Trio Whole-Genome Sequencing for Diagnosis of Neonates with Multiple Congenital Anomalies

Genetic disorders are major causes of morbidity and mortality in neonatal intensive care units (NICUs), yet traditional tests like karyotyping and chromosomal microarray (CMA) often fail to provide a diagnosis, resulting in a "diagnostic odyssey". Trio whole-genome sequencing (WGS) has emerged as a comprehensive tool capable of detecting a wide range of variants beyond protein-coding regions, potentially offering higher diagnostic yields and rapid, actionable results for critical care management.

This prospective study evaluated 16 neonates with multiple congenital anomalies who had previously received negative results from conventional genetic investigations. Trio WGS was performed using the Illumina NovaSeq 6000 platform, targeting a mean coverage of ×30. A stringent bioinformatics pipeline and a three-step filtering process were applied to prioritize rare functional variants (MAF <0.1%) and genes specifically curated for NICU-associated conditions.

A molecular diagnosis was achieved in 37.5% (6/16) of the cases within the first 6 months of age. Pathogenic or likely pathogenic variants were identified in genes including SCN4A, DLL1, ACTA1, KLHL40, CHD7, and CNTNAP1, covering both autosomal recessive and autosomal dominant inheritance patterns. These findings provided definitive diagnoses for complex conditions such as arthrogryposis, congenital myopathy, and CHARGE syndrome.

Trio WGS is a highly effective and feasible diagnostic tool for neonates with clinically heterogeneous anomalies, successfully identifying causes where standard-of-care testing fails. Its implementation in the NICU facilitates targeted medical management, informs prognosis, and provides essential data for family planning.