Disorders linked to DLL1 (OMIM: 618709) exhibit diverse neurodevelopmental symptoms, including developmental delays, intellectual disabilities, autism, attention deficits, stereotypical behaviors, and brain anomalies like hydrocephalus and dysplasia. Common symptoms also include seizures, hypotonia, joint hyperextension, ataxia, scoliosis/kyphosis, and cognitive impairment (Fischer-Zirnsak et al., 2019). Overlapping microdeletions in the 6q27 region, affecting genes like DLL1, THBS2, PHF10, and ERMARD, linked to developmental delay, intellectual disability, and brain malformations (Fischer-Zirnsak et al., 2019). During embryogenesis, DLL1, expressed in neural precursor cells, regulates differentiation through oscillatory Notch signaling, affecting brain development by inhibiting differentiation in adjacent cells (Fischer-Zirnsak et al., 2019; Louvi et al., 2006; Cao et al., 2019; Shimojo et al., 2011; Artavanis-Tsakonas et al., 1999). Research has shown that the Notch ligand DLL1 is crucial for developing the central nervous system, somites, and lymphocytes (Gray et al., 1999; Hrabĕ de Angelis et al., 1997; Marklund et al., 2010; Hiraoka et al., 2013; Dunwoodie et al., 1997; Okigawa et al., 2014; Mahler et al., 2010). Among these, DLL1 is most likely intolerant to loss of function (LoF), suggesting its deficiency may contribute to neurodevelopmental disorders (Fischer-Zirnsak et al., 2019; Karczewski et al., 2020; Peddibhotla et al., 2015). Given DLL1’s role in neurodevelopment, we investigated whether 6q terminal deletions involving DLL1 underlie prenatal brain anomalies detected by ultrasound/MRI. To delineate genomic causes, we applied CNV-seq to all cases and performed reflex WES when CNV-seq was negative.

In this study, analyze five cases of cerebral dysplasia within a familial context for genetic diagnosis. All fetuses exhibited a cerebellar diameter smaller than expected for their gestational age, as determined by US, 4/5 cases underwent MRI. Common complications included mild lateral ventricle dilatation (5/5 cases), corpus callosum abnormalities (1/5 cases) and low positioning of the conus medullaris (2/5 cases). Among them, fetus 4 was first found at 21 + 4 weeks of gestation with multiple system malformations, Included were fetal growth restriction, double outlet right ventricle, Nuchal fold thickened, scoliosis, hand deformities. We applied CNV-seq to all cases. CNV-seq identified the 6q terminal deletion in fetuses 1–4, however, in fetus 5, a deletion missed by CNV-seq was detected by WES, analysis revealed a heterozygous deletion of 0.14 Mb in the Chr6:g.170460966-170604541 region, likely terminal coverage drop/binning limit.

The study of DLL1 and prenatal fetal phenotypes is relatively limited. Research by Sanlaville (Lesieur-Sebellin et al., 2022) explored the relationship between DLL1 and fetal phenotypes, revealing that fetuses with a DLL1 deletion exhibited altered brain structures. The most prevalent abnormalities were cerebellar dysplasia, increased ventricle width, and corpus callosum anomalies (observed in over 70% of cases). Gyration abnormalities were identified in 46% of the fetuses. Less common phenotypes included heterotopia cerebri, vertebral malformations, and renal abnormalities. In this study, five fetuses exhibited bilateral lateral ventricle expansion, and the cerebellar transverse diameter was smaller than expected for the gestational age, compared to typical clinical phenotypes. Two cases showed isolated chamber abnormalities.

Notably, fetus number 4 had the largest terminal deletion on chromosome 6q (21.99 Mb) and presented with the most severe phenotype. In addition to bilateral ventriculomegaly and a cerebellar transverse diameter smaller than expected for the gestational age, the condition was also associated with spinal abnormalities, including scoliosis, intrauterine growth retardation, and abnormal development of both arms. When the 6q terminal deletion extends to 7.1 Mb, the majority of the patient’s clinical features can be attributed to the DLL1. Deletions exceeding 7.1 Mb result in a more severe phenotype (Engwerda et al., 2023). DLL1 haploinsufficiency plausibly explains prenatal anomalies.

CNV-Seq analysis can detect aneuploidies as well as pathogenic/likely pathogenic CNVs, significantly improving abnormality detection rates (Zhang C et al., 2018). Currently, CNV-seq achieves a resolution limit of about 100 kb. In this study, CNV-seq successfully identified 6q terminal deletions in fetuses 1–4, but failed to detect the 0.14 Mb deletion in fetus 5, a size within its theoretical detection range. This miss may be due to insufficient coverage of the 6q terminal region during sequencing. WES has been widely used clinically for diagnosing monogenic disorders (Zhang et al., 2021), can detect single-nucleotide variants, small insertions/deletions (≤20 bp), certain exon-level CNVs, and copy number changes. Fetus 5, the CNV-seq false negative was subsequently identified by WES. Thus, in prenatal cases with findings such as hydrocephalus, ventriculomegaly, or a small cerebellar diameter, WES successfully identified the deletion that CNV-seq had missed. As of May 2024, the ClinVar database lists 78 pathogenic/likely pathogenic CNVs, 25 frameshift, 7 nonsense (across 15 variants), and 4 splicing/missense mutations related to this region. Therefore, if CNV testing is negative in fetuses with these phenotypes, further analysis for single-base changes and small indels should be pursued to exclude monogenic disorders.

This study was a retrospective study with a small sample size, no postnatal phenotyping due to pregnancy terminations, imaging variability. Future work should prioritize the establishment of prospective registries, harmonized imaging metrics, capture of postnatal outcomes when pregnancies continue.

Fetuses with reduced transverse cerebellar diameter, hydrocephalus, or bilateral ventriculomegaly should be evaluated for 6q terminal deletions involving DLL1. Reflex WES following negative CNV-seq reduces missed diagnoses and improves prenatal counseling.