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1.
Nat Commun ; 10(1): 2884, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253780

RESUMO

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 × 10-5). 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.


Assuntos
Ciliopatias/genética , Proteínas do Olho/genética , Predisposição Genética para Doença , Doenças Retinianas/genética , Elementos Alu/genética , Grupo com Ancestrais do Continente Asiático/genética , Genômica , Humanos , Japão , Mutação , Linhagem
2.
Ophthalmic Genet ; 40(2): 177-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31012789

RESUMO

BACKGROUND: Inherited retinal degenerations (IRDs) encompass a wide spectrum of genetic ocular diseases characterized by considerable genetic and clinical heterogeneity. METHODS: Complete ophthalmic examination and next-generation sequencing. RESULTS: We describe a patient with no family history of vision loss, who at the age of 28 years developed visual impairment consistent with a severe form of retinitis pigmentosa. Genetic testing by means of whole exome sequencing identified a homozygous variant in the gene IDH3A. To date, only three papers have reported mutations in IDH3A, in families with early-onset retinal degeneration with or without the presence of macular pseudocoloboma. CONCLUSION: This study highlights the importance of including this rarely-mutated gene in the molecular diagnostic set-ups for IRDs, and further delineates the phenotypic spectrum elicited by mutations in IDH3A.

3.
J Cell Biol ; 217(1): 151-161, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29237719

RESUMO

Primary cilia have pivotal roles as organizers of many different signaling pathways, including platelet-derived growth factor receptor α (PDGFRα) signaling, which, when aberrantly regulated, is associated with developmental disorders, tumorigenesis, and cancer. PDGFRα is up-regulated during ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor is subsequently ubiquitinated and internalized. In contrast, in IFT20-depleted cells, PDGFRα localizes aberrantly to the plasma membrane and is overactivated after ligand stimulation because of destabilization and degradation of c-Cbl and Cbl-b.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células 3T3 , Animais , Linhagem Celular , Cílios/metabolismo , Células HEK293 , Humanos , Camundongos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Interferência de RNA , Transdução de Sinais/genética , Ubiquitinação/fisiologia
4.
Am J Hum Genet ; 99(3): 770-776, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27588451

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/genética , Cílios/patologia , Distrofias de Cones e Bastonetes/complicações , Distrofias de Cones e Bastonetes/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Mutação/genética , Idoso , Alelos , Animais , Cadáver , Proteínas de Ciclo Celular/metabolismo , Estudos de Coortes , Distrofias de Cones e Bastonetes/patologia , Distrofias de Cones e Bastonetes/fisiopatologia , Exoma/genética , Olho/embriologia , Olho/metabolismo , Proteínas do Olho/metabolismo , Feminino , Fibroblastos/patologia , Grécia , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Íntrons/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Linhagem , Ligação Proteica , RNA Mensageiro/análise , Suécia , Transcriptoma , Síndromes de Usher/patologia
5.
Am J Hum Genet ; 99(2): 470-80, 2016 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-27486781

RESUMO

Inherited retinal dystrophies (iRDs) are a group of genetically and clinically heterogeneous conditions resulting from mutations in over 250 genes. Here, homozygosity mapping and whole-exome sequencing (WES) in a consanguineous family revealed a homozygous missense mutation, c.973C>T (p.His325Tyr), in RCBTB1. In affected individuals, it was found to segregate with retinitis pigmentosa (RP), goiter, primary ovarian insufficiency, and mild intellectual disability. Subsequent analysis of WES data in different cohorts uncovered four additional homozygous missense mutations in five unrelated families in whom iRD segregates with or without syndromic features. Ocular phenotypes ranged from typical RP starting in the second decade to chorioretinal dystrophy with a later age of onset. The five missense mutations affect highly conserved residues either in the sixth repeat of the RCC1 domain or in the BTB1 domain. A founder haplotype was identified for mutation c.919G>A (p.Val307Met), occurring in two families of Mediterranean origin. We showed ubiquitous mRNA expression of RCBTB1 and demonstrated predominant RCBTB1 localization in human inner retina. RCBTB1 was very recently shown to be involved in ubiquitination, more specifically as a CUL3 substrate adaptor. Therefore, the effect on different components of the CUL3 and NFE2L2 (NRF2) pathway was assessed in affected individuals' lymphocytes, revealing decreased mRNA expression of NFE2L2 and several NFE2L2 target genes. In conclusion, our study puts forward mutations in RCBTB1 as a cause of autosomal-recessive non-syndromic and syndromic iRD. Finally, our data support a role for impaired ubiquitination in the pathogenetic mechanism of RCBTB1 mutations.


Assuntos
Alelos , Fatores de Troca do Nucleotídeo Guanina/genética , Mutação de Sentido Incorreto/genética , Distrofias Retinianas/genética , Ubiquitinação/genética , Adolescente , Adulto , Idade de Início , Criança , Consanguinidade , Proteínas Culina/metabolismo , Exoma/genética , Feminino , Efeito Fundador , Genes Recessivos , Haplótipos/genética , Homozigoto , Humanos , Linfócitos/metabolismo , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Linhagem , Fenótipo , RNA Mensageiro/genética , Retina/metabolismo , Síndrome , Turquia
6.
Hum Mol Genet ; 25(20): 4546-4555, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28173158

RESUMO

Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also displayed a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.


Assuntos
Proteínas de Transporte/genética , Distrofias de Cones e Bastonetes/enzimologia , Expressão Gênica , Infertilidade Masculina/enzimologia , Mutação , Adolescente , Adulto , Idoso , Animais , Distrofias de Cones e Bastonetes/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Proteínas do Olho/genética , Feminino , Homozigoto , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Especificidade de Órgãos , Linhagem , Células Fotorreceptoras de Vertebrados/enzimologia , Ratos , Motilidade Espermática , Espermatozoides/enzimologia , Testículo/enzimologia
7.
Hum Mol Genet ; 24(12): 3359-71, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25749990

RESUMO

Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal disease.


Assuntos
Centrossomo/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Genes Recessivos , Complexo de Golgi/metabolismo , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Animais , Proteínas de Transporte , Bovinos , Linhagem Celular , Citoesqueleto/metabolismo , Humanos , Espaço Intracelular/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido
8.
PLoS One ; 9(11): e112142, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25392995

RESUMO

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.


Assuntos
Morte Celular/fisiologia , Degeneração Retiniana/fisiopatologia , Animais , Animais Geneticamente Modificados , Calpaína/fisiologia , Morte Celular/genética , GMP Cíclico/fisiologia , Modelos Animais de Doenças , Histona Desacetilases/fisiologia , Transdução de Sinal Luminoso/genética , Camundongos , Mutação , Poli Adenosina Difosfato Ribose/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Ratos , Degeneração Retiniana/genética
9.
J Neurochem ; 127(6): 868-79, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23796581

RESUMO

Retinitis Pigmentosa involves a hereditary degeneration of photoreceptors by as yet unresolved mechanisms. The secretable protein α-Klotho has a function related to ageing processes, and α-Klotho-deficient mice have reduced lifespan and declining functions in several tissues. Here, we studied Klotho in connection with inherited photoreceptor degeneration. Increased nuclear immunostaining for α-Klotho protein was seen in degenerating photoreceptors in four different Retinitis Pigmentosa models (rd1, rd2 mice; P23H, S334ter rhodopsin mutant rats). Correspondingly, in rd1 retina α-Klotho mRNA expression was significantly up-regulated. Moreover, immunostaining for another Klotho family protein, ß-Klotho, also co-localized with degenerating rd1 photoreceptors. The rd1 retina displayed reduced levels of fibroblast growth factor 15, a member of the fibroblast growth factor subfamily for which Klotho acts as a co-receptor. Exogenous α-Klotho protein added to retinal explant cultures did not affect cell death in rd1 retinae, but caused a severe layer disordering in wild-type retinae. Our study suggests Klotho as a novel player in the retina, with a clear connection to photoreceptor cell death as well as with an influence on retinal organization.


Assuntos
Glucuronidase/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retinite Pigmentosa/metabolismo , Animais , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Células Fotorreceptoras de Vertebrados/patologia , Isoformas de Proteínas/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/patologia , Técnicas de Cultura de Tecidos , Regulação para Cima
10.
PLoS One ; 6(7): e22181, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21765948

RESUMO

Retinitis pigmentosa (RP) is a heterogeneous group of inherited neurodegenerative diseases affecting photoreceptors and causing blindness. Many human cases are caused by mutations in the rhodopsin gene. An important question regarding RP pathology is whether different genetic defects trigger the same or different cell death mechanisms. To answer this question, we analysed photoreceptor degeneration in P23H and S334ter transgenic rats carrying rhodopsin mutations that affect protein folding and sorting respectively. We found strong activation of calpain and poly(ADP-ribose) polymerase (PARP) in both mutants, concomitant with calpastatin down-regulation, increased oxidative DNA damage and accumulation of PAR polymers. These parameters were strictly correlated with the temporal progression of photoreceptor degeneration, mirroring earlier findings in the phosphodiesterase-6 mutant rd1 mouse, and suggesting execution of non-apoptotic cell death mechanisms. Interestingly, activation of caspases-3 and -9 and cytochrome c leakage-key events in apoptotic cell death--were observed only in the S334ter mutant, which also showed increased expression of PARP-1. The identification of the same metabolic markers triggered by different mutations in two different species suggests the existence of common cell death mechanisms, which is a major consideration for any mutation independent treatment.


Assuntos
Calpaína/metabolismo , Mutação/genética , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Rodopsina/genética , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Morte Celular , Forma Celular , Citocromos c/metabolismo , Dano ao DNA , Ativação Enzimática , Humanos , Marcação In Situ das Extremidades Cortadas , Estresse Oxidativo , Poli Adenosina Difosfato Ribose/metabolismo , Transporte Proteico , Ratos , Ratos Mutantes , Ratos Transgênicos , Coloração e Rotulagem
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