CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the
CEP78
gene cause retinal cone-rod dystrophy associated with hearing loss. However, the ...mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing
CEP78
p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1
VPRBP
E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78
L150S
mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.
Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and ...severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.
Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual impairment and are usually transmitted as a Mendelian trait. Pathogenic mutations can occur in any of the ...100 or more disease genes identified so far, making molecular diagnosis a rather laborious process. In this work we explored the use of whole exome sequencing (WES) as a tool for identification of RD mutations, with the aim of assessing its applicability in a diagnostic context.
We ascertained 12 Spanish families with seemingly recessive RD. All of the index patients underwent mutational pre-screening by chip-based sequence hybridization and resulted to be negative for known RD mutations. With the exception of one pedigree, to simulate a standard diagnostic scenario we processed by WES only the DNA from the index patient of each family, followed by in silico data analysis. We successfully identified causative mutations in patients from 10 different families, which were later verified by Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic DNA variants (∼50% novel mutations) in the genes RP1, USH2A, CNGB3, NMNAT1, CHM, and ABCA4, responsible for retinitis pigmentosa, Usher syndrome, achromatopsia, Leber congenital amaurosis, choroideremia, or recessive Stargardt/cone-rod dystrophy cases.
Despite the absence of genetic information from other family members that could help excluding nonpathogenic DNA variants, we could detect causative mutations in a variety of genes known to represent a wide spectrum of clinical phenotypes in 83% of the patients analyzed. Considering the constant drop in costs for human exome sequencing and the relative simplicity of the analyses made, this technique could represent a valuable tool for molecular diagnostics or genetic research, even in cases for which no genotypes from family members are available.
Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we ...identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e., a 3.5-fold enrichment; p-value = 9.2 × 10
). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation in trans with the Alu insertion, but might also associate with disease in combination with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.
L1 elements are autonomous retrotransposons that can cause hereditary diseases. We have previously identified a full-length L1 insertion in the CHM (choroideremia) gene of a patient with ...choroideremia, an X-linked progressive eye disease. Because this L1 element, designated L1CHM, contains two 3′-transductions, we were able to delineate a retrotransposition path in which a precursor L1 on chromosome 10p15 or 18p11 retrotransposed to chromosome 6p21 and subsequently to the CHM gene on chromosome Xq21. A cell culture retrotransposition assay showed that L1CHM is one of the most active L1 elements in the human genome. Most importantly, analysis of genomic DNA from the CHM patient's relatives indicated somatic and germ-line mosaicism for the L1 insertion in his mother. These findings provide evidence that L1 retrotransposition can occur very early in human embryonic development.
Introduction
Full-thickness macular hole (FTMH) formation is rarely seen in patients with retinitis pigmentosa (RP) and can have an adverse impact on their residual visual function. The underlying ...mechanisms are unknown, and clinical experience is limited regarding surgical outcomes. Here, we describe the surgical management of FTMH in a young patient with genetically confirmed Usher syndrome, the most common form of syndromic RP.
Case Report
A 28-year-old woman presented with blurred vision in her right eye (RE). She had a history of RP and bilateral hearing impairment since childhood. Fundoscopy and spectral-domain optical coherence tomography revealed a FTMH in the RE along with typical RP features bilaterally. After pars plana vitrectomy (PPV) with internal limiting membrane peel and gas tamponade, the FTMH closed. Six months after PPV the patient underwent cataract surgery in the affected eye, and the visual acuity remained stable compared to baseline. The clinical diagnosis of Usher syndrome was genetically confirmed by whole exome sequencing (WES), which revealed the presence of two pathogenic nucleotide variants
in trans
(compound heterozygosity) in the gene
USH2A
.
Conclusion
We report a rare case of successful closure of a FTMH in a patient with Usher syndrome. Surgical treatment of FTMH can help preserve the central vision in RP patients, whose peripheral vision is severely affected.
Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly ...differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs∗18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.
Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous retinal disorder characterized by abnormal vascularisation of the peripheral retina, often accompanied by retinal ...detachment. To date, mutations in three genes (FZD4, LRP5, and NDP) have been shown to be causative for FEVR. In two large Dutch pedigrees segregating autosomal-dominant FEVR, genome-wide SNP analysis identified an FEVR locus of ∼40 Mb on chromosome 7. Microsatellite marker analysis suggested similar at risk haplotypes in patients of both families. To identify the causative gene, we applied next-generation sequencing in the proband of one of the families, by analyzing all exons and intron-exon boundaries of 338 genes, in addition to microRNAs, noncoding RNAs, and other highly conserved genomic regions in the 40 Mb linkage interval. After detailed bioinformatic analysis of the sequence data, prioritization of all detected sequence variants led to three candidates to be considered as the causative genetic defect in this family. One of these variants was an alanine-to-proline substitution in the transmembrane 4 superfamily member 12 protein, encoded by TSPAN12. This protein has very recently been implicated in regulating the development of retinal vasculature, together with the proteins encoded by FZD4, LRP5, and NDP. Sequence analysis of TSPAN12 revealed two mutations segregating in five of 11 FEVR families, indicating that mutations in TSPAN12 are a relatively frequent cause of FEVR. Furthermore, we demonstrate the power of targeted next-generation sequencing technology to identify disease genes in linkage intervals.
To describe the clinical and genetic spectrum of RP1-associated retinal dystrophies.
In this multicenter case series, we included 22 patients with RP1-associated retinal dystrophies from 19 families ...from The Netherlands and Japan. Data on clinical characteristics, visual acuity, visual field, ERG, and retinal imaging were extracted from medical records over a mean follow-up of 8.1 years.
Eleven patients were diagnosed with autosomal recessive macular dystrophy (arMD) or autosomal recessive cone-rod dystrophy (arCRD), five with autosomal recessive retinitis pigmentosa (arRP), and six with autosomal dominant RP (adRP). The mean age of onset was 40.3 years (range 14-56) in the patients with arMD/arCRD, 26.2 years (range 18-40) in adRP, and 8.8 years (range 5-12) in arRP patients. All patients with arMD/arCRD carried either the hypomorphic p.Arg1933* variant positioned close to the C-terminus (8 of 11 patients) or a missense variant in exon 2 (3 of 11 patients), compound heterozygous with a likely deleterious frameshift or nonsense mutation, or the p.Gln1916* variant. In contrast, all mutations identified in adRP and arRP patients were frameshift and/or nonsense variants located far from the C-terminus.
Mutations in the RP1 gene are associated with a broad spectrum of progressive retinal dystrophies. In addition to adRP and arRP, our study provides further evidence that arCRD and arMD are RP1-associated phenotypes as well. The macular involvement in patients with the hypomorphic RP1 variant suggests that macular function may remain compromised if expression levels of RP1 do not reach adequate levels after gene augmentation therapy.
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