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Importance: Exfoliation syndrome is a systemic disorder characterized by progressive accumulation of abnormal fibrillar protein aggregates manifesting clinically in the anterior chamber of the eye. This disorder is the most commonly known cause of glaucoma and a major cause of irreversible blindness. Objective: To determine if exfoliation syndrome is associated with rare, protein-changing variants predicted to impair protein function. Design, Setting, and Participants: A 2-stage, case-control, whole-exome sequencing association study with a discovery cohort and 2 independently ascertained validation cohorts. Study participants from 14 countries were enrolled between February 1999 and December 2019. The date of last clinical follow-up was December 2019. Affected individuals had exfoliation material on anterior segment structures of at least 1 eye as visualized by slit lamp examination. Unaffected individuals had no signs of exfoliation syndrome. Exposures: Rare, coding-sequence genetic variants predicted to be damaging by bioinformatic algorithms trained to recognize alterations that impair protein function. Main Outcomes and Measures: The primary outcome was the presence of exfoliation syndrome. Exome-wide significance for detected variants was defined as P < 2.5 × 10-6. The secondary outcomes included biochemical enzymatic assays and gene expression analyses. Results: The discovery cohort included 4028 participants with exfoliation syndrome (median age, 78 years [interquartile range, 73-83 years]; 2377 [59.0%] women) and 5638 participants without exfoliation syndrome (median age, 72 years [interquartile range, 65-78 years]; 3159 [56.0%] women). In the discovery cohort, persons with exfoliation syndrome, compared with those without exfoliation syndrome, were significantly more likely to carry damaging CYP39A1 variants (1.3% vs 0.30%, respectively; odds ratio, 3.55 [95% CI, 2.07-6.10]; P = 6.1 × 10-7). This outcome was validated in 2 independent cohorts. The fi

Syne-2 (also known as Nesprin-2) is a member of a family of proteins that are found primarily in the outer nuclear membrane, as well as other subcellular compartments. Syne-2 contains a C-terminal KASH transmembrane domain and is part of a protein network that associates the nuclear envelope to the cytoskeleton via the binding to actin filaments. Syne-2 plays a role in nuclear migration, nuclear positioning during retinal development, and in ciliogenesis. In a previous study, we showed a connection between Syne-2 and the multifunctional scaffold protein Pericentrin (Pcnt). The elimination of the interaction of Syne-2 and Pcnt showed defects in nuclear migration and the formation of outer segments during retinal development, as well as disturbances in centrosomal migration at the beginning of ciliogenesis in general. In this study, the Syne-2 KO mouse model Nesprin-2oABD (Syne-2(tm1Ngl), MGI) with special attention to Pcnt and ciliogenesis was analyzed. We show reduced expression of Syne-2 in the retina of the Syne-2 KO mouse but found no significant structural-and only a minor functional-phenotype. For the first time, detailed expression analyses showed an expression of a Syne-2 protein larger than 400 kDa (similar to 750 kDa) in the Syne-2/Nesprin-2 KO mouse. In conclusion, the lack of an overt phenotype in Syne-2/Nesprin-2 KO mice suggests the usage of alternative translational start sites, producing Syne-2 splice variants with an intact Pcnt interaction site. Nevertheless, deletion of the actin-binding site in the Syne-2/Nesprin-2 KO mouse revealed a high variability in scotopic oscillatory potentials assuming a novel function of Syne-2 in synchronizing inner retinal processes.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptorconnecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt-Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

Pericentrin (Pcnt) is a multifunctional scaffold protein and mutations in the human PCNT gene are associated with several diseases, including ciliopathies. Pcnt plays a crucial role in ciliary development in olfactory receptor neurons, but its function in the photoreceptor-connecting cilium is unknown. We downregulated Pcnt in the retina ex vivo and in vivo via a virus-based RNA interference approach to study Pcnt function in photoreceptors. ShRNA-mediated knockdown of Pcnt impaired the development of the connecting cilium and the outer segment of photoreceptors, and caused a nuclear migration defect. In protein interaction screens, we found that the outer nuclear membrane protein Syne-2 (also known as Nesprin-2) is an interaction partner of Pcnt in photoreceptors. Syne-2 is important for positioning murine photoreceptor cell nuclei and for centrosomal migration during early ciliogenesis. CRISPR/Cas9-mediated knockout of Syne-2 in cell culture led to an overexpression and mislocalization of Pcnt and to ciliogenesis defects. Our findings suggest that the Pcnt–Syne-2 complex is important for ciliogenesis and outer segment formation during retinal development and plays a role in nuclear migration.

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20-40% of wild-type levels) of all three subunits alpha(olf), beta(1) and gamma(13) of the olfactory heterotrimeric G-protein, G(olf), in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per$^{WT}$), mutant peripherin-2 (per$^{MT}$), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to per$^{WT}$ and Rom-1. Furthermore, both mutants are preferentially forming non-covalent per$^{MT}$-per$^{MT}$, per$^{WT}$-per$^{MT}$, and Rom-1-per$^{MT}$ dimers. However, only per$^{WT}$-per$^{MT}$, but not per$^{MT}$-per$^{MT}$ or Rom-1-per$^{MT}$ complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent per$^{WT}$-per$^{MT}$ dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of per$^{WT}$ and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals.