June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
A precision genomics approach to validating NOLC1 as the cause of a unique nanophthalmos-microcephaly syndrome
Author Affiliations & Notes
  • Sairah Yousaf
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Blake Carrington
    National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Zhiyu Li
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Mones Abu-Asab
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Laryssa Huryn
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Raman Sood
    National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Robert B. Hufnagel
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Sairah Yousaf None; Blake Carrington None; Zhiyu Li None; Mones Abu-Asab None; Laryssa Huryn None; Raman Sood None; Robert Hufnagel None
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 4525. doi:
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      Sairah Yousaf, Blake Carrington, Zhiyu Li, Mones Abu-Asab, Laryssa Huryn, Raman Sood, Robert B. Hufnagel; A precision genomics approach to validating NOLC1 as the cause of a unique nanophthalmos-microcephaly syndrome. Invest. Ophthalmol. Vis. Sci. 2023;64(8):4525.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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