Background
Historically, prenatal screening has focused primarily on the detection of fetal aneuploidies. Cell-free DNA now enables noninvasive screening for subchromosomal copy number variants, including 22q11.2 deletion syndrome (or DiGeorge syndrome), which is the most common microdeletion and a leading cause of congenital heart defects and neurodevelopmental delay. Although smaller studies have demonstrated the feasibility of screening for 22q11.2 deletion syndrome, large cohort studies with confirmatory postnatal testing to assess test performance have not been reported.
Objective
This study aimed to assess the performance of single-nucleotide polymorphism–based, prenatal cell-free DNA screening for detection of 22q11.2 deletion syndrome.
Study Design
Patients who underwent single-nucleotide polymorphism–based prenatal cell-free DNA screening for 22q11.2 deletion syndrome were prospectively enrolled at 21 centers in 6 countries. Prenatal or newborn DNA samples were requested in all cases for genetic confirmation using chromosomal microarrays. The primary outcome was sensitivity, specificity, positive predictive value, and negative predictive value of cell-free DNA screening for the detection of all deletions, including the classical deletion and nested deletions that are ≥500 kb, in the 22q11.2 low-copy repeat A-D region. Secondary outcomes included the prevalence of 22q11.2 deletion syndrome and performance of an updated cell-free DNA algorithm that was evaluated with blinding to the pregnancy outcome.
Results
Of the 20,887 women enrolled, a genetic outcome was available for 18,289 (87.6%). A total of 12 22q11.2 deletion syndrome cases were confirmed in the cohort, including 5 (41.7%) nested deletions, yielding a prevalence of 1 in 1524. In the total cohort, cell-free DNA screening identified 17,976 (98.3%) cases as low risk for 22q11.2 deletion syndrome and 38 (0.2%) cases as high risk; 275 (1.5%) cases were nonreportable. Overall, 9 of 12 cases of 22q11.2 were detected, yielding a sensitivity of 75.0% (95% confidence interval, 42.8–94.5); specificity of 99.84% (95% confidence interval, 99.77–99.89); positive predictive value of 23.7% (95% confidence interval, 11.44–40.24), and negative predictive value of 99.98% (95% confidence interval, 99.95–100). None of the cases with a nonreportable result was diagnosed with 22q11.2 deletion syndrome. The updated algorithm detected 10 of 12 cases (83.3%; 95% confidence interval, 51.6–97.9) with a lower false positive rate (0.05% vs 0.16%; P <.001) and a positive predictive value of 52.6% (10/19; 95% confidence interval, 28.9–75.6).
Conclusion
Noninvasive cell-free DNA prenatal screening for 22q11.2 deletion syndrome can detect most affected cases, including smaller nested deletions, with a low false positive rate.
Introduction
Prenatal screening for genetic disorders has traditionally focused on screening for Down syndrome (T21) and other aneuploidies (T13 and T18) in the fetus. However, such chromosomal aneuploidies constitute a relatively small proportion of the total number of genetic conditions that contribute to adverse infant and childhood outcomes. In recent years, noninvasive prenatal screening based on sequencing of circulating cell-free DNA (cfDNA) in maternal blood has introduced the potential to target any region of the genome, including an option to screen for subchromosomal copy number variants such as chromosomal microdeletions.
Why was this study conducted?
22q11.2 deletion syndrome (22q11.2DS or DiGeorge syndrome) is the most common microdeletion and a leading cause of congenital heart defects and neurodevelopmental delay. Although cell-free DNA (cfDNA) prenatal screening for 22q11.2DS is feasible, data on test performance are limited.
Key findings
Based on genetic confirmation in all cases, the cohort prevalence of 22q11.2DS was 1 in 1524. Single-nucleotide polymorphism–based cfDNA screening identified most cases of 22q11.2DS including both classical and nested deletions that are ≥500 kb. The test false positive rate was 0.15%, which is similar to the false positive rate seen with cfDNA aneuploidy screening.
What does this add to what is known?
This study presents new and comprehensive information on the performance of cfDNA screening for 22q11.2DS, with results based on genetic confirmation in all cases. The findings in this study demonstrate that cfDNA screening for 22q11.2 can be added to aneuploidy screening without a significant increase in the screen positive rate.
Although individually rare, in aggregate, chromosomal microdeletions and duplications are more prevalent than the common trisomies, and because their birth incidence is not associated with increasing maternal age, they are more common than T21 in women <30 years of age. , The most common of these is the 22q11.2 deletion syndrome (22q11.2DS), also known as DiGeorge or velocardiofacial syndrome. This condition is characterized by variable features including congenital heart defects and developmental delay in most patients, a cleft palate or velopharyngeal insufficiency, hypocalcemia, immunodeficiency, autism, and psychiatric disorders. The 22q11.2DS has been estimated to affect approximately 1 in 3000 to 6000 live births and is therefore one of the most common causes of developmental delay and congenital heart anomalies. These mostly de novo deletions are caused by meiotic recombination events in 4 hot spot regions known as A-D low-copy repeats (LCR) on the long arm of chromosome 22 ( Figure 1 ). In approximately 85% of affected individuals, the entire 2.5 to 3 Mb LCR A-D region is deleted, whereas others have smaller nested deletions within this region. ,
In addition to providing parents with important information about their pregnancy, antenatal diagnosis of 22q11.2DS has the potential to improve short- and long-term outcomes for these children. Prenatal detection of congenital heart defects enables delivery at a center capable of caring for these neonates and providing timely treatment for neonatal hypocalcemia and immunodeficiency, which has been shown to improve outcomes. , Despite these benefits, the limited data on test performance have precluded prenatal screening for the syndrome from being routinely offered. Screening for 22q11.2DS has been evaluated in a few studies involving either artificially derived plasma mixtures or plasma samples from women with a high probability of having a fetus with a genetic abnormality. Retrospective analyses of clinical cohorts reported positive predictive values (PPVs) but have not performed full-cohort confirmatory genetic testing to determine test sensitivity and specificity.
We therefore, sought to assess the performance of single-nucleotide polymorphism (SNP)–based cfDNA screening for 22q11.2DS in a large prospective study with genetic confirmation in all pregnancies.
Materials and Methods
Study design and participants
This was a multicenter, prospective observational study. Women with singleton gestations who underwent SNP-based cfDNA screening for aneuploidy and 22q11.2DS were enrolled at 21 centers in the United States, Europe, and Australia. ( Supplemental Materials and Methods ). The study was registered with ClinicalTrials.gov (identifier: NCT02381457; SNP-based Microdeletion and Aneuploidy RegisTry or SMART) and approved by each site’s institutional review board. All participants provided written consent. Eligible women were ≥18 years old, at ≥9 weeks’ gestation, had a singleton pregnancy, and planned to deliver at a study site–affiliated hospital. Women were excluded if they received a cfDNA result before enrollment, underwent organ transplantation, conceived using ovum donation, or were unable to provide a newborn sample. Women who previously underwent traditional serum screening for aneuploidy or sonographic detection of fetal anomalies were eligible for inclusion. Participants did not receive remuneration for enrolling and were not charged for the 22q11.2DS analysis. Screening results were utilized as part of clinical care.
Genetic outcomes were assessed by analysis of prenatal (chorionic villus sampling, amniocentesis, products of conception) or infant (cord blood, buccal swab or newborn blood spot) samples. In all cases, a sample was requested at the end of pregnancy for chromosomal microarray analysis (CMA), regardless of previous prenatal testing. The postnatal CMA was performed by an independent laboratory (Center for Applied Genomics, Children’s Hospital of Philadelphia, PA) that was blinded to the clinical or laboratory results. If postnatal CMA confirmation was not available, results from clinical testing with prenatal CMA, fluorescence in situ hybridization (FISH), bacterial artificial chromosomes (BACs)-on-beads, or multiplex ligation-dependent probe amplification (MLPA), if available, were used for genetic confirmation.
Outcomes
The primary outcome was test performance of cfDNA screening for detection of 22q11.2 deletions ≥500 kb in the LCR A-D region. Secondary outcomes included the prevalence of 22q11.2DS and performance of an updated screening algorithm that was assessed after enrollment completion.
Procedures
Sample preparation and analysis of cfDNA were performed as previously described (Natera Inc, San Carlos, CA). Results indicating a risk of ≥1 in 100 for 22q11.2DS were categorized as high risk and those indicating a risk of <1 in 100 were categorized as low risk. In cases with nonreportable results, patients were offered repeat testing and results obtained after a second blood sample collection were included; a third sample was not requested. During enrollment, the cfDNA laboratory protocol was modified once. , Results from both periods were combined for analysis. After enrollment completion, a third updated algorithm was developed by the laboratory, optimized to identify both the full and nested deletions using a deep neural network (DNN) component and reflex testing of high-risk calls with deeper sequencing. A deep learning (TensorFlow v1.15, Google Brain, Mountain View, CA) approach was used to optimally model noise using a deep mixture of experts neural network with multiple independent networks, combining the results into a probability score. The self-supervised algorithm leveraged 1.6 million sequenced mixtures of mother and fetus cfDNA samples, learning to harness the linkage among the SNPs to improve call confidence. This updated protocol was assessed with blinding to the outcomes.
For confirmatory CMA analysis, DNA was prepared from the neonates’ cord blood, buccal smear, or, predominantly, dried blood spot. Copy number variants, including aneuploidies and 22q11.2DS, were identified using the Illumina (San Diego, CA) SNP-based Infinium Global Screening Array (GSA) platform. Samples were genotyped in standard versions (GSA-V1.0, GSA-V2.0, GSAMD-V1.0, or GSAMD-V2.0) or in a custom-designed SMARTArray in which additional SNPs were added to the GSA backbone. Within the 22q11 region of interest (chr22:18,950,000-21,500,000; hg19), the GSA backbone contains 600 SNPs, whereas the custom SMARTArray has 1963 SNPs including those in the backbone. A positive 22q11.2DS was confirmed if a deletion ≥500 kb was identified within the LCR A–D interval. Positive samples underwent confirmation with the Omni 2.5-8V1-3 array and were reviewed by a clinical cytogeneticist before the results were generated.
Because neonatal DNA samples were obtained from different sources, mostly from dry blood spots that were collected by state health departments for routine neonatal screening, we developed a concordance test for quality assurance purposes. The concordance test was designed to confirm that the cfDNA results and newborn samples were correctly paired by using alignment between SNPs in the 2 samples; any samples that could not be paired were excluded.
Data collection
Onsite research coordinators recorded information using a secured computerized tracking system developed and managed by The Biostatistics Center at George Washington University, Washington DC. Data that were collected included patient and obstetrical data, imaging reports, aneuploidy serum screening, and prenatal diagnosis results. After delivery, information on pregnancy complications, genetic testing or ultrasound findings, newborn features suggestive of a genetic abnormality, major malformations, and other adverse outcomes was collected.
Study oversight
This study was a collaboration between the clinical investigators and the sponsor (Natera, Inc, San Carlos, CA). The first and last authors designed the protocol in collaboration with the sponsor and had a majority vote in study design and data interpretation. There were no confidentiality agreements among the authors, sites, or sponsor. All laboratory analyses were conducted with blinding to the outcome data. Clinical and laboratory results were managed by the data coordinating center, which independently matched the information and de-identified and analyzed the results.
Patient and public involvement
Patients and the public were not involved in the design of the study protocol, in establishing the research question, or in the outcome measures. No patients or members of the public were involved in the recruitment process or the conduct of the study. Finally, no patients or members of the public were or will be involved in the interpretation or dissemination of the study’s results.
Statistical analysis
Originally, a sample size of 10,000 participants was planned based on 22q11.2DS prevalence estimates that ranged from 1 in 300 to 1 in 2000. , , During the trial, concerns arose that the prevalence of the 22q11.2DS may be lower and prior to unblinding, the sample size was increased to 20,000, which allowed for a higher level of precision to assess performance. The sensitivity, specificity, PPV, and negative predictive value (NPV) of the cfDNA results were assessed and exact (Clopper-Pearson) 95% confidence intervals (CIs) were reported. Participants without cfDNA results or genetic confirmation were excluded from the test performance analysis. SAS Studio 9.04 software (SAS Institute, Cary, NC) was used for analysis. Continuous variables were compared using the Wilcoxon test and categorical variables were compared using chi-square or Fisher exact tests as appropriate. McNemar test was used for paired analyses.
Results
Study participants
From April 2015 through January 2019, we screened 25,892 women and enrolled 20,887 ( Figure 2 ). Overall, 54.8% were enrolled in the United States and 45.2% in Europe or Australia. Of the enrolled participants, 296 (1.4%) had a pregnancy loss without genetic confirmation, 1110 (5.3%) were lost to follow-up and therefore the pregnancy outcome is unknown, for 811 (3.9%), a confirmatory sample was not obtained, 94 (0.5%) withdrew consent, and for 287 (1.4%), the confirmation test failed laboratory quality control. The latter group included 49 cases that failed the concordance quality assurance test and for which the neonatal sample could not be genetically paired with a cfDNA sample. After exclusions, the study cohort included 18,289 (87.6%) participants who had both cfDNA and DNA confirmation results for 22q11.2DS.
The median maternal and gestational ages at enrollment were 34.5 years and 12.6 weeks, respectively ( Table 1 ). Overall, 108 (0.6%) underwent cfDNA screening after detection of a fetal anomaly on ultrasound, 95 (0.5%) after diagnosis of a cystic hygroma or nuchal translucency ≥3 mm, and 623 (3.4%) following a high-risk result on serum analyte screening for aneuploidy.
| Variable | Study cohort (n=18,289) |
|---|---|
| Maternal and gestational characteristics | |
| Maternal age (y), median (IQR) | 34.5 (30.4–37.5) |
| Nulliparity, n/total, n (%) | 8022/18,248 (44.0) |
| BMI (kg/m 2 ), median (IQR) b , c | 24.9 (22.3–29.0) |
| Race and ethnicity, n (%) d | |
| Asian | 1542 (8.4) |
| Black | 1554 (8.5) |
| White | 11,272 (61.6) |
| Hispanic | 3309 (18.1) |
| Other or unknown | 612 (3.3) |
| Gestational age at enrollment (wk), median (IQR) | 12.6 (11.6–13.9) |
| Pregnancy through assisted reproductive technology, n (%) | 959 (5.2) |
| Current smoker, n/total, n (%) | 321/18,211 (1.8) |
| Enrolled at a US site, n (%) | 10,005 (54.7) |
| Prenatal screening and testing | |
| Positive first trimester screen before enrollment, n (%) | 518 (2.8) |
| Nuchal translucency ≥3 mm or cystic hygroma before enrollment, n (%) | 95 (0.5) |
| Positive second trimester or integrated screen before enrollment, n (%) | 105 (0.6) |
| Major anomaly before testing, n (%) | 107 (0.6) |
| Fetal fraction (%), mean±SD c | 9.9±4.1 |
| Diagnostic testing, n (%) | 420 (2.3) |
| Pregnancy and delivery outcome | |
| Miscarriage, n/total, n (%) | 5/18,281 (0.03) |
| Pregnancy termination, n/total, n (%) | 41/18,281 (0.2) |
| Live birth, n/total, n (%) | 18,224/18,281 (99.7) |
| Stillbirth, n/total, n (%) | 11/18,281 (0.06) |
| Neonatal death, n/total, n (%) | 24/18,281 (0.1) |
| Aneuploidy (T13, 18, 21), n (%) | 36 (0.2) |
| Gestational age at delivery (wk), median (IQR) c | 39.4 (38.6–40.3) |
| PTB <37 weeks’ gestation, n/total, n (%) | 1311/18,230 (7.2) |
| Preeclampsia, n/total, n (%) | 735/18,230 (4.1) |
| Birthweight (g), mean (SD) c | 3361±555 |
| Birthweight <10% percentile, n/total, n (%) | 1578/18,042 (8.8) |
| Days to newborn discharge, median (IQR) c | 2 (2–3) |
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