ARF1-Related Diseases in China: The Initial Study of Phenotype and Molecular Profile

Abstract

Background: The ADP-ribosylation factor 1 (ARF1) gene encodes a protein which plays a critical role in intra-Golgi transport. Clinical evidence suggests that individuals harbouring variants in the ARF1 gene display a consistent set of phenotypic features, including intellectual disability, microcephaly, epilepsy, and periventricular nodular heterotopia (PVNH).

Methods: This study describes the case of a 6-year and 5-month-old female presenting with focal seizures on a fixed side that were resistant to various anti-seizure medications. The genetic aetiology was elucidated through exome sequencing of the pedigree. The pathogenicity of the identified variant was subsequently assessed using molecular dynamics structural analysis, western blotting, and co-immunoprecipitation techniques.

Results: A de novo variant, c.509T > C (p.Leu170Pro), was detected in the ARF1 gene, and functional analysis demonstrated that this modification is anticipated to hinder its association with the Golgi-localising, γ-adaptin ear homology domain and ARF-binding protein, thereby playing a role in the pathogenesis of the disease.

Conclusion: This study introduces the initial instance of ARF1-related disease in China, wherein the patient is without the presence of PVNH. The findings add novel clinical phenotypes to the range of ARF1-related diseases, and an investigation into the potential pathogenic mechanisms of this condition was conducted by confirming the deleterious impacts of the variant.

Keywords: ADP‐ribosylation factor 1; drug‐refractory epilepsy; exome sequencing; gene function analysis.

FIGURE 1

Phenotype of our patient (A and B). Genealogy map of the patient's family (C). VEEG from February 21, 2022, when the patient was 4 years old (D–G, abnormal discharge in the left central, parietal and temporal regions of the brain). MRI and PCT‐CT fusion images of the patient (H and I). MRI, magnetic resonance imaging; PCT‐CT, Positron emission tomography–computed Tomograph VEEG, video electroencephalograph..

FIGURE 2

Molecular dynamics simulation of the complex structure of ARF1 and GGA3 proteins showed that the Leu170Pro variant in ARF1 resulted in the change of the α‐helix structure to the Loop structure (shown in the red box), and the variant also destroyed the hydrogen bonding between the adjacent Tyr167 residue and the GGA3 protein Thr706, Thr697 and Lys695 residues.

FIGURE 3

Western blot results showed that the expression of Flag‐tagged ARF1‐MUT group was a little low, which was significantly lower than that of Flag‐tagged ARF1‐WT group, about 70% (****p < 0.0001).

FIGURE 4

Wild type and mutant ARF1 were co‐transfected into 293 T cells with GGA3, and Flag protein was immunoprecipitated. The interaction between ARF1 and GGA3 was detected by western blotting, and results suggest that ARF1 variation may affect the interaction with GGA3.