Current controversies in prenatal diagnosis 2: should a fetal exome be used in the assessment of a dysmorphic or malformed fetus?

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1 DOI: /pd.4718 PRESENTATIONS FROM THE 2015 ISPD MEETING IN WASHINGTON, DC Current controversies in prenatal diagnosis 2: should a fetal exome be used in the assessment of a dysmorphic or malformed fetus? Lyn S. Chitty 1, Jan M. Friedman 2 and Sylvie Langlois 2 * 1 Genetics and Genomic Medicine, UCL Institute of Child Health and Great Ormond Street NHS Foundation Trust, London, England 2 Department of Medical Genetics, University of British Columbia, Vancouver, Canada *Correspondence to: Sylvie Langlois. slanglois@cw.bc.ca This written debate summarizes the oral presentations made at the 2015 International Society for Prenatal Diagnosis meeting in Washington, DC, USA. It does not necessarily reflect the personal opinions of each of the authors. All authors contributed equally to this work. Funding sources: None Conflicts of interest: None declared INTRODUCTION (SYLVIE LANGLOIS) Major advances in DNA sequencing technology and bioinformatics make it possible to do large-scale sequencing for diagnostic purposes as opposed to the traditional approach of sequencing individual gene(s) responsible for a given phenotype. One such new large-scale sequencing approach is whole-exome sequencing (WES), which consists of using massively parallel sequencing to determine the sequence of the exome, the component of the genome that predominantly encodes protein. The exome comprises 1% of the genome and is, so far, the component most likely to include interpretable mutations that result in abnormal clinical phenotypes. 1 A fetal exome would consist of performing WES of fetal DNA obtained through chorionic villus sampling or amniocentesis. Interest in performing fetal exomes has come from studies of WES showing a diagnostic yield of 25 to 30% in pediatric and adult patients suspected to have a genetic condition but no specific diagnosis after the traditional diagnostic process. 2,3 Despite progress made in prenatal ultrasound and magnetic resonance imaging, making the diagnosis of a genetic syndrome is constrained by our ability to define the full phenotype. In pregnancies with a fetal abnormality, prenatal chromosomal microarray analysis (CMA) has increased diagnostic yield by up to 6% over conventional karyotyping 4,butdespiteits implementation in practice, in a significant proportion of fetuses with a single or multiple anomalies, no specific diagnosis is made prenatally. For example, the finding of a tetralogy of Fallot in a fetus may represent an isolated birth defect, but it also raises the possibility of trisomy 21, a copy number variant (e.g. microdeletion 22q11.2), and a number of syndromes because of a mutation in a single gene (e.g. Alagille syndrome, Adams Oliver, CHARGE). Therefore, if a chromosomal abnormality is excluded, a number of potential diagnoses still remain and the current approach of sequencing individual genes does not allow a cost-effective, time-sensitive way to confirm or exclude these diagnostic possibilities. WES would seem like an appealing solution. We therefore asked the question, Should a fetal exome be used in the assessment of a dysmorphic or malformed fetus? FOR (LYN CHITTY) To answer the question, we also need to consider whether or not exome sequencing will assist in making a definitive prenatal diagnosis. Thus, I will address three issues: 1. Will exome sequencing contribute to the diagnosis? 2. Will it subsequently contribute to the counseling? 3. Will it contribute to the management? Fetal anomalies affect around 3 5% of all pregnancies. When detected in pregnancy, prenatal diagnosis is required to offer appropriate counseling and allay parental anxiety where possible, discuss continuing or terminating the pregnancy and, for ongoing pregnancies, to offer appropriate treatment, be it prenatal or postnatal, and arrange for the most appropriate place of delivery. It also informs genetic counseling and reproductive decision making. 5 Rapid aneuploidy testing using quantitative fluorescent polymerase chain reaction or FISH will identify trisomy as the underlying etiology in around 30% of dysmorphic fetuses, including those with a nuchal translucency of 3.5 mm (data from the UK Evaluation of Array Comparative genomic Hybridization and non-invasive prenatal diagnosis using cell free fetal DNA in prenatal diagnosis of fetal anomalies (EACH) study Traditional karyotyping, which offers resolution of 5 to 10 Mb, will identify a pathogenic unbalanced chromosomal rearrangement as the underlying pathology in a further 5% of cases. Microarray analysis can have a resolution of around 0.2 Mb, allowing detection of pathogenic imbalances of 200 Kb. It has been shown to detect

2 16 L. Chitty et al. between 3.5 and 6% of additional pathogenic rearrangements in dysmorphic fetuses with a normal karyotype. 4,6,7 Thus, using a combination of these technologies, at best, we can identify the underlying etiology in up to 40% of cases, leaving around 60% of cases undiagnosed. This results in an inability to adequately counsel the parents, leads to uninformed decisions regarding termination of pregnancy and promotes a frequently protracted postnatal diagnostic course and long-term parental anxiety. Exome sequencing has much greater resolution than arrays, down to the base pair level. It can therefore detect mutations in single genes. Postnatally, there are increasing data demonstrating the clinical utility of WES for the detection of the molecular genetic etiology in dysmorphic patients with or without developmental delay. In series of 2000 patients, predominantly pediatric cases with diverse phenotypes, the Baylor group showed that WES could identify the molecular diagnosis in 504/2000 (25.2%), with 34.3% of those having an autosomal recessive and 12.3% X-linked recessive mode of inheritance. 8 Another study from the USA showed that WES yielded a diagnosis in 6/32 (19%) patients previously investigated with microarrays. 9 In the Deciphering Development Delay study in the UK, which is undertaking exome sequencing in 12,000 dysmorphic individuals with developmental delay, analysis of the first 317 has identified probable pathogenic variants in 113 (28%). 10 Evidence of effectiveness in the prenatal setting remains limited, but there is a growing body of evidence accumulating. There are several case reports in the literature, but these clearly represent a rather biased cohort. In their series of 250 cases, Yang et al. reported one of four fetuses in which WES identified a pathogenic mutation. 2 In their larger series published 2 years later, they reported diagnostic pathogenic changes in 11 (54.5%) terminated fetuses. 8 Filges and Friedman also made a good case for exome sequencing to diagnose lethal conditions in pregnancies that ended in fetal loss. 11 Furthermore, in 2014, a UK group used WES to detect three (10%) causative mutations in a total of 30 cases tested. 12 More evidence was presented at the International Society for Prenatal Diagnosis meeting in Washington by a group who detected five causative mutations in 25 fetuses tested prenatally (25%). 13 This group also identified known mutations in recessive genes in two further cases, indicating a likely recessive etiology. Finally, a study presented at the European Society of Human Genetics reported results using an exome panel with 4800 known Mendelian genes, the Mendeliome, which identified PTPN11 mutations in 4/53 (7.5%) fetuses with an increased nuchal translucency, a new novel gene mutation in 1/6 fetuses with intrauterine fetal growth restriction or other anomalies and autosomal recessive pathogenic mutations in 3/12 (25%) aborted dysmorphic fetuses (P. Joset oral communication Glasgow June 2015). Together, these data, while relating to relatively small and selected series, make it clear that exome sequencing will contribute significantly to the diagnosis of the dysmorphic fetus in up to 25% of cases, much along the lines of the added value we now recognize in the postnatal setting. However, adding to the diagnosis is one thing, but what about clinical utility? The latter can be generally defined as test results that inform decision making, influence pregnancy management to improve outcome or lead to any outcome considered important to individuals. Take the example of a case seen at 16 weeks of gestation with slightly short long bones, mild bowing of the femora and humeri, a slightly hypomineralized skull but a normal thorax. The parents were advised of a possible diagnosis of osteogenesis imperfecta. They elected to have an invasive test, and exome sequencing was performed on DNA extracted from amniocytes. This showed a homozygous mutation in LEPRE1 c g>t. The parents elected to terminate this pregnancy before this result was available, and so, it could be argued that there was no clinical utility as it did not affect pregnancy management. However, having this result enabled early and informative postnatal genetic counseling when parents were advised of the 25% recurrence risk. It also informs prenatal management in future pregnancies. Other examples in which potential for clinical utility during pregnancy can be demonstrated have been described, including diagnoses of Millroy syndrome and Freeman Sheldon to allow appropriate counseling regarding expected pregnancy outcome. 13 With increasing use of fetal exomes, we will see evidence of increasing clinical utility as accurate diagnosis inevitably informs accurate parental counseling and informed decision making with regard to pregnancy management. Many would argue that exome sequencing takes too long to be offered in the prenatal setting. However, if Saunders and colleagues can deliver a rapid diagnosis within 50 h using WES in the neonatal unit, 14 rapid exome sequencing must be achievable in the prenatal setting. Indeed, in our unit s routine workflow, we are now able to deliver a rapid exome result following extraction of DNA from amniocytes in just over 2 weeks. Some would argue that the cost is too great given the need to analyze the trio of fetus and parents, for accurate and timely interpretation of the results. However, is it really more expensive to do this in the prenatal setting? In a busy molecular genetic laboratory offering exome sequencing postnatally, it should be easy enough to find the space to sequence occasional fetal samples without setting up runs specifically for that purpose at extra cost. In our unit in the UK, the cost of exome sequencing would be the same whether performed prenatally or postnatally. Furthermore, the advantage of doing it prenatally is that the results may inform prenatal management, and even in situations where there is no change in management, it may end the diagnostic odyssey that frequently occurs postnatally with serial genetic tests being performed thus reducing costs of multiple serial testing, both molecular, imaging and other pathological investigations some of which may be invasive, and potentially reducing extended neonatal hospital stay while awaiting a diagnosis. Those in the laboratory would argue that for accurate interpretation of exome sequencing results, it is necessary to have full and detailed phenotyping and that this is not possible prenatally. Obviously, we are unable to detect developmental delay or autistic spectrum disorders before birth, but one can argue that this also applies to exome sequencing performed in the neonatal unit. I would argue that with modern ultrasound equipment and the addition of 3D and 4D scanning, the identification of dysmorphic features is increasingly achievable. This is evidenced by the cases already reported in the literature in which exome sequencing results were highly compatible with the fetal dysmorphic features detected by prenatal ultrasound examination. 12,13

3 Fetal exome in the assessment of a dysmorphic of malformed fetus 17 There are two final points that need addressing before concluding that exome sequencing does indeed contribute to the counseling and management of the dysmorphic or malformed fetus. The first is the argument that exome sequencing will identify many incidental findings and that management of these in the prenatal setting is complex. However, those of us expert in the diagnosis and management of the dysmorphic fetus have been dealing with these issues for years. In the 1980s, karyotyping frequently identified marker chromosomes, with microarray analysis we are faced with interpreting and reporting variants of unknown significance (VOUS), and with fetal ultrasound examination we regularly deal with challenging situations such as mild ventriculomegaly. While we may be faced with uncertainty more often with exome sequencing, there are approaches to limit this. Where possible, we should apply targeted panels or targeted interpretation of data to minimize the identification of incidental findings. When reporting results of parental exome sequencing, we should apply the postnatal guidelines that have been developed 15 for which there is a large degree of concordance among clinicians. 16 For the fetus, we should report results that are compatible with the phenotype. This brings me to the second and final point; to apply these guidelines, careful pretest counseling is required so that parents make informed choices about the results they wish to receive and understand that exome sequencing will not detect all pathogenic mutations as it does not give complete coverage of all known genes. In conclusion, it is clear that exome sequencing can aid the diagnosis in up to 25% of dysmorphic fetuses in a timely fashion and at no greater cost than if performed postnatally. Exome sequencing therefore contributes to the counseling and management of the dysmorpic fetus. We should listen to parents, 83% of whom say exome sequencing should be offered, 17 and remove the barriers to this testing that are being experienced in societies with private and insurance-based health care 18 to improve the care we offer to parents confronted with the unexpected diagnosis of a dysmorphic fetus. AGAINST (JAN FRIEDMAN) If we practice evidence-based medicine, the answer to this question must be no. The clinical validity (defined as accuracy of detection of presence or absence of disease) and cost effectiveness (defined as value for money) of fetal exome sequencing have not been established for any indication. There have been case reports and small series in which interesting results have been obtained by fetal exome sequencing, but these anecdotes do not establish the clinical utility of this test. Most of the anecdotal successes have occurred in research studies, 11,19,20 in which the goal is the discovery of new knowledge, and interpretation is open-ended with respect to time and clinical implications. In contrast, the goal of clinical exome sequencing is diagnosis to improve clinical management. Diagnostic accuracy and speed in returning the results are critical for clinical testing but are serious challenges for fetal exome sequencing. Exome sequencing can survey tens of thousands of genes all at once, but when used as a clinical test,itcanonlyidentifyknown or very likely pathogenic sequence variants of the protein-coding portions of known mendelian disease genes Exome sequencing is not a good test for the detection of genomic imbalance (copy number variants), structural chromosomal rearrangements, or sequence mutations involving more than one or a few nucleotides (e.g. trinucleotide repeat expansions that cause fragile X syndrome). Exome sequencing is also not useful for diagnosing most mutations that lie outside of the proteincoding regions of genes nor birth defects that are multifactorial in origin, such as most cases of neural tube defects or autism. Exome sequencing does not even provide information on the entire protein-coding portion of the genome. As a result of technical limitations of exome capture, the coverage of 3 10% of the exome is usually insufficient to permit confident diagnosis of variants. Coverage of the protein-coding segments of genes included in multigene panels is often more complete than that of the same genes obtained by exome sequencing. Current technology permits obtaining exome sequencing results in a few days, but this usually requires testing both parents as well as the fetus (trio sequencing) and is substantially more expensive than routine clinical exome sequencing performed postnatally on a child. The biggest challenge to using exome sequencing to diagnose serious genetic disease in a fetus is interpretation of the results The diploid human genome consists of more than 6,000,000 base pairs of DNA, and every person (or fetus) has millions of differences in comparison with the normal reference genome. A typical exome from a person who does not have a mendelian disease includes more than 100 novel or rare variants that are predicted to alter protein structure. Clinical diagnosis of a mendelian disease from exome sequence data requires recognition of the one or two variants that actually cause the disease in this large background of other variants that are present but have nothing to do with the disease. The relationship between pathogenic genetic variants and disease is often complex. Mutations of more than one, and sometimes many, different genes may cause indistinguishable clinical pictures. Different mutations of a single genetic locus may cause different diseases, and exactly the same mutation may cause disease manifestations of strikingly different severity, or no disease at all, in different individuals. This variability can make predicting the clinical effects of a potentially pathogenic genetic variant difficult. The problem of genotype phenotype correlation is exacerbated when the patient is a fetus because information about structural alterations is usually limited to what can be seen on detailed ultrasound examination, and information about function of the brain and other important organs is frequently unavailable. Exome sequencing of either the fetus or a parent (if trio sequencing is performed) may also produce incidental findings genetic variants of potential clinical importance that are unrelated to the disease for which the testing was performed The frequency with which incidental findings are encountered can range from a few percent of patients to every single family tested, depending on how the data are analyzed and what kinds of incidental findings the testing laboratory reports. The accuracy of clinical interpretation of exome sequencing data has not been rigorously studied, but false-positive and false-negative reports of pathogenic variants have occurred Such errors are more likely in circumstances in which rigorous genotype phenotype correlation is not possible, as often occurs

4 18 L. Chitty et al. in fetal exome sequencing. The difficulty of clinical interpretation of fetal exome data and the possibility of incidental findings emphasize the importance of genetic counseling before and after exome sequencing for fetal diagnosis. Fetal exome sequencing for clinical diagnosis may sometimes be indicated when well-defined fetal malformations can be interpreted in the context of the full phenotype, e.g. as part of an autopsy following fetal death or stillbirth or when delivery is imminent, and the result will influence neonatal management. In other clinical circumstances, e.g. in a fetus found to have malformations on ultrasound examination in the first half of pregnancy, other genetic diagnostic tests CMA or testing of individual genes or targeted panels of genes suspected of being pathogenic are usually more appropriate, given our current state (and lack) of knowledge. CONCLUSIONS (SYLVIE LANGLOIS) Both debaters bring strong arguments in favor and against using exome sequencing in the management of the dysmorphic or malformed fetus. Dr. Chitty advocates doing so as it will increase the diagnostic yield thus allowing appropriate counseling to parents, informed reproductive choices, and for ongoing pregnancies, facilitate prenatal and postnatal management and avoid protracted postnatal investigations. She argues that our current use of exome sequencing in the neonatal intensive care unit, with the ability to achieve a rapid turnaround time provides support for the use of such testing in the prenatal setting. She provides examples where making a definite diagnosis allowed clarification of recurrence risks and options for prenatal diagnosis in future pregnancies. Dr. Friedman, however, acknowledges that case reports and small series of fetal exomes show interesting results but points out that the clinical validity and cost effectiveness of a fetal exome have not been established, and thus, the clinical utility of this test has not been demonstrated for any prenatal indication. He argues that there are still limitations inherent to the exome sequencing process itself and that the need for a trio analysis (fetal exome and parental DNA) for rapid results makes this test substantially more expensive than routine clinical exome sequencing in a child. He advocates that this test should not be used to investigate the dysmorphic or malformed fetus in most prenatal circumstances and argues that other kinds of genetic testing are usually more appropriate. As Drs. Chitty and Friedman made their arguments, it became clear that both agreed on a number of points: (1) Evidence of effectiveness in the prenatal setting is limited and based on case reports and small series, thus potentially representing biased cohorts; (2) interpretation of results and establishing pathogenicity of the variants detected in the prenatal setting brings an added complexity because of the incomplete delineation ofthephenotype;(3)exomesequencingoftrioswillidentify incidental findings, but there are strategies that may be applied to limit the counseling difficulties these may pose; (4) fetal exome sequencing, should it be performed, requires careful pretest counseling so that parents make informed choices. From the arguments made, it would appear that fetal exome sequencing has the potential to increase diagnostic yield in the prenatal setting, but research is needed to answer the question of clinical utility. Data must be gathered to determine if the yield is significant only in the presence of multiple anomalies or if there are specific fetal abnormalities, even if isolated, that justify its use. The Prenatal Assessment of Genome and Exome project, a UK-funded research project that will perform exome sequencing in 1000 fetal : parent trios in pregnancies with a fetal structural anomaly, aims to answer the question of whether or not and how exome sequencing should be used to investigate the dysmorpic or malformed fetus. 13,20 This study is crucial because we must not be guided by what is technically possible but by what is clinically beneficial. We must ensure that testing is implemented in a way that benefits families without creating too much anxiety. The use of fetal exome sequencing is in its infancy, and, from points made by both debaters, while it seems clear that it will add to diagnosing the underlying etiology of prenatally detected abnormalities, there are significant hurdles to implementing fetal exomes in the routine assessment of the dysmorphic and malformed fetus. In selected cases with a high suspicion of a genetic single gene disorder, the use of targeted sequencing panels or targeted interpretation of data from a fetal exome may be a more prudent approach to minimize the identification of uninterpretable variants of uncertain significance or incidental findings. However, given that the use of fetal exome sequencing in the investigation of the dysmorphic or malformed fetus is likely to become clinical practice as data become available on appropriate indications, clinical guidelines addressing the reporting of incidental findings in the prenatal setting need to be developed. WHAT S ALREADY KNOWN ABOUT THIS TOPIC? Whole-exome sequencing is a comprehensive genetic test with a diagnostic yield of 25 30% in postnatal cohorts of undiagnosed patients suspected of having a genetic disorder. Reports on the use of whole-exome sequencing prenatally are limited to case reports and small series. WHAT DOES THIS STUDY ADD? This report summarizes an oral debate presented at the 19th International Conference on Prenatal Diagnosis and Therapy in Washington, DC, USA on 14 July The benefits, limitations and challenges of performing fetal exome sequencing to investigate a pregnancy presenting with a dysmorphic or malformed fetus are discussed. REFERENCES 1. ACMG Board of Directors. Points to consider in the clinical application of genomic sequencing. Genet Med 2012;14(8): Yang Y, Muzny DM, Reid JG, et al. Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013; 369(16): Farwell KD, Shahmirzadi L, El-Khechen D, et al. Enhanced utility of family-centered diagnostic exome sequencing with inheritance modelbased analysis: results from 500 unselected families with undiagnosed genetic conditions. Genet Med 2015;17(7):

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