Sequencing Following Ultrasound Improves Prenatal Diagnosis

Nonsevere fetal malformations and structural abnormalities are relatively frequent findings of routine, prenatal ultrasounds. Yet, these findings provide a diagnostic challenge for clinicians, as they can vary by identifiable genetic cause and present with varying clinical significance. Often the lack of a precise diagnosis precludes accurate reproductive and fetal risk assessment. However, several small studies are showing the potential for sequencing-based testing to improve prenatal diagnosis.

Targeted-Exome Sequencing

The “Fetalis” targeted-exome sequencing strategy provided a definitive or highly likely diagnosis in 43 percent of cases of fetus with “troubling but non-extreme” abnormalities detected through prenatal ultrasound examination, according to a small study published in PeerJ. The targeted approach provided rapid, clinical diagnosis with few uncertain findings.

“Although this figure is obviously a very preliminary assessment from a limited number of cases, it nonetheless provides an initial proof-of-principle regarding the merits of the approach,” write the authors led by Constantinos Pangalos, from InterGenetics in Greece, in the April 26 issue of PeerJ. “The volume of data … is an order of magnitude less than the corresponding whole-exome sequencing data and … affords a highly cost-effective, more simplified and timely diagnosis (even less than 1 week) during the course of pregnancy.”

Typically, prenatal genetic diagnosis is limited to the investigation of possible chromosomal imbalances. Even array comparative genomic hybridization can only detect the underlying genetic cause in less than 15 percent of cases. While technically feasible, whole-exome or whole-genome sequencing (WES or WGS) yields complex results requiring time to analyze and are plagued with incidental findings and variants of unknown significance.

In the present study, the researchers used an expanded exome sequencing-based test, coupled to a bioinformatics-driven prioritization algorithm that targeted gene disorders (n=758 genes) presenting with abnormal prenatal ultrasound findings. DNA samples were extracted directly from uncultured amniotic fluid or chorionic villi sampling samples from 14 euploid fetuses (11 from on-going pregnancies and three products of abortion), all with various abnormalities or malformations detected on prenatal ultrasound examination.

The researchers made a definitive or highly likely diagnosis in six of 14 cases—in all three abortuses and in three out of 11 on-going pregnancies. In an additional on-going pregnancy case a ZIC1 variant of unknown clinical significance was detected, while in the seven remaining cases testing did not reveal any pathogenic variant(s). These eight on-going pregnancies were followed to birth. One neonate was found to harbor the PROKR2 mutation, presenting with isolated, minor structural cardiac abnormalities, while the remaining seven appeared as healthy neonates.

On average, a total of approximately 2,200 variants per case were detected following gene prioritization. Following variant filtering through the Fetalis algorithm, approximately one to three potentially significant variants/mutations were left for manual inspection and final clinical evaluation.

Whole-Exome Sequencing

More recently, researchers found that WES can identify the cause of 20 percent of sonographic abnormalities, according to an abstract published in Ultrasound in Obstetrics & Gynecology in conjunction with the 26th World Congress on Ultrasound in Obstetrics and Gynecology (Italy; Sept. 24–28).

The researchers aimed to develop a strategy to use WES for trio analysis of multiple genes for rapid molecular diagnosis to inform pregnancy management. Amniotic fluid or CVS samples, along with germline DNA from both parents, was analyzed using microarray followed by next-generation sequencing for normal microarray samples. Variants were identified using a combination of published and in-house bioinformatics pipelines and potentially pathogenic mutations were confirmed by Sanger sequencing.

Of six cases analyzed, half had pathogenic mutations, all indicating recessive inheritance and high recurrence risks. Results were available within three weeks.

Takeaway: Sequencing-based testing holds promise to rapidly clarify diagnosis of abnormal prenatal ultrasound findings for pregnancy management.