Congenital cone-rod synaptic disorder (CRSD), also known as incomplete congenital stationary night blindness (iCSNB), is a non-progressive inherited retinal disease (IRD) characterized by night blindness, photophobia, and nystagmus, and distinctive electroretinographic features. Here, we report bi-allelic RIMS2 variants in seven CRSD-affected individuals from four unrelated families. Apart from CRSD, neurodevelopmental disease was observed in all affected individuals, and abnormal glucose homeostasis was observed in the eldest affected individual. RIMS2 regulates synaptic membrane exocytosis. Data mining of human adult bulk and single-cell retinal transcriptional datasets revealed predominant expression in rod photoreceptors, and immunostaining demonstrated RIMS2 localization in the human retinal outer plexiform layer, Purkinje cells, and pancreatic islets. Additionally, nonsense variants were shown to result in truncated RIMS2 and decreased insulin secretion in mammalian cells. The identification of a syndromic stationary congenital IRD has a major impact on the differential diagnosis of syndromic congenital IRD, which has previously been exclusively linked with degenerative IRD.
In the originally published version of this article, the accession number given for RIMS2a was incorrect. It should have been NM_001348484.1. The number has now been corrected online. The authors regret the error.
Inactivating variants as well as a missense variant in the centrosomal CEP78 gene have been identified in autosomal recessive cone-rod dystrophy with hearing loss (CRDHL), a rare syndromic inherited retinal disease distinct from Usher syndrome. Apart from this, a complex structural variant (SV) implicating CEP78 has been reported in CRDHL. Here we aimed to expand the genetic architecture of typical CRDHL by the identification of complex SVs of the CEP78 region and characterization of their underlying mechanisms. Approaches used for the identification of the SVs are shallow whole-genome sequencing (sWGS) combined with quantitative polymerase chain reaction (PCR) and long-range PCR, or ExomeDepth analysis on whole-exome sequencing (WES) data. Targeted or whole-genome nanopore long-read sequencing (LRS) was used to delineate breakpoint junctions at the nucleotide level. For all SVs cases, the effect of the SVs on CEP78 expression was assessed using quantitative PCR on patient-derived RNA. Apart from two novel canonical CEP78 splice variants and a frameshifting single-nucleotide variant (SNV), two SVs affecting CEP78 were identified in three unrelated individuals with CRDHL: a heterozygous total gene deletion of 235 kb and a partial gene deletion of 15 kb in a heterozygous and homozygous state, respectively. Assessment of the molecular consequences of the SVs on patient’s materials displayed a loss-of-function effect. Delineation and characterization of the 15-kb deletion using targeted LRS revealed the previously described complex CEP78 SV, suggestive of a recurrent genomic rearrangement. A founder haplotype was demonstrated for the latter SV in cases of Belgian and British origin, respectively. The novel 235-kb deletion was delineated using whole-genome LRS. Breakpoint analysis showed microhomology and pointed to a replication-based underlying mechanism. Moreover, data mining of bulk and single-cell human and mouse transcriptional datasets, together with CEP78 immunostaining on human retina, linked the CEP78 expression domain with its phenotypic manifestations. Overall, this study supports that the CEP78 locus is prone to distinct SVs and that SV analysis should be considered in a genetic workup of CRDHL. Finally, it demonstrated the power of sWGS and both targeted and whole-genome LRS in identifying and characterizing complex SVs in patients with ocular diseases.
North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. With the identification of non-coding single nucleotide variants (SNVs) near PRDM13 and duplications overlapping a DNase I hypersensitive site (DHS) near PRDM13 or IRX1 as its underlying genetic cause, we hypothesize that NCMD is a retinal enhanceropathy. Here we aim to provide insight into the cis-regulatory mechanisms of NCMD by integrating multi-omics profiling of human retina with in vitro and in vivo enhancer assays.First, we established RegRet (http://genome.ucsc.edu/s/stvdsomp/RegRet), a genome-wide multi-omics retinal database. Next, UMI-4C profiling was performed on adult human retina to fine-map chromatin interactions of cis-regulatory elements (CREs) with the PRDM13 and IRX1 promoters. Multi-omics analysis including the UMI-4C data revealed sixteen candidate CREs (cCREs), seven for the PRDM13 and nine for the IRX1 region. Subsequently, the activity of cCREs was investigated by in vitro luciferase assays and by in vivo enhancer assays in Xenopus laevis and tropicalis. Four cCREs showed in vivo eye- and brain-specific activity in Xenopus. Furthermore, we expanded the genetic architecture of NCMD with two novel non-coding SNVs (V15, V16) with a likely effect on PRDM13 regulation. Luciferase assays showed that the non-coding SNVs that are located in the two hotspot regions of PRDM13 have an effect on cCRE activity. Interestingly, cCRE4 in which V16 is located was shown to interact with the PRDM13 promoter and demonstrated in vivo activity in Xenopus. This cCRE is active at a specific developmental stage (d103) compatible with the timepoint when retinal progenitor cells of the central retina exit mitosis. Mining of single-cell (sc) transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to emerge and begin to synapse with retinal ganglion cells. This supports the hypothesis that altered PRDM13 or IRX1 expression impairs synaptic interactions between amacrine and ganglion cells during retinogenesis. Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that NCMD is a retinal enhanceropathy.
Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubuleassociated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.
Ciliopathies often comprise retinal degeneration since the photoreceptor’s outer segment is an adapted primary cilium. CEP162 is a distal end centriolar protein required for proper transition zone assembly during ciliogenesis and whose loss causes ciliopathy in zebrafish. CEP162 has so far not been implicated in human disease. Here, we identified a homozygous CEP162 frameshift variant, c.1935dupA (p.(E646R*5)), in retinitis pigmentosa patients from two unrelated Moroccan families, likely representing a founder allele. We found that even though mRNA levels were reduced, the truncated CEP162-E646R*5 protein was expressed and localized to the mitotic spindle during mitosis, but not at the basal body of the cilium. In CEP162 knockdown cells, expression of the truncated CEP162-E646R*5 protein is unable to restore ciliation indicating its loss of function at the cilium. In patient fibroblasts, cilia overcome the absence of CEP162 from the primary cilium by delaying ciliogenesis through the persistence of CP110 at the mother centriole. The patient fibroblasts are ultimately able to extend some abnormally long cilia that are missing key transition zone components. Defective transition zone formation likely disproportionately affects the long-living ciliary outer segment of photoreceptors resulting in retinal dystrophy. CEP162 is expressed in human retina, and we show that wild-type CEP162, but not truncated CEP162-E646R*5, specifically localizes to the distal end of centrioles of mouse photoreceptor cilia. Together, our genetic, cell-based, and in vivo modeling establish that CEP162 deficiency causes retinal ciliopathy in humans.SIGNIFICANCE STATEMENTCiliopathies often comprise retinal degeneration since the photoreceptor’s outer segment is an adapted primary cilium. CEP162 is a basal body protein required for proper transition zone assembly during ciliogenesis and has so far not been implicated in human disease. Using genetic, cell-based, and in vivo studies, we show that a biallelic mutation of CEP162 causes late-onset human retinal degeneration. This mutation specifically hinders CEP162 function at the cilia, leading to impaired transition zone assembly and delayed formation of dysmorphic cilia. Studies in mouse photoreceptors support that absence of CEP162 could lead to defective outer segments. The discovery of novel ciliopathy genes, such as CEP162, advances our insight into cell-specific functions of the primary cilium.
Engrailed 1 (EN1) is a conserved transcription factor essential for programming, survival, and maintenance of midbrain dopaminergic neurons. En1-hemizygosity (En1+/-) leads to a spontaneous Parkinson's disease-like (PD-like) progressive nigrostriatal degeneration as well as motor impairment and depressive-like behavior in SwissOF1 (OF1-En1+/-) mice. This phenotype is absent in C57Bl/6j (C57-En1+/-) mice. Here we studied PD-like phenotypes and early transcriptome profiles in OF1 wild-type (WT) and OF1-En1+/- male mice and compare to that of C57 WT and C57-En1+/- male mice. To detect transcriptional changes prior to dopaminergic cell loss, we performed RNA-seq of 1-week old mice substantia nigra pars compacta (SNpc). Histology and stereology were used to assess dopaminergic nigrostriatal pathology in 4 and 16 weeks old mice. OF1-En1+/- mice showed an increase (+-61617;79%) in dopaminergic striatal axonal swellings from 4 to 16 weeks and a loss (+-61617;23%) of dopaminergic neurons in the SNpc at 16 weeks compared to OF1 WT. Axonal swellings were also present in C57-En1+/- mice but did not increase over time. 52 differentially expressed genes (DEGs) were observed between the C57-WT and the C57-En1+/- mice, while 198 DEGs were observed in the OF1 strain. Enrichment analysis revealed that the neuroprotective phenotype of C57-En1+/- mice was associated with an overexpression of oxidative phosphorylation-related genes compared to both C57 WT and to OF1- En1+/- mice. These results highlight the importance of considering genetic background in PD models and provide valuable insight on how expression of mitochondrial proteins before the onset of neurodegeneration is associated to vulnerability of nigrostriatal dopaminergic neurons.
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