Altered centriolar cohesion by CEP250 and appendages impact outcome of patients with pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the leading cause of cancer death worldwide. PDACs are characterized by centrosome aberrations, but whether centrosome-related genes influence patient outcomes has not been tested. METHODS: Publicly available RNA-sequencing data of patients diagnosed with PDAC were interrogated with unsupervised approaches to identify centrosome protein-encoding genes with prognostic relevance. Candidate genes were validated by immunohistochemistry and multiplex immunofluorescence in a set of clinical PDAC and normal pancreatic tissues. RESULTS: Results showed that two genes CEP250 and CEP170, involved in centrosome linker and centriolar subdistal appendages, were expressed at high levels in PDAC tissues and were correlated with prognosis of PDAC patients in independent databases. Large clustered γ-tubulin-labelled centrosomes were linked together by aberrant circular and planar-shaped CEP250 arrangements in CEP250-high expressing PDACs. Furthermore, PDACs displayed prominent centrosome separation and reduced CEP164-centrosomal labelling associated with acetylated-tubulin staining compared to normal pancreatic tissues. Interestingly, in a small validation cohort, CEP250-high expressing patients had shorter disease free- and overall-survival and almost none of those who received gemcitabine plus nab-paclitaxel first-line therapy achieved a clinical response. In contrast, weak CEP250 expression was associated with long-term survivors or responses to medical treatments. CONCLUSIONS: Alteration of the centriolar cohesion and appendages has effect on the survival of patients with PDAC.

RNA-Seq Analysis Reveals an Essential Role of the cGMP-PKG-MAPK Pathways in Retinal Degeneration Caused by Cep250 Deficiency.

Usher syndrome (USH) is characterised by degenerative vision loss known as retinitis pigmentosa (RP), sensorineural hearing loss, and vestibular dysfunction. RP can cause degeneration and the loss of rod and cone photoreceptors, leading to structural and functional changes in the retina. Cep250 is a candidate gene for atypical Usher syndrome, and this study describes the development of a Cep250 KO mouse model to investigate its pathogenesis. OCT and ERG were applied in Cep250 and WT mice at P90 and P180 to access the general structure and function of the retina. After recording the ERG responses and OCT images at P90 and P180, the cone and rod photoreceptors were visualised using an immunofluorescent stain. TUNEL assays were applied to observe the apoptosis in Cep250 and WT mice retinas. The total RNA was extracted from the retinas and executed for RNA sequencing at P90. Compared with WT mice, the thickness of the ONL, IS/OS, and whole retina of Cep250 mice was significantly reduced. The a-wave and b-wave amplitude of Cep250 mice in scotopic and photopic ERG were lower, especially the a-wave. According to the immunostaining and TUNEL stain results, the photoreceptors in the Cep250 retinas were also reduced. An RNA-seq analysis showed that 149 genes were upregulated and another 149 genes were downregulated in Cep250 KO retinas compared with WT mice retinas. A KEGG enrichment analysis indicated that cGMP-PKG signalling pathways, MAPK signalling pathways, edn2-fgf2 axis pathways, and thyroid hormone synthesis were upregulated, whereas protein processing in the endoplasmic reticulum was downregulated in Cep250 KO eyes. Cep250 KO mice experience a late-stage retinal degeneration that manifests as the atypical USH phenotype. The dysregulation of the cGMP-PKG-MAPK pathways may contribute to the pathogenesis of cilia-related retinal degeneration.

Functional characterization of CEP250 variant identified in nonsyndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common manifestation of inherited retinal diseases with high degree of genetic, allelic, and phenotypic heterogeneity. CEP250 encodes the C-Nap1 protein and has been associated with various retinal phenotypes. Here, we report the identification of a mutation (c.562C>T, p.R188*) in the CEP250 in a consanguineous family with nonsyndromic RP. To gain insights into the molecular pathomechanism underlying CEP250 defects and the functional relevance of CEP250 variants in humans, we conducted a functional characterization of CEP250 variant using a novel Cep250 knockin mouse line. Remarkably, the disruption of Cep250 resulted in severe impairment of retinal function and significant retinal morphological alterations. The homozygous knockin mice showed significantly reduced retinal thickness and ERG responses. This study not only broadens the spectrum of phenotypes associated with CEP250 mutations, but also, for the first time, elucidates the function of CEP250 in photoreceptors using a newly established animal model.

Novel Variant in CEP250 Causes Protein Mislocalization and Leads to Nonsyndromic Autosomal Recessive Type of Progressive Hearing Loss.

Genetic hearing loss is the most common hereditary sensorial disorder. Though more than 120 genes associated with deafness have been identified, unveiled causative genes and variants of diverse types of hearing loss remain. Herein, we identified a novel nonsense homozygous variant in CEP250 (c.3511C>T; p.Gln1171Ter) among the family members with progressive moderate sensorineural hearing loss in nonsyndromic autosomal recessive type but without retinal degeneration. CEP250 encodes C-Nap1 protein belonging to the CEP protein family, comprising 30 proteins that play roles in centrosome aggregation and cell cycle progression. The nonsense variant in CEP250 led to the early truncating protein of C-Nap1, which hindered centrosome localization; heterologous expression of CEP250 (c.3511C>T) in NIH3T3 cells within cilia expression condition revealed that the truncating C-Nap1 (p.Gln1171Ter) was not localized at the centrosome but was dispersed in the cytosol. In the murine adult cochlea, Cep250 was expressed in the inner and outer hair cells. Knockout mice of Cep250 showed significant hair cell degeneration and progressive hearing loss in auditory brainstem response. In conclusion, a nonsense variant in CEP250 results in a deficit of centrosome localization and hair cell degeneration in the cochlea, which is associated with the progression of hearing loss in humans and mice.

Expanding the clinical phenotype in patients with disease causing variants associated with atypical Usher syndrome.

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG, and ABHD12.Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG, or ABHD12.CEP78-related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12-related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe.This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH.

Involvement of NEK2 and its interaction with NDC80 and CEP250 in hepatocellular carcinoma.

BACKGROUND: NEK2 has an established involvement in hepatocellular carcinoma (HCC) but the roles of NEK2 and its interacting proteins in HCC have not been systematically explored. METHODS: This study examined NEK2 and its interacting proteins in HCC based on multiple databases. RESULTS: NEK2 mRNA was highly expressed in HCC tissues compared with normal liver tissues. The survival of HCC patients with high NEK2 mRNA expression was shorter than those with low expression. MAD1L1, CEP250, MAPK1, NDC80, PPP1CA, PPP1R2 and NEK11 were the interacting proteins of NEK2. Among them, NDC80 and CEP250 were the key interacting proteins of NEK2. Mitotic prometaphase may be the key pathway that NEK2 and its interacting proteins contributed to HCC pathogenesis. NEK2, NDC80 and CEP250 mRNAs were highly expressed in HCC tissues compared with normal liver tissues. The mRNA levels of NEK2 were positively correlated with those of NDC80 or CEP250. Univariate regression showed that NEK2, NDC80 and CEP250 mRNA expressions were significantly associated with HCC patients' survival. Multivariate regression showed that NDC80 mRNA expression was an independent predictor for HCC patients' survival. Methylations and genetic alterations of NEK2, NDC80 and CEP250 were observed in HCC samples. The alterations of NEK2, NDC80 and CEP250 genes were co-occurrence. Patients with high mRNA expression and genetic alterations of NEK2, NDC80 and CEP250 had poor prognosis. CONCLUSIONS: NEK2 and its interacting proteins NDC80 and CEP250 play important roles in HCC development and progression and thus may be potentially used as biomarkers and therapeutic targets of HCC.

ALMS1 and Alström syndrome: a recessive form of metabolic, neurosensory and cardiac deficits.

Alström syndrome (AS) is characterised by metabolic deficits, retinal dystrophy, sensorineural hearing loss, dilated cardiomyopathy and multi-organ fibrosis. Elucidating the function of the mutated gene, ALMS1, is critical for the development of specific treatments and may uncover pathways relevant to a range of other disorders including common forms of obesity and type 2 diabetes. Interest in ALMS1 is heightened by the recent discovery of its involvement in neonatal cardiomyocyte cell cycle arrest, a process with potential relevance to regenerative medicine. ALMS1 encodes a ~ 0.5 megadalton protein that localises to the base of centrioles. Some studies have suggested a role for this protein in maintaining centriole-nucleated sensory organelles termed primary cilia, and AS is now considered to belong to the growing class of human genetic disorders linked to ciliary dysfunction (ciliopathies). However, mechanistic details are lacking, and recent studies have implicated ALMS1 in several processes including endosomal trafficking, actin organisation, maintenance of centrosome cohesion and transcription. In line with a more complex picture, multiple isoforms of the protein likely exist and non-centrosomal sites of localisation have been reported. This review outlines the evidence for both ciliary and extra-ciliary functions of ALMS1.

CEP250 mutations associated with mild cone-rod dystrophy and sensorineural hearing loss in a Japanese family.

BACKGROUND: CEP250 encodes the C-Nap1 protein which belongs to the CEP family of proteins. C-Nap1 has been reported to be expressed in the photoreceptor cilia and is known to interact with other ciliary proteins. Mutations of CEP250 cause atypical Usher syndrome which is characterized by early-onset sensorineural hearing loss (SNHL) and a relatively mild retinitis pigmentosa. This study tested the hypothesis that the mild cone-rod dystrophy (CRD) and SNHL in a non-consanguineous Japanese family was caused by CEP250 mutations. METHODS: Detailed ophthalmic and auditory examinations were performed on the proband and her family members. Whole exome sequencing (WES) was used on the DNA obtained from the proband. RESULTS: Electrophysiological analysis revealed a mild CRD in two family members. Adaptive optics (AO) imaging showed reduced cone density around the fovea. Auditory examinations showed a slight SNHL in both patients. WES of the proband identified compound heterozygous variants c.361C>T, p.R121*, and c.562C>T, p.R188* in CEP250. The variants were found to co-segregate with the disease in five members of the family. CONCLUSIONS: The variants of CEP250 are both null variants and according to American College of Medical Genetics and Genomics (ACMG) standards and guideline, these variants are classified into the very strong category (PVS1). The criteria for both alleles will be pathogenic. Our data indicate that mutations of CEP250 can cause mild CRD and SNHL in Japanese patients. Because the ophthalmological phenotypes were very mild, high-resolution retinal imaging analysis, such as AO, will be helpful in diagnosing CEP250-associated disease.