A Novel DLL4 Missense Mutation in a Chinese Patient with Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare genetic disorder characterized by congenital scalp defects (cutis aplasia) and terminal transverse limb malformations. Initially described in 1945, the condition has an estimated prevalence of 1 in 225,000 live births. Clinical features extend beyond the hallmark symptoms, with approximately 20% of patients exhibiting cardiovascular anomalies, vascular irregularities, or central nervous system defects. The genetic basis of AOS is heterogeneous, with six genes currently implicated in its pathogenesis. Autosomal-dominant forms are linked to mutations in ARHGAP31, RBPJ, NOTCH1, and DLL4, while autosomal-recessive forms are associated with DOCK6 and EOGT. Among these, NOTCH1 mutations account for the largest proportion of cases (10%), followed by DLL4 (6%) in European cohorts. This article details the identification of a novel DLL4 missense mutation in a Chinese patient with AOS, expanding the mutational spectrum and phenotypic understanding of this disorder.

Clinical Presentation of the Case

The proband was a 3-year-old female referred for genetic evaluation at the McKusick-Zhang Center for Genetic Medicine. At birth, she presented with a 3 cm × 4 cm cutaneous and cranial defect at the scalp vertex, covered by a thin mucosal layer. By four months of age, the lesion had healed with residual scarring and localized alopecia [Figure 1A, 1B]. Limb abnormalities included brachydactyly and hypoplastic or absent nails on both hands and feet. Radiographic imaging revealed agenesis of the distal phalanges in the 2nd to 5th fingers bilaterally [Figure 1C]. In the lower extremities, the left foot demonstrated single phalanges or ossification centers in the 1st to 4th toes, with complete absence of the 5th toe phalanges. The right foot exhibited similar defects in the 3rd to 5th toes, along with absent phalanges in the 1st and 2nd toes [Figure 1D]. Echocardiography ruled out congenital heart defects, and metabolic screening tests (amino acid/acylcarnitine profiles) were negative. The child exhibited normal cognitive and physical development, with no internal organ involvement. Family history was unremarkable for similar anomalies, and parental consanguinity was denied. Chromosomal analysis identified a pericentric inversion of chromosome 9 [46, XX, inv(9)], a common polymorphic variant considered unrelated to the phenotype.

Genetic Analysis and Mutation Identification

Targeted sequencing of AOS-associated genes (ARHGAP31, DOCK6, RBPJ, EOGT, NOTCH1, DLL4) revealed a heterozygous missense mutation in DLL4 (c.1346G>C), resulting in the substitution of cysteine by serine at position 449 (p.Cys449Ser). This variant was absent in both parents, suggesting a de novo origin or germline mosaicism. The mutation was not reported in public databases (dbSNP150, 1000 Genomes, ExAC, HGMD, ClinVar) and was absent in 200 ethnically matched controls.

The affected residue lies within the epidermal growth factor (EGF)-like 7 domain of DLL4, a cysteine-rich region critical for structural integrity. Multiple sequence alignment demonstrated evolutionary conservation of Cys449 across species from rhesus macaques to lampreys, underscoring its functional importance. In silico tools uniformly predicted pathogenicity: Polyphen-2 classified the variant as “damaging,” SIFT as “deleterious,” and MutationTaster as “disease-causing.” Splice-site prediction algorithms (HSF) confirmed the mutation does not disrupt canonical splicing signals.

DLL4 Structure-Function Relationships and Mutational Landscape

DLL4 maps to chromosome 15q15.1 and encodes a transmembrane ligand for the NOTCH1 receptor, pivotal in embryonic vascular development, angiogenesis, and cell fate determination. The DLL4 protein comprises five domains: an N-terminal MNNL domain, a Delta/Serrate/Lag-2 (DSL) domain, eight EGF-like repeats, a transmembrane segment, and a cytoplasmic tail. Structural studies reveal that NOTCH1 activation requires interactions between its EGF-like repeats 11–12 and the DLL4 DSL/MNNL domains [Figure 1F]. Disruption of these interfaces impairs NOTCH signaling, leading to vascular dysregulation—a plausible mechanism for AOS pathogenesis.

To date, 14 DLL4 mutations have been linked to AOS, including 12 missense and 2 nonsense variants [Figure 1F]. Most cluster within EGF-like domains (8/14), often altering conserved cysteine residues essential for disulfide bonding. For example, mutations like p.Cys404Tyr and p.Cys469Arg perturb EGF-like domain 5 and 8, respectively. The current p.Cys449Ser variant similarly disrupts EGF-like 7, likely destabilizing the protein’s tertiary structure. Two truncating mutations (p.Gly544, p.Arg589) occur in the cytoplasmic domain, though their functional impact remains unclear. Notably, the p.Ala121Pro substitution in the MNNL domain correlates with severe phenotypes, including growth hormone deficiency and cardiac defects, whereas DSL domain mutations (e.g., p.Arg217Gln) manifest milder features.

Genotype-Phenotype Correlations and Variability

Congenital cutis aplasia is nearly universal in DLL4-related AOS, observed in 13/14 reported cases. Terminal limb defects, however, exhibit incomplete penetrance, with severity ranging from nail hypoplasia to complete digit aplasia. Cardiovascular anomalies (e.g., atrial septal defects, pulmonary stenosis) occur in approximately 30% of cases. The proband’s lack of cardiac involvement aligns with the variable expressivity of DLL4 mutations. Environmental modifiers, epigenetic factors, or genetic background may explain this heterogeneity. Additionally, haploinsufficiency of DLL4 in mice causes embryonic lethality due to vascular remodeling defects, suggesting that hypomorphic mutations in humans permit survival but disrupt specific developmental pathways.

Mechanistic Insights and Therapeutic Implications

The NOTCH-DLL4 axis is indispensable for angiogenesis, particularly in arterial specification and tip/stalk cell selection during vascular sprouting. Loss of DLL4 impairs endothelial cell migration and promotes excessive branching, as seen in murine models. In AOS, dysregulated NOTCH signaling may perturb cranial vasculogenesis, leading to scalp defects, or impair limb bud angiogenesis, resulting in terminal hypoperfusion and transverse reductions. The p.Cys449Ser mutation likely diminishes DLL4-NOTCH1 binding affinity, compromising signal transduction. Future studies using cellular models (e.g., endothelial cells expressing mutant DLL4) could quantify NOTCH activation deficits and test therapeutic strategies to augment residual signaling.

Conclusion

This study reports the first DLL4 mutation (p.Cys449Ser) in a Chinese AOS patient, highlighting the global relevance of DLL4 in this disorder. The mutation’s localization within a conserved cysteine residue of the EGF-like 7 domain reinforces the importance of structural integrity in DLL4 function. Although genotype-phenotype correlations remain tentative, the absence of cardiac defects in this case underscores the influence of modifying factors. Expanding mutational screening in diverse populations will refine diagnostic algorithms and deepen insights into NOTCH pathway biology.

doi.org/10.1097/CM9.0000000000000316

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