Exome Sequencing Improves Diagnosis of Fetal Structural Anomalies

Prenatal diagnostics have evolved from conventional cytogenetic analysis to increasingly chromosomal microarray analysis, within the last 10 years. While whole-exome sequencing (WES) has shown utility in identifying genetic causes of developmental disorders in children, up until now there has been limited evidence evaluating the usefulness of WES for the diagnosis and clinical management of ultrasound-detected fetal anomalies.

Two recent studies, both published Jan. 31 in The Lancet, show that the addition of whole-exome sequencing (WES) improves genetic diagnosis of fetal structural anomalies, which, in turn, can facilitate better determination of fetal prognosis and risk of recurrence in future pregnancies.

It is estimated that roughly 3 percent of pregnancies will have a fetal structural anomaly detectible on ultrasound, which can range from a single minor defect to severe, fatal multisystem anomalies. Currently, when fetal structural anomalies are detected, a routine workup would include testing for aneuploidy and copy number variations (CNVs). The two Lancet studies, which both included fetuses pre-screened for pathogenic chromosomal abnormalities or CNVs but with a wide range of structural anomalies, consistently demonstrated clinically significant genetic variants in 8.5 percent to 10.3 percent of fetuses and that the majority of these diagnostic variants in both studies were from de-novo mutations.

“We suggest that these studies serve as the introduction of WES into prenatal testing and document a compelling justification for its adoption,” write Michael Talkowski and Heidi Rehm, both from Massachusetts General Hospital (Boston) in an accompanying commentary. “The technology is mature, the data are reproducible, and the processes are established in many clinical laboratories.”

Technically WES creates “voids,” experts say, in its inability to screen for CNVs, balanced chromosomal abnormalities, and complex structural variation, meaning that a combination of WES and molecular cytogenetic methods represents the most “comprehensive” viable approach for routine prenatal genetic testing.

The U.K. PAGE Study
The Prenatal Assessment of Genomes and Exomes (PAGE) study recruited women with fetuses with structural anomalies between Oct. 22, 2014, and June 29, 2017, from 34 fetal medicine units in England and Scotland. Samples were taken from fetuses without aneuploidy and large CNVs and their parents (596 fetus–parent trios, 14 fetus–parent dyads). Sequencing results were interpreted using a targeted virtual gene panel consisting of 1,628 genes implicated in developmental disorders (n = 1,511) and other prenatal findings (n = 117). Genetic results were returned post-pregnancy.

Following bioinformatic filtering and prioritization according to allele frequency and effect on protein and inheritance pattern, the researchers identified 321 genetic diagnostic genetic variants, representing 255 potential diagnoses (0.42 potential diagnoses per fetus). A multidisciplinary clinical panel reviewed variants.

Overall, a clinically significant genetic variant was identified in 8.5 percent of fetuses, with an additional 3.9 percent of fetuses having variants of possible clinical significance. The proportion of fetuses with diagnostic genetic variants varied by phenotype: 3.2 percent in fetuses with increased nuchal translucency, 11.1 percent in those with cardiac anomalies, and 15.4 percent in those with skeletal or multisystem fetal structural anomalies. Just over one-third of diagnostic genetic variants had a high recurrence risk.

The authors, led by Jenny Lord, Ph.D., from the Wellcome Sanger Institute in the United Kingdom, acknowledged that although WES increases the frequency of identification of genetic causes of structural anomalies in fetuses more than cytogenetics or chromosomal microarray alone, the overall frequency of diagnostic genetic may have been lower than expected. Based on their experience, the researchers advise the use of

• Fetal-parental trio analysis rather than fetus-only WES to speed identification of de-novo variants in monoallelic developmental disorder genes
• Selected subgroups (e.g., those with multiple congenital anomalies) or after genetic review
• Curating the developmental disorders gene to phenotype list to remove genes not associated with fetal structural anomalies and using smaller, phenotype-specific virtual gene panels, to reduce the number of variants of unknown significance that are irrelevant to the fetal structural anomalies.

A U.S.-Based Study
In the second study, researchers from Columbia University in New York screened consecutive fetuses with structural anomalies (April 2015 to April 2017) and performed WES on 234 fetus–parent trios. WES was offered to all patients, regardless of the anomaly, but a substantial portion of parents (24 percent) declined testing or did not consent to WES.

This study used a tiered interpretation approach incorporated bioinformatic signatures. This allows 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. While this approach increases sensitivity it also increases interpretation workload. The researchers identified 1,182 qualifying genotypes that warranted consideration (a mean of 4.8 qualifying genotypes per fetus, which is ten times the number identified in the PAGE study). After review by the multidisciplinary panel, this interpretation approach yielded molecular diagnoses in 2 percent of the 1,182 variants considered.

Overall, a diagnostic genetic variant was identified in 10.3 percent of fetuses. Further, mutations suggestive of pathogenicity, but with insufficient evidence to be considered diagnostic, were identified in 20 percent of fetuses.

Similar to the PAGE study, the proportions of fetuses with a diagnostic variant varied by phenotype: 5 percent with cardiac, 16 percent of fetuses with renal, 22 percent with central nervous system, and 24 percent of fetuses with lymphatic/effusion or skeletal anomalies.

A genetic diagnosis was achieved three times more often in fetuses with several anomalies versus those with one anomaly (19 percent versus 6 percent). In fetuses with three or more anomalies, a diagnostic genetic variant was identified in more than 30 percent of cases.

The authors, led by Slavé Petrovski, Ph.D., from Columbia University in New York, recommend that

• Interpretation of prenatal sequencing involves collaboration of a multidisciplinary team, including clinical and molecular geneticists, genetic counselors, and fetal imaging specialists
• Workflow must be optimized to minimize turnaround time so that results are returned in a timeframe conducive to decision-making in the “time-compressed” prenatal setting.

Takeaway: WES can improve upon the diagnostic capabilities of karyotyping and microarray analysis to find a diagnostic variant responsible for ultrasound-detected structural anomalies in fetuses. While WES is both clinically useful and feasible, some important questions remain regarding fetal selection, the number and type of genes to be interrogated, how to improve turnaround time to deliver results in an actionable timeframe, and which results to return and when.