Exome Sequencing in the Evaluation of the Fetus With Structural Anomalies

Acknowledgments

With thanks to Elizabeth Young (Clinical Scientist, West Midlands Regional Genetics Laboratory) for her input in the section on cfDNA-based (noninvasive) prenatal testing.

Funding Sources

Elizabeth Quinlan-Jones is funded through the Department of Health, Wellcome Trust, Health Innovation Challenge Fund (Award Number HICF-R7-396) as a research midwife for the PAGE and PAGE2 research studies.

Introduction

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

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