Objective The purpose of this study was to explore the diagnostic yield and clinical utility of trio‐based rapid whole exome sequencing (rWES) in pregnancies of fetuses with a wide range of congenital anomalies detected by ultrasound imaging. Methods In this observational study, we analyzed the first 54 cases referred to our laboratory for prenatal rWES to support clinical decision making, after the sonographic detection of fetal congenital anomalies. The most common identified congenital anomalies were skeletal dysplasia (n = 20), multiple major fetal congenital anomalies (n = 17) and intracerebral structural anomalies (n = 7). Results A conclusive diagnosis was identified in 18 of the 54 cases (33%). Pathogenic variants were detected most often in fetuses with skeletal dysplasia (n = 11) followed by fetuses with multiple major fetal congenital anomalies (n = 4) and intracerebral structural anomalies (n = 3). A survey, completed by the physicians for 37 of 54 cases, indicated that the rWES results impacted clinical decision making in 68% of cases. Conclusions These results suggest that rWES improves prenatal diagnosis of fetuses with congenital anomalies, and has an important impact on prenatal and peripartum parental and clinical decision making.

Objective: Conventional genetic tests (quantitative fluorescent‐PCR [QF‐PCR] and single nucleotide polymorphism‐array) only diagnose ~40% of fetuses showing ultrasound abnormalities. Rapid exome sequencing (rES) may improve this diagnostic yield, but includes challenges such as uncertainties in fetal phenotyping, variant interpretation, incidental unsolicited findings, and rapid turnaround times. In this study, we implemented rES in prenatal care to increase diagnostic yield.Methods: We prospectively studied 55 fetuses. Inclusion criteria were: (a) two or more independent major fetal anomalies, (b) hydrops fetalis or bilateral renal cysts alone, or (c) one major fetal anomaly and a first‐degree relative with the same anomaly. In addition to conventional genetic tests, we performed trio rES analysis using a custom virtual gene panel of ~3850 Online Mendelian Inheritance in Man (OMIM) genes.Results: We established a genetic rES‐based diagnosis in 8 out of 23 fetuses (35%) without QF‐PCR or array abnormalities. Diagnoses included MIRAGE (SAMD9), Zellweger (PEX1), Walker‐Warburg (POMGNT1), Noonan (PTNP11), Kabuki (KMT2D), and CHARGE (CHD7) syndrome and two cases of Osteogenesis Imperfecta type 2 (COL1A1). In six cases, rES diagnosis aided perinatal management. The median turnaround time was 14 (range 8‐20) days.Conclusion: Implementing rES as a routine test in the prenatal setting is challenging but technically feasible, with a promising diagnostic yield and significant clinical relevance.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway — KYNU, HAAO, and NADSYN1 — have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain–specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1^+/– mothers, any B₃ vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1^–/– mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Background: Adding rapid exome sequencing (rES) to conventional genetic tests improves the diagnostic yield of pregnancies showing ultrasound abnormalities but also carries a higher chance of unsolicited findings. We evaluated how rES, including pre‐ and post‐test counseling, was experienced by parents investigating its impact on decision‐making and experienced levels of anxiety.Methods: A mixed‐methods approach was adopted. Participating couples (n = 46) were asked to fill in two surveys (pre‐test and post‐test counseling) and 11 couples were approached for an additional interview.Results: All couples accepted the rES test‐offer with the most important reason for testing emphasizing their hope of finding an underlying diagnosis that would aid decision‐making. The actual impact on decision‐making was low, however, since most parents decided to terminate the pregnancy based on the major and multiple fetal ultrasound anomalies and did not wait for their rES results. Anxiety was elevated for most participants and decreased over time.Conclusion: Major congenital anomalies detected on ultrasound seem to have more impact on prenatal parental decision‐making and anxiety then the offer and results of rES. However, the impact of rES on reproductive decision‐making and experienced anxiety requires further investigation, especially in pregnancies where less (severe) fetal anomalies are detected on ultrasound.

Background: In prenatal genetic diagnostics, the detection of single-gene defects relies on chorionic villus sampling (CVS) and amniocentesis, which carry a miscarriage risk of 0.2–0.3%. To mitigate this risk, fetal trophoblasts have been isolated from a Papanicolaou smear using Trophoblast Retrieval and Isolation from the Cervix (TRIC). However, this method is labor-intensive and has been shown to be challenging to implement in clinical practice. Here, we describe our experiences in using semi-automated immunomagnetic cell sorting for isolating trophoblasts from clinically obtained Papanicolaou smears during ongoing pregnancies. Methods: Using HLA-G-positive Jeg-3 and HLA-G-negative HeLa cell lines in 10%, 1%, and 0.1% dilutions, we tested and optimized the isolation of HLA-G-positive cells using FACS and semi-automated immunomagnetic cell sorting. We used the latter technique for isolation of HLA-G-positive cells from Papanicolaou smears collected from 26 pregnant women, gestational age between 6 and 20 weeks, who underwent CVS. Results: In four independent dilution series, the mean percentages of Jeg-3 cells went from 7.1% to 53.5%, 0.9% to 32.6%, and 0.4% to 2.6% (7.5, 36, and 6.5-fold enrichment, respectively) using immunomagnetic cell sorting. After sorting of the Papanicolaou smears, HLA-G-positive cells were moderately increased in the positive (14.61 vs. 11.63%) and decreased in the negative fraction (7.87 vs. 11.63%) compared to baseline pre-sorting. However, we could not identify fetal cells using XY-chromosomal FISH in a male sample. Conclusions: Our study supports previous findings that careful sampling of fetal cells from Papanicolaou smears in a clinical context poses significant challenges to cell retrieval.