Future of Dutch NGS-Based Newborn Screening: Exploring the Technical Possibilities and Assessment of a Variant Classification Strategy.

In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation.

Genome sequencing as a generic diagnostic strategy for rare disease.

BACKGROUND: To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. METHODS: We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels < 50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥ 98% was considered the threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected. RESULTS: Overall, 95% (1206/1271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79-100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected. CONCLUSIONS: GS can capture clinically relevant germline variants in a 'GS-first strategy' for the majority of clinical indications in a genetics diagnostic lab.

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing.

BACKGROUND: Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. RESULTS: We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. CONCLUSION: We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques.

Rapid exome sequencing as a first-tier test in neonates with suspected genetic disorder: results of a prospective multicenter clinical utility study in the Netherlands.

The introduction of rapid exome sequencing (rES) for critically ill neonates admitted to the neonatal intensive care unit has made it possible to impact clinical decision-making. Unbiased prospective studies to quantify the impact of rES over routine genetic testing are, however, scarce. We performed a clinical utility study to compare rES to conventional genetic diagnostic workup for critically ill neonates with suspected genetic disorders. In a multicenter prospective parallel cohort study involving five Dutch NICUs, we performed rES in parallel to routine genetic testing for 60 neonates with a suspected genetic disorder and monitored diagnostic yield and the time to diagnosis. To assess the economic impact of rES, healthcare resource use was collected for all neonates. rES detected more conclusive genetic diagnoses than routine genetic testing (20% vs. 10%, respectively), in a significantly shorter time to diagnosis (15 days (95% CI 10-20) vs. 59 days (95% CI 23-98, p < 0.001)). Moreover, rES reduced genetic diagnostic costs by 1.5% (€85 per neonate). CONCLUSION:  Our findings demonstrate the clinical utility of rES for critically ill neonates based on increased diagnostic yield, shorter time to diagnosis, and net healthcare savings. Our observations warrant the widespread implementation of rES as first-tier genetic test in critically ill neonates with disorders of suspected genetic origin. WHAT IS KNOWN: • Rapid exome sequencing (rES) enables diagnosing rare genetic disorders in a fast and reliable manner, but retrospective studies with neonates admitted to the neonatal intensive care unit (NICU) indicated that genetic disorders are likely underdiagnosed as rES is not routinely used. • Scenario modeling for implementation of rES for neonates with presumed genetic disorders indicated an expected increase in costs associated with genetic testing. WHAT IS NEW: • This unique prospective national clinical utility study of rES in a NICU setting shows that rES obtained more and faster diagnoses than conventional genetic tests. • Implementation of rES as replacement for all other genetic tests does not increase healthcare costs but in fact leads to a reduction in healthcare costs.

Newborn screening for Cerebrotendinous Xanthomatosis: A retrospective biomarker study using both flow-injection and UPLC-MS/MS analysis in 20,000 newborns.

BACKGROUND AND AIMS: Cerebrotendinous Xanthomatosis (CTX) is a treatable disorder of bile acid synthesis caused by deficiency of 27-sterol hydroxylase -encoded by CYP27A1- leading to gastrointestinal and progressive neuropsychiatric symptoms. Biochemically, CTX is characterized by accumulation of the bile alcohol cholestanetetrol glucuronide (GlcA-tetrol) and the deficiency of tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA). MATERIALS AND METHODS: To ascertain the feasibility of CTX newborn screening (NBS) we performed a study with deidentified Dutch dried blood spots using reagents and equipment that is frequently used in NBS laboratories. 20,076 deidentified newborn blood spots were subjected to flow-injection (FIA)-MS/MS and UPLC-MS/MS analysis to determine the concentration of GlcA-tetrol and calculate the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios. RESULTS: Using UPLC-MS/MS analysis both GlcA-tetrol concentration and/or metabolite ratios GlcA-tetrol/t-CDCA proved to be informative biomarkers; newborn DBS results did not overlap with those of the CTX patients. For FIA-MS/MS, GlcA-tetrol also was an excellent marker but when using the combination of the GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios also did not yield any screen positives. CONCLUSION: Newborn screening for CTX using only metabolite ratios following the measurement of three CTX biomarkers is possible using either FIA-MS/MS or UPLC-MS/MS, which paves the way for introduction of CTX NBS.

Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease-associated genes.

PURPOSE: Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. METHODS: Optical genome mapping was performed to improve SV detection in short-read genome sequencing-negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. RESULTS: In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. CONCLUSION: Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.

The performance of genome sequencing as a first-tier test for neurodevelopmental disorders.

Genome sequencing (GS) can identify novel diagnoses for patients who remain undiagnosed after routine diagnostic procedures. We tested whether GS is a better first-tier genetic diagnostic test than current standard of care (SOC) by assessing the technical and clinical validity of GS for patients with neurodevelopmental disorders (NDD). We performed both GS and exome sequencing in 150 consecutive NDD patient-parent trios. The primary outcome was diagnostic yield, calculated from disease-causing variants affecting exonic sequence of known NDD genes. GS (30%, n = 45) and SOC (28.7%, n = 43) had similar diagnostic yield. All 43 conclusive diagnoses obtained with SOC testing were also identified by GS. SOC, however, required integration of multiple test results to obtain these diagnoses. GS yielded two more conclusive diagnoses, and four more possible diagnoses than ES-based SOC (35 vs. 31). Interestingly, these six variants detected only by GS were copy number variants (CNVs). Our data demonstrate the technical and clinical validity of GS to serve as routine first-tier genetic test for patients with NDD. Although the additional diagnostic yield from GS is limited, GS comprehensively identified all variants in a single experiment, suggesting that GS constitutes a more efficient genetic diagnostic workflow.

Lessons learned from rapid exome sequencing for 575 critically ill patients across the broad spectrum of rare disease.

Introduction: Rapid exome sequencing (rES) has become the first-choice genetic test for critically ill patients, mostly neonates, young infants, or fetuses in prenatal care, in time-sensitive situations and when it is expected that the genetic test result may guide clinical decision making. The implementation of rES has revolutionized medicine by enabling timely identification of genetic causes for various rare diseases. The utilization of rES has increasingly been recognized as an essential diagnostic tool for the identification of complex and undiagnosed genetic disorders. Methods: We conducted a retrospective evaluation of our experiences with rES performed on 575 critically ill patients from various age groups (prenatal to adulthood), over a four-year period (2016-2019). These patients presented with a wide spectrum of rare diseases, including but not limited to neurological disorders, severe combined immune deficiency, and cancer. Results: During the study period, there was a significant increase in rES referrals, with a rise from a total of two referrals in Q1-2016 to 10 referrals per week in Q4-2019. The median turnaround time (TAT) decreased from 17 to 11 days in the period 2016-2019, with an overall median TAT of 11 days (IQR 8-15 days). The overall diagnostic yield for this cohort was 30.4%, and did not significantly differ between the different age groups (e.g. adults 22.2% vs children 31.0%; p-value 0.35). However, variability in yield was observed between clinical entities: craniofacial anomalies yielded 58.3%, while for three clinical entities (severe combined immune deficiency, aneurysm, and hypogonadotropic hypogonadism) no diagnoses were obtained. Discussion: Importantly, whereas clinical significance is often only attributed to a conclusive diagnosis, we also observed impact on clinical decision-making for individuals in whom no genetic diagnosis was established. Hence, our experience shows that rES has an important role for patients of all ages and across the broad spectrum of rare diseases to impact clinical outcomes.

Diagnostic analysis of the highly complex OPN1LW/OPN1MW gene cluster using long-read sequencing and MLPA.

Pathogenic variants in the OPN1LW/OPN1MW gene cluster are causal for a range of mild to severe visual impairments with color deficiencies. The widely utilized short-read next-generation sequencing (NGS) is inappropriate for the analysis of the OPN1LW/OPN1MW gene cluster and many patients with pathogenic variants stay underdiagnosed. A diagnostic genetic assay was developed for the OPN1LW/OPN1MW gene cluster, consisting of copy number analysis via multiplex ligation-dependent probe amplification and sequence analysis via long-read circular consensus sequencing. Performance was determined on 50 clinical samples referred for genetic confirmation of the clinical diagnosis (n = 43) or carrier status analysis (n = 7). A broad range of pathogenic haplotypes were detected, including deletions, hybrid genes, single variants and combinations of variants. The developed genetic assay for the OPN1LW/OPN1MW gene cluster is a diagnostic test that can detect both structural and nucleotide variants with a straightforward analysis, improving diagnostic care of patients with visual impairment.

Reanalysis of exome negative patients with rare disease: a pragmatic workflow for diagnostic applications.

BACKGROUND: Approximately two third of patients with a rare genetic disease remain undiagnosed after exome sequencing (ES). As part of our post-test counseling procedures, patients without a conclusive diagnosis are advised to recontact their referring clinician to discuss new diagnostic opportunities in due time. We performed a systematic study of genetically undiagnosed patients 5 years after their initial negative ES report to determine the efficiency of diverse reanalysis strategies. METHODS: We revisited a cohort of 150 pediatric neurology patients originally enrolled at Radboud University Medical Center, of whom 103 initially remained genetically undiagnosed. We monitored uptake of physician-initiated routine clinical and/or genetic re-evaluation (ad hoc re-evaluation) and performed systematic reanalysis, including ES-based resequencing, of all genetically undiagnosed patients (systematic re-evaluation). RESULTS: Ad hoc re-evaluation was initiated for 45 of 103 patients and yielded 18 diagnoses (including 1 non-genetic). Subsequent systematic re-evaluation identified another 14 diagnoses, increasing the diagnostic yield in our cohort from 31% (47/150) to 53% (79/150). New genetic diagnoses were established by reclassification of previously identified variants (10%, 3/31), reanalysis with enhanced bioinformatic pipelines (19%, 6/31), improved coverage after resequencing (29%, 9/31), and new disease-gene associations (42%, 13/31). Crucially, our systematic study also showed that 11 of the 14 further conclusive genetic diagnoses were made in patients without a genetic diagnosis that did not recontact their referring clinician. CONCLUSIONS: We find that upon re-evaluation of undiagnosed patients, both reanalysis of existing ES data as well as resequencing strategies are needed to identify additional genetic diagnoses. Importantly, not all patients are routinely re-evaluated in clinical care, prolonging their diagnostic trajectory, unless systematic reanalysis is facilitated. We have translated our observations into considerations for systematic and ad hoc reanalysis in routine genetic care.

Towards Next-Generation Sequencing (NGS)-Based Newborn Screening: A Technical Study to Prepare for the Challenges Ahead.

Newborn screening (NBS) aims to identify neonates with severe conditions for whom immediate treatment is required. Currently, a biochemistry-first approach is used to identify these disorders, which are predominantly inherited meta1bolic disorders (IMD). Next-generation sequencing (NGS) is expected to have some advantages over the current approach, for example the ability to detect IMDs that meet all screening criteria but lack an identifiable biochemical footprint. We have now designed a technical study to explore the use of NGS techniques as a first-tier approach in NBS. Here, we describe the aim and set-up of the NGS-first for the NBS (NGSf4NBS) project, which will proceed in three steps. In Step 1, we will identify IMDs eligible for NGS-first testing, based on treatability. In Step 2, we will investigate the feasibility, limitations and comparability of different technical NGS approaches and analysis workflows for NBS, eventually aiming to develop a rapid NGS-based workflow. Finally, in Step 3, we will prepare for the incorporation of this workflow into the existing Dutch NBS program and propose a protocol for referral of a child after a positive NGS test result. The results of this study will be the basis for an additional analytical route within NBS that will be further studied for its applicability within the NBS program, e.g., regarding the ethical, legal, financial and social implications.

Clinical exome sequencing-Mistakes and caveats.

Massive parallel sequencing technology has become the predominant technique for genetic diagnostics and research. Many genetic laboratories have wrestled with the challenges of setting up genetic testing workflows based on a completely new technology. The learning curve we went through as a laboratory was accompanied by growing pains while we gained new knowledge and expertise. Here we discuss some important mistakes that have been made in our laboratory through 10 years of clinical exome sequencing but that have given us important new insights on how to adapt our working methods. We provide these examples and the lessons that we learned to help other laboratories avoid to make the same mistakes.

A multi-platform reference for somatic structural variation detection.

Accurate detection of somatic structural variation (SV) in cancer genomes remains a challenging problem. This is in part due to the lack of high-quality, gold-standard datasets that enable the benchmarking of experimental approaches and bioinformatic analysis pipelines. Here, we performed somatic SV analysis of the paired melanoma and normal lymphoblastoid COLO829 cell lines using four different sequencing technologies. Based on the evidence from multiple technologies combined with extensive experimental validation, we compiled a comprehensive set of carefully curated and validated somatic SVs, comprising all SV types. We demonstrate the utility of this resource by determining the SV detection performance as a function of tumor purity and sequence depth, highlighting the importance of assessing these parameters in cancer genomics projects. The truth somatic SV dataset as well as the underlying raw multi-platform sequencing data are freely available and are an important resource for community somatic benchmarking efforts.

Optical genome mapping identifies a germline retrotransposon insertion in SMARCB1 in two siblings with atypical teratoid rhabdoid tumors.

In a subset of pediatric cancers, a germline cancer predisposition is highly suspected based on clinical and pathological findings, but genetic evidence is lacking, which hampers genetic counseling and predictive testing in the families involved. We describe a family with two siblings born from healthy parents who were both neonatally diagnosed with atypical teratoid rhabdoid tumor (ATRT). This rare and aggressive pediatric tumor is associated with biallelic inactivation of SMARCB1, and in 30% of the cases, a predisposing germline mutation is involved. Whereas the tumors of both siblings showed loss of expression of SMARCB1 and acquired homozygosity of the locus, whole exome and whole genome sequencing failed to identify germline or somatic SMARCB1 pathogenic mutations. We therefore hypothesized that the insertion of a pathogenic repeat-rich structure might hamper its detection, and we performed optical genome mapping (OGM) as an alternative strategy to identify structural variation in this locus. Using this approach, an insertion of ~2.8 kb within intron 2 of SMARCB1 was detected. Long-range PCR covering this region remained unsuccessful, but PacBio HiFi genome sequencing identified this insertion to be a SINE-VNTR-Alu, subfamily E (SVA-E) retrotransposon element, which was present in a mosaic state in the mother. This SVA-E insertion disrupts correct splicing of the gene, resulting in loss of a functional allele. This case demonstrates the power of OGM and long-read sequencing to identify genomic variations in high-risk cancer-predisposing genes that are refractory to detection with standard techniques, thereby completing the clinical and molecular diagnosis of such complex cases and greatly improving counseling and surveillance of the families involved. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Long-read technologies identify a hidden inverted duplication in a family with choroideremia.

The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read techniques have complementary strengths to overcome these limitations, with a particular strength in identifying structural variants. By using optical genome mapping and long-read sequencing, we aimed to identify the pathogenic variant in a large family with X-linked choroideremia. In this family, aberrant splicing of exon 12 of the choroideremia gene CHM was detected in 2003, but the underlying genomic defect remained elusive. Optical genome mapping and long-read sequencing approaches now revealed an intragenic 1,752 bp inverted duplication including exon 12 and surrounding regions, located downstream of the wild-type copy of exon 12. Both breakpoint junctions were confirmed with Sanger sequencing and segregate with the X-linked inheritance in the family. The breakpoint junctions displayed sequence microhomology suggestive for an erroneous replication mechanism as the origin of the structural variant. The inverted duplication is predicted to result in a hairpin formation of the pre-mRNA with the wild-type exon 12, leading to exon skipping in the mature mRNA. The identified inverted duplication is deemed the hidden pathogenic cause of disease in this family. Our study shows that optical genome mapping and long-read sequencing have significant potential for the identification of (hidden) structural variants in rare genetic diseases.

Long-read trio sequencing of individuals with unsolved intellectual disability.

Long-read sequencing (LRS) has the potential to comprehensively identify all medically relevant genome variation, including variation commonly missed by short-read sequencing (SRS) approaches. To determine this potential, we performed LRS around 15×-40× genome coverage using the Pacific Biosciences Sequel I System for five trios. The respective probands were diagnosed with intellectual disability (ID) whose etiology remained unresolved after SRS exomes and genomes. Systematic assessment of LRS coverage showed that ~35 Mb of the human reference genome was only accessible by LRS and not SRS. Genome-wide structural variant (SV) calling yielded on average 28,292 SV calls per individual, totaling 12.9 Mb of sequence. Trio-based analyses which allowed to study segregation, showed concordance for up to 95% of these SV calls across the genome, and 80% of the LRS SV calls were not identified by SRS. De novo mutation analysis did not identify any de novo SVs, confirming that these are rare events. Because of high sequence coverage, we were also able to call single nucleotide substitutions. On average, we identified 3 million substitutions per genome, with a Mendelian inheritance concordance of up to 97%. Of these, ~100,000 were located in the ~35 Mb of the genome that was only captured by LRS. Moreover, these variants affected the coding sequence of 64 genes, including 32 known Mendelian disease genes. Our data show the potential added value of LRS compared to SRS for identifying medically relevant genome variation.

Presence of Genetic Variants Among Young Men With Severe COVID-19.

Importance: Severe coronavirus disease 2019 (COVID-19) can occur in younger, predominantly male, patients without preexisting medical conditions. Some individuals may have primary immunodeficiencies that predispose to severe infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objective: To explore the presence of genetic variants associated with primary immunodeficiencies among young patients with COVID-19. Design, Setting, and Participants: Case series of pairs of brothers without medical history meeting the selection criteria of young (age <35 years) brother pairs admitted to the intensive care unit (ICU) due to severe COVID-19. Four men from 2 unrelated families were admitted to the ICUs of 4 hospitals in the Netherlands between March 23 and April 12, 2020. The final date of follow-up was May 16, 2020. Available family members were included for genetic variant segregation analysis and as controls for functional experiments. Exposure: Severe COVID-19. Main Outcome and Measures: Results of rapid clinical whole-exome sequencing, performed to identify a potential monogenic cause. Subsequently, basic genetic and immunological tests were performed in primary immune cells isolated from the patients and family members to characterize any immune defects. Results: The 4 male patients had a mean age of 26 years (range, 21-32), with no history of major chronic disease. They were previously well before developing respiratory insufficiency due to severe COVID-19, requiring mechanical ventilation in the ICU. The mean duration of ventilatory support was 10 days (range, 9-11); the mean duration of ICU stay was 13 days (range, 10-16). One patient died. Rapid clinical whole-exome sequencing of the patients and segregation in available family members identified loss-of-function variants of the X-chromosomal TLR7. In members of family 1, a maternally inherited 4-nucleotide deletion was identified (c.2129_2132del; p.[Gln710Argfs*18]); the affected members of family 2 carried a missense variant (c.2383G>T; p.[Val795Phe]). In primary peripheral blood mononuclear cells from the patients, downstream type I interferon (IFN) signaling was transcriptionally downregulated, as measured by significantly decreased mRNA expression of IRF7, IFNB1, and ISG15 on stimulation with the TLR7 agonist imiquimod as compared with family members and controls. The production of IFN-γ, a type II IFN, was decreased in patients in response to stimulation with imiquimod. Conclusions and Relevance: In this case series of 4 young male patients with severe COVID-19, rare putative loss-of-function variants of X-chromosomal TLR7 were identified that were associated with impaired type I and II IFN responses. These preliminary findings provide insights into the pathogenesis of COVID-19.

Rapid whole exome sequencing in pregnancies to identify the underlying genetic cause in fetuses with congenital anomalies detected by ultrasound imaging.

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.