneuploidy screening, many women who previously would have had invasive testing are choosing to avoid these procedures because of the small risk of pregnancy loss.
Submicroscopic genomic alterations are harder to detect noninvasively because of their small size. A small proportion may be identified incidentally through traditional serum and ultrasound screening, but these tests were not designed to screen for these anomalies. The introduction of a highly accurate noninvasive prenatal screening test that would identify women who are at high risk for microdeletions or duplications therefore would be useful. Recently, proof-of-principle studies that used shotgun or whole-genome sequencing reported the detection of subchromosomal microdeletions and microduplications. However, these approaches were limited by the requirement for exceptionally high sequence reads, and interpretation was complicated by the identification of variants of unknown clinical significance. Here, we used a targeted SNP-based approach to detect the larger deletions that underlie 5 microdeletion syndromes with clinically severe phenotypes.
Materials and Methods
Initial validation studies were performed with genomic DNA that had been isolated from 40 characterized cell lines to demonstrate that the SNP-targeted assay was capable of detecting the presence or absence of 22q11.2, 1p36, cri-du-chat, Prader-Willi, and Angelman deletions. These cell lines included 7 with 22q11.2 deletions, 19 with 5p deletions (cri-du-chat syndrome), 10 with 15q11-13 deletions (3 with Angelman syndrome and 7 with Prader-Willi syndrome), and 4 with no deletions.
After validation of the SNP-targeted assay, a cohort of 469 test samples was evaluated ( Table 1 ). This included 6 maternal plasma samples from pregnant women in which the fetus had a microdeletion (3 with 22q11.2 deletions, 2 with 5p deletions, and 1 with a 1p36 deletion), 352 unaffected pregnancy plasmas, and 111 artificial DNA mixtures (PlasmArts). Seventy-three of the PlasmArts were generated from DNA derived from 2 individuals with 22q11.2 deletions, 1 with a 5p deletion, and one unaffected child, each of which was diluted into matched maternal DNA. Thirty-eight samples were generated from genomic DNA isolated from two 15q-cell lines (1 Angelman, and 1 Prader-Willi) and the corresponding maternal cell lines. All cell lines were obtained from the Coriell Cell Repository (Camden, NJ). Patients who provided samples were enrolled at prenatal and postnatal care centers under institutional review board–approved protocols (Western Institutional Review Board protocol number: 12-014-NPT), pursuant to local regulations.
| Samples | Sample deletion size | n |
|---|---|---|
| Pregnancy samples | ||
| DiGeorge deletion | arr[hg18] 22q11.21(17,010,000-20,130,000)x1 | 1 |
| DiGeorge deletion | arr[hg18] 22q11.21(17,020,000-20,130,000)x1 | 1 |
| DiGeorge deletion | 46,XX.nuc ish(HIRAx1) | 1 |
| Cri-du-chat deletion | 46,XX,del(5)(p15.1p15.3) | 1 |
| Cri-du-chat deletion | 46,XY,del(5)(p14.2) | 1 |
| 1p36 deletion | 46,XY,del(1)(p36.1) | 1 |
| 46,XX and 46,XY | 352 | |
| PlasmArt samples: born triads | ||
| DiGeorge deletion | arr[hg18] 22q11.2(17,270,000- 19,810,000)x1 | 22 |
| DiGeorge deletion | arr[hg18] 22q11.2(16,950,000-20,250,000)x1 | 22 |
| Cri-du-chat deletion | arr[hg18] 5p15.33p14.1(91,100-29,500,000)x1 | 22 |
| 46,XX and 46,XY | 7 | |
| PlasmArt samples: cell lines | ||
| Prader-Willi deletion | arr[hg18] 15q11.2q13.1(20,310,000-27,130,000)x1 | 16 |
| Angelman deletion | arr[hg18] 15q11.2q13.1(20,310,000-27,220,000)x1 | 22 |
Genomic DNA for PlasmArt mixtures was isolated from the buffy coats from mother and child pairs or from paired mother and child cell lines. These DNA preparations were cleaved into internucleosomal fragments of roughly 150 base pairs and multiples thereof with the use of a proprietary reaction that included micrococcal nuclease (New England Biolabs, Ipswich, MA). Because fetal cfDNA exists in vivo mainly as mononucleosomal fragments, child DNA of approximately 150 base pairs was isolated using Solid Phase Reversible Immobilization beads (Agencourt Biosciences, Beverly, MA). Maternal genomic DNA was not size purified because maternal cfDNA exists as a nucleosomal ladder. Child DNA was titrated into the corresponding maternal DNA to achieve artificial mixtures with “fetal” fractions that ranged from 3.8-33%, which was a similar distribution to that observed in maternal plasma clinical samples. The “fetal” fraction distribution of these samples is shown in Figure 1 ; for comparison, the fetal fraction distribution from 19,910 consecutive maternal plasma samples from women at 10-16 weeks of gestation is also shown.
All samples, including maternal and (when available) paternal samples, underwent targeted multiplex polymerase chain reaction and were sequenced; the data were analyzed with the Next-Generation Aneuploidy Test Using SNPs (NATUS) algorithm as described previously, with the following alterations: a unique set of primers was designed to amplify 4128 SNPs in the regions-of-interest (672 SNPs targeting 2.91 Mb in the 22q11.2 region and 1152 SNPs in each of the other regions, targeting 5.85 Mb in the Prader-Willi/Angelman region, 10.0 Mb in the 1p36 region, and 20.0 Mb in the cri-du-chat region). The assay was not validated for the smaller, less-frequent deletions that are associated with these disorders because positive control samples were not available. The estimated relative prevalence of the targeted deletions in the 22q11.2, Prader-Willi/Angelman, 1p36, and cri-du-chat regions were 87%, 28%, 60%, and 65%, respectively. Samples were analyzed with the NATUS algorithm as previously described, and all samples that passed quality control (QC) were included in this cohort. The NATUS algorithm was then used to predict fetal copy number (1, 2, or ≥3 copies) for the microdeletion regions-of-interest. The algorithm was blinded to sample status, and all calls were reported as predicted by the algorithm without subjective modification by laboratory personnel.
Results
Algorithm validation using genomic samples
Validation experiments confirmed that the SNP-based technology and the microdeletion-specific primer pools could detect the microdeletions accurately in the 5 syndromes described. Heterozygous SNPs clearly were absent in all affected regions and were present in all unaffected regions; Figure 2 shows the graphic representations of the sequencing data that were obtained from genomic DNA that had been isolated from one cell line with a 22q11.2 deletion. The plots are described in detail in the legend of Figure 2 . Briefly, the absence of the central green cluster in the 22q11.2 (DiGeorge) region indicated a lack of heterozygous SNPs, from which it is possible to infer a deletion of one copy of the DNA in this region.
Pregnancy plasma cohort
Of the 358 pregnancy samples, 335 samples passed QC metrics. The algorithm did not return a result for 23 of 358 of the samples (6.4%); all of these were unaffected. The detection rates and false-positive rates for those samples that passed QC are listed in Table 2 . Of the 6 affected pregnancy plasmas, 1 false negative was reported (22q11.2). Of the 335 unaffected pregnancy plasmas that passed QC, 4 false positives were reported (3 for the 22q11.2 deletion and 1 for the deletion associated with cri-du-chat syndrome). Figure 3 shows a sample with a fetal fraction of 33% having a cri-du-chat deletion on the maternally inherited chromosome 5. In this sample, 2 green clusters in the cri-du-chat region indicate a deletion; 3 green clusters in the 1p36, Prader-Willi/Angelman, and 22q11.2 regions indicate that 2 copies of the fetal chromosomes are present. The patterns are described in detail in the legend of Figure 2 .
| Disorder | Affected (n = 6 plasma; 108 PlasmArt samples) | Unaffected (n = 335 plasma; 108 PlasmArt samples) | ||||||
|---|---|---|---|---|---|---|---|---|
| Pregnancy plasma, n/N | PlasmArt, n/N | Total, n/N | Analytic detection rate, % (95% CI) | Pregnancy plasma, n/N | PlasmArt, n/N | Total, n/N | False-positive rate, % (95% CI) | |
| 22q11.2 del | 2/3 | 43/43 | 45/46 | 97.8 (88.5–99.9) | 3/332 | 0/65 | 3/397 | 0.76 (0.1–2.2) |
| Prader-Willi | 15/15 | 15/15 | 100 | 0/335 | 0/93 | 0/428 | 0 | |
| Angelman | 21/21 | 21/21 | 100 | 0/335 | 0/87 | 0/422 | 0 | |
| 1p36 del | 1/1 | 1/1 | 100 | 0/334 | 0/108 | 0/442 | 0 | |
| Cri-du-chat | 2/2 | 22/22 | 24/24 | 100 | 1/333 | 0/86 | 1/419 | 0.24 |
| Larger deletions combined | 3/3 | 58/58 | 61/61 | 100 (94.1–100) | 1/1337 | 0/374 | 1/1711 | 0.06 (0.0–0.3) |
Artificial mixtures (PlasmArt)
In the cohort of 111 PlasmArt samples, 108 samples passed QC metrics. The 3 samples that did not pass (1 Angelman, 1 22q11.2 deletion, 1 Prader-Willi) were due to low algorithm-generated confidence for the chromosome region of interest (1 Angelman), no-call for the chromosome region of interest (1 22q11.2 deletion), or a fetal fraction below the threshold where the algorithm makes a high-confidence copy number call (1 Prader-Willi). The detection rates and false-positive rates for the samples that passed QC are presented in Table 2 .
Figure 4 shows a 22q11.2 deletion on the paternal copy of chromosome 22 that was detected from a set of PlasmArt samples with fetal fractions that ranged from 25.9–4.8%. The absence of the peripheral red and blue clusters where the maternal genotype is homozygous (AA or BB) is the hallmark pattern of a deletion on the paternal copy of the chromosome. The deletion is detectable visually as low as 4.8% fetal fraction ( Figure 4 ).
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