Prenatal Cell-free DNA Screening in Pregnancies With Diverse Genetic Risk Profiles Utilizing Targeted and Whole-exome Sequencing

Genetic Disease

This multicenter study aims to recruit a minimum of 1,600 pregnant women, encompassing individuals with varying levels of genetic risk. The study particularly focuses on cases with increased fetal nuchal translucency (NT ≥3.5 mm), additional ultrasound markers, and/or fetal structural anomalies. Peripheral blood samples of eligible participants will be collected for two state-of-the-art cfDNA tests based on coordinative allele-aware target enrichment sequencing (COATE-seq): (1) a targeted panel to screen for frequent chromosomal aneuploidies, microdeletions/duplications, and dominant single-gene conditions, and (2) comprehensive whole-exome cfDNA sequencing for aneuploidies, microdeletions/duplications, monogenic variants (both dominant and recessive variants), uniparental disomy, and hydatidiform moles. The results of both cfDNA tests will be compared with those from invasive or postnatal diagnostic testing. Pregnancy outcome will be followed up to six weeks postpartum. The primary goal is to determine the clinical validity of targeted and whole exome cfDNA analyses, assessed through sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) relative to diagnostic standards. Secondary goal is to assess efficacy across diverse genetic risk populations by analyzing detection rates of pathogenic variants associated with fetal indications. The clinical utility of cfDNA screening will also be evaluated by its impact on clinical management decisions, including follow-up diagnostic procedures or prenatal/perinatal interventions.

This is a prospective, multicenter clinical study evaluating the clinical validity and utility of a next-generation non-invasive prenatal screening platform, COATE-seq (Coordinative Allele-Aware Target Enrichment Sequencing). COATE-seq is a cfDNA-based approach that integrates hybrid capture panel sequencing with whole-exome sequencing (WES) from a single maternal plasma sample, enabling detection of a broad spectrum of fetal genetic conditions. These include common and rare aneuploidies, clinically significant microdeletions, monogenic pathogenic variants, uniparental disomy (UPD), and molar pregnancies. The platform leverages coordinated allelic modeling and deep sequencing to enhance sensitivity for low-allelic-fraction variants, including those in autosomal recessive and dominant genes. The panel-based component targets 56 monogenic conditions selected using the SEPH framework (Severe, Early-onset, Prevalent, High detectability), while the WES component interrogates approximately 19,000 protein-coding genes to enable secondary and exploratory analyses of ultra-rare or novel variants. Variant interpretation adheres to ACMG/AMP guidelines, and only pathogenic or likely pathogenic variants associated with severe outcomes are reported. The study is designed to benchmark the performance of this integrated assay against conventional diagnostic standards, including karyotyping, chromosomal microarray (CMA), targeted gene sequencing, whole-exome sequencing, and whole-genome sequencing. Reference testing is performed prenatally or postnatally, and in select cases, UPD is confirmed via methylation-specific PCR or short tandem repeat (STR) analysis. Data collected across the study include: Clinical history and prenatal imaging findings cfDNA sequencing results from both targeted panels and exome-based assays Confirmatory diagnostic results Pregnancy outcomes and postnatal phenotypes, where available Performance metrics including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) will be calculated in subjects with complete cfDNA and diagnostic data. Additional analyses will assess detection rates stratified by clinical indication (such as abnormal ultrasound findings), quantify the incremental yield of WES compared to panel-only testing, and evaluate the downstream clinical impact on decision-making and pregnancy management. COATE-seq incorporates a rigorously validated bioinformatics pipeline for fetal fraction estimation, detection of allelic imbalance, chromosomal dosage, copy number variation (CNV) analysis, and pathogenic variant annotation. Quality assurance procedures are applied throughout sample processing, sequencing, and data interpretation, including safeguards during plasma handling, DNA extraction, target capture, library construction, and analytical review. The study complies with Good Clinical Practice (GCP) and relevant regulatory and ethical guidelines. Institutional Review Board (IRB) approval is obtained at all participating sites, and informed consent is secured prior to enrollment. The results of this study will inform best practices for expanded cfDNA screening in prenatal care and contribute to the evidence base supporting the clinical integration of comprehensive molecular testing for fetal genetic conditions. Scientific findings will be disseminated through peer-reviewed publications and conference presentations.

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China, Hong Kong