Introduction

Non-invasive prenatal testing (NIPT) using cell-free DNA from maternal plasma has altered the landscape of prenatal screening, owing to its improved performance over traditional screening techniques [1, 2]. Clinical adoption of prenatal screening for commonly observed aneuploidies has been rapid since its introduction in 2011 [2]. Recently, NIPT coverage has expanded to include a range of sub-chromosomal abnormalities with high penetrance and severe phenotypes, including the 22q11.2 deletion syndrome (22q11.2 DS also known as DiGeorge or velocardiofacial syndrome) [3–5].

With a reported population-wide frequency of 1 in 3,000–6,000 live births, 22q11.2 DS is the most common microdeletion syndrome [6, 7]. Several reports suggest a higher prenatal prevalence, with an estimated frequency of approximately 1 in 1000 [4, 8, 9]. 22q11.2 DS is characterized by a spectrum of clinical manifestations with varying degrees of severity, including congenital heart defects, immune dysfunction, hypocalcemia, mild-to-severe learning disabilities, and an increased risk of mental health disorders in adulthood [10]. Due to the variability in presentation, diagnosis based on clinical findings is challenging, and 22q11.2 DS remains undiagnosed in approximately one third of affected infants. For those cases diagnosed prenatally, appropriate prenatal counselling addressing the variable phenotype of the condition offers expectant couples the opportunity to make informed reproductive decisions and plan for prenatal and neonatal patient management. Patients who do not receive a perinatal diagnosis often undergo a lengthy process of evaluation by multiple specialist providers before a definitive diagnosis by molecular cytogenetics is reached [10].

For individuals with 22q11.2 DS pregnancy, changes in management such as delivery at specialized neonatal care centers may be advised and early therapeutic intervention can positively affect clinical outcomes [11]. For example, the majority (~87%) of deaths associated with the syndrome are attributed to congenital heart defects, [10] but appropriate care at birth can reduce related mortality by ~12% [12]. Likewise, hypocalcemia-induced seizures can be optimally managed by close monitoring of calcium levels and providing calcium supplementation as needed [13, 14]. Currently there is a paucity of information about the phenotype for 22q11.2 deletion cases that are ascertained through NIPT and how detection translates into improved long-term outcomes.

Previously, validation of a targeted single–nucleotide-polymorphism (SNP)-based test targeting the 22q11.2 deletion (~3 Mb A–D region) demonstrated an analytical detection rate of 97.8% and a false positive rate of 0.76% [8]. In that study, only three affected pregnancy plasma samples were analyzed along with 43 artificially generated mixture samples. The paucity of pregnancy plasma samples was considered a key limitation of the study [15]. Herein, we present results from an analytical validation of a revised version of the 22q11.2 deletion assay in a cohort consisting entirely of pregnancy plasma samples.

Methods

Results

To validate the performance of the updated SNP-based technology in detecting the 22q11.2 deletion, 419 pregnancy plasma samples with known fetal copy number for the 22q11.2 region were evaluated. The study included 10 samples with a confirmed affected fetus (Table 1) and 409 samples with an unaffected fetus for the 22q11.2 deletion, as determined by chromosomal microarray (5 affected samples), fluorescence in situ hybridization (FISH) (4 affected samples), or massively multiplexed PCR (mmPCR)/NGS analysis (1 affected, 409 unaffected samples) [8] on fetal genetic material obtained from an invasive procedure. For all samples, the mother was determined to be disomic based on analysis of the maternal circulating free DNA (cfDNA).

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Table 1. Clinical information for affected samples.

https://doi.org/10.1371/journal.pone.0193476.t001

Among the 10 affected samples, the deletion was confirmed by array or mmPCR to occur in the common A–D region in six samples and deletion sizes ranged from 2.55–3.16 Mb. The remaining four affected samples were confirmed by FISH and were conservatively treated as having the full ~3 Mb A–D deletion (Table 1). In 8 of the 10 affected samples, the blood draw for NIPT was performed ≥2 days following the invasive procedure; in the other 2 cases, blood was drawn prior to the invasive procedure (Table 1). Of the 409 unaffected pregnancy plasma samples, 19 (4.6%) received a no-call due to low fetal fraction (fetal fraction <2.8%; n = 10), or inability of the algorithm to make a high confidence call (n = 9).

The mean maternal age for the affected samples was 28.1 weeks compared to 26.0 weeks for unaffected samples (p-value of 0.351). The gestational age range was higher (mean 21.7 weeks, range 13.4–37.6) for the affected samples compared to the unaffected samples (mean 12.8 weeks, range 9.0–27.0) with a p-value of 0.005. The affected samples also had a higher fetal fraction distribution (mean 16.6%, 7.9–39.7%) compared to the unaffected samples (mean 9.4%, 2.8–31.0%) with a p-value of 0.04 (Table 2).

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Table 2. Patient characteristics of study samples.

https://doi.org/10.1371/journal.pone.0193476.t002

Of the 10 affected pregnancy samples, 9 were identified as high-risk (test positive) and 1 was identified as low-risk (test negative), generating a sensitivity of 90% (9/10; 95% confidence interval, 55.5–99.75%) (Table 3). All high-risk calls were further evaluated for both paternal and maternal haplotypes to identify the parental source of the 22q11.2 deletion. Of the 9 samples that received a high-risk call, 6 were found to be deleted on the maternally inherited chromosome and 3 on the paternally inherited chromosome (Table 1). Of the 390 unaffected samples, one false positive case was reported (gestational age 15 weeks; fetal fraction 8.5%; no evidence of chromosomal anomalies in the CVS sample), generating an observed specificity of 99.74% (389/390; 95% confidence interval, 98.58%–99.99%) (Table 3) with a corresponding false positive rate of 0.26% (95% confidence interval, 0.01–1.42%). Low-risk calls for 99 unaffected cases with fetal fraction ranging from 2.8–6.5% were based on the analysis of only the paternal allele. Based on a prevalence of 1/1442 in the clinical pregnancy population for the A–D 22q11.2 deletion [16], the estimated PPV is 19.6% (Table 3).

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Table 3. Sensitivity, specificity, and estimated positive predictive value for 22q11.2 A–D deletions.

https://doi.org/10.1371/journal.pone.0193476.t003

Discussion

Previously, we reported that the SNP-based NIPT detects the fetal 22q11.2 deletion (~3 Mb A–D deletion) with a sensitivity of 97.8% and a false positive rate of 0.76% [8]. To reduce the false positive rate and lower the barrier to clinical adoption, the assay was revised in three ways. First, the number of targeted SNPs in the 22q11.2 region was increased from 672 to 1,351. Second, the algorithm’s positive-call confidence threshold was raised from 0.90 to 0.95 [16]. Finally, reflex sequencing of “high-risk” calls with maternally-deleted haplotypes at higher depth of read was implemented [4]. Data from this validation of the revised test on 400 pregnancy plasma samples (which included 10 affected samples) demonstrated a sensitivity of 90% and a false positive rate of 0.26%, in line with expected assay performance given the methodological improvements and previous reports [8, 16]. Of note, this SNP-based test is designed to detect the large A–D 22q11.2 deletion that is associated with 22q11.2 DS[8, 16]. Performance of the assay has not yet been assessed for smaller deletions within the A-D region. Thus, a conservative estimate of the detection rate under the assumption that none of these smaller deletions are identifiable would be approximately 78.3% [the proportion of all 22q11.2 DS that consist of full A–D deletions (87%) x the measured sensitivity of the assay for the full A–D deletions (90%)]. Based on a prenatal test referral prevalence of 1/1442 for full A–D deletions (or 1/1225 for all significant deletions in A–D deletions) the PPV was 19.6%. Alternately, using a population-wide prevalence number of 1/4,000 [6, 7], the estimated PPV of the assay would be 8.1%. The PPV of this assay was directly measured in a clinical population previously and was found to be 44.2% [16].

The false positive rate with the revised methodology was 0.26% compared with 0.76% in the previous validation study [8]. However, precise measurement of a false positive rate close to zero requires a very large study cohort. A clinical experience study reporting results in more than 70,000 samples tested for fetal 22q11.2 deletion yielded an estimated false positive rate of 0.07% when using the same revised protocol [16]. Although clinical follow-up was not available for all women in that study, an estimate of the false positive rate was made using the conservative assumption that all “high-risk” cases lost to follow-up were false positives, yielding an upper-bound false positive rate estimate of 0.09% and a corresponding specificity of 99.91%. Caution should be exercised, however, when interpreting false positive rates—false positive cases may harbor smaller deletions in 22q11.2 that could remain undetected by FISH, which is often used as the confirmatory test for subchromosomal imbalances. Confined placental mosaicism might also contribute to the false positive rate [18].

A limitation of this study is the higher gestational age distribution of the affected pregnancy cases compared with those typically observed in a clinical cohort. The higher gestational age is attributed to the delay in identifying and procuring samples from affected pregnancies. As gestational age and fetal fraction are positively correlated, higher fetal fractions among the affected pregnancies would be expected to affect the assay’s performance. Note that in our previous validation study, which used mixture samples, the fetal fraction distribution of the affected samples was specifically designed to approximate that of a clinical cohort [8], and in that study, the sensitivity was comparable to the sensitivity observed in this study. Another factor that may affect fetal fraction is the time between the invasive procedure (CVS or amniocentesis) and the blood draw for NIPT. Although it has been suggested that fetal fraction may rise immediately following an invasive procedure [19], another study reported no evidence of an increase in fetal fraction 24 hours following a CVS procedure [20]. All blood samples in this study were either drawn prior to, or >24 hours after invasive procedures.

The relative rarity of the 22q11.2 deletion makes it challenging to assemble a patient cohort closely resembling a clinical population for use in a validation study. Historically, validation studies for rare conditions required alternative approaches—e.g., testing maternal plasma diluted with DNA from an affected individual [8, 21]. As NIPT coverage expands to include rarer conditions, indication-specific analytic validations using affected pregnancy plasma samples, as obtained for this study, will prove increasingly difficult to do, and alternative validation approaches will become necessary. It is therefore notable that in this study, consisting entirely of pregnancy plasma samples, the demonstrated assay performance was comparable to that reported in a previous study that used a substantial number of simulated pregnancy samples [8]. Another recent study reported the performance of a microarray-based cfDNA test for the detection of the 22q11.2 deletion that included approximately 120 affected lab-generated pregnancy plasma samples [22], further supporting the use of laboratory-generated samples as substitutes for clinical samples.

Subchromosomal abnormalities occur in >1% of pregnancies and are often associated with severe phenotypes [23]. NIPT has recently expanded its coverage of testing to include a subset of clinically significant microdeletions. The 22q11.2 DS is a well-defined, severe condition, and improved clinical outcomes have been demonstrated with prenatal diagnosis [10]. The data presented in this study add to the growing body of evidence demonstrating the ability of the SNP-based NIPT to detect 22q11.2 deletions with high sensitivity and specificity [4, 8].

Acknowledgments

The authors thank Dr. Peter Benn for critical input on the manuscript, Marlene Shapira for excellent clinical support, Akshita Kalyan and Varsha Vijayan for their assistance with data analysis, and Dr. Stephanie Kareht for editorial support.