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 .

Existing Evidence

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:

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  Elucidate the relative contribution of different forms of genetic variation to prenatal structural anomalies

  
  
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  Design cost-effective genome sequencing assays for improved prenatal diagnosis of structural anomalies

  
  
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  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.

Case Example

Technical Issues

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.

Managing Findings

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:

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  Class 1—clearly not pathogenic, common polymorphism, not reported

  
  
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  Class 2—unlikely to be pathogenic, diagnosis not confirmed molecularly, not reported

  
  
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  Class 3—VUS, uncertain pathogenicity, does not confirm or exclude diagnosis, local team to determine whether to report

  
  
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  Class 4—likely to be pathogenic, consistent with diagnosis, reported

  
  
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  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.