Abstract
Purpose :
Impaired ribosome biogenesis results in tissue-specific malformations, in particular neurodevelopmental and craniofacial syndromes. Here, we report a novel neuro-ocular syndrome including nanophthalmos, microcephaly, global developmental delay, and short stature.
Methods :
Exome sequencing was used to identify the NOLC1 (Nucleolar & coiled-body phosphoprotein 1) variant in affected individuals. Western blot assay, immunohistochemistry, and transmission electron microscopy (TEM) studies were used to assess the functional impact of identified variant in patient-derived cells. Expression of nolc1 in zebrafish was detected by whole mount in situ hybridization. CRISPR/Cas9 technique was used to generate zebrafish null (knock-out, KO) & missense (knock-in, KI) lines. Homozygous (hmz) mutant larvae at 7 days post fertilization (dpf) were evaluated for head & eye abnormalities through gross morphology analysis, histology, and TEM.
Results :
A novel homozygous missense variant (NM_004741.5: c.146C>T, p.Ser49Phe) in NOLC1 segregated with a novel nanophthalmos-microcephaly syndrome. NOLC1 protein was significantly reduced in patient-derived cells, suggesting a loss-of-function mechanism. TEM of patient fibroblasts demonstrated fewer nucleolar dense fibrillar components & malformed tripartite nucleolar organization compared to controls. Embryonic zebrafish expression of nolc1 was localized to the developing brain and eyes. Next, nolc1-null KO fish (NM_001256652.1:c.1371_1377delACCCACC, p.Pro458Argfs*54 homozygotes) recapitulated human phenotypes, namely reduced brain (p<0.0005), and eye size (p<0.0001). Similarly, microcephaly (p<0.0001) and nanophthalmos (p<0.0001) were observed in a CRISPR-generated nolc1 missense KI model (NM_001256652.1:c.136A>T & c.137G>T, p.Ser46Phe) harboring the human-equivalent NOLC1 variant. Both nolc1 KO and KI hmz mutants lead to 99 and 92% lethality by 12 dpf, respectively. TEM analysis revealed less developed nucleoli in the retinal outer nuclear layer & retinal pigment epithelium of both KO & KI lines.
Conclusions :
Human in vivo & in vitro phenotyping coupled to human-equivalent zebrafish genetic models support the pathogenic nature of this NOLC1 biallelic variant. Insights into the nucleolar mechanism of this novel ribosomopathy may unlock next-generation therapeutic strategies for this family of disorders.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.