Kelly L. Gilmore

Current clinical guidelines recommend three genetic tests for the assessment of fetal structural anomalies: karyotype to detect microscopically-visible balanced and unbalanced chromosomal rearrangements, chromosomal microarray (CMA) to detect sub-microscopic copy number variants (CNVs), and exome sequencing (ES) to identify individual nucleotide changes in coding sequence. Advances in genome sequencing (GS) analysis suggest that it is poised to displace the sequential application of all three conventional tests to become a single diagnostic approach for the assessment of fetal structural anomalies. However, systematic benchmarking is required to assure that GS can capture the full mutational spectrum associated with fetal structural anomalies and to accurately quantify the added diagnostic yield of GS. We applied a novel GS analytic framework that included the discovery, filtration, and interpretation of nine classes of genomic variation to 7,195 individuals. We assessed the sensitivity of GS to detect diagnostic variants (pathogenic or likely pathogenic) from three standard-of-care tests using 1,612 autism spectrum disorder quartet families (ASD; n=6,448) with matched GS, ES, and CMA data, and validated these findings in 46 fetuses with a clinically reportable variant originally identified by karyotype, CMA, or ES. We then assessed the added diagnostic yield of GS in 249 trios (n=747) comprising a fetus with a structural anomaly detected by ultrasound and two unaffected parents that were pre-screened with a combination of all three standard-of-care tests. Across both cohorts, our GS analytic framework identified 98.2% of all diagnostic variants detected by standard-of-care tests, including 100% of those originally detected by CMA (n=88) and ES (n=61), as well as 78.6% (n=11/14) of the chromosomal rearrangements identified by karyotype. The diagnostic yield from GS was 7.8% across all 1,612 ASD probands, almost two-fold more than CMA (4.4%) and three-fold more than ES (3.0%). We also demonstrated that the yield of ES can approach that of GS when CNVs are captured with high sensitivity from exome data (7.4% vs. 7.8%, respectively). In 249 pre-screened fetuses with structural anomalies, GS provided an additional diagnostic yield of 0.4% beyond the combination of all three tests (karyotype, CMA, and ES). Applying our benchmarking results to existing data indicates that GS can achieve an overall diagnostic yield of 46.1% in unselected fetuses with fetal structural anomalies, providing an estimated 17.2% increase in diagnostic yield over karyotype, 14.1% over CMA, and 36.1% over ES when sequence variants are assessed, and 4.1% when CNVs are also identified from exome data. In this study we demonstrate that GS is sensitive to the detection of almost all pathogenic variation captured by karyotype, CMA, and ES, provides a superior diagnostic yield than any individual test by a wide margin, and contributes a modest increase in diagnostic yield beyond the combination of all three tests. We also outline several strategies to aid the interpretation of GS variants that are cryptic to conventional technologies, which we anticipate will be increasingly encountered as comprehensive variant identification from GS is performed. Taken together, these data suggest GS warrants consideration as a first-tier diagnostic approach for fetal structural anomalies.

What’s already known about this topic?: Sequencing-based noninvasive testing can detect large copy number abnormalities and some auto-somal dominant single-gene disorders Exome sequencing (ES) on fetal samples provides 20% diagnostic yield for structural abnormalities after normal karyotype & microarray What does this study add?: ES on cell-free DNA in three gravid patients with suspected genetic disease in the fetus We demonstrate broad sequencing approaches are limited by sampling and technical difficulties, concluding broad sequencing is currently inappropriate for noninvasive testing

Objective: To evaluate associations between prenatal trio exome sequencing (trio-ES) and psychological outcomes among women with an anomalous pregnancy. Methods: Trio-ES study enrolling patients with major fetal anomaly and normal microarray. Women completed self-reported measures and free response interviews at two time points, pre- (1) and post- (2) sequencing. Pre-sequencing responses were compared to post-sequencing responses; post-sequencing responses were stratified by women who received trio-ES results that may explain fetal findings, secondary findings (medically actionable or carrier couple status), or negative results. Free responses were content analyzed. Results: 115 trios were enrolled. Of those, 41/115 (35.7%) received results from trio-ES, including 36 (31.3%) who received results that may explain the fetal phenotype. These women had greater post-sequencing distress compared to women who received negative results, including generalized distress (p=0.03) and test-related distress (p=0.2); they also had worse psychological adaptation to results (p=0.001). Genomic knowledge did not change from pre- to post-sequencing (p=0.51). Major themes from content analyses included closure, future pregnancy, altruism, anxiety , and gratitude . Conclusions: Women show more distress after receiving trio-ES results compared to those who do not.