A Tiered Genetic Diagnostic Approach in Newborns With Major Congenital Anomalies: Experience From a Tertiary NICU

Major congenital anomalies (MCAs) are a leading cause of neonatal morbidity and mortality and frequently reflect underlying chromosomal, copy number, or single-gene defects. In neonates with multiple MCAs, early molecular diagnosis is critical but remains challenging due to phenotypic heterogeneity, overlapping developmental pathways, and limitations in selecting the most appropriate genetic test.

This prospective study included 146 neonates with congenital anomalies who were referred for genetic consultation from a tertiary neonatal intensive care unit between April 2022 and May 2025. A major congenital anomaly was defined as a structural abnormality present at birth with significant medical, functional, or cosmetic consequences, while multiple congenital anomalies were defined as two or more major anomalies involving different organ systems. Clinical features were systematically annotated using Human Phenotype Ontology terms and classified according to organ system and embryological origin. A tiered diagnostic strategy was applied, including conventional cytogenetic analysis, chromosomal microarray analysis (CMA), whole-exome sequencing (WES), and rapid-WES (rWES) in selected critically ill neonates.

Cardiovascular anomalies were the most frequent (45.9%), and 88 of 146 neonates (60.3%) had two or more MCAs. Cytogenetic testing identified numerical chromosomal abnormalities in eight neonates. CMA detected pathogenic or likely pathogenic copy number variants in 6 of 65 tested cases (9.1%). In CMA-negative patients, WES achieved a diagnostic yield of 41.4%, identifying variants in genes involved in multisystem developmental pathways. Rapid WES established definitive molecular diagnoses in two of three critically ill neonates within 72 h, directly influencing clinical management. Embryological analysis revealed recurrent co-occurrence patterns, particularly cardiovascular-craniofacial and cardiovascular-genitourinary associations.

A structured, tiered genomic approach enhances diagnostic yield in neonates with congenital anomalies. Integration of cytogenetics, CMA, and CNV-aware exome sequencing provides a pragmatic and clinically actionable framework for molecular diagnosis in NICU settings and supports developmentally informed interpretation of complex phenotypes.