Diagnostic Yield After Postnatal Reanalysis of Prenatal Exome Sequencing Results

Abstract

Objective:

Analysis of exome sequencing (ES) relies on correlation with phenotypic features, but fetal phenotyping is often incomplete. The additional yield of postnatal follow-up in cases with negative or inconclusive prenatal ES has not been demonstrated. Our objective was to assess the incremental diagnostic yield of ES reanalysis after initially negative prenatal ES for congenital anomalies incorporating features identified postnatally.

Methods:

This was a secondary analysis of two prospective cohort studies of ES for fetal anomalies. We included cases in which initial ES utilizing the prenatal phenotype was not diagnostic. The primary outcome was incremental diagnostic yield of ES when incorporating postnatal findings.

Results:

Eighty-seven cases with negative or inconclusive prenatal ES and postnatal follow-up available were included. Of those, 56 (64%) had new findings postnatally. There was an incremental yield of 2% in the entire cohort, and 7% in those with new postnatal findings. In two additional cases, postnatal evaluation suggested a specific genetic diagnosis that was not detectable with ES.

Conclusion:

Among pregnancies with fetal anomalies and no clear diagnosis identified by prenatal ES, postnatal follow-up is recommended. Reanalysis of ES results can result in a genetic diagnosis in 7% of cases with new findings.

1 |. Introduction

Prenatal exome sequencing (ES) concurrently assesses thousands of genes for disease-causing variants, and is particularly useful when fetal anomalies are identified but the phenotype does not clearly suggest a specific genetic etiology [1–5]. However, accurate interpretation of ES data in part relies on correlation with phenotypic features, many of which cannot be identified in the prenatal setting. Many postnatal features commonly associated with genetic disorders, such as neurodevelopmental delay, seizures, or hearing or vision loss, are typically missed in utero. Studies comparing prenatal imaging findings with both autopsy and postnatal imaging suggest that prenatal imaging may fail to detect as many as 50% of disease findings [6–11]. This limited ability to completely characterize a phenotype in utero, in part, explains why the yield of ES in the prenatal setting is significantly lower than in children and adults [12].

Variant filtering and ranking pipelines frequently rely on an individual’s phenotype to prioritize the variants identified [13–18]. Human Phenotype Ontology, or HPO, terms describe standardized phenotypic features and are commonly used to aid in variant filtering and ranking in both the prenatal and pediatric settings [19, 20]. Given the limitations of prenatal phenotyping, postnatal follow-up and ES reanalysis using additional identified findings may be of utility to increase diagnostic yield when prenatal ES is negative (for congenital anomalies was not diagnostic). One small case series reported that in cases of fetal anomalies with negative prenatal ES results, reanalysis incorporating postnatal findings led to a positive result in 20% of cases [21]. However, the yield of ES reanalysis has not been systematically studied in larger cohorts, and there is a lack of guidance regarding if and when postnatal follow-up, and ES reanalysis should be performed for infants with congenital anomalies and negative or inconclusive prenatal ES results.

Given that many important features commonly associated with genetic disorders cannot be detected by prenatal imaging, it is possible that including such potential phenotypes in prenatal ES could mimic neonatal follow-up and improve the diagnostic yield of prenatal ES. How incorporation of these potential phenotypes into prenatal ES analysis may impact diagnostic yield is unknown.

There remains a lack of available literature to understand when ES reanalysis should be performed for cases with negative or inconclusive results from prenatal ES, and the utility of adding potentially missed phenotypes for accurate disease identification is unstudied. We aimed to assess the incremental yield of ES by incorporating additional postnatal phenotypic features, the incremental yield of ES when potentially missed phenotypes are incorporated into bioinformatic analyses, and the frequency and types of additionally identified postnatal phenotypes. We hypothesized that both approaches to reanalysis would lead to a significant incremental diagnostic yield of prenatal ES for pregnancies with fetal anomalies.

2 |. Materials and Methods

This is a secondary analysis of data from two prospective cohort studies of ES for fetal anomalies [1, 12]. Participants were eligible for enrollment in the primary studies if one or more fetal structural anomalies, fetal effusions, or nonimmune hydrops were diagnosed prenatally and chromosomal microarray (or karyotype in a small proportion) was not diagnostic. Patients underwent detailed ultrasound as well as fetal echocardiogram or MRI as clinically indicated and had ES with variant interpretation performed during pregnancy based on prenatal phenotypic features. The majority of enrolled participants were evaluated through the UCSF Fetal Treatment Center or Prenatal Diagnosis Center and thus had imaging reports available internally. A smaller number of patients were referred by other centers; in such cases, local prenatal imaging reports were collected and reviewed for analysis. Participants were enrolled in the primary studies between August 2017 and January 2022. The primary studies were approved by the UCSF Institutional Review Board (#17-21662 and #17-22420).

Participants were eligible for inclusion in this secondary analysis if ES utilizing the prenatal findings alone did not clearly provide an explanation for the fetal anomaly or anomalies. Cases excluded from this secondary analysis were those with pathogenic (P), likely pathogenic (LP), or highly suspicious variants of uncertain significance (VUS) in genes that have been associated with features consistent with the prenatal presentation. Only those with secondary findings (meaning no genetic diagnosis explaining the prenatal presentation) were included in this analysis.

The primary outcome of this study was the incremental diagnostic yield of ES when reanalysis was performed, including additional findings identified in the postnatal period. Secondary outcomes were the incremental diagnostic yield of ES when reanalysis was performed, including potentially missed phenotypes associated with genetic disease that are not possible to identify in utero, and the frequency and types of actual additional features in the postnatal period.

Given that reanalysis can result in variant reclassification due to new medical knowledge over time, we first reanalyzed each case utilizing the prenatal phenotypes only [22–24]. If negative or inconclusive, we then incorporated any new postnatal phenotypes for a second reanalysis, in order to assess how inclusion of postnatal phenotypes impacted variant identification and ES yield. We then performed a third analysis, in which we included prenatal phenotypes as well as seven neurodevelopmental HPO terms that could be associated with genetic disorders but are generally not detectable in utero (seizures, hypotonia, hearing impairment, visual impairment, autism, intellectual disability, and neurodevelopmental delay). These were applied to all cases regardless of fetal presentation, given the inability to detect these features prenatally. The reanalysis process for this study is described in Figure 1.

FIGURE 1 |.

Investigations performed as part of this secondary analysis.

All ES was performed at the University of California, San Francisco Genomic Medicine Laboratory (UCSF GML). Sequencing was performed utilizing an Illumina NovaSeq 6000 or HiSeq 2500 Sequencer on Rapid Run mode. Trio ES, including DNA samples from both biological parents, was performed whenever possible. Duo ES or proband-only ES were performed in rare circumstances when samples from both biological parents were unavailable. Human Phenotype Ontology (HPO) terms were used to describe phenotypic features in a standardized manner to optimize variant prioritization and interpretation [19, 20]. Variants were ranked by Moon (Diploid, Invitae), a software program that utilizes HPO terms to prioritize variants in genes that have been associated with diseases consistent with the prenatal presentation. Moon generates a list of potential provisional diagnoses by sequentially filtering and ranking variants with decision trees, Bayesian models, neural networks, and natural language processing using the provided HPO terms. The filtering pipeline was designed to minimize false negatives. For single-nucleotide variant analysis, Moon excluded low-quality and common variants [> 2% in Genome Aggregation Database (gnomAD)] and known likely benign/benign variants in ClinVar. Only variants located within coding sequences and splice site regions were retained, together with previously reported pathogenic variants in noncoding regions. A disease annotation was added to the remaining variants on the basis of a proprietary disorder model which includes natural language processing of the genetics literature to automatically extract associations between diseases, disease genes, inheritance patterns, specific clinical features, and other metadata on an ongoing basis. Details of the method have been published elsewhere [25, 26].

Variants were adjudicated according to the American College of Medical Genetics & Genomics (ACMG) criteria [16]. Classification of all variants identified was discussed and agreed upon during weekly meetings of the multidisciplinary exome board, attended by laboratory geneticists, bioinformaticians, pediatric and reproductive geneticists, genetic counselors, ethicists, and others [16]. The patient history and presenting findings were provided during the discussion for each case to aid in the interpretation of the clinical significance of any variants identified. As part of the primary study, clinical reports were generated for each case and results were disclosed to patients within 4 weeks for ongoing pregnancies and 3 months for non-continuing pregnancies.

Postnatal follow-up was collected for pregnancies resulting in miscarriage, stillbirth, or neonatal demise, including autopsy, postmortem imaging, and placental pathology when available.

For live births, postnatal follow-up included physical examination, developmental assessment, imaging studies, laboratory studies, and pathology studies. This follow-up was through discharge of the infant from the birth hospitalization; if additional follow-up was available for review, this was also included. Postmortem and postnatal clinical evaluations were guided by the clinical care team and were not protocolized by the study. Postnatal record review was performed to confirm the prenatally identified features as well as any additional neonatal or postmortem findings.

Bivariable analyses were performed using Fisher’s exact and Chi-square tests as appropriate for categorical variables, and Wilcoxon rank sum compared median values for nonparametric continuous variables. Statistical analysis was performed using Stata version 15.1 (College Station, TX).

3 |. Results

There were 329 cases of prenatal ES performed for fetal anomalies that were assessed for inclusion in this study. Of these, 86 (26%) had a genetic diagnosis utilizing the prenatal phenotype only and were therefore excluded. Demographics of participants included in this secondary analysis are described in Table 1.

TABLE 1 |.

Demographics of study participants.

	Participants (87)
Maternal age	33.5 (30–36)
Self-reported race/ethnicity
Asian	11 (13)
Black	5 (6)
Latina/Hispanic	24 (28)
Native American	0 (0)
Native Hawaiian/Pacific Islander	2 (2)
White	39 (45)
More than one	4 (5)
Not reported/unknown	2 (2)
Insurance status
Private	57 (74)
Government	20 (26)
State of residence
California	78 (90)
Outside of California	9 (10)
Prenatal phenotype
One or more structural anomalies, no effusion	45 (52)
One or more fetal effusions with one or more structural anomalies	22 (25)
One or more fetal effusions with no structural anomalies	20 (23)
Pregnancy outcome
Live birth	72 (83)
Stillbirth or miscarriage	3 (3)
Termination	12 (14)

Note: Data presented as n (%) or median (interquartile range).

Of the remaining 243 cases, 87 (36%) had postnatal follow-up available. The majority of cases without postnatal follow-up available did not result in a live birth. Reasons for exclusion are outlined in Figure 1. Among the 87 cases with follow-up, most (n = 56, 64%) had new findings identified after birth. The affected organ systems for these new findings were neurologic (n = 26), gastrointestinal (n = 14), craniofacial (n = 11), hematologic (n = 8), and others. Neonatal neurologic findings included hypotonia, encephalopathy, stroke, hearing loss, developmental delay, and speech delay. New postnatal findings by organ system are presented in Table 2.

TABLE 2 |.

Frequency of new findings by organ system on postnatal assessment after negative or inconclusive prenatal exome sequencing for fetal anomalies.

Organ system	Study participants n = 87
Neurologic	26 (30)
Gastrointestinal	14 (16)
Craniofacial	11 (13)
Hematologic	8 (9)
Cardiac	7 (8)
Skeletal	7 (8)
Renal/Genitourinary	5 (6)
Pulmonary	4 (5)
Endocrine	3 (4)
Lymphatic	3 (4)
Immunologic	2 (2)
Neoplastic	1 (1)

Note: Data presented as no. (%) 24 individuals had new findings in more than one organ system.

The first reanalysis, based on the prenatal phenotype alone without incorporating new findings in order to account for the passage of time and advances in the scientific literature, did not result in reclassification in any of the 243 cases. ES reanalysis incorporating actual additional postnatal findings resulted in the identification of one or more pathogenic or likely pathogenic variants in 4 cases. This represented an incremental yield of 2% (4/243) among all previously unsolved cases, and an incremental yield of 7% among the cases with new findings identified in the postnatal period (4/56) (Table 3). Three of the cases with newly detected genetic variants resulted in live birth, while one resulted in pregnancy termination. In one of these cases, the genetic variant identified (homozygous OTOA c.1880+1G>A) was associated with non-syndromic hearing loss and likely represented an incidental finding unrelated to the prenatal phenotype of congenital diaphragmatic hernia. The other three cases were thought to explain the prenatal findings.

TABLE 3 |.

Detailed information for newly identified genetic diseases using actual additional postnatal phenotypes.

Prenatal presentation	Additional postnatal features	Gene involved	Associated disorder	Variant(s)	Zygosity	Inheritance
Nonimmune hydrops fetalis, AV canal defect	Microcystic renal disease identified on autopsy	NPHP3 (NM_153240.5)	Meckel syndrome 7, nephronophthisis 3, renal-hepatic pancreatic dysplasia 1	c.2694-2_2694-1del (splice site) (P)	Heterozygous	Maternal
				c.3156dup (p.Ser1053fs) (P)	Heterozygous	Paternal
Nonimmune hydrops fetalis, macrosomia	Atypical facial features, arthrogryposis, pectus excavatum	ACTA1 (NM_001100.4)	Congenital myopathy 2	c.521C>G (p.Pro174Arg) (LP)	Heterozygous	de novo
Pericardial effusion, fetal anemia	Atypical facial features, developmental delay	ANKRD11 (NM_013275.6)	KBG syndrome	c.4087C>T (p.Arg1363Ter) (P)	Heterozygous	de novo
Congenital diaphragmatic hernia	Bilateral sensorineural hearing loss, developmental delay, chronic constipation	OTOA (NM_144672.4)	Autosomal recessive deafness	c.1880+1G>A (splice site) (LP)	Hemizygous (in trans with deletion)	Paternal

Abbreviations: LP, likely pathogenic; P, pathogenic.

In 2 cases that resulted in live births, postnatal evaluation suggested a genetic diagnosis for which testing aside from ES is indicated. One case presented with hypotonia and methylation testing identified Prader-Willi syndrome. The second case presented with different facial features, anemia, thrombocytopenia, and coagulopathy, and urine glycosaminoglycans followed by deletion/duplication testing confirmed mucopolysaccharidosis VII. At the time of initial ES, copy number variants were not detected on the platform used, so the homozygous deletions leading to a diagnosis of mucopolysaccharidosis were missed. Including these additional 2 cases in which other genetic testing modalities were performed based on postnatal findings, a genetic diagnosis was made in 11% of those with new findings in the postnatal period (6/56).

Finally, including seven neurodevelopmental HPO terms for phenotypes that cannot be detected in utero led to reclassification in seven cases (P/LP variants in three cases, and a suspicious VUS in an additional four cases). Two of these cases were also captured by inclusion of postnatal findings (ANRKD11 and OTOA). This corresponds to an incremental yield of 3% (7/243), including highly suspicious variants of uncertain significance as well as incidental findings unrelated to the prenatal phenotype (Table 4). However, this approach also led to the deprioritization of genetic variants and, thus, failure to identify previously detected variants in four cases due to a lack of overlap with the updated phenotype (Table 4).

TABLE 4 |.

Detailed information for genetic diseases identified and missed with inclusion of hypothetical neurodevelopmental phenotypes on exome sequencing analysis.

Prenatal presentation	Gene involved	Associated disorder	Variant(s) (classifcation)	Zygosity	Inheritance
Identified only with inclusion of potentially missed phenotypes not detected in uteroa
Nonimmune hydrops fetalis, polyhydramnios	CTNNB1 (NM_001904.4)	CTNNB1 neurodevelopmental disorder	c.542del (p.Lys181fs) (P)	Heterozygous	Unknown (father unavailable)
Pericardial effusion, fetal anemia	ANKRD11 (NM_013275.6)	KBG syndrome	c.4087C>T (p.Arg1363Ter) (P)	Heterozygous	de novo
Congenital diaphragmatic hernia	OTOA (NM_144672.4)	Autosomal recessive deafness	c.1880+1G>A (splice site) (LP)	Hemizygous (in trans with deletion)	Paternal
Omphalocele, aortic coarctation	MAST1 (NM_014975.2)	Mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations	c.2296T>C (p.Trp766Arg) (VUS)	Heterozygous	de novo
Enlarged nuchal translucency, macrosomia	WDFY3 (NM_014991.4)	Autosomal dominant primary microcephaly 18	c.8474T>G (p.Phe2825Cys) (VUS)	Heterozygous	de novo
Lower urinary tract obstruction, echogenic kidney, single umbilical artery	ATRX (NM_000489.6)	Alpha-thalassemia/impaired intellectual development syndrome	c.6241A>G (p.Ille2081Val) (VUS)	Hemizygous	Maternal
Intrahemispheric cyst, ventriculomegaly, hypoplastic corpus collosum	KMT2A (NM_001197104.1)	Wiedemann-Steiner syndrome	c.11756G>A (p.Arg3919Gln) (VUS)	Heterozygous	de novo
Identified only using the actual prenatal phenotype
Cystic hygroma with progression to hydrops fetalis, echogenic kidneys	FOXC2 (NM_005251.3)	Lymphedema-distichiasis syndrome	c.1027G>T (p.Glu343Ter) (LP)	Heterozygous	Maternal
Hypoplastic long bones, abnormal vertebrae	FLNB (NM_001457.4)	Atelosteogenesis	c.4943T>A (p.Val1648Glu) (LP)	Heterozygous	de novo
Hypoplastic and demineralized long bones	DYNC2H1 (NM_001377.3)	Short-rib thoracic dysplasia 3 with or without polydactyly	c.4267C>T p.Arg1423Cys (LP)	Compound heterozygous	Maternal
			c.4718A>C p.Gln1573Pro (VUS)		Paternal
Nonimmune hydrops fetalis, tetralogy of Fallot	MYH6 (NM_002471.4)	Cardiomyopathy	c.4150C>T (p.Arg1384Trp) (VUS)	Heterozygous	de novo

Abbreviations: LP, likely pathogenic; P, pathogenic; VUS, variant of uncertain significance.

^aIncludes seizure, hypotonia, hearing impairment, visual impairment, autistic behavior, intellectual disability, and neurodevelopmental delay.

4 |. Comment

4.1 |. Principal Findings

In this secondary analysis of a prospective cohort study, new phenotypic features were identified after birth in 64% of cases with fetal anomalies and negative or inconclusive prenatal ES, with neurologic features being the most common. Among the cases with new phenotypic features identified after birth, the incremental yield of ES was 7%. In the absence of new findings, in contrast, reanalysis after birth did not increase the diagnostic yield of ES, although it is likely that this would change with a greater time interval that allows for improved knowledge of genetic variants leading to disease. Finally, adding potentially undetectable phenotypic features, such as neurodevelopmental findings, to the prenatal analysis in an attempt to identify important variants did reveal some pathogenic findings but also led to other variants not being reported. Had these been included in the initial analysis, a diagnosis likely would have been missed.

4.2 |. Results in the Context of What Is Known

The high frequency of additional findings in the neonates is consistent with previously reported studies and highlights the limitations of prenatal imaging [7–12, 27]. The incremental yield of ES after reanalysis using postnatal findings was significantly lower than in a previously reported study, which suggested an incremental yield of 20% [21]. There are likely several reasons for this. First, the prior study included only 20 patients, as compared to 86 patients who had reanalysis in this report. In our study, the original ES as well as the reanalyses were performed at the same in-house lab, whereas in the prior study, initial ES was performed at a commercial lab and follow up performed at the in-house lab. It is possible that differences in variant interpretation, reporting criteria, and perhaps most importantly, access to clinical information may have informed different yields between the two labs. Additionally, for the current analysis, ES utilizing only the prenatal phenotype was followed by immediate reanalysis incorporating the additional postnatal findings. In the prior study, there was some time delay, typically several months, between the two analyses, such that updates to the medical literature and changes to variant classification may have also informed differences in the yield. Other studies assessing the yield of ES reanalysis in the pediatric and adult settings have shown significant impact of the passage of time and greater understanding of genetic disease, with reported yields of 10% or more due primarily to these factors rather than development of new phenotypes with advancing age of the individual [22–24, 27]. Taken together, these differences in study design allowed our study to focus primarily on the incremental yield of postnatal ES reanalysis specifically related to additional phenotypic features identified in the postnatal setting.

4.3 |. Clinical Implications

While it is well known that additional findings in the neonate after birth can sometimes be identified, the high frequency in this study—64%—is important for patient counseling. In some cases, these phenotypes may have also been appreciable prenatally. For example, hypotonia may be suggested by evaluating fetal movement on ultrasound, soliciting perception of fetal movement from the pregnant person, and assessing for polyhydramnios that can represent impaired swallowing. While other additional phenotypes such as hearing loss are simply not possible to detect in utero at present, the high frequency of additional postnatal features we identified highlights the importance of deep prenatal phenotyping to uncover clues to an underlying genetic diagnosis.

The 7% incremental yield of ES reanalysis using additional postnatal phenotypes suggests that this step of reanalyzing the prenatal ES data can be important when new findings are detected in the infant, and highlights the importance of careful postnatal phenotyping. Identifying a genetic diagnosis is helpful not only to the family but also to the clinical team caring for the infant, and a diagnosis informs recurrence risk along with many decisions about future pregnancies. In the United States, most commercial ES laboratories will provide one reanalysis without charge to the patient, and reanalysis does not require resequencing or a new DNA sample.

The use of potential, but not detectable, phenotypes in the initial ES analysis is more controversial and is not routine. However, many neurodevelopmental phenotypes cannot be appreciated prenatally, and many may not be appreciated until the child is several years old. Given the importance of making a diagnosis prenatally, bioinformaticians may consider including these potential neurodevelopmental HPO terms in the ranking process in order to capture potentially relevant gene variants that may otherwise be missed. However, given that this approach also resulted in the exclusion of some relevant variants that were not associated with neurodevelopmental phenotypes, close collaboration with the clinical team should be used if this is considered. A stepwise approach should be considered, first incorporating only the known features and, if not diagnostic, a second analysis including potentially undetectable features could be performed. Further research is needed to understand the potential utility of this approach, including long-term follow-up to determine how often the undetected prenatal phenotypes are ultimately identified in the infant or child and whether or not inclusion of these HPO terms led to more accurate prenatal diagnosis.

Finally, it is important to highlight that ES cannot identify all disease-causing genetic variants, which was the case for two infants in our cohort. Clinicians should be aware of the limitations of ES and consider whether alternative testing strategies may be appropriate based on the differential diagnosis.

4.4 |. Research Implications

Ongoing research should focus on improving phenotyping in the prenatal setting and exploring additional modalities for deep phenotyping such as fetal MRI. Ongoing work assessing the unique fetal presentations of genetic diseases will further improve our prenatal phenotyping, incorporating not only structural abnormalities but also features such as placental appearance, amniotic fluid volume, growth trajectories, and fetal movement and tone. Further work exploring bioinformatics pipelines and the inclusion of potential but undetectable features is needed before recommending this approach. Finally, further research is needed to understand the time interval that is recommended for reanalysis of prenatal ES data when new postnatal features are not identified, allowing for sufficient time and increased medical knowledge that might then uncover a previously undiagnosed genetic disease.

4.5 |. Strengths and Limitations

There are multiple strengths of this study. This represents a large cohort of patients undergoing ES for fetal anomalies, and a diverse patient population with regard to the indications for testing and patient characteristics, including self-identified race/ethnicity. Our study design allowed us to utilize many important details of the prenatal and postnatal clinical phenotype and to maximize consistency by performing all analyses through the same laboratory. The study also has limitations. Postnatal assessment was at the discretion of the pediatric care team rather than protocolized, such that participants did not receive standardized imaging or laboratory evaluations. In some cases, postnatal evaluation was limited to the initial weeks after birth, and some postnatal outcomes such as developmental delay or seizures may not be detected until later in infancy or childhood. Finally, while many cases did not have follow up postnatal information available, most of these resulted in a fetal demise or termination of pregnancy, and thus may have been less likely than surviving cases to contribute new phenotypes such as seizures that were missed in utero.

5 |. Conclusions

In conclusion, prenatal phenotyping in the setting of fetal anomalies is often incomplete, such that new postnatal features are identified in nearly 2/3 of cases. A majority of these new findings are neurologic, and reanalysis with incorporation of these new findings can meaningfully increase the diagnostic yield of prenatal ES. Further research to optimize prenatal phenotyping, understand if undetectable prenatal phenotypes should be included in analyses, and clarify the most appropriate time intervals for reanalyses in the absence of new postnatal findings will be key for advancing prenatal diagnosis using ES.

Highlights.

• What is already known?

  • Detailed phenotype is key for accurate interpretation of exome sequencing data. Many features of genetic disease cannot be detected prenatally, but there is limited data to understand how exome sequencing reanalysis utilizing postnatal phenotypes impacts diagnostic yield.
• What does this study add?

  • Postnatal reanalysis utilizing additional anomalies identified after birth increased the yield of prenatal exome sequencing by 7%.
  • Analyses considering potentially missed phenotypes in utero increased diagnostic yield by 3% but also resulted in the exclusion of initially detected variants. This study highlights the limitations of prenatal phenotyping, and how methods such as postnatal reanalysis or inclusion of potentially missed phenotypes in utero may improve diagnostic yield.

Data Availability Statement

Deidentified data will be made available upon request.