Prenatal whole exome sequencing (WES) in the fetus with isolated or multiple structural anomalies has increased potential to identify a genetic diagnosis above standard genetic testing methods. Fetal DNA extracted from chorionic villi or amniocytes is currently required, but in time WES will be possible on cell-free DNA in maternal blood. Prenatal WES can increase understanding of the genetic etiology of congenital malformation and enable individualized pregnancy management. WES and whole genome sequencing are increasingly being used to investigate perinatal loss and enable genomic-informed treatment in critically ill newborns with undiagnosed genetic disease. Prenatal WES presents additional technical, ethical, and resource-related challenges, and professional guidance alongside patient views and discussions on ethics are emerging. This chapter summarizes the evidence for WES in the fetus with ultrasound evident congenital anomalies, as well as the available literature on patient and provider views, ethical implications, and the development of sequencing applications in the perinatal and neonatal context, alongside the prenatal utility of this developing technology.
KeywordsExome sequencing, Fetal structural anomaly, Prenatal genetic diagnosis, WES, WGS, TEC, Clinical exome, NGS, Secondary findings, Incidental findings, PAGE Study
Incidence of Fetal Structural Anomalies
Ultrasound detects structural anomalies in the fetus in up to 3% of pregnancies and those are associated with a significant global burden of disease . Pregnancy outcome is variable depending on the number and type of abnormalities, and the underlying genetic etiology . After identification of a fetal structural anomaly, genetic testing is available for parents. Recent advances in molecular genetics enable increasingly detailed genetic testing leading to an accurate prenatal diagnosis in more cases . This prenatal information on the genetic causes of fetal anomalies is relevant for prognosis for the affected fetus and recurrence risk for subsequent pregnancies . Such prenatal genetic diagnosis is of significant value to parents to inform decisions on continuation or termination and for their future reproductive planning, and for caregivers for optimal perinatal management . Within these decisions are strong moral and ethical considerations.
Prenatal Genetic Testing Methods
In the United Kingdom (UK), prenatal genetic diagnosis is available for individuals with an increased risk of aneuploidy based on history or routine antenatal screening results, or following a diagnosis of fetal ultrasound anomaly. Such testing involves increasingly routine quantitative-fluorescence polymerase chain reaction (QF-PCR) to identify the most common aneuploidies, followed by chromosomal microarray (CMA) to detect copy number variations (CNVs) and microscopic insertions/deletions (indels) in cases with normal QF-PCR . Indels are small (typically < 5bases) alterations in DNA sequence and differ from larger CNVs classified as microdeletions and microduplications. Some genetic laboratories continue to use fluorescence in situ hybridization and G-banded (conventional) karyotyping to determine structural chromosomal rearrangements. Targeted exome capture (TEC) also called clinical exome sequencing of specific exonic regions of interest, and whole exome sequencing (WES) of all exonic regions, are used to detect single nucleotide variants (SNVs) associated with various monogenic disorders, but these modalities have limited potential to identify CNVs . TEC enables filtering against a prespecified gene list or panel with known disease association such as Noonan syndrome. WES, on the other hand, allows for all exonic regions to be filtered according to an expanded list (typically thousands) of potentially relevant genes to look for alterations that may be associated with multiple genetic disorders. The exome encompasses only 1.5% of the genome but harbors 85% of the variants that cause single-gene disorders. Whole genome sequencing (WGS), in theory more powerful than WES, is also beginning to be used, as tools for interpretation improve and data sources are developed. It permits genome-wide (entire exonic and intronic) filtering against a comprehensive list of potentially relevant genes to identify all types of disease-causing mutations. More detailed information relating to the various prenatal genetic testing methods can be found in Chapters 2 and 3 .
Next-Generation Sequencing Applications
Next-generation Sequencing (NGS) applications (TEC, WES, and WGS) are broadening the scope of prenatal diagnosis to identify the genetic etiology of sporadic and inherited disease and are changing current practice . Sequencing analysis of trio DNA (fetus and both parents) aids the assignment of pathogenicity and improves timeliness of interpretation. Fetal or placental DNA is currently obtained by amniocentesis or chorionic villus sampling, but in time testing will be possible on placental cell-free DNA (cfDNA) in maternal blood . WES identifies SNVs and small indels and captures the regions of the genome that encode proteins . As a technique, it is useful for the diagnosis of known genetic disease and for the discovery of novel disorder genes . It is increasingly being used to diagnose rare Mendelian conditions in fetuses with a single major anomaly or anomalies in multiple organ systems, when standard tests results are normal .
The use of WES in prenatal diagnosis enables more accurate prospective risk assessment, focused genetic counseling, and personalized pregnancy care. For future pregnancies reproductive genetic counseling, preventative-assisted reproduction approaches, such as preimplantation genetic diagnosis, or invasive or noninvasive prenatal diagnosis will help the family to avoid recurrence . Prenatal NGS can present challenges around the interpretation of results. Not all genomic alterations have been linked to a phenotype and the significance of some findings may be uncertain. Challenges of all genetic tests, but of CMA, WES, and WGS in particular, are “incidental findings” (ICFs), “variants of uncertain significance” (VUS) and susceptibility loci. The prenatal detection of these types of findings may have significant emotional effects for parents and their relatives and further complicate prenatal decision-making.
Cell-Free DNA-Based Noninvasive Prenatal Testing and Noninvasive Prenatal Diagnosis
The development of massively parallel sequencing has enabled extremely accurate detection of common chromosomal aneuploidies, i.e., trisomy 13, 18, and 21 in cfDNA within maternal plasma . Cell-free DNA-based NIPT (cfDNA NIPT) is widely available for this purpose, but the technology is also increasingly being used to identify indels and various single-gene alterations for Noninvasive Prenatal Diagnosis (NIPD). Uptake of cfDNA NIPT for aneuploidy has increased considerably due to the improved accuracy of the technology when compared with conventional screening methods, leading to significant reductions in the number of invasive diagnostic procedures alongside a concomitant decrease in the procedure-related pregnancy loss rate . The analysis process is technically challenging, however, and false-positive results are inevitable because cfDNA originates from the cytotrophoblast and may be subject to confined placental mosaicism. Confirmatory testing on amniotic fluid cells or cytotrophoblast and mesenchymal culture of chorionic villi is required when cfDNA-based prenatal testing indicates an aneuploidy or a CNV. NIPD for single-gene disorders is available for parents with recurrence risks—a concept explored further in Chapter 9 . The potential for diagnostic sequencing analysis using cfDNA samples is currently being explored in a research capacity as part of the PAGE (prenatal assessment of genomes and exomes) Study and final results are pending. As NGS technology improves and the cost of sequencing falls, it is likely that analysis of cfDNA will play an increasingly important role not only in prenatal screening but also in prenatal diagnosis in cases of ultrasound-detected congenital anomalies .
NGS in Perinatal Loss
Various sequencing approaches have recently been used to investigate perinatal loss . Armes et al. performed trio WGS in 16 cases of fetal, perinatal, and early neonatal death where postmortem information was available . In all cases conventional cytogenetic and single nucleotide polymorphism (SNP) array analysis was normal, and in some cases targeted gene testing was undertaken and reported as negative. A likely genetic diagnosis was made in 2 cases. In the first case compound heterozygous variants (a paternally inherited splice-site variant, and a maternally inherited missense variant) in RYR1 were detected and assessed as pathogenic and causative of RYR1-associated congenital myopathy. This was in keeping with the phenotype of arthrogryposis multiplex congenita with fetal akinesia deformation sequence and pulmonary hypoplasia. The second case involved a heterozygous, de novo, splice-site variant in COL2A1 associated with Kniest dysplasia, also in keeping with the phenotype of respiratory failure secondary to Pierre Robin sequence and skeletal dysplasia (Kniest type). These data demonstrate that WGS is useful for genetic diagnosis but the alterations described could also have been detected with WES, a likely more cost-effective approach with a potentially quicker turnaround time. Shebab et al. similarly used WGS to identify the genetic etiology of recurrent male intrauterine fetal death (~ 19) in a large multigenerational pedigree . Sequence analysis of 5 healthy obligatory carrier females and an unaffected male offspring demonstrated an X-linked frame-shift mutation in FOXP3 associated with IPEX syndrome in all of the tested females and absent in the tested male. In DNA extracted from paraffin embedded tissue derived from an intrauterine demised male fetus in this family the same FOXP3 variant was confirmed. This finding was consistent with the prenatal phenotype of hydrops fetalis, and although in this case the variant was identified on WGS, it could have been detected on WES as well. A WES approach was employed by Shamseldin et al. in a cohort of 44 families with a history of intrauterine fetal death, nonimmune hydrops fetalis, or congenital malformation resulting in termination of pregnancy . All cases were reported as having normal chromosome analysis although SNP array analysis was not performed. Where fetal DNA was available solo WES was carried out, otherwise duo WES on both parents was carried out to identify shared heterozygous variants under an autosomal recessive model of inheritance. The authors report that pathogenic or likely pathogenic variants were identified in 22 families (50%). Given that pathogenic findings in the fetus could not be confirmed as having arisen de novo (as trio analysis was not performed), and as it was not possible to directly confirm candidate variants in the fetus after duo-exome analysis in the parents, it is not possible to accurately determine the diagnostic yield of WES in this cohort. In addition, postmortem information was not available in any case and thus the lack of a genotype–phenotype correlation is a limitation of this study. Yates et al. carried out WES in 84 deceased fetuses with ultrasound diagnosed anomalies: 29 cases as solo fetal analysis, 4 maternal–fetal duos, 45 fetal–parent trios, and 6 fetal–parent plus sibling quads . Diagnostic yield in this cohort was 20%, 24% for trio cases, and 14% for fetus-only cases. There was postmortem information available for some cases to inform on genotype–phenotype correlation, and for other cases the fetal phenotype was deducted from prenatal ultrasound information. Research to evaluate WES in cases of perinatal mortality as part of the PAGE Study is currently underway. A retrospective cohort of ~ 100 fetus/parental trios with normal QF-PCR/CMA/NIPT results will undergo WES to investigate the potential diagnostic yield of this approach. DNA extracted from postmortem fetal tissue will be sequenced alongside parental DNA extracted from blood or saliva samples. Preliminary data indicates that WES has the potential to determine the underlying genetic etiology in approximately 30% of perinatal mortality cases above standard cytogenetic methods. The results of this research are expected toward the end of 2018.
NGS in Critically Ill Newborns
In critically ill newborns most commonly NGS panel sequencing (also called clinical exome sequencing or TEC) is used. This technique focuses on specific diseases or phenotypes to identify disease-causing gene variants. The technique enables simultaneous analysis of hundreds of genes with comprehensive coverage for defined phenotypes such as cardiac defects, skeletal dysplasias, mitochondrial disorders, and Noonan syndrome. Disease-focused analysis is generally less expensive than WES or WGS and can be used alongside other technologies such as CMA, to identify alterations, e.g., CNVs that are more challenging to detect with some NGS methods . The turnaround time for panel analysis can be prolonged depending on the need for sample send-away to specialist national/international laboratories, delaying the time to diagnosis with potential implications for clinical management. Studies are currently underway to compare cost and time to diagnosis between traditional diagnostic pathways and “whole exome or whole genome sequencing first.” Turnaround times for TEC and WES are becoming shorter: a mean turnaround time of 13 days was recently reported . These techniques will therefore likely become standard practice for clinical diagnosis of neonates with rare unknown genetic disorders . A few papers have recently been published . Meng et al. performed clinical exome sequencing on 278 unrelated infants as proband-only (~ 176) or as trio analysis (~ 102) with a median turnaround time of 13 days and reported an overall diagnostic yield of 36.7%. Obtaining a genetic diagnosis in this cohort enabled improved medical management of the affected neonate in 52% of cases, leading the authors to suggest that clinical exome sequencing should be considered as a first-line test for neonates with suspected monogenic disease. WES as first-tier diagnostics in children with congenital or early onset disorders showed potential to achieve high diagnostic yields . Use of trio WES in early diagnostic workup will potentially enable reductions in the use of financial resources and costs related to days of admission and facilitate individualized clinical management.
Prenatal WES: The Potential
WES in patients with suspected Mendelian disease has a diagnostic yield in the order of 25% thus it is likely that WES will also be valuable in prenatal diagnosis . Research on the use of genetic sequencing for prenatal diagnosis is limited but feasibility has been demonstrated in small case series . Pangalos et al. performed WES on fetal DNA targeting 758 genes associated with fetal malformation in 11 ongoing pregnancies all with various anomalies identified on prenatal ultrasound examination . A definitive or highly likely diagnosis was made in 3 (27%) cases: Citrullinemia, Noonan Syndrome, and PROKR2-related Kallmann syndrome. Trio WES analysis in 30 nonaneuploid fetuses and neonates with diverse ultrasound-detected structural anomalies was reported by Carss et al. and showed a diagnostic yield of 10% . All findings were de novo mutations with low recurrence risks in FGFR3, COL1A1, and OFD1. Drury et al. also used a prenatal WES approach in 24 euploid fetuses with abnormal ultrasound findings. They sequenced fetal DNA only in the first 14 cases and performed trio analysis in the 10 subsequent cases . A genetic diagnosis was made in 5 cases (5%): Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman–Sheldon syndrome, and Baraitser–Winter syndrome.
More recently, the potential for trio WES to increase genetic diagnosis in structurally abnormal fetuses, with normal QF-PCR and CMA analysis, has been reported in two large prospective prenatal cohorts . Wapner et al. have reported on 166 parent–fetus trios where WES was carried out following ultrasound detection of varying fetal structural anomalies. This resulted in a genetic diagnosis in 13 fetuses (7.8%) . Fetuses with multiple anomalies affecting different anatomical systems had a higher diagnosis rate (14.9%) than those with isolated anomalies involving a single anatomical system (5%). McMullan et al. have similarly performed trio WES in 406 structurally abnormal fetuses as part of the PAGE Study . They demonstrated an overall diagnostic yield of 6.2% . Here again the highest yield (16%) was reached in the group of fetuses with multisystem disorders. These studies have demonstrated that the application of WES can substantially improve prenatal genetic diagnosis in fetuses with congenital abnormalities identified on ultrasound assessment.
Successful implementation of WES for prenatal diagnosis will require health economic assessment, and clinical utility will among others depend upon the development of comprehensive and rapid analytical and interpretation pipelines . Meaningful results need to be available within a timeframe that allows for timely informing the parents and give the opportunity for altering pregnancy management. Also, a robust system for relating the genetic results to a potential syndrome or disease, the so-called variant calling procedure, needs to be in place . The challenge of prenatal WES will be the integration of sequencing analysis into prenatal diagnostics as part of a responsible and ethical framework for clinical practice .
Prenatal Assessment of Genomes and Exomes Study
The PAGE consortium project funded by the Department of Health, Wellcome Trust, Health Innovation Challenge Fund, is currently recruiting parent/fetus trios across the UK to investigate the use of WES as a diagnostic tool in cases of structural anomaly identified on prenatal ultrasound scan . The study will analyze ~ 1000 trio whole exomes with three primary objectives:
Elucidate the relative contribution of different forms of genetic variation to prenatal structural anomalies
Design cost-effective genome sequencing assays for improved prenatal diagnosis of structural anomalies
Catalyze the adoption, by the National Health Service (NHS), of prenatal diagnostic sequencing through translation of acquired knowledge, rigorous health economic assessment, and establishment of an ethical social science framework for clinical implementation
As a secondary objective, the consortium will also explore the feasibility of targeted sequencing of cfDNA from maternal plasma in a selected series of positive control study samples to promote rapid translation of the technology to noninvasive prenatal diagnostics. Prospective recruitment of cases is currently ongoing with findings expected toward the end of 2018.
Prenatal WES: The Clinical Utility
In the context of rare disease, the time taken to establish a genetic diagnosis can be extremely protracted. This process, sometimes referred to as a “diagnostic odyssey,” is attributed to the heterogeneity of genetic disorders, the diversity of disease manifestations, and a lack of clinical genetic knowledge among some healthcare professionals . In the absence of an accurate diagnosis, it is not always possible for clinicians to develop an appropriate plan of care for the individual concerned; hence opportunities for early intervention and effective treatment may be missed. The availability of comprehensive prenatal diagnostic assessment, including genomic sequence analysis, can help to avoid this delay in postnatal diagnosis and negate the need for prolonged admissions and serial testing and thus reduce associated costs . Prenatal diagnosis of a lethal or severe genetic condition enables parents to make informed choices around termination of pregnancy, pregnancy and delivery management, and immediate clinical management of the neonate . Genetic diagnosis can also enable parents to choose reproductive genetic technologies such as preimplantation genetic diagnosis, or targeted prenatal diagnosis, in a subsequent pregnancy . Finally, genomic-driven prenatal diagnosis has the potential to improve perinatal outcome through fetal treatment, and immediate and long-term clinical management of the neonate as demonstrated by de Koning and Brison . Fetal therapy is further explored in Chapter 20 . In cases where in utero fetal therapy is an option, WES may help select cases that are most likely to benefit from intervention, based on the prognostic value of prenatal genomic analysis . Finally, the ability to perform prenatal WES in fetuses affected with various congenital anomalies is leading to increased understanding of early human development and the multiple prenatal phenotypes, both lethal and nonlethal that may be associated with a given mutation. WES has already, and will likely continue to enable the identification of new genes pivotal for normal human development, and also provide a means to further classify known causative genes associated with fetal structural malformation.
Background : G1P0, age 24 years, no relevant family history, fit and well, referred for fetal medicine opinion at 22 weeks gestation. Ultrasound and MRI findings: 22/40 USS—macrocephaly and associated asymmetry of the cerebral hemispheres, localized lymphangiomata of the lower abdomen, 23/40 MRI—extensive subcutaneous swelling in both fetal flanks and marked enlargement of the left cerebral hemisphere with advanced sulcation, 24,28,32,34 week USS—similar appearances. Normal QF-PCR, chromosomal microarray, and maternal hypermethylation studies for Beckwith–Wiedemann syndrome
Presumptive diagnosis : PROS disorder
Targeted genetic testing : Mutation screen of seven genes associated with fetal overgrowth syndromes on cultured amniocytes identified a pathogenic de novo missense variant (c.1633G > A p.Glu545Lys) in PIK3CA estimated to be present at a level of 36% mosaicism confirmed on Sanger sequencing.
Outcome : Livebirth by LSCS at 36 + 6 weeks. Girl with a birthweight of 3334 g, born in good condition with no resuscitation required. Asymmetrical discoloration and swelling on the skin of the left upper abdomen noted alongside mildly splayed toes bilaterally. Cranial and abdominal USS confirmed left-sided mild hemimegalencephaly and marked multiloculated and heavily septated areas of fluid within the abdomen
Key point : Prenatal ultrasound phenotype enabled prospective diagnosis of PROS disorder which was then confirmed with targeted genetic sequencing of relevant genes associated with fetal overgrowth disorders. This informed the parents and facilitated multidisciplinary perinatal management
Citation : Quinlan-Jones E, Williams D, Bell C, et al. Prenatal detection of PIK3CA-related overgrowth spectrum in cultured amniocytes using long-range PCR and next-generation sequencing. Pediatr Dev Pathol 2017;20(1):54–57.
Prenatal WES: The challenges
From a technical perspective prenatal WES presents various challenges. Currently, invasive testing by CVS or amniocentesis is required to obtain a suitable sample for DNA extraction. This limits the quantity of available material. Maternal cell contamination (MCC) affects the purity of DNA and is estimated to occur in approximately 0.3%–0.5% of amniotic fluid samples , and in 1%–2.5% of CVS specimens . Discarding of the first few milliliters of amniotic fluid is useful to reduce the presence of maternal cells. MCC is also decreased through cell culture by selectively enhancing fetal cell growth . The prenatal clinical pipeline from sample retrieval to reportable results is currently lengthy (approximately 9–12 months) and relevant findings cannot be used to inform decision-making and management of the index pregnancy. Turnaround times can be reduced if variants are filtered according to a defined group of genes related to a specific phenotype, but adopting a targeted approach can miss potentially relevant genes and prevent diagnosis . Trio analysis can expedite interpretation if a de novo variant is detected, or if parental samples are needed to confirm the phase of heterozygous alterations in a single gene . It is also feasible to avoid the need for confirmation with Sanger sequencing with good quality metrics for high confidence true positive variants and adequate sample mix-up controls . Improved genome library preparation protocols amenable to small DNA amounts that utilize direct tissue as a starting material can remove the requirement for lengthy culture times . Furthermore, it is not possible to cover all genomic regions equally, and some regions, e.g., those with high GC content, pseudogenes, and repetitive sequences, may not be covered at all. The accuracy of WES is determined by the depth of coverage, which if too low (generally < 30-fold), will reduce the mutational detection rate. And finally, there is variability between sequencing platforms.
Implications for Counseling
Prospective parents are entitled to pretest counseling that includes the benefits and limitations of WES for genetic diagnosis, the types of findings that may be revealed, the inclusion or exclusion of findings unrelated to the primary indication and variants of uncertain significance (VUS) in the results disclosure, and the strategies for sample and data storage and reanalysis . Given the amount and complexity of information trained healthcare professionals should be responsible for this counseling as well as for returning results . Prenatal testing may not necessarily be the best option in all circumstances, thus genetic counseling that assists parents to make informed choices is essential to ensure that parents have realistic expectations . The aim of post-test counseling is to inform on the significance of the results in terms of diagnosis, prognosis, and management options, and to facilitate the decisions parents can make. It is also geared toward providing information on risk of recurrence and potential ways to avoid recurrence . Depending on the results it may also be necessary to organize carrier testing in family members.
Accurate interpretation and classification of WES findings are complicated and time-consuming aspects of molecular prenatal genetic diagnosis . In the prenatal setting, WES result interpretation requires a multidisciplinary team approach involving clinical scientists and geneticists, genetic counselors, and fetal medicine specialists with expert knowledge in prenatal dysmorphology . Currently, there is no existing comprehensive fetal variant database, or international registry of fetal phenotypes and associated variants, to assist genetic scientists/clinicians with the interpretation of sequence findings. There is a need for a formal curation of prenatally relevant variants given the increasing use of NGS methods in the prenatal space . UK practice guidance for the evaluation of pathogenicity and reporting of sequence variants in clinical molecular genetics have been disseminated by the Association of Clinical Genetic Science . They recommend use of a five-class system which they consider to be essential for the standardization of report wording and follow-up studies. According to these guidelines Sequence findings are classified as follows:
Class 1—clearly not pathogenic, common polymorphism, not reported
Class 2—unlikely to be pathogenic, diagnosis not confirmed molecularly, not reported
Class 3—VUS, uncertain pathogenicity, does not confirm or exclude diagnosis, local team to determine whether to report
Class 4—likely to be pathogenic, consistent with diagnosis, reported
Class 5—clearly pathogenic, predicted to be pathogenic, result confirms diagnosis, reported.
Joint American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology standards and guidance for the interpretation of sequence variants have been formally adopted for use in UK laboratories to assist clinical scientists with classification of sequence variants identified prenatally.
Variants of uncertain significance
WES may lead to the identification of VUS; these are variants with unclear pathogenicity. Their frequency is currently unknown. VUS pose complex counseling challenges prenatally as well as postnatally . When the multidisciplinary team decides to disclose the VUS to the patient, they may experience confusion and anxiety, and their decisions relating to the pregnancy become more complex especially if the information is unexpected. Parental anxiety may be further compounded by the need to initiate additional testing and follow-up. This impacts on the healthcare system as well . Appropriate pre- and post-test counseling that addresses the various implications of identifying VUS is crucial to reduce this negative emotional impact on parents who choose to have WES for prenatal diagnosis. Although parents are generally keen to have access to as much information as possible about the genetics of their fetus, the majority feel cautious about receiving findings that have an uncertain meaning . One of the options for parents is to receive VUS at a later time, or not at all, depending on the judgment of a multidisciplinary team. As our understanding of prenatal genomics improves the likelihood and burden of VUS will decrease . The final clinical utility of WES and WGS depends on our knowledge and understanding of the role of genetic variants in disease.
Secondary (or additional) findings are genetic alterations considered to be medically actionable but unrelated to the primary indication for testing (e.g., BRCA1 indicating increased risk for breast cancer). Currently in the UK there is no existing guidance that recommends the prospective identification of sequence variants of secondary significance in known disease-associated genes when testing is performed for a prenatal indication. Guidance issued by the ACMG relating to the use of genetic sequencing performed postnatally recommends that diagnostic laboratories actively look for and report on pathogenic variants in 59 genes predominantly related to cancer predisposition and cardiac disease . Patients recruited to the UK 100,000 Genome Project have been given the option to receive information on additional findings. Primary results are currently being returned to participants in this study, and in time secondary findings will also be fed back to participants who “opted in” to receiving this information. Insight into how this process is managed, and the implications for the families involved, will hopefully inform future best practice guidance relating to the management of secondary findings in the context of prenatal diagnostics.
ICFs are unexpected discoveries that are unrelated to the primary indication for testing and not medically actionable (e.g., childhood disorders). Currently, ICFs are unlikely to be reported, although guidance for the management of ICFs identified as a result of prenatal testing is not yet available. The use of trio prenatal sequencing approaches also has the potential to reveal both nonpaternity and parental consanguinity, adding a further dimension on the complexity of prenatal WES.