Real-World Application of Trio-Based Exome Sequencing in Prenatal Genetic Diagnosis

Real-World Application of Trio-Based Exome Sequencing in Prenatal Genetic Diagnosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Real-World Application of Trio-Based Exome Sequencing in Prenatal Genetic Diagnosis Juan Antonio Jiménez-Barceló, Alexander Damián Heine-Suñer, María Rosa Martorell-Riera, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8038718/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Whole exome sequencing (WES) is increasingly employed in the prenatal setting to identify genetic causes of fetal anomalies, particularly when cytogenetic tests such as karyotyping and chromosomal microarray (CMA) return normal results. In this prospective study, we assessed the diagnostic yield of trio-based WES (fetus and both parents) in 249 pregnancies with sonographic findings and prior negative results from quantitative fluorescent PCR (QF-PCR), conducted in parallel with CMA. A molecular diagnosis was established in 26.9% (67/249) of cases. Among these, 58.2% were de novo autosomal-dominant variants, 29.9% were autosomal-recessive (compound heterozygous or homozygous), 9.0% were inherited dominant, and 3.0% were X-linked. Diagnostic yield varied by anomaly category, ranging from 37.5% in growth disorders to 6.3% in gastrointestinal anomalies. The median turnaround time was 12 days, notably shorter than the average turnaround time of 20 days typically of other reports. Identification of a pathogenic variant had a direct impact on clinical management, with pregnancy termination occurring in 54.7% of diagnosed cases compared to 18.1% among those without a diagnosis. Prenatal parallel trio-based WES and CMA following negative QF-PCR results could represent an appropriate and actionable strategy in the current prenatal diagnostic landscape. This approach provides a significantly improved diagnostic yield and faster turnaround time, especially in structurally anomalous pregnancies, thereby delivering meaningful support to families facing complex prenatal decision-making. Chromosomal microarray analysis (CMA) decision-making prenatal diagnostic yield trio-based sequencing ultrasound anomalies whole exome sequencing (WES) Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Prenatal anomalies encompass structural or functional abnormalities that arise during fetal development. Genetic disorders are a major underlying cause, accounting for a substantial proportion of pregnancy complications and congenital defects (DeSilva et al., 2016 ). These conditions pose significant concerns during pregnancy due to their potential impact on fetal health and development. Advances in medical genetics have enabled the development of increasingly sophisticated prenatal testing methods, allowing for earlier and more accurate detection of these anomalies. Invasive prenatal diagnostic tests are performed on chorionic villus samples or amniotic fluid (Gabriel et al., 2022 ). Traditionally, most laboratories conduct genetic tests such as quantitative fluorescent PCR (QF-PCR) for rapid aneuploidy screening, G-banded karyotyping for chromosomal abnormalities, and chromosomal microarray analysis (CMA) for the detection of copy number variations (CNVs) (Best et al., 2018 ; Fu et al., 2022; Gabriel et al., 2022 ; Sabbagh & Van den Veyver, 2019 ; Tran Mau-Them et al., 2023). QF-PCR has demonstrated a diagnostic yield of around 15% (Janicki et al., 2023 ; Shin et al., 2016 ; Zhang et al., 2019 ), while CMA detects abnormalities in approximately 13% of cases overall(Miller et al., 2010) and uncovers additional pathogenic copy-number variants in about 6% of fetuses with a normal karyotype (Callaway et al., 2013 ; Margiotti et al., 2024 ; R. J. Wapner et al., 2012). Next-generation sequencing (NGS) has revolutionized prenatal diagnosis by providing a cost-effective, high-resolution method for analysing fetal DNA (Margiotti et al., 2024 ; Sabbagh & Van den Veyver, 2019 ). Depending on the level of genomic analysis required there are different genetic tests based on NGS that vary in their scope and genes analysed. Clinical exome sequencing (CES) targets a specific set of disease-associated genes, while whole-exome sequencing (WES) examines all protein-coding regions. Whole-genome sequencing (WGS) provides the most comprehensive analysis by covering both coding and non-coding regions, enabling the detection of a broader spectrum of genetic variations. Among these approaches, WES is currently more widely adopted than WGS, as it captures most of known pathogenic variants while requiring less complex data analysis (Monaghan et al., 2020 ). WES has demonstrated strong diagnostic utility in postnatal settings, particularly for uncovering the genetic basis of congenital anomalies, with reported diagnostic yields around 25% (Fu et al., 2018 ; Slavotinek et al., 2023). However, data on its use in the prenatal diagnostic setting remains limited with yields that can vary from approximately 6% to 80%. This variability is largely attributable to differences in study design, including sample size, the use of singleton versus trio-based sequencing strategies, and the criteria used for case selection (Best et al., 2018 ; Ferretti et al., 2019 ; Mellis et al., 2022 ; Tran Mau-Them et al., 2023). Trio-based WES, which involves sequencing the fetus alongside both parents, offers significant advantages for variant interpretation. It allows for confident detection of de novo variants, distinction between cis and trans configurations in biallelic recessive variants, and precise determination of the parental origin of inherited pathogenic alleles (Margiotti et al., 2024 ). In summary, although WES is increasingly used in prenatal care to enhance diagnostic yield, additional studies are needed to evaluate its performance and clinical utility in this specific setting. This study investigates the effectiveness of trio-based WES for prenatal genetic diagnosis by assessing its diagnostic yield in cases with fetal ultrasound abnormalities and previously negative results from QF-PCR. Additionally, we examine the association between a positive NGS result and the decision to pursue a legal termination of pregnancy. Materials and Methods Study design and participants Genetic testing within the public health system (IB-Salut) of the Balearic Islands (Spain), which serves a population of 1.1 million, is conducted at the Molecular Diagnostics and Clinical Genetics Unit (UDMGC) at Hospital Universitari Son Espases (HUSE). Prenatal genetic testing was performed on samples collected from obstetric services across multiple hospitals in the Balearic Islands, including Hospital Universitari Son Espases, Hospital Universitari Son Llàtzer, Hospital d’Inca, and Hospital de Manacor in Mallorca, as well as Hospital Mateu Orfila in Menorca and Hospital Can Misses in Eivissa. Informed consent was obtained from all participating couples in accordance with institutional review board policies. Between 2020 and 2024, both a clinical exome panel and a whole exome panel were utilized for trio-exome sequencing. This analysis was performed on fetal DNA and parental DNA from pregnancies between 11 and 39 weeks of gestation, where ultrasound anomalies were detected, and prior QF-PCR and chromosomal microarray (array-CGH) results were normal or concurrent. The classification of the clinical pathogenicity of the genetic variants was conducted according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the guidelines for NGS procedures applied to prenatal diagnosis by the Spanish Society of Gynecology and Obstetrics and the Spanish Association of Prenatal Diagnosis (Abulí et al., 2024 ; Monaghan et al., 2020 ; Richards et al., 2015 ). Variants classified as pathogenic or likely pathogenic were reported, while variants of uncertain significance (VUS), as well as those classified as benign or likely benign, were excluded from the final results. Procedures Fetal genomic DNA was extracted from amniotic fluid or chorionic villi following standard protocols, while parental genomic DNA was obtained from peripheral blood leukocytes. Exome sequencing was performed using the Illumina DNA Prep with Exome v2.5 Enrichment, covering a total of 37.5 Mb. Library preparation followed the Nextera Flex for Enrichment protocol (Illumina), which utilizes hybridization-based capture of target regions. Sequencing was carried out on an Illumina NovaSeq 6000DX platform, achieving an average coverage depth of 80×, with 95% of bases sequenced at a depth of at least 20×. Data processing included base annotation, alignment to the human reference genome (hg19, GRCh37), filtering of low-quality reads, and variant annotation using the latest version of DRAGEN (Illumina). Only variants sequenced at a read depth > 20× and meeting quality thresholds (> Q30, > 20% of reads, and visual inspection) were considered. Variant analysis and filtering were conducted using the Geneyx Analysis platform (Geneyx, Tel Aviv, Israel). Variants were assessed using multiple classification databases, including dbSNP, GNOMAD, OMIM, Locus-Specific Mutation Databases, ClinVar, HGMD, Varsome, Franklin, LOVD, and UCSC. Additionally, only variants with a minor allele frequency (MAF) below 1% in the 1000 Genomes Project and GnomAD were considered. QF-PCR was performed using the Devyser Compact (Devyser) and Chromosomal microarray analysis was performed using the qChip® Pre v1.1 Complete platform (qGenomics). The data were analyzed with Genomic Workbench 7.0 software and qGenviewer v2.1.1. Results This study was conducted through prenatal molecular diagnosis using CMA and trio-based WES simultaneously in cases of fetuses with structural anomalies. Between 2020 and 2024, WES and CMA were performed on 249 fetuses with ultrasound-detected anomalies. Figure 1 shows the annual diagnostic yield of trio-based exome sequencing. A molecular diagnosis, defined as the identification of pathogenic or likely pathogenic variants, was established in 26.9% (67/249) of cases, while 73.1% (182/249) remained without a definitive genetic diagnosis (Figure 2A). Of the 182 fetuses with negative WES results, 4.4% (8/182) received a positive diagnosis through CMA. Interestingly, CMA revealed a de novo 3.43 Mb duplication at 4p15.1–p14, in a fetus with two compound heterozygous pathogenic (SNV) variants in the PKHD1 gene identified by WES, highlighting a rare case of dual diagnosis through complementary genomic approaches (NM_138694.3: c.5912G>A p.Gly1971Asp; c.5895dupA p.Leu1966ThrfsTer4, Table S2). In total, after CMA and trio-based WES analysis 174 fetuses remained without a genetic diagnosis. Among the cases diagnosed by trio-based WES, 58.2% (39/67) involved de novo variants in autosomal dominant genes, and 29.9% (20/67) were due to biallelic variants (compound heterozygous or homozygous) in autosomal recessive genes. Meanwhile, 9.0% (6/67) of fetuses harbored inherited pathogenic variants in autosomal dominant genes, mainly those with incomplete penetrance and/or variable expressivity, such as DSTYK or EPHB4 , where the affected parent had not been previously diagnosed. Additionally, 3.0% (2/67) carried X-linked pathogenic variants inherited from asymptomatic mothers, involving genes such as FLNA and FOXP3 (Figure 2B). Detailed descriptions of specific de novo and autosomal recessive variants are provided in Supplementary Tables S1 and S2. The average turnaround time from sample receipt to diagnosis was 12 days. Notably, the number of WES analyses performed in clinical practice increased progressively over the course of the study period. However, from 2021 onward, the annual number of diagnosed cases with pathogenic variants remained relatively stable at 14 to 15 per year. In contrast, the number of undiagnosed cases increased substantially, rising from 15 in 2020 to over 60 in 2024. This trend has led to a widening gap between diagnosed and undiagnosed cases, particularly in the most recent years. To further assess the diagnostic utility of trio-based WES in the prenatal setting, each case was classified according to the primary physiological system most affected based on ultrasound findings (Syngelaki et al., 2019). Using this classification, skeletal abnormalities were identified in 51 cases, with a diagnostic yield of 37.3% (19/51). In 56 cases presenting with intrauterine growth restriction (IUGR), hydrops fetalis, or suspected rasopathies, 37.5% (21/56) harbored pathogenic variants. Central nervous system anomalies were observed in 55 cases, with a yield of 20% (11/55). Genitourinary anomalies were present in 17 cases, with pathogenic variants identified in 35.3% (6/17). Cardiovascular anomalies were found in 54 cases, yielding a diagnostic rate of 16.7% (9/54). Gastrointestinal anomalies were the least likely to result in a genetic diagnosis, with only 6.3% (1/16) of the 16 cases showing pathogenic variants (Figure 3A and 3B). To evaluate the impact of WES findings on clinical decision-making, we compared the detection rate of pathogenic variants between pregnancies in which legal termination of pregnancy (TOP) was chosen and those that continued (No TOP). Among ongoing pregnancies, a pathogenic variant was identified in 18.1% (32/177) of cases. In contrast, 54.7% (35/64) of terminated pregnancies had a confirmed pathogenic variant explaining the ultrasound anomaly (Figure 4). Discussion In recent years, WES has been established as a valuable tool in prenatal diagnosis and its diagnostic performance has been evaluated in a series of studies involving fetuses with structural anomalies. Nevertheless, information on its adoption in hospitals within a public health system is still incomplete, particularly in the context of the trio study, primarily due to its high cost. Additionally, successful implementation requires robust laboratory and bioinformatic infrastructure. In routine clinical practice, standard prenatal genetic testing, such as karyotyping, QF-PCR, and CMA, is primarily performed to exclude chromosomal abnormalities. However, a recent meta-analysis has demonstrated that WES provides an additional diagnostic yield of approximately 31% over CMA or karyotyping alone in fetal structural anomalies (Mellis et al., 2022). Diagnostic yields vary considerably, influenced by factors such as the type and number of anomalies, cohort size, and case selection criteria (Best et al., 2018; Ferretti et al., 2019; Mellis et al., 2022; Richards et al., 2015; Tran Mau-Them et al., 2023). Among these factors, cohort selection appears to have one of the greatest impacts on diagnostic yield. Cohorts are generally classified as either unselected or selected, depending on the criteria used for case inclusion. Selected cohorts typically comprise fetuses with specific types of malformations known to be associated with genetic disorders, a family history of similar conditions, or other risk factors suggestive of a monogenic etiology. (Best et al., 2018; Mellis et al., 2022; Pratt et al., 2020). The aim of this study was to assess the effectiveness of trio-based WES for prenatal genetic diagnosis in fetuses with ultrasound anomalies, performed in parallel with CMA following negative results from QF-PCR. We found that our trio-based WES achieved a diagnostic yield around 27%, however, it varies considerably when cases are grouped according to the phenotype observed ecographically. In this respect, high yields were observed in intrauterine growth restriction, fetal hydrops, and skeletal abnormalities, and lower rates in cardiovascular and gastrointestinal conditions. Consistent with previous studies, skeletal abnormalities show a high likelihood of genetic diagnosis, though our yield (37.3%) is slightly lower than reported rates of 40-60% (Gabriel et al., 2022; Mellis et al., 2022). Similarly, our yield for intrauterine growth restriction, fetal hydrops, and rasopathies aligns with prior reports of 15-25% (Best et al., 2018; Ferretti et al., 2019; Fu et al., 2022; Gabriel et al., 2022; Lord et al., 2019; Mellis et al., 2022; Petrovski et al., 2019; R. Wapner et al., 2017) (Figure 3B). The majority of diagnostic findings involved de novo variants, followed by pathogenic variants in autosomal recessive genes. Identifying these variants accurately requires knowledge of their inheritance patterns, which is crucial for proper classification. This highlights the importance of trio-based sequencing, as such insights cannot be achieved through analysis of fetal DNA alone. In our cohort, de novo variants were identified in 58.2% (39/67) of diagnosed cases. Among these, interrogation of public databases such as ClinVar ( ClinVar , 2025), gnomAD ( GnomAD , 2025), and Franklin ( Franklin , 2025) indicated that 41% (16/39) of these variants had either never been characterized or were classified as variants of uncertain significance. The usefulness of trio-based WES lies precisely in its ability to evaluate inheritance patterns during exome analysis, enabling the reclassification of certain variants as likely pathogenic (Abulí et al., 2024; Monaghan et al., 2020; Richards et al., 2015). Without the inclusion of parental data, these variants would have remained classified as variants of uncertain significance (VUS) and, consequently, would not have been reported, resulting in a negative study outcome. Similarly, the biallelic inheritance of compound heterozygoses for recessively inherited genes are quickly identified as we can distinguish a cis from a trans conformation of both variants. Over the years, particularly in the last one, the number of requests for prenatal exome studies was found to increase significantly in our center. However, this rise in testing has not been accompanied by a proportional increase in the detection of pathogenic variants that explain the ultrasound findings, resulting in a lower overall diagnostic yield. This underscores the importance and need for testing cases with a higher likelihood of an underlying monogenic etiology (Mellis et al., 2022) to achieve higher diagnostic yields and avoid futile tests. One possible way to achieve this would be to evaluate pretest the convenience of genetic testing by expert committees with genetic and obstetric expertise, something we have begun to implement. Beyond diagnostic yield, exome sequencing results impact decision making during ongoing pregnancies. However, significant challenges remain, such as interpreting variants of uncertain significance, reporting pathogenic and secondary findings, and providing genetic counselling for ambiguous results (McInnes-Dean et al., 2024; Tolusso et al., 2021). Rapid prenatal diagnosis is essential for early interventions and decision making especially with respect to pregnancy termination in accordance with the laws of each country(Chen et al., 2020; Normand et al., 2018). The results of our approach, in which trio-exome sequencing and CMA are performed in parallel following a negative QF-PCR result, differ from the previously proposed stepwise strategy QF-PCR/CMA and, subsequently, exome sequencing, in which CMA was recommended as the first-tier test for prenatal diagnosis (Liu et al., 2022). This parallel testing model allows results to be delivered within 12 days from the receipt of the sample in the laboratory and enables timely reporting to the patient reducing clinical risks and allowing earlier counseling for decision making, whereas previous publications reported turnaround times of 14 to 41 days, with a median of 20 days (Chen et al., 2020; Gabriel et al., 2022; Habiba et al., 2009; Mellis et al., 2022; Normand et al., 2018). A particularly instructive case underscored the complementary value of performing WES and CMA in parallel. The fetus presented with a severe prenatal phenotype, including anhydramnios, nephromegaly, and absence of both the stomach and bladder. CMA identified a de novo 3.43 Mb duplication at 4p15.1–p14, a region previously implicated in various congenital anomalies. However, this structural variant alone did not fully account for the constellation of clinical findings. Crucially, because WES was performed concurrently, it revealed compound heterozygous pathogenic variants in PKHD1 (NM_138694.3: p.Gly1971Asp and p.Leu1966ThrfsTer4), a gene associated with autosomal recessive polycystic kidney disease. These variants provided a more comprehensive and mechanistically plausible explanation for the observed ultrasound phenotype. This dual diagnostic outcome emphasizes the importance of integrated genomic testing strategies in prenatal settings. The case highlights how the concurrent application of CMA and WES can enhance diagnostic yield, prevent misinterpretation of isolated findings, and enable more accurate and informed genetic counseling. Reliance on CMA alone, without follow-up WES, could have resulted in an incomplete diagnosis and a misinformed assessment of recurrence risk, potentially leading to inappropriate reproductive counselling. In contrast, the combined approach allowed for a precise identification of the underlying genetic cause, thereby providing reliable guidance for future pregnancies. Therefore, we consider that our parallel trio-exome/CMA strategy remains the most appropriate approach until there is sufficient evidence to support the use of WGS over QF-PCR, CMA, and exome sequencing. In this regard, a recent review has indicated that WGS may require less DNA compared to other stepwise testing strategies and has the potential to deliver results within a shorter timeframe, thus facilitating clinical decision-making. However, it is also associated with a higher rate of variants of uncertain significance (VUS) (Shreeve et al., 2024). By delineating the molecular basis of fetal ultrasound abnormalities, this approach enables families to understand the implications for the current pregnancy and any future gestations, thereby reducing uncertainty and alleviating associated psychological stress (Clift et al., 2015; Halverson et al., 2016; Talati et al., 2021). The presence of a confirmed pathogenic variant exerts a measurable influence on parental decision-making. In our cohort, 54.7% (35/64) of women who opted for legal TOP had received a genetic diagnosis that explained the sonographic findings, whereas 18.1% (32/177) of those who continued their pregnancies were also found to carry a pathogenic or likely pathogenic variant consistent with the fetal phenotype. These findings align with previous studies that explore the experiences of families undergoing prenatal exome-based diagnosis (Diderich et al., 2024; McInnes-Dean et al., 2024), reporting similar trends. In conclusion, our findings support the implementation of parallel trio-based WES and CMA following a negative QF-PCR result as a feasible and effective strategy for prenatal diagnosis within the public health system. This approach offers a faster turnaround time and allows for timely clinical decision-making. Trio analysis is critical for accurate variant interpretation, enabling the reclassification of variants and improving diagnostic confidence. Furthermore, the presence of a definitive genetic diagnosis significantly impacts parental decision-making, reinforcing the clinical value of this approach beyond diagnostic metrics alone. While WGS shows promise as a future first-tier test, current limitations suggest that parallel trio-WES and CMA remains the most appropriate and actionable strategy in the present landscape. Moving forward, expert pre-test case selection and the integration of comprehensive genetic counselling is essential to maximize diagnostic yield, optimize resource use, and provide meaningful support to families navigating complex prenatal decisions. Declarations Funding This work was supported by SOIB Qualificats Sector Públic, UIB i Entitats Locals 2024 grant to Juan Antonio Jiménez Barceló. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution Statement Juan Antonio Jiménez-Barceló and Víctor José Asensio-Landa performed statistical analysis, interpretation of results and drafted the original manuscript. Alexander Damián Heine-Suñer, María Rosa Martorell Riera, Angeles Pérez-Granero, Laura Torres-Juan, Iciar Martinez-Lopez, Susana Renee Avella-Klaassen, Maria Carmen Prado-Farnós, and Dora Noguera-Benbassat, members of the Molecular diagnostics and clinical genetics unit (UDMGC), performed the interpretation of pathogenic variants, clinical genomic diagnosis and designed the study. Fernando Santos-Simarro and Maria Garcia de Paso Mora were responsible for pediatric management, including clinical care and follow-up of the patients. Maria Victoria Llull-Albertí and Marc Ventayol-Guirado performed the statistical analysis and the interpretation of the results. Rosa Ruiz de Gopegui, María Vila-Cortes, Andrea Alegre-García, Celia Garrido-Palmer, Albert Tubau Navarra, and Aina Ruiz-Romero were responsible for the obstetric management, including the clinical care and follow-up of the patients, and other clínical procedures. All authors read and approved the final version of the manuscript and agree to be accountable for all aspects of the work, ensuring integrity and accuracy. Data Availability Statement The datasets generated during the current study comprise individual‐level clinical and genetic information. As these data contain potentially identifiable patient information and consent for public deposition was not obtained, they cannot be made openly available in a public repository. Individual-level data may be made available from the corresponding author upon reasonable request. Ethics approval A request has been submitted to the Ethics Committee for a waiver of informed consent in relation to the publication of a scientific article. References Abulí, A., Antolín, E., Borrell, A., Garcia-Hoyos, M., García Santiago, F., Gómez Manjón, I., Maíz, N., González González, C., Rodríguez-Revenga, L., Valenzuena Palafoll, I., & Suela, J. (2024). 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Supplementary Files SuplementaryTableS1.docx SuplementaryTableS2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 20 Mar, 2026 Reviews received at journal 13 Mar, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviews received at journal 05 Dec, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers invited by journal 17 Nov, 2025 Editor assigned by journal 05 Nov, 2025 Submission checks completed at journal 05 Nov, 2025 First submitted to journal 05 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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14:14:46","extension":"png","order_by":33,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12781,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/cd9fc42161cbe62efcf2d561.png"},{"id":96847783,"identity":"28b01857-c739-4cbe-b7f4-37ae3a7cecb1","added_by":"auto","created_at":"2025-11-26 16:51:59","extension":"png","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11092,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/3d9bf4a707be26f3b1ffbc1b.png"},{"id":96920063,"identity":"c92cd14d-1131-40db-9f16-eccb0120d59f","added_by":"auto","created_at":"2025-11-27 14:14:44","extension":"xml","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":116429,"visible":true,"origin":"","legend":"","description":"","filename":"17e5b5937f6146038a9bddb9ee450fdf1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/6fbd08449c8c38e70eeb7dc2.xml"},{"id":96847773,"identity":"abf8c550-bc59-4d63-af79-659bc8962ff7","added_by":"auto","created_at":"2025-11-26 16:51:59","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128042,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/3ce546cd48018a155a260490.html"},{"id":96847742,"identity":"0d9f08ac-b505-490e-ac99-646a6e6ff6e6","added_by":"auto","created_at":"2025-11-26 16:51:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":98420,"visible":true,"origin":"","legend":"\u003cp\u003eYearly distribution of pathogenic and undiagnosed cases from 2020 to 2024. Pathogenic results included pathogenic (P) and likely pathogenic (LP) variants and without finding results include variants of uncertain significance (VUS), benign (B) and likely benign (LB).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/042963830671521c66b9518d.png"},{"id":96918641,"identity":"3053b293-6c56-42de-a7da-eef39a4b30fa","added_by":"auto","created_at":"2025-11-27 14:12:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eProportion of pathogenic and undiagnosed results obtained by trio-based WES. AD: autosomal dominant; AR: autosomal recessive; XL: X-linked. Pathogenic results include pathogenic (P) and likely pathogenic (LP) variants. Undiagnosed results include variants of uncertain significance (VUS), benign (B), and likely benign (LB) variants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eDistribution of pathogenic variants according to mode of inheritance. AD: autosomal dominant; AR: autosomal recessive; XL: X-linked. Pathogenic results include pathogenic (P) and likely pathogenic (LP) variants. Undiagnosed results include variants of uncertain significance (VUS), benign (B), and likely benign (LB) variants.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/ac1caefbd1ee7d9f228b3c95.png"},{"id":96917791,"identity":"bff55672-c534-4d5d-8191-4b3331065d02","added_by":"auto","created_at":"2025-11-27 14:10:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":89580,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003ePercentage distribution of pathogenic and undiagnosed results according to the ultrasound abnormalities observed. Pathogenic results include pathogenic (P) and likely pathogenic (LP) variants. Undiagnosed results include variants of uncertain significance (VUS), benign (B), and likely benign (LB) variants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Absolute number of pathogenic and undiagnosed results for each ultrasound abnormality observed. Pathogenic results include pathogenic (P) and likely pathogenic (LP) variants. Undiagnosed results include variants of uncertain significance (VUS), benign (B), and likely benign (LB) variants.\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of WES findings on clinical decision-making, we compared the detection rate of pathogenic variants between pregnancies in which legal termination of pregnancy (TOP) was chosen and those that continued (No TOP). Among ongoing pregnancies, a pathogenic variant was identified in 18.1% (32/177) of cases. In contrast, 54.7% (35/64) of terminated pregnancies had a confirmed pathogenic variant explaining the ultrasound anomaly (Figure 4).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/031acbe21899987788074634.png"},{"id":96919175,"identity":"946dc68e-af99-4a86-ae04-fd55a8f6f09f","added_by":"auto","created_at":"2025-11-27 14:13:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54315,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of pathogenic and undiagnosed results in cases where a termination of pregnancy (TOP) was performed and in cases where it was not performed (No TOP). Pathogenic results include pathogenic (P) and likely pathogenic (LP) variants. Undiagnosed results include variants of uncertain significance (VUS), benign (B), and likely benign (LB) variants.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/67d51f58e7b885e8990c87f9.png"},{"id":96923337,"identity":"1c02784f-d325-4dca-9902-50c272b225ec","added_by":"auto","created_at":"2025-11-27 14:21:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":943631,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/55112f1f-56a2-4ba8-9ce1-7de4641e5087.pdf"},{"id":96847743,"identity":"c7d1b5ce-058a-4d40-af49-55a56f07cd17","added_by":"auto","created_at":"2025-11-26 16:51:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18185,"visible":true,"origin":"","legend":"","description":"","filename":"SuplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/f723e372a29784a666c26068.docx"},{"id":96920204,"identity":"4c7c353d-c0f0-456f-954d-ad046928803e","added_by":"auto","created_at":"2025-11-27 14:14:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16705,"visible":true,"origin":"","legend":"","description":"","filename":"SuplementaryTableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8038718/v1/a3b1605e79cc40b8910c5251.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Real-World Application of Trio-Based Exome Sequencing in Prenatal Genetic Diagnosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePrenatal anomalies encompass structural or functional abnormalities that arise during fetal development. Genetic disorders are a major underlying cause, accounting for a substantial proportion of pregnancy complications and congenital defects (DeSilva et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These conditions pose significant concerns during pregnancy due to their potential impact on fetal health and development. Advances in medical genetics have enabled the development of increasingly sophisticated prenatal testing methods, allowing for earlier and more accurate detection of these anomalies.\u003c/p\u003e\u003cp\u003eInvasive prenatal diagnostic tests are performed on chorionic villus samples or amniotic fluid (Gabriel et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Traditionally, most laboratories conduct genetic tests such as quantitative fluorescent PCR (QF-PCR) for rapid aneuploidy screening, G-banded karyotyping for chromosomal abnormalities, and chromosomal microarray analysis (CMA) for the detection of copy number variations (CNVs) (Best et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fu et al., 2022; Gabriel et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sabbagh \u0026amp; Van den Veyver, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tran Mau-Them et al., 2023). QF-PCR has demonstrated a diagnostic yield of around 15% (Janicki et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), while CMA detects abnormalities in approximately 13% of cases overall(Miller et al., 2010) and uncovers additional pathogenic copy-number variants in about 6% of fetuses with a normal karyotype (Callaway et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Margiotti et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; R. J. Wapner et al., 2012).\u003c/p\u003e\u003cp\u003eNext-generation sequencing (NGS) has revolutionized prenatal diagnosis by providing a cost-effective, high-resolution method for analysing fetal DNA (Margiotti et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sabbagh \u0026amp; Van den Veyver, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Depending on the level of genomic analysis required there are different genetic tests based on NGS that vary in their scope and genes analysed. Clinical exome sequencing (CES) targets a specific set of disease-associated genes, while whole-exome sequencing (WES) examines all protein-coding regions. Whole-genome sequencing (WGS) provides the most comprehensive analysis by covering both coding and non-coding regions, enabling the detection of a broader spectrum of genetic variations. Among these approaches, WES is currently more widely adopted than WGS, as it captures most of known pathogenic variants while requiring less complex data analysis (Monaghan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWES has demonstrated strong diagnostic utility in postnatal settings, particularly for uncovering the genetic basis of congenital anomalies, with reported diagnostic yields around 25% (Fu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Slavotinek et al., 2023). However, data on its use in the prenatal diagnostic setting remains limited with yields that can vary from approximately 6% to 80%. This variability is largely attributable to differences in study design, including sample size, the use of singleton versus trio-based sequencing strategies, and the criteria used for case selection (Best et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ferretti et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mellis et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tran Mau-Them et al., 2023).\u003c/p\u003e\u003cp\u003eTrio-based WES, which involves sequencing the fetus alongside both parents, offers significant advantages for variant interpretation. It allows for confident detection of \u003cem\u003ede novo\u003c/em\u003e variants, distinction between cis and trans configurations in biallelic recessive variants, and precise determination of the parental origin of inherited pathogenic alleles (Margiotti et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn summary, although WES is increasingly used in prenatal care to enhance diagnostic yield, additional studies are needed to evaluate its performance and clinical utility in this specific setting. This study investigates the effectiveness of trio-based WES for prenatal genetic diagnosis by assessing its diagnostic yield in cases with fetal ultrasound abnormalities and previously negative results from QF-PCR. Additionally, we examine the association between a positive NGS result and the decision to pursue a legal termination of pregnancy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy design and participants\u003c/h2\u003e\u003cp\u003eGenetic testing within the public health system (IB-Salut) of the Balearic Islands (Spain), which serves a population of 1.1\u0026nbsp;million, is conducted at the Molecular Diagnostics and Clinical Genetics Unit (UDMGC) at Hospital Universitari Son Espases (HUSE). Prenatal genetic testing was performed on samples collected from obstetric services across multiple hospitals in the Balearic Islands, including Hospital Universitari Son Espases, Hospital Universitari Son Ll\u0026agrave;tzer, Hospital d\u0026rsquo;Inca, and Hospital de Manacor in Mallorca, as well as Hospital Mateu Orfila in Menorca and Hospital Can Misses in Eivissa.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e\u003cp\u003ewas obtained from all participating couples in accordance with institutional review board policies. Between 2020 and 2024, both a clinical exome panel and a whole exome panel were utilized for trio-exome sequencing. This analysis was performed on fetal DNA and parental DNA from pregnancies between 11 and 39 weeks of gestation, where ultrasound anomalies were detected, and prior QF-PCR and chromosomal microarray (array-CGH) results were normal or concurrent.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe classification of the clinical pathogenicity of the genetic variants was conducted according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the guidelines for NGS procedures applied to prenatal diagnosis by the Spanish Society of Gynecology and Obstetrics and the Spanish Association of Prenatal Diagnosis (Abul\u0026iacute; et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Monaghan et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Richards et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Variants classified as pathogenic or likely pathogenic were reported, while variants of uncertain significance (VUS), as well as those classified as benign or likely benign, were excluded from the final results.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProcedures\u003c/h3\u003e\n\u003cp\u003eFetal genomic DNA was extracted from amniotic fluid or chorionic villi following standard protocols, while parental genomic DNA was obtained from peripheral blood leukocytes. Exome sequencing was performed using the Illumina DNA Prep with Exome v2.5 Enrichment, covering a total of 37.5 Mb. Library preparation followed the Nextera Flex for Enrichment protocol (Illumina), which utilizes hybridization-based capture of target regions. Sequencing was carried out on an Illumina NovaSeq 6000DX platform, achieving an average coverage depth of 80\u0026times;, with 95% of bases sequenced at a depth of at least 20\u0026times;.\u003c/p\u003e\u003cp\u003eData processing included base annotation, alignment to the human reference genome (hg19, GRCh37), filtering of low-quality reads, and variant annotation using the latest version of DRAGEN (Illumina). Only variants sequenced at a read depth\u0026thinsp;\u0026gt;\u0026thinsp;20\u0026times; and meeting quality thresholds (\u0026gt;\u0026thinsp;Q30, \u0026gt;\u0026thinsp;20% of reads, and visual inspection) were considered.\u003c/p\u003e\u003cp\u003eVariant analysis and filtering were conducted using the Geneyx Analysis platform (Geneyx, Tel Aviv, Israel). Variants were assessed using multiple classification databases, including dbSNP, GNOMAD, OMIM, Locus-Specific Mutation Databases, ClinVar, HGMD, Varsome, Franklin, LOVD, and UCSC. Additionally, only variants with a minor allele frequency (MAF) below 1% in the 1000 Genomes Project and GnomAD were considered.\u003c/p\u003e\u003cp\u003eQF-PCR was performed using the Devyser Compact (Devyser) and Chromosomal microarray analysis was performed using the qChip\u0026reg; Pre v1.1 Complete platform (qGenomics). The data were analyzed with Genomic Workbench 7.0 software and qGenviewer v2.1.1.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThis study was conducted through prenatal molecular diagnosis using CMA and trio-based WES simultaneously in cases of fetuses with structural anomalies. Between 2020 and 2024, WES and CMA were performed on 249 fetuses with ultrasound-detected anomalies. Figure 1 shows the annual diagnostic yield of trio-based exome sequencing. A molecular diagnosis, defined as the identification of pathogenic or likely pathogenic variants, was established in 26.9% (67/249) of cases, while 73.1% (182/249) remained without a definitive genetic diagnosis (Figure 2A). Of the 182 fetuses with negative WES results, 4.4% (8/182) received a positive diagnosis through CMA. Interestingly, CMA revealed a \u003cem\u003ede novo\u003c/em\u003e 3.43 Mb duplication at 4p15.1\u0026ndash;p14, in a fetus with two compound heterozygous pathogenic (SNV) variants in the \u003cem\u003ePKHD1\u003c/em\u003e gene identified by WES, highlighting a rare case of dual diagnosis through complementary genomic approaches (NM_138694.3: c.5912G\u0026gt;A p.Gly1971Asp; c.5895dupA p.Leu1966ThrfsTer4, Table S2). In total, after CMA and trio-based WES analysis 174 fetuses remained without a genetic diagnosis.\u003c/p\u003e\n\u003cp\u003eAmong the cases diagnosed by trio-based WES, 58.2% (39/67) involved \u003cem\u003ede novo\u003c/em\u003e variants in autosomal dominant genes, and 29.9% (20/67) were due to biallelic variants (compound heterozygous or homozygous) in autosomal recessive genes. Meanwhile, 9.0% (6/67) of fetuses harbored inherited pathogenic variants in autosomal dominant genes, mainly those with incomplete penetrance and/or variable expressivity, such as \u003cem\u003eDSTYK\u003c/em\u003e or \u003cem\u003eEPHB4\u003c/em\u003e, where the affected parent had not been previously diagnosed. Additionally, 3.0% (2/67) carried X-linked pathogenic variants inherited from asymptomatic mothers, involving genes such as \u003cem\u003eFLNA\u003c/em\u003e and \u003cem\u003eFOXP3\u003c/em\u003e (Figure 2B). Detailed descriptions of specific \u003cem\u003ede novo\u003c/em\u003e and autosomal recessive variants are provided in Supplementary Tables S1 and S2.\u003c/p\u003e\n\u003cp\u003eThe average turnaround time from sample receipt to diagnosis was 12 days. Notably, the number of WES analyses performed in clinical practice increased progressively over the course of the study period. However, from 2021 onward, the annual number of diagnosed cases with pathogenic variants remained relatively stable at 14 to 15 per year. In contrast, the number of undiagnosed cases increased substantially, rising from 15 in 2020 to over 60 in 2024. This trend has led to a widening gap between diagnosed and undiagnosed cases, particularly in the most recent years.\u003c/p\u003e\n\u003cp\u003eTo further assess the diagnostic utility of trio-based WES in the prenatal setting, each case was classified according to the primary physiological system most affected based on ultrasound findings (Syngelaki et al., 2019). Using this classification, skeletal abnormalities were identified in 51 cases, with a diagnostic yield of 37.3% (19/51). In 56 cases presenting with intrauterine growth restriction (IUGR), hydrops fetalis, or suspected rasopathies, 37.5% (21/56) harbored pathogenic variants. Central nervous system anomalies were observed in 55 cases, with a yield of 20% (11/55). Genitourinary anomalies were present in 17 cases, with pathogenic variants identified in 35.3% (6/17). Cardiovascular anomalies were found in 54 cases, yielding a diagnostic rate of 16.7% (9/54). Gastrointestinal anomalies were the least likely to result in a genetic diagnosis, with only 6.3% (1/16) of the 16 cases showing pathogenic variants (Figure 3A and 3B).\u003c/p\u003e\n\u003cp\u003eTo evaluate the impact of WES findings on clinical decision-making, we compared the detection rate of pathogenic variants between pregnancies in which legal termination of pregnancy (TOP) was chosen and those that continued (No TOP). Among ongoing pregnancies, a pathogenic variant was identified in 18.1% (32/177) of cases. In contrast, 54.7% (35/64) of terminated pregnancies had a confirmed pathogenic variant explaining the ultrasound anomaly (Figure 4).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, WES has been established as a valuable tool in prenatal diagnosis and its diagnostic performance has been evaluated in a series of studies involving fetuses with structural anomalies. Nevertheless, information on its adoption in hospitals within a public health system is still incomplete, particularly in the context of the trio study, primarily due to its high cost. Additionally, successful implementation requires robust laboratory and bioinformatic infrastructure.\u003c/p\u003e\n\u003cp\u003eIn routine clinical practice, standard prenatal genetic testing, such as karyotyping, QF-PCR, and CMA, is primarily performed to exclude chromosomal abnormalities. However, a recent meta-analysis has demonstrated that WES provides an additional diagnostic yield of approximately 31% over CMA or karyotyping alone in fetal structural anomalies (Mellis et al., 2022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiagnostic yields vary considerably, influenced by factors such as the type and number of anomalies, cohort size, and case selection criteria (Best et al., 2018; Ferretti et al., 2019; Mellis et al., 2022; Richards et al., 2015; Tran Mau-Them et al., 2023). Among these factors, cohort selection appears to have one of the greatest impacts on diagnostic yield. Cohorts are generally classified as either unselected or selected, depending on the criteria used for case inclusion. Selected cohorts typically comprise fetuses with specific types of malformations known to be associated with genetic disorders, a family history of similar conditions, or other risk factors suggestive of a monogenic etiology. (Best et al., 2018; Mellis et al., 2022; Pratt et al., 2020).\u003c/p\u003e\n\u003cp\u003eThe aim of this study was to assess the effectiveness of trio-based WES for prenatal genetic diagnosis in fetuses with ultrasound anomalies, performed in parallel with CMA following negative results from QF-PCR. We found that our trio-based WES achieved a diagnostic yield around 27%, however, it varies considerably when cases are grouped according to the phenotype observed ecographically. In this respect, high yields were observed in intrauterine growth restriction, fetal hydrops, and skeletal abnormalities, and lower rates in cardiovascular and gastrointestinal conditions. Consistent with previous studies, skeletal abnormalities show a high likelihood of genetic diagnosis, though our yield (37.3%) is slightly lower than reported rates of 40-60% (Gabriel et al., 2022; Mellis et al., 2022). Similarly, our yield for intrauterine growth restriction, fetal hydrops, and rasopathies aligns with prior reports of 15-25% (Best et al., 2018; Ferretti et al., 2019; Fu et al., 2022; Gabriel et al., 2022; Lord et al., 2019; Mellis et al., 2022; Petrovski et al., 2019; R. Wapner et al., 2017) (Figure 3B).\u003c/p\u003e\n\u003cp\u003eThe majority of diagnostic findings involved \u003cem\u003ede novo\u003c/em\u003e variants, followed by pathogenic variants in autosomal recessive genes. Identifying these variants accurately requires knowledge of their inheritance patterns, which is crucial for proper classification. This highlights the importance of trio-based sequencing, as such insights cannot be achieved through analysis of fetal DNA alone. In our cohort, \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003evariants were identified in 58.2% (39/67) of diagnosed cases. Among these, interrogation of public databases such as ClinVar (\u003cem\u003eClinVar\u003c/em\u003e, 2025), gnomAD (\u003cem\u003eGnomAD\u003c/em\u003e, 2025), and Franklin (\u003cem\u003eFranklin\u003c/em\u003e, 2025) indicated that 41% (16/39) of these variants had either never been characterized or were classified as variants of uncertain significance. The usefulness of trio-based WES lies precisely in its ability to evaluate inheritance patterns during exome analysis, enabling the reclassification of certain variants as likely pathogenic (Abul\u0026iacute; et al., 2024; Monaghan et al., 2020; Richards et al., 2015). Without the inclusion of parental data, these variants would have remained classified as variants of uncertain significance (VUS) and, consequently, would not have been reported, resulting in a negative study outcome. Similarly, the biallelic inheritance of compound heterozygoses for recessively inherited genes are quickly identified as we can distinguish a cis from a trans conformation of both variants.\u003c/p\u003e\n\u003cp\u003eOver the years, particularly in the last one, the number of requests for prenatal exome studies was found to increase significantly in our center. However, this rise in testing has not been accompanied by a proportional increase in the detection of pathogenic variants that explain the ultrasound findings, resulting in a lower overall diagnostic yield. This underscores the importance and need for testing cases with a higher likelihood of an underlying monogenic etiology (Mellis et al., 2022)\u0026nbsp;to achieve higher diagnostic yields and avoid futile tests. One possible way to achieve this would be to evaluate pretest the convenience of genetic testing by expert committees with genetic and obstetric expertise, something we have begun to implement.\u003c/p\u003e\n\u003cp\u003eBeyond diagnostic yield, exome sequencing results impact decision making during ongoing pregnancies. However, significant challenges remain, such as interpreting variants of uncertain significance, reporting pathogenic and secondary findings, and providing genetic counselling for ambiguous results (McInnes-Dean et al., 2024; Tolusso et al., 2021). Rapid prenatal diagnosis is essential for early interventions and decision making especially with respect to pregnancy termination in accordance with the laws of each country(Chen et al., 2020; Normand et al., 2018). The results of our approach, in which trio-exome sequencing and CMA are performed in parallel following a negative QF-PCR result, differ from the previously proposed stepwise strategy QF-PCR/CMA and, subsequently, exome sequencing, in which CMA was recommended as the first-tier test for prenatal diagnosis (Liu et al., 2022). This parallel testing model allows results to be delivered within 12 days from the receipt of the sample in the laboratory and enables timely reporting to the patient reducing clinical risks and allowing earlier counseling for decision making, whereas previous publications reported turnaround times of 14 to 41 days, with a median of 20 days (Chen et al., 2020; Gabriel et al., 2022; Habiba et al., 2009; Mellis et al., 2022; Normand et al., 2018).\u003c/p\u003e\n\u003cp\u003eA particularly instructive case underscored the complementary value of performing WES and CMA in parallel. The fetus presented with a severe prenatal phenotype, including anhydramnios, nephromegaly, and absence of both the stomach and bladder. CMA identified a \u003cem\u003ede novo\u003c/em\u003e 3.43 Mb duplication at 4p15.1\u0026ndash;p14, a region previously implicated in various congenital anomalies. However, this structural variant alone did not fully account for the constellation of clinical findings. Crucially, because WES was performed concurrently, it revealed compound heterozygous pathogenic variants in PKHD1 (NM_138694.3: p.Gly1971Asp and p.Leu1966ThrfsTer4), a gene associated with autosomal recessive polycystic kidney disease. These variants provided a more comprehensive and mechanistically plausible explanation for the observed ultrasound phenotype. This dual diagnostic outcome emphasizes the importance of integrated genomic testing strategies in prenatal settings. The case highlights how the concurrent application of CMA and WES can enhance diagnostic yield, prevent misinterpretation of isolated findings, and enable more accurate and informed genetic counseling. Reliance on CMA alone, without follow-up WES, could have resulted in an incomplete diagnosis and a misinformed assessment of recurrence risk, potentially leading to inappropriate reproductive counselling. In contrast, the combined approach allowed for a precise identification of the underlying genetic cause, thereby providing reliable guidance for future pregnancies.\u003c/p\u003e\n\u003cp\u003eTherefore, we consider that our parallel trio-exome/CMA strategy remains the most appropriate approach until there is sufficient evidence to support the use of WGS over QF-PCR, CMA, and exome sequencing. In this regard, a recent review has indicated that WGS may require less DNA compared to other stepwise testing strategies and has the potential to deliver results within a shorter timeframe, thus facilitating clinical decision-making. However, it is also associated with a higher rate of variants of uncertain significance (VUS) (Shreeve et al., 2024).\u003c/p\u003e\n\u003cp\u003eBy delineating the molecular basis of fetal ultrasound abnormalities, this approach enables families to understand the implications for the current pregnancy and any future gestations, thereby reducing uncertainty and alleviating associated psychological stress (Clift et al., 2015; Halverson et al., 2016; Talati et al., 2021). The presence of a confirmed pathogenic variant exerts a measurable influence on parental decision-making. In our cohort, 54.7% (35/64) of women who opted for legal TOP had received a genetic diagnosis that explained the sonographic findings, whereas 18.1% (32/177) of those who continued their pregnancies were also found to carry a pathogenic or likely pathogenic variant consistent with the fetal phenotype. These findings align with previous studies that explore the experiences of families undergoing prenatal exome-based diagnosis (Diderich et al., 2024; McInnes-Dean et al., 2024), reporting similar trends.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our findings support the implementation of parallel trio-based WES and CMA following a negative QF-PCR result as a feasible and effective strategy for prenatal diagnosis within the public health system. This approach offers a faster turnaround time and allows for timely clinical decision-making. Trio analysis is critical for accurate variant interpretation, enabling the reclassification of variants and improving diagnostic confidence. Furthermore, the presence of a definitive genetic diagnosis significantly impacts parental decision-making, reinforcing the clinical value of this approach beyond diagnostic metrics alone. While WGS shows promise as a future first-tier test, current limitations suggest that parallel trio-WES and CMA remains the most appropriate and actionable strategy in the present landscape. Moving forward, expert pre-test case selection and the integration of comprehensive genetic counselling is essential to maximize diagnostic yield, optimize resource use, and provide meaningful support to families navigating complex prenatal decisions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u003cem\u003e\u0026nbsp;SOIB Qualificats Sector P\u0026uacute;blic, UIB i Entitats Locals 2024\u0026nbsp;\u003c/em\u003egrant to Juan Antonio Jim\u0026eacute;nez Barcel\u0026oacute;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuan Antonio Jim\u0026eacute;nez-Barcel\u0026oacute; and V\u0026iacute;ctor Jos\u0026eacute; Asensio-Landa performed statistical analysis, interpretation of results and drafted the original manuscript. Alexander Dami\u0026aacute;n Heine-Su\u0026ntilde;er, Mar\u0026iacute;a Rosa Martorell Riera, Angeles P\u0026eacute;rez-Granero, Laura Torres-Juan, Iciar Martinez-Lopez, Susana Renee Avella-Klaassen, Maria Carmen Prado-Farn\u0026oacute;s, and Dora Noguera-Benbassat, members of the Molecular diagnostics and clinical genetics unit (UDMGC), performed the interpretation of pathogenic variants, clinical genomic diagnosis and designed the study. Fernando Santos-Simarro and Maria Garcia de Paso Mora were responsible for pediatric management, including clinical care and follow-up of the patients. Maria Victoria Llull-Albert\u0026iacute; and Marc Ventayol-Guirado performed the statistical analysis and the interpretation of the results. Rosa Ruiz de Gopegui, Mar\u0026iacute;a Vila-Cortes, Andrea Alegre-Garc\u0026iacute;a, Celia Garrido-Palmer, Albert Tubau Navarra, and Aina Ruiz-Romero\u003csup\u003e\u0026nbsp;\u003c/sup\u003ewere responsible for the obstetric management, including the clinical care and follow-up of the patients, and other cl\u0026iacute;nical procedures.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript and agree to be accountable for all aspects of the work, ensuring integrity and accuracy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study comprise individual‐level clinical and genetic information. As these data contain potentially identifiable patient information and consent for public deposition was not obtained, they cannot be made openly available in a public repository. Individual-level data may be made available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA request has been submitted to the Ethics Committee for a waiver of informed consent in relation to the publication of a scientific article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbul\u0026iacute;, A., Antol\u0026iacute;n, E., Borrell, A., Garcia-Hoyos, M., Garc\u0026iacute;a Santiago, F., G\u0026oacute;mez Manj\u0026oacute;n, I., Ma\u0026iacute;z, N., Gonz\u0026aacute;lez Gonz\u0026aacute;lez, C., Rodr\u0026iacute;guez-Revenga, L., Valenzuena Palafoll, I., \u0026amp; Suela, J. (2024). 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(2023). Prenatal diagnosis by trio exome sequencing in fetuses with ultrasound anomalies: A powerful diagnostic tool. \u003cem\u003eFrontiers in Genetics\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e. https://doi.org/10.3389/FGENE.2023.1099995.\u003c/li\u003e\n\u003cli\u003eWapner, R. J., Martin, C. L., Levy, B., Ballif, B. C., Eng, C. M., Zachary, J. M., Savage, M., Platt, L. D., Saltzman, D., Grobman, W. A., Klugman, S., Scholl, T., Simpson, J. L., McCall, K., Aggarwal, V. S., Bunke, B., Nahum, O., Patel, A., Lamb, A. N., \u0026hellip; Jackson, L. (2012). Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis. \u003cem\u003eNew England Journal of Medicine\u003c/em\u003e, \u003cem\u003e367\u003c/em\u003e(23), 2175\u0026ndash;2184. https://doi.org/10.1056/NEJMOA1203382.\u003c/li\u003e\n\u003cli\u003eWapner, R., Petrovski, S., Brennan, K., Bier, L., Wou, K., \u0026amp; Goldstein, D. (2017). 8: Whole exome sequencing in the evaluation of fetal structural anomalies: A prospective study of sequential patients. \u003cem\u003eAmerican Journal of Obstetrics and Gynecology\u003c/em\u003e, \u003cem\u003e216\u003c/em\u003e(1), S5\u0026ndash;S6. https://doi.org/10.1016/j.ajog.2016.11.009.\u003c/li\u003e\n\u003cli\u003eZhang, Z., Hu, T., Wang, J., Li, Q., Wang, H., \u0026amp; Liu, S. (2019). Prenatal Diagnostic Value of Chromosomal Microarray in Fetuses with Nuchal Translucency Greater than 2.5 mm. \u003cem\u003eBioMed Research International\u003c/em\u003e, \u003cem\u003e2019\u003c/em\u003e. https://doi.org/10.1155/2019/6504159. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chromosomal microarray analysis (CMA), decision-making, prenatal diagnostic yield, trio-based sequencing, ultrasound anomalies, whole exome sequencing (WES)","lastPublishedDoi":"10.21203/rs.3.rs-8038718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8038718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhole exome sequencing (WES) is increasingly employed in the prenatal setting to identify genetic causes of fetal anomalies, particularly when cytogenetic tests such as karyotyping and chromosomal microarray (CMA) return normal results. In this prospective study, we assessed the diagnostic yield of trio-based WES (fetus and both parents) in 249 pregnancies with sonographic findings and prior negative results from quantitative fluorescent PCR (QF-PCR), conducted in parallel with CMA. A molecular diagnosis was established in 26.9% (67/249) of cases. Among these, 58.2% were \u003cem\u003ede novo\u003c/em\u003e autosomal-dominant variants, 29.9% were autosomal-recessive (compound heterozygous or homozygous), 9.0% were inherited dominant, and 3.0% were X-linked. Diagnostic yield varied by anomaly category, ranging from 37.5% in growth disorders to 6.3% in gastrointestinal anomalies. The median turnaround time was 12 days, notably shorter than the average turnaround time of 20 days typically of other reports. Identification of a pathogenic variant had a direct impact on clinical management, with pregnancy termination occurring in 54.7% of diagnosed cases compared to 18.1% among those without a diagnosis. Prenatal parallel trio-based WES and CMA following negative QF-PCR results could represent an appropriate and actionable strategy in the current prenatal diagnostic landscape. This approach provides a significantly improved diagnostic yield and faster turnaround time, especially in structurally anomalous pregnancies, thereby delivering meaningful support to families facing complex prenatal decision-making.\u003c/p\u003e","manuscriptTitle":"Real-World Application of Trio-Based Exome Sequencing in Prenatal Genetic Diagnosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 16:51:53","doi":"10.21203/rs.3.rs-8038718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T06:27:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T21:05:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-13T15:15:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315328735979342281505249983490796959475","date":"2026-02-18T14:19:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286950300493056616296994225903459800684","date":"2026-02-18T08:04:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-05T11:16:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32902537401213967834326297651354593440","date":"2025-11-24T10:18:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-17T05:47:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-06T02:25:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-06T02:24:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Genetics","date":"2025-11-05T12:58:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huge","sideBox":"Learn more about [Human Genetics](https://www.springer.com/journal/439)","snPcode":"439","submissionUrl":"https://submission.nature.com/new-submission/439/3","title":"Human Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2c667496-a567-4d98-9fe6-44b5c4fac1b3","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T09:53:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-26 16:51:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8038718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8038718","identity":"rs-8038718","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}