Introduction of genomics into prenatal diagnostics
Authors: Michael E Talkowski, Heidi L Rehm
Journal: The Lancet (2019)

The introduction of new technologies presents unique challenges for invasive prenatal genetic testing. Clinical phenotyping is indirect, many traits cannot be assessed in utero, and rapid turnaround times are crucial. In their Articles in The Lancet, Jenny Lord and colleagues1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar and Slavé Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar explore the relative value of whole-exome sequencing (WES) in the prenatal assessment of fetal structural anomalies. These studies will have immediate translational implications; they provide roadmaps for clinical interpretation of WES and crucial benchmarks of diagnostic yields for health-care consumers, providers, and payers alike. Karyotyping to visualise megabase-scale copy number variants (CNVs) and balanced chromosomal abnormalities was the mainstay of cytogenetic testing for several decades. In the early 2000s, microarray technology enabled access to submicroscopic CNVs,3Lucito R West J Reiner A et al.Detecting gene copy number fluctuations in tumor cells by microarray analysis of genomic representations.Genome Res. 2000; 10: 1726-1736Crossref PubMed Scopus (80) Google Scholar, 4Sebat J Lakshmi B Troge J et al.Large-scale copy number polymorphism in the human genome.Science. 2004; 305: 525-528Crossref PubMed Scopus (1971) Google Scholar, 5Iafrate AJ Feuk L Rivera MN et al.Detection of large-scale variation in the human genome.Nat Genet. 2004; 36: 949-951Crossref PubMed Scopus (2309) Google Scholar and discoveries over the subsequent decade demonstrated the clinical usefulness of this technology.6Miller DT Adam MP Aradhya S et al.Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies.Am J Hum Genet. 2010; 86: 749-764Summary Full Text Full Text PDF PubMed Scopus (1963) Google Scholar A seminal study7Wapner RJ Martin CL Levy B et al.Chromosomal microarray versus karyotyping for prenatal diagnosis.N Engl J Med. 2012; 367: 2175-2184Crossref PubMed Scopus (913) Google Scholar in 2012 marked a transition point for prenatal testing, in which microarray of 4406 pregnancies revealed a 6% yield of clinically significant CNVs in fetal structural anomalies; however, the improved resolution came at a cost since microarray cannot detect balanced chromosomal abnormalities.8Warburton D De novo balanced chromosome rearrangements and extra marker chromosomes identified at prenatal diagnosis: clinical significance and distribution of breakpoints.Am J Hum Genet. 1991; 49: 995-1013PubMed Google Scholar, 9Talkowski ME Rosenfeld JA Blumenthal I et al.Sequencing chromosomal abnormalities reveals neurodevelopmental loci that confer risk across diagnostic boundaries.Cell. 2012; 149: 525-537Summary Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 10Redin C Brand H Collins RL et al.The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies.Nat Genet. 2017; 49: 36-45Crossref PubMed Scopus (175) Google Scholar At about the same time, WES transformed gene discovery research by providing a method to capture and sequence most of the protein-coding genome.11Gnirke A Melnikov A Maguire J et al.Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing.Nat Biotechnol. 2009; 27: 182-189Crossref PubMed Scopus (1040) Google Scholar, 12Ng SB Turner EH Robertson PD et al.Targeted capture and massively parallel sequencing of 12 human exomes.Nature. 2009; 461: 272-276Crossref PubMed Scopus (1540) Google Scholar These two independent studies by Lord and colleagues1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar and Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar now apply WES in the prenatal setting, and they arrive at almost identical conclusions: routine WES will enable more molecular diagnoses of causal genetic variation in fetuses with structural anomalies relative to conventional cytogenetic methods. Early studies13Best S Wou K Vora N Van der Veyver IB Wapner R Chitty LS Promises, pitfalls and practicalities of prenatal whole exome sequencing.Prenat Diagn. 2018; 38: 10-19Crossref PubMed Scopus (206) Google Scholar of WES in fetuses with structural anomalies reported diagnostic variants (ie, genetic alterations likely to be associated with the observed ultrasound anomaly) in 6·2–80% of these fetuses, dependent on the selection of fetal anomalies being studied and interpretation criteria applied. In the presence of such variability, the studies by Lord and colleagues1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar and Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar are notable for their systematic designs and transparent analyses. The larger study from the PAGE Consortium1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar evaluated WES in a prospective cohort of 610 fetuses with structural anomalies that were detected by ultrasound (596 fetus–parent trios, 14 fetus–parent dyads). The second study from Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar screened consecutive fetuses with structural anomalies at Columbia University from 2015 to 2017 and performed WES on 234 fetus–parent trios. Both studies pre-screened fetuses for pathogenic chromosomal abnormalities or CNVs, and plausible pathogenic variants from WES were reviewed by multidisciplinary teams to determine final molecular diagnoses. These carefully designed studies found remarkably consistent results: the PAGE study1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar reported a clinically significant genetic variant in 8·5% of fetuses, with an additional 3·9% of fetuses harbouring variants of possible clinical significance (12·4% total), and the study by Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar found a diagnostic genetic variant in 10·3% of fetuses. In the PAGE study,1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar the proportion of fetuses with phenotype-specific diagnostic genetic variants were found to range from 3·2% (in fetuses with increased nuchal translucency) to 15·4% (in those with multisystem fetal structural anomalies). Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar found that these proportions ranged from 6·3% in fetuses with a single affected anatomical system to 18·9% in fetuses with multisystem fetal structural anomalies. The greatest source of pathogenic variation was dominant de-novo mutations in both studies (representing 32 [61·5%] of 52 diagnostic variants in the PAGE study and 15 [63%] of 24 diagnostic variants in the Petrovski and colleagues' study). Both studies also discuss topics related to parental decisions, clinical outcomes, and incidental findings. The PAGE study1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar provided pregnancy outcome data for 474 (78%) of 610 fetuses, and they revealed that parents opted for termination in 142 (30%) pregnancies, 14 (3%) ended in miscarriage, and there were 22 (5%) stillbirths, 14 (3%) neonatal deaths, and 282 (59%) liveborn babies. The proportion of fetuses with pathogenic or likely pathogenic genetic variants that did not survive beyond birth was significantly higher than those that were liveborn (14·1% vs 7·1%; p=0·0181). The study by Petrovski and colleagues2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar reported outcomes for 220 (94%) of 234 fetuses: 57% of pregnancies resulted in livebirths, 33% were terminations, and 3% were stillborn or died in utero. Incidental findings were also reported for four (2%) fetuses, in which the pathogenic variant was presumed to be unrelated to the structural anomaly. Both studies also considered the sensitive issue of return of results. In the PAGE study, no results were provided during the current pregnancy, but results related to the ultrasound findings were subsequently reported to parents, whereas the study by Petrovski and colleagues reported diagnoses during the pregnancy. Interpretation from WES remains a source of variability, and the underlying data between studies highlight instructive differences in their approaches. The PAGE study restricted interpretation to a virtual panel of 1628 genes that are implicated in developmental disorders and prenatal findings. In this narrowed search space, 255 potential diagnoses required manual review, which was a mean of 0·42 potential diagnoses per fetus. Otherwise stated, 33·6% of all fetuses had a variant that warranted consideration and 8·5% received a diagnosis. The design of the study by Petrovski and colleagues was more expansive; this study included all genes and used methods that are often applied to annotate variants in association studies of neurodevelopmental disorders.14Adzhubei I Jordan DM Sunyaev SR Predicting functional effect of human missense mutations using PolyPhen-2.Curr Protoc Hum Genet. 2014; 76: 20.1-2041Google Scholar, 15Deciphering Developmental Disorders StudyLarge-scale discovery of novel genetic causes of developmental disorders.Nature. 2015; 519: 223-228Crossref PubMed Scopus (721) Google Scholar, 16Gussow AB Petrovski S Wang Q Allen AS Goldstein DB The intolerance to functional genetic variation of protein domains predicts the localization of pathogenic mutations within genes.Genome Biol. 2016; 17: 9Crossref PubMed Scopus (83) Google Scholar, 17Petrovski S Wang Q Heinzen EL Allen AS Goldstein DB Genic intolerance to functional variation and the interpretation of personal genomes.PLoS Genet. 2013; 9: e1003709Crossref PubMed Scopus (643) Google Scholar, 18Samocha KE Robinson EB Sanders SJ et al.A framework for the interpretation of de novo mutation in human disease.Nat Genet. 2014; 46: 944-950Crossref PubMed Scopus (615) Google Scholar This tiered interpretation incorporated so-called bioinformatic signatures, meaning that it allowed variants in genes not yet linked to disease to be considered if they had similar properties to variants that occur at higher frequency in developmental disorders relative to their occurrence in the general population. This approach should increase sensitivity, as well as interpretation workload, which bears out in their identification of 1182 qualifying genotypes that warranted consideration, which was a mean of 4·8 qualifying genotypes per fetus (ie, more than ten times as many as those identified in the PAGE study). After review by the multidisciplinary panel, this approach yielded molecular diagnoses in 24 (2%) of 1182 variants considered. There was justification for this increased interpretation burden since some variants in novel genes were re-interpreted as pathogenic with support from emerging literature. In one fetus, a bioinformatic signature was not reported to the parents because of insufficient evidence at the time of analysis, and a subsequent pregnancy revealed a recurrent fetal anomaly. These data underscore the potential clinical value of computational prediction to prioritise variants when genes without a known phenotypic match are included in analyses, and they highlight the delicate balance between conservative interpretation and under-reporting of uncertain results. Nonetheless, it remains important to look in detail at phenotype-matching genetic variants, regardless of signatures, since two molecular diagnoses derived from de-novo variants in the study by Petrovski and colleagues were not captured by the bioinformatic signatures. Both studies emphasise the crucial need for manual review and re-interpretation over time. As more sophisticated methods are developed to leverage population-scale references and more complete functional annotations, this process can become standardised for routine clinical practice. We suggest that these studies serve as the introduction of WES into prenatal testing and document a compelling justification for its adoption. The technology is mature, the data are reproducible, and the processes are established in many clinical laboratories. There remain sobering limitations to the technology since adoption of WES as a single test could create voids in the screening of CNVs, balanced chromosomal abnormalities, and complex structural variation. It is of note that both studies pre-screened fetuses for these variants with conventional technologies. Although CNV detection from WES is feasible, and was applied in the PAGE study, it remains a bioinformatic challenge and has not been widely adopted by clinical diagnostic labs. Implementation of WES as a first-tier screen will thus leave clinicians and patients with some blind spots, and the time-compressed prenatal setting likely precludes serial reflexive testing for structural variations. Whole-genome sequencing holds promise to displace these methods because it can capture coding and non-coding point mutations as well as CNVs and balanced chromosomal abnormalities in a single test.19Talkowski ME Ordulu Z Pillalamarri V et al.Clinical diagnosis by whole-genome sequencing of a prenatal sample.N Engl J Med. 2012; 367: 2226-2232Crossref PubMed Scopus (138) Google Scholar, 20Werling DM Brand H An JY et al.An analytical framework for whole-genome sequence association studies and its implications for autism spectrum disorder.Nat Genet. 2018; 50: 727-736Crossref PubMed Scopus (142) Google Scholar However, until its arrival for routine genetic testing, it is likely that a combination of WES and molecular cytogenetic methods will represent the most comprehensive approach. The data from these studies1Lord J McMullan DJ Eberhardt RY et al.Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)31940-8Summary Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 2Petrovski S Aggarwal V Giordano JL et al.Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study.Lancet. 2019; (published online Jan 31.)http://dx.doi.org/10.1016/S0140-6736(18)32042-7Summary Full Text Full Text PDF PubMed Scopus (267) Google Scholar indicate that such a combination will offer substantial improvements in diagnostic precision and clinical management of fetal anomalies. MET and HLR are supported by grants related to this work from the National Institutes of Health (MET: HD081256, GM061354, MH115957; HLR: UM1HG008900, U41HG006834). MET has collaborated with authors of the two Articles that this Comment discusses. HLR is employed by Massachusetts General Hospital and the Broad Institute of MIT and Harvard, which both offer fee-for-service genomic testing. Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort studyWES facilitates genetic diagnosis of fetal structural anomalies, which enables more accurate predictions of fetal prognosis and risk of recurrence in future pregnancies. However, the overall detection of diagnostic genetic variants in a prospectively ascertained cohort with a broad range of fetal structural anomalies is lower than that suggested by previous smaller-scale studies of fewer phenotypes. WES improved the identification of genetic disorders in fetuses with structural abnormalities; however, before clinical implementation, careful consideration should be given to case selection to maximise clinical usefulness. Full-Text PDF Open AccessWhole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort studyOur analysis of WES data in a prospective cohort of unselected fetuses with structural anomalies shows the value added by WES following the use of routine genetic tests. Our findings suggest that, in cases of fetal anomalies in which assessment with karyotype testing and chromosomal microarray fail to determine the underlying cause of a structural anomaly, WES can add clinically relevant information that could assist current management of a pregnancy. The unique challenges of WES-based prenatal diagnostics require analysis by a multidisciplinary team of perinatal practitioners and laboratory specialists. Full-Text PDF