Variants in NR6A1 cause a novel oculo-vertebral-renal (OVR) syndrome

Abstract

Colobomatous microphthalmia is a potentially blinding congenital ocular malformation that can present either in isolation or together with other syndromic features. Despite a strong genetic component to disease, many cases lack a molecular diagnosis. We describe a novel autosomal dominant oculo-vertebral-renal (OVR) syndrome in six independent families characterized by colobomatous microphthalmia, missing vertebrae and congenital kidney abnormalities. Genome sequencing identified six rare variants in the orphan nuclear receptor gene NR6A1 in these families. We performed in silico, cellular and zebrafish experiments to demonstrate the NR6A1 variants were pathogenic or likely pathogenic for OVR syndrome. Knockdown of either or both zebrafish paralogs of NR6A1 results in abnormal eye and somite development, which was rescued by wild-type but not variant NR6A1 mRNA. Illustrating the power of genomic ascertainment in medicine, our study establishes NR6A1 as a critical factor in eye and vertebral development and a pleiotropic gene responsible for OVR syndrome.

Fig. 2:. Subcellular localization of wild-type (WT) and mutant forms of NR6A1.

NR6A1 variant localization pattern was studied by overexpression in HEK293 cells and representative high magnification (63X) images are shown from three different trials (A). Scale bar = 10 μm. The localization pattern for the WT and the two variant isoforms was observed to be consistent across three transfection experiments. (Cells counted: WT=387, R92W=350 and R436C=217). Scale bar = 100 μm.

Fig. 3:

A. Comparative levels of NR6A1 from publicly available bulk human tissue RNA-sequencing (RNA-Seq) datasets accessed on the eyeIntegration website (https://eyeintegration.nei.nih.gov/). On average, expression is higher in embryonic and induced pluripotent stem cells (ESC, iPSC, respectively) than in adult ocular tissues. B. In adult retina, expression of NR6A1 is highest in horizontal cells (HC) compared to other cell types in the Human Retinal Cell Atlas (HRCA) (AC, amacrine cell; BC, bipolar cell; MG, Müller glia; RGC, retinal ganglion cell; RPE, retinal pigment epithelium). C. Correlation of NR6A1 expression with fetal retina and RPE RNA-Seq data demonstrates association with several known coloboma associated genes (boxed labels). D. Among systemic tissues, NR6A1 is expressed most highly in bone marrow and testes.

Fig. 4:. Expression pattern of nr6a1a and nr6a1b paralogs in zebrafish.

nr6a1a is expressed ubiquitously at 11 hours post-fertilization (hpf) (A). By 16–19 hpf (B, C) expression is present in the somites (S), neural tube (NT), and notochord (N). At 24 hpf, expression remains in the NT but is decreased in the S and N. Expression in the lens (L) is first noted at 19 hpf and is particularly prominent by 24 hpf (D-D”). nr6a1b expression at 11 hpf is anterior trunk, localizing to neural tube and somites from 16 hpf (F) and 19 hpf (G). At 24hpf (H-H”) it remains expressed in the neural tube and somites, with faint expression can be seen in the lens. All embryos are oriented in a lateral view, anterior to the left and dorsal up, except D’ and H’ shown in dorsal views. Scale bar = 100 μM. e-epiphysis, l-lens, p-pronephros, n-notochord, s-somite, nt-neural tube, ad-anterior diencephalon, tg-tegementum.

Fig. 5:. Rescue of nr6a1+nr6a1b zebrafish morphant phenotypes with wildtype and mutant human NR6A1 mRNA:

Controls (A, A’) have a straight body axis and the optic fissure (OF) is closed. The nr6a1+nr6a1b morphants that have a mild phenotype (B, B’) have close to a normal body with microphthalmia and heart edema; a moderate phenotype (C, C’) with a slightly bent body axis with smaller eyes, coloboma and a severe heart edema; and severe morphants (D, D”) have a curved body axis with smaller eyes, coloboma and heart edema. The morphant phenotype was rescued when the morpholinos were co-injected along with the human-NR6A1-wild type mRNA. However, there was no significant rescue in the morphant phenotype when the morpholinos were injected with either R92W or R436C human disease-causing variants (E). Morpholinos were injected at 0.75 ng each (1.5 ng total). Scale bar = 100μM