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

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


| INTRODUCTION
Fetal congenital anomalies are detected in 2% to 5% of pregnancies by routine ultrasound. 1,2 The occurrence of these anomalies can cause significant distress for the expecting parents and have a major impact on perinatal mortality and long-term morbidity. 3,4 The underlying etiology of these anomalies is diverse and includes genetic factors. Current routine prenatal genetic testing strategies often include molecular rapid aneuploidy testing (RAD) and chromosomal microarray analysis (CMA), designed to detect numerical and structural chromosome abnormalities, respectively, which show a combined diagnostic yield of approximately 40%. 5,6 However, this means that for the large majority of cases, the underlying cause of the identified congenital anomalies remains unknown. The latter is most prominent for congenital anomalies that are a result of monogenic disorders caused by point mutations and/or small insertion deletion events. Whole exome sequencing (WES) in a postnatal setting has shown to increase diagnostic yield for genetically heterogeneous (monogenic) disorders to up to 58%, depending on the clinical preselection of the cohort and subset(s) of genes analyzed. [7][8][9] The turn-around times (TATs) of routine WES, being several months, has so far always hampered this assay to be implemented in routine prenatal diagnostics. A decrease of this TAT may help to diagnose those fetuses with congenital anomalies in which the genetic diagnosis remained elusive using routine prenatal procedures.
Rapid whole exome sequencing (rWES), with TATs varying from 4 days to several weeks, has been shown to contribute to clinical decision making in pediatric and neonatal critical care. [10][11][12][13] It is very likely that rWES has the same potential for prenatal clinical decision making. In a recent study on the use of rWES for fetuses presenting with skeletal anomalies, 81% of cases were genetically diagnosed. 14 Although this increase in diagnoses enabled more accurate prediction of pregnancy outcome, providing parents more certainty in prenatal decision making, the contribution of skeletal anomalies only accounts for around 30% of all fetal congenital anomalies. 15,16 The efficacy of adopting rWES as a first tier test for the full spectrum of fetal congenital anomalies detected during routine ultrasound imaging has also been recently studied in a few pilot studies. [17][18][19][20] The vast majority of these studies focused on the diagnostic yield and TAT as outcome parameters, rather than focus-

| Patient eligibility and selection for prenatal rWES
Since January 2016, rWES has been offered as a routine diagnostic test at the Radboudumc for cases whose medical management could be directly impacted by a genetic diagnosis. For prenatal cases, rWES was offered following the detection of multiple fetal congenital anomalies suggestive of a possible genetic etiology detected by ultrasound imaging in level III academic centers, executed or supervised by Maternal Fetal Medicine specialists. In case of an isolated major anomaly or (multiple) soft markers, 21 rWES was only offered if there was a high suspicion of a genetic cause. A detailed case by case description of the clinical presentation is provided in Appendix S1. Fetal materials derived from a pregnancy that had ended in fetal death, or from a termination of pregnancy (TOP) were not included in this study.

| Informed consent and counseling
Patients received pre-and posttest rWES counseling by a clinical geneticist. Diagnostic informed consent was identical to our routine What's already known about this topic?
• Several pilot studies report on an added value of prenatal rapid whole exome sequencing (rWES), when routine techniques fail to identify a genetic diagnosis in fetuses with congenital anomalies detected by ultrasound imaging.
What does this study add?
• We determined the diagnostic yield for rWES in 54 cases with fetal congenital anomalies detected by ultrasound imaging in pregnancies being 33% and show its impact on clinical decision making.
• Rapid aggregation of prenatal molecular and clinical information into a conclusive diagnosis is challenging and requires cooperation of a dedicated team. postnatal procedure, and comprised of a two-tiered process to limit the chance of uncovering incidental findings. In tier 1, interpretation is focused toward gene variants in dedicated (in silico) disease gene panel(s), which are selected by the clinical geneticist based on the clinical presentation of the fetus. In this study, 12 different in silico disease-gene panels were used, in size ranging between 48 genes for "Fetal Akinesia" and 1158 for "Intellectual Disability" (Table 1, Appendix S1). To allow immediate interpretation of all genes with a known disease phenotype in tier 1, "The Mendeliome Panel" (also referred to as clinical exome) can be requested, containing 3605 genes with wellestablished genotype-phenotype associations. In tier 2, generally only performed after a negative (or possible) result for the in silico disease gene panel(s), interpretation is extended to the Mendeliome as well as all other protein-coding genes with currently unknown disease-relationships. In this study, tier 2 analysis was performed for 12 cases, of whom eight cases already had had a negative Mendeliome analysis in tier 1 (Appendix S1). An overview of all genes included in the in silico disease-gene panels, as well as our policy for disclosing of incidental findings, can be found online in https://order.radboudumc.nl/en/ genetics/rapid-exome-sequencing. Other: anomalies such as congenital diaphragmatic hernia or fetal akinesia.

| rWES
c Analytical details per case are listed in Appendix S1. Abbreviation: rWES, rapid whole exome sequencing.

| Variant classification
Classification of variant pathogenicity for single nucleotide variants (SNVs) or copy number variations (CNVs) was based on European guidelines. 23,24 If insufficient clinical information, or potential discrepancy between molecular and clinical causality was noted, variants were discussed in a multidisciplinary team, consisting of a clinical laboratory geneticist, a clinical geneticist and a fetal maternal specialist. Overall, this resulted in reporting of (likely) pathogenic variants (class 4 and 5) related to the fetal phenotype, as well as the reporting of variants of unknown significance (VUS; class 3) if the multidisciplinary team concluded that the VUS was likely to contribute to the fetal phenotype. additional data related to, amongst others, the reason for the rWES request, the pregnancy outcome, and management adaptations (Appendix S1). Impact was defined as to influence the decision:

| Primary end points
• To opt for a TOP before 24 weeks of gestation, which-in the Netherlands-is the legal limit for a TOP; and/or • To request for a late TOP after 24 weeks of pregnancy, which-in the Netherlands-is only allowed when a severe fetal outcome is imminent; and/or • To continue the pregnancy; and/or • To adjust peripartum management.
To assess the representativeness of the responses for the total cohort, we compared the diagnostic yield in responders and nonresponders using a Fisher's exact test.

| Cohort characteristics
From May 2016 to November 2018, we received 54 requests for prenatal rWES after the identification of fetal congenital anomalies. The median gestational age was 21 weeks and 5 days (range: 16 + 5 to 38 + 1 weeks) and the median maternal age was 30 years (range: 20-38 years).
The most common clinical indications were skeletal dysplasia (n = 20; 37%), multiple major fetal congenital anomalies (n = 17; 31%) and intracerebral structural anomalies (n = 7; 13%). Ten cases presented with other congenital anomalies, such as congenital diaphragmatic hernia, fetal akinesia and ambiguous genitalia. An overview of the cohort characteristics is provided in

| Diagnostic yield of rWES
A conclusive diagnosis was obtained in 18 of the 54 cases (33%) ( Figure 1; Table 2; Appendix S1). The highest diagnostic yield was obtained for skeletal dysplasia (61%, n = 11/18), followed by multiple major fetal congenital anomalies (22%, n = 4/18) and intracerebral structural anomalies (17%, n = 3/18; Figure 1). In the ten cases with other congenital anomalies, no molecular diagnoses were made ( Figure 1). Among the 18 diagnoses, autosomal dominant (AD) disorders accounted for 72% (n = 13), of which the majority was caused by a de novo variant (n = 11/13; 85%). Autosomal recessive (AR) disorders were diagnosed in the remaining five (28%). Interestingly, in case #7, a homozygous pathogenic variant was detected in ERCC5, which had resulted from maternal segmental uniparental isodisomy of the distal part of chromosome 13q. The isodisomic segment was confirmed after re-analysis of the CMA data, which was performed prior to the rWES and was reported as normal.
The main reason to request rWES (n = 21; 57%) was to support clinical decision making before 24 weeks of gestation ( Figure 2   Abbreviations: AD, autosomal dominant; AR, Autosomal recessive; MFCA, multiple fetal congenital anomalies; rWES, rapid whole exome sequencing. In 6 of 37 cases (16%), rWES was requested in relation to a late TOP ( Figure 2; Appendix S1). In three of them (#2, #15 and #22), rWES results had impact on this decision. For case #15, late TOP was initially rejected, but rWES revealed a severe skeletal dysplasia in the spectrum of Antley-Bixler syndrome or Pfeiffer syndrome, which led to a review of the initial request. In the other two cases (#2 and 22), the lack of a (severe) genetic diagnosis, however, provided an additional argument for the parents to continue the pregnancy. In the other three cases, rWES results did not impact decision making. In two of them (#22 and #28) no diagnosis was obtained and in the third one (#17), late TOP was already initiated abroad because of the severity of the identified skeletal anomalies prior to the return of the positive rWES results for Kniest dysplasia.
Lastly, in the remaining 10 cases the rWES was requested to guide peripartum rather than prenatal management (Figure 2; Appendix S1). In four cases, the rWES result was unable to have an impact on peripartum management, either because of the absence of a genetic diagnosis (n = 2), or fetal loss (n = 2) before the rWES report returned. Also no genetic cause was identified in the latter two cases.

| DISCUSSION
In this study, we aimed to determine the use of rWES in ongoing pregnancies of fetuses with congenital anomalies detected by ultrasound imaging. Using our diagnostic rWES set-up, we identified the underlying genetic cause in 33% of cases with a median TAT of 10 days. In 68% of the cases, the rWES result contributed to parental and clinical decision making, even when no genetic cause could be identified.
The diagnostic yield of 33% by rWES found in our study is comparable to other studies reporting a diagnostic yield between 18% and 40% when applied to all prenatal abnormalities and without preselecting for certain phenotypes. [17][18][19][20] However, if such pre-selecting is performed, even higher diagnostic yields can be obtained, as was shown for fetuses presenting with skeletal anomalies, in whom a diagnosis was reached in 13/16 cases (81%). 14 Whereas this percentage may seem to be higher than the 55% (11/20)  presentation.
One of the most important reasons for the introduction of rWES in a prenatal setting is the possibility to impact prenatal and peripartum clinical decision making. Previously, rWES has already proven to impact clinical decision making for critically ill children, but supportive evidence for prenatal cases is still limited. [10][11][12][13][14]17,28 Needless to say, prenatal counseling guided by the severity of congenital anomalies alone is often sufficient for parents to opt for TOP, as was also noted for 6 of 37 cases for whom the impact on clinical decision making was determined. In this study, we, however, now also show that rWES outcomes strengthened parental and clinical decision making in 68%, mostly because of the more accurate predictions on the prognosis for parents after the identification of a genetic disorder, and precision medicine for peripartum management. Importantly, impact was obtained for 44% of the cases in whom no causative mutation(s) could be identified. The fact that also a negative rWES has impact on parental and clinical decision making indicates that the efficacy of prenatal rWES should be evaluated by more variables than diagnostic yield alone. This is particularly the case for the clinical subcohort in this study defined as "Others": in this subcohort no diagnoses were made, which is significantly lower than in the other subcohorts. The latter may suggest that this "Other" cohort does not benefit from rWES.
Yet, impact on clinical decision making was imminent in four of six cases for whom the impact was assessed: for the parents of these cases, it was the relief that most monogenic disorders were largely excluded reinforcing their decision to continue the pregnancy.
Recently, two large prospective studies for prenatal rWES in unselected cohorts of fetuses with structural anomalies also showed that rWES can indeed add clinically relevant information to assist current management of a pregnancy, but also highlighted that careful consideration should be given to case selection to maximize clinical usefulness. 29,30 Based on our experience, the majority of eligible patients were included in our study, however, we did not investigate how often parents did not give consent for rWES. We were able to report trends when comparing different subgroups of fetal congenital anomalies and based on these small numbers we can suggest that performing rWES in skeletal dysplasia, intracerebral structural anomalies and multiple major fetal congenital anomalies are beneficial. This seems in line with the results from Lord et al. en Petrovski et al, 29,30 and thus that our results contribute to improved patient selection and enhance the clinical utility of rWES for prenatal diagnostics.

| CONCLUSION
We performed rWES in 54 cases of pregnancies in which fetal congenital anomalies by ultrasound imaging were detected, with a median TAT of 10 days. The diagnostic yield in this cohort was 33%. Genetic diagnoses were identified in fetuses who presented with skeletal dysplasia, intracerebral structural anomalies and/or multiple major fetal congenital anomalies. In the majority of cases (68%), the rWES result contributed to clinical decision making, even when no genetic cause could be identified.