Parallel Sequencing of Fetal Genome and RNA in the Presence of Ultrasound Warning Signs: a Complementary Approach for the Prenatal Diagnosis of Rare Diseases.

Parallel Sequencing of the Fetal Genome and RNA in the Presence of Ultrasound Warning Signs: a Complementary Approach for the Prenatal Diagnosis of Rare Diseases.

Prenatal exome sequencing (ES) is increasingly used for fetuses with ultrasound-detected anomalies but yields 10-15% variants of uncertain significance (VUS), limiting diagnostic performance, particularly in prenatal settings with incomplete phenotypes.

This study aims to evaluate the added value of combined prenatal genome sequencing (GS) and RNA sequencing (RNA-Seq), which are not currently part of routine care. Conducted at AP-HP, it will compare the diagnostic yield of GS + RNA-Seq with the current standard approach (chromosomal microarray analysis + ES), according to variant type (coding, non-coding, and structural). The contribution of systematic RNA-Seq to rapid VUS resolution will be specifically assessed.

Overall, this project will assess the feasibility, diagnostic performance, and clinical utility of implementing GS + RNA-Seq in prenatal diagnosis, supporting future integration into routine care in France.

Couples will be enrolled during a "pre-test" genetic consultation by a physician or a research-trained collaborator (genetic counselor or another investigator).

As part of routine clinical care: chromosomal microarray analysis (CMA) and exome sequencing (ES) will be performed locally, with interpretation by a biologist from the site providing follow-up to the couple.

For research purposes, after obtaining written consent from both the pregnant woman and her partner: samples will be prepared for pre-analytical processing at Pitié-Salpêtrière and sequenced at SeqOIA. Interpretation will be conducted by a biologist at a site different from the one performing the exome analysis, using the Gleaves-P interface dedicated to the project (MOABI).

Transcriptomic analysis (RNA-Seq) will be performed on RNA extracted from amniotic fluid cultures, with and without emetine (a NMD inhibitor). Sequencing will be carried out after Agilent capture at the Genetics Laboratory of Necker-Enfants Malades Hospital (AP-HP). Bioinformatics analysis will be performed by the Imagine platform. Study biologists will interpret results using the PolyRNASEQ interface (qualitative splice junction analysis) and IGV (semi-quantitative analysis and expression).

All results from CMA + ES and GS + RNA-Seq will be reviewed during weekly meetings. In cases of discordant or unexpected results (anticipated for ~10 couples), a reference method will be applied to provide clinically validated results through an accredited prenatal diagnostic center, ensuring no loss of care opportunity.

Results will be discussed in a multidisciplinary prenatal diagnosis meeting (RCP) and integrated into the prognostic discussion. Couples will be seen during a "post-test" genetic consultation for communication of results.

For couples included at Necker-Enfants Malades Hospital only: two additional maternal blood tubes will be collected during routine pregnancy follow-up before amniocentesis. Circulating DNA will be extracted from maternal plasma and subjected to genome sequencing under the same conditions as the amniotic DNA. The generated data will be analysed using bioinformatic approaches integrating artificial intelligence methods to improve the distinction between fetal- and maternal-derived DNA fragments. Interpretation will be performed via the Gleaves-P interface. Non-invasive prenatal genome sequencing results are strictly for research purposes: they will not be communicated to couples and will not be used for clinical decision-making. Their objective is to evaluate the feasibility, accuracy, and performance of non-invasive genome sequencing.

Inclusion Criteria:

• Participating couple aged ≥ 18 years
• Ongoing pregnancy for which the indication for exome sequencing has been discussed and validated by a Multidisciplinary Prenatal Diagnosis Center (CPDPN).

While a strict list of indications is not appropriate in the prenatal setting, examples include multiple anomalies not related to a malformation sequence, persistent increased nuchal translucency, hydrops fetalis (anasarca), cleft palate, multiple contractures/arthrogryposis, skeletal dysplasia, bowed femurs, or brain anomalies.

• Sequencing performed on an amniotic fluid sample (chorionic villus sampling is excluded)
• Attendance at a genetic counseling consultation
• Written informed consent obtained for study participation

Exclusion Criteria:

• Couple not covered by the social security system
• Couple deprived of liberty or under legal guardianship or curatorship
• Monoparental pregnancies