J Korean Med Sci.  2024 Sep;39(36):e250. 10.3346/jkms.2024.39.e250.

Diagnostic Utility of Whole Genome Sequencing After Negative Karyotyping/Chromosomal Microarray in Infants Born With Multiple Congenital Anomalies

• Yang M
• Kim JA
• Jo HS
• Park JH
• Ahn SY
• Sung SI
• Park WS
• Cho HW
• Kim JM
• Park MH
• Park HY
• Jang JH
• Chang YS

• KMID: 2559773
• DOI: http://doi.org/10.3346/jkms.2024.39.e250

Abstract

Background
Achieving a definitive genetic diagnosis of unexplained multiple congenital anomalies (MCAs) in neonatal intensive care units (NICUs) infants is challenging because of the limited diagnostic capabilities of conventional genetic tests. Although the implementation of whole genome sequencing (WGS) has commenced for diagnosing MCAs, due to constraints in resources and faculty, many NICUs continue to utilize chromosomal microarray (CMA) and/or karyotyping as the initial diagnostic approach. We aimed to evaluate the diagnostic efficacy of WGS in infants with MCAs who have received negative results from karyotyping and/or CMA.
Methods
In this prospective study, we enrolled 80 infants with MCAs who were admitted to a NICU at a single center and had received negative results from CMA and/or karyotyping. The phenotypic characteristics were classified according to the International Classification of Diseases and the Human Phenotype Ontology. We assessed the diagnostic yield of trioWGS in infants with normal chromosomal result and explored the process of diagnosing by analyzing both phenotype and genotype. Also, we compared the phenotype and clinical outcomes between the groups diagnosed with WGS and the undiagnosed group. Results: The diagnostic yield of WGS was 26% (21/80), of which 76% were novel variants. There was a higher diagnostic yield in cases of craniofacial abnormalities, including those of the eye and ear, and a lower diagnostic yield in cases of gastrointestinal and genitourinary abnormalities. In addition, higher rates of rehabilitation therapy and gastrostomy were observed in WGS-diagnosed infants than in undiagnosed infants.
Conclusion
This prospective cohort study assessed the usefulness of trio-WGS following chromosomal analysis for diagnosing MCAs in the NICU and revealed improvements in the diagnostic yield and clinical utility of WGS.

Keyword

Congenital Abnormalities; Whole Genome Sequencing; Microarray Analysis; Karyotyping; Infants; Intensive Care Units, Neonatal

Figure

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  Fig. 1 Study population.NICU = neonatal intensive care unit, CMA = chromosomal microarray, WGS = whole genome sequencing.

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  Fig. 2 Genetic diagnosis using RT-PCR and cDNA analysis. (A) RT-PCR amplifications of the mRNA of case 17. The lower band (386 bp) represents the aberrant NIPBL gene transcript. (B) Diagram showing regions of the NIPBL where the splicing variant described was identified as NM_133433.3(NIPBL):c.5808G>A. The cDNA sequence shows skipping of exon 31.RT-PCR = reverse transcription polymerase chain reaction.

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  Fig. 3 Genetic diagnosis using gap PCR and direct sequencing. (A) Electrophoresis of gap-PCR amplifications of Case 12. (B) Diagram showing a 59 kb deletion on chromosome 6p25.3 including the entire FOXC1 gene and part of the adjacent GMDS gene. The band was sequenced, and the breakpoint was delineated as NC_000006.11:g.1596640_1655705delinsGAG.PCR = polymerase chain reaction.

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  Fig. 4 Distribution of infants by organ system based on HPO term.HPO = Human Phenotype Ontology, WGS = whole genome sequencing.

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