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Summary Background Identification of chromosomal aneuploidies and copy number variants that are associated with fetal structural anomalies has substantial value. Although whole-exome sequencing (WES) has been applied to case series of a few selected prenatal cases, its value in routine clinical settings has not been prospectively assessed in a large unselected cohort of fetuses with structural anomalies. We therefore aimed to determine the incremental diagnostic yield (ie, the added value) of WES following uninformative results of standard investigations with karyotype testing and chromosomal microarray in an unselected cohort of sequential pregnancies showing fetal structural anomalies. Methods In this prospective cohort study, the parents of fetuses who were found to have a structural anomaly in a prenatal ultrasound were screened for possible participation in the study. These participants were predominantly identified in or were referred to the Columbia University Carmen and John Thain Center for Prenatal Pediatrics (New York, NY, USA). Fetuses with confirmed aneuploidy or a causal pathogenic copy number variant were excluded from WES analyses. By use of WES of the fetuses and parents (parent–fetus trios), we identified genetic variants that indicated an underlying cause (diagnostic genetic variants) and genetic variants that met the criteria of bioinformatic signatures that had previously been described to be significantly enriched among diagnostic genetic variants. Findings Between April 24, 2015, and April 19, 2017, 517 sequentially identified pregnant women found to have fetuses with a structural anomaly were screened for their eligibility for inclusion in our study. 71 (14%) couples declined testing, 87 (17%) trios were missing at least one DNA sample (from either parent or the fetus), 69 (13%) trios had a clinically relevant abnormal karyotype or chromosomal microarray finding, 51 (10%) couples did not consent to WES or withdrew consent, and five (1%) samples were not of good enough quality for analysis. DNA samples from 234 (45%) eligible trios were therefore used for analysis of the primary outcome. By use of trio sequence data, we identified diagnostic genetic variants in 24 (10%) families. Mutations with bioinformatic signatures that were indicative of pathogenicity but with insufficient evidence to be considered diagnostic were also evaluated; 46 (20%) of the 234 fetuses assessed were found to have such signatures. Interpretation Our analysis of WES data in a prospective cohort of unselected fetuses with structural anomalies shows the value added by WES following the use of routine genetic tests. Our findings suggest that, in cases of fetal anomalies in which assessment with karyotype testing and chromosomal microarray fail to determine the underlying cause of a structural anomaly, WES can add clinically relevant information that could assist current management of a pregnancy. The unique challenges of WES-based prenatal diagnostics require analysis by a multidisciplinary team of perinatal practitioners and laboratory specialists. Funding Institute for Genomic Medicine (Columbia University Irving Medical Center).

Objective To identify pregnancies at increased risk for trisomy 13, trisomy 18 or triploidy attributable to low fetal fraction (FF). Methods A FF‐based risk (FFBR) model was built using data from more than 165 000 singleton pregnancies referred for single‐nucleotide polymorphism (SNP)‐based non‐invasive prenatal testing (NIPT). Based on maternal weight and gestational age (GA), FF distributions for normal, trisomy 13, trisomy 18 and triploid pregnancies were constructed and used to adjust prior risks for these abnormalities. A risk cut‐off of ≥ 1% was chosen to define pregnancies at high risk for trisomy 13, trisomy 18 or triploidy (high FFBR score). The model was evaluated on an independent blinded set of pregnancies for which SNP‐based NIPT did not return a result, and for which pregnancy outcome information was gathered retrospectively. Results The evaluation cohort comprised 1148 cases, of which approximately half received a high FFBR score. Compared with rates expected based on maternal age (MA) and GA, cases with a high FFBR score had a significantly increased rate of trisomy 13, trisomy 18 or triploidy combined (5.7% vs 0.7%; P

Single-nucleotide polymorphism (SNP)-based non-invasive prenatal testing (NIPT) can currently predict a subset of submicroscopic abnormalities associated with severe clinical manifestations. We retrospectively analyzed the performance of SNP-based NIPT in 80 449 referrals for 22q11.2 deletion syndrome and 42 326 referrals for 1p36, cri-du-chat, Prader-Willi, and Angelman microdeletion syndromes over a 1-year period, and compared the original screening protocol with a revision that reflexively sequenced high-risk calls at a higher depth of read. The prevalence of these microdeletion syndromes was also estimated in the referral population. The positive predictive value of the original test was 15.7% for 22q11.2 deletion syndrome, and 5.2% for the other 4 disorders combined. With the revised protocol, these values increased to 44.2% for 22q11.2 and 31.7% for the others. The 0.33% false-positive rate (FPR) for 22q11.2 deletion syndrome decreased to 0.07% with the revised protocol. Similarly, the FPR for the other 4 disorders combined decreased from 0.56% to 0.07%. Minimal prevalences were estimated to be 1 in 1255 for 22q11.2 deletion syndrome and 1 in 1464 for 1p36, cri-du-chat, and Angelman syndromes combined. Our results show that these microdeletions are relatively common in the referral population, and that the performance of SNP-based NIPT is improved with high-depth resequencing.

In the nearly 60 years since prenatal diagnosis for genetic disease was first offered, the field of prenatal diagnosis has progressed far past rudimentary uterine puncture to provide fetal material to assess gender and interpret risk. Concurrent with the improvements in invasive fetal sampling came technological advances in cytogenetics and molecular biology that widened both the scope of genetic disorders that could be diagnosed and also the resolution at which the human genome could be interrogated. Nowadays, routine blood work available to all pregnant women can determine the risk for common chromosome abnormalities; chorionic villus sampling (CVS) and amniocentesis can be used to diagnose nearly all conditions with a known genetic cause; and the genome and/or exome of a fetus with multiple anomalies can be sequenced in an attempt to determine the underlying etiology. This chapter will discuss some of the major advances in prenatal sampling and prenatal diagnostic laboratory techniques that have occurred over the past six decades.

Objectives To evaluate the performance of a single-nucleotide polymorphism (SNP)-based non-invasive prenatal test (NIPT) for the detection of fetal 22q11.2 deletion syndrome in clinical practice, assess clinical follow-up and review patient choices for women with high-risk results. Methods In this study, 21 948 samples were submitted for screening for 22q11.2 deletion syndrome using a SNP-based NIPT and subsequently evaluated. Follow-up was conducted for all cases with a high-risk result. Results Ninety-five cases were reported as high risk for fetal 22q11.2 deletion. Diagnostic testing results were available for 61 (64.2%) cases, which confirmed 11 (18.0%) true positives and identified 50 (82.0%) false positives, resulting in a positive predictive value (PPV) of 18.0%. Information regarding invasive testing was available for 84 (88.4%) high-risk cases: 57.1% (48/84) had invasive testing and 42.9% (36/84) did not. Ultrasound anomalies were present in 81.8% of true-positive and 18.0% of false-positive cases. Two additional cases were high risk for a maternal 22q11.2 deletion; one was confirmed by diagnostic testing and one had a positive family history. There were three pregnancy terminations related to screening results of 22q11.2 deletion, two of which were confirmed as true positive by invasive testing. Conclusions Clinical experience with this SNP-based non-invasive screening test for 22q11.2 deletion syndrome indicates that these deletions have a frequency of approximately 1 in 1000 in the referral population with most identifiable through this test. Use of this screening method requires the availability of counseling and other management resources for high-risk pregnancies. © 2015 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd. on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Objective To estimate performance of a single-nucleotide-polymorphism–based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations upon single venopuncture.

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.

ObjectiveWe sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism–based noninvasive prenatal aneuploidy test in high- and low-risk women.Study DesignAll samples received from March through September 2013 and drawn ≥9 weeks’ gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth.ResultsSamples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged

Objective We sought to determine the ability of single-nucleotide polymorphism–based noninvasive prenatal testing (NIPT) to identify triploid, unrecognized twin, and vanishing twin pregnancies. Study Design The study included 30,795 consecutive reported clinical cases received for NIPT for fetal whole-chromosome aneuploidies; known multiple gestations were excluded. Cell-free DNA was isolated from maternal blood samples, amplified via 19,488-plex polymerase chain reaction, and sequenced. Sequencing results were analyzed to determine fetal chromosome copy number and to identify the presence of additional fetal haplotypes. Results Additional fetal haplotypes, indicative of fetal triploidy, vanishing twin, or undetected twin pregnancy, were identified in 130 (0.42%) cases. Clinical confirmation (karyotype for singleton pregnancies, ultrasound for multifetal pregnancies) was available for 58.5% (76/130) of cases. Of the 76 cases with confirmation, 42.1% were vanishing twin, 48.7% were viable twin, 5.3% were diandric triploids, and 3.9% were nontriploid pregnancies that lacked evidence of co-twin demise. One pregnancy had other indications suggesting triploidy but lacked karyotype confirmation. Of the 5 vanishing twin cases with a known date of demise, 100% of losses occurred in the first trimester; up to 8 weeks elapsed between loss and detection by NIPT. Conclusion This single-nucleotide polymorphism–based NIPT successfully identified vanished twin, previously unrecognized twin, and triploid pregnancies. As vanishing twins are more likely to be aneuploid, and undetected residual cell-free DNA could bias NIPT results, the ability of this method to identify additional fetal haplotypes is expected to result in fewer false-positive calls and prevent incorrect fetal sex calls.