Supari, N. | Search bioRxiv

bioRxiv2026

Patient-specific zebrafish models reveal a complex phenotypic spectrum in NBAS-associated Atypical Osteogenesis Imperfecta

Neuroblastoma amplified sequence gene (NBAS) variants are associated with short stature, optic atrophy, and Pelger-Huet anomaly (SOPH) syndrome. We previously identified compound heterozygous variants in NBAS to cause atypical Osteogenesis Imperfecta (OI), with these patients presenting with short stature, developmental delay and recurrent long-bone fractures. However, skeletal disease progression due to these variants and the disease mechanisms underlying NBAS-associated OI remain poorly understood. Here, we provide a clinical update on previously identified patients and investigate the role of NBAS during skeletal development using zebrafish knockout and patient-specific missense variant zebrafish models. Homozygous knockout larvae exhibited delayed operculum development, reduced bone ossification, and defects in Meckels cartilage morphology and its underlying cellular structure. Homozygous missense larvae displayed milder cartilage defects without any major defects to early skeletal structures. Seemingly opposing phenotypes were observed in compound heterozygous zebrafish carrying the knockout and missense alleles in trans, with no obvious phenotypes seen in the Meckels cartilage and accelerated operculum development observed. Together, our results demonstrate that different nbas variants differentially affect skeletal development, suggesting complex spectrums of phenotypic and pathogenic mechanisms in NBAS-associated atypical OI.

genetics↗

bioRxiv2026

Functional surrogacy enables Vascular Ehlers-Danlos Syndrome modelling in zebrafish in the absence of a COL3A1 ortholog

Pathogenic variants in COL3A1 cause Vascular Ehlers-Danlos syndrome (vEDS), a rare connective tissue disorder characterised by vascular fragility, increasing the risk of arterial ruptures/dissection. Advances in genomic sequencing have led to an increasing number of COL3A1 variants where the clinical significance is unclear, with these being termed variants of uncertain significance (VUS). VUS creates challenges for diagnosis and clinical management. Thus major efforts have been made to reclassify these to either pathogenic or benign variants in disease causality. Functional data from model systems can provide significant evidence to clinicians on the pathogenicity of a variant. To address the increasing numbers of VUS in COL3A1, we developed a fast pipeline using F0 crispant zebrafish to provide functional evidence for variant classification despite there being no direct orthologue of COL3A1 in zebrafish. Loss of col5a1 resulted in cardiac defects, dysmorphic blood vessel structures and delayed angiogenic sprouting. Trunk haemorrhage prevalence under physical stress increased in col5a1 knockout zebrafish, recapitulating vEDS patients. Remarkably, co-injection of F0 col5a1 knockout crispants with human wildtype COL3A1 mRNA partially rescued cardiac and vascular phenotypes, indicating a level of functional conservation between zebrafish type V and human type III collagen. These findings establish a tractable in vivo platform for functional assessment of COL3A1 VUS. Phenotypic rescue with wildtype COL3A1 provides a benchmark against which the pathogenicity of variants can be evaluated, generating functional evidence for VUS reclassification. Our model provides both a valuable tool for investigating vEDS disease mechanisms and a clinically relevant platform to improve diagnoses for patients with suspected vEDS.

genetics↗