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1.
PLoS Genet ; 16(4): e1008583, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236127

RESUMO

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.


Assuntos
Olho/anatomia & histologia , Olho/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Eletrorretinografia , Anormalidades do Olho/genética , Feminino , Deleção de Genes , Mutação com Perda de Função , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão/genética , Ligação Proteica , Transporte Proteico , Epitélio Pigmentado da Retina/anormalidades , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
2.
Genet Med ; 23(3): 479-487, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33100333

RESUMO

PURPOSE: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. METHODS: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. RESULTS: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. CONCLUSION: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8.


Assuntos
Albinismo Oculocutâneo , Peixe-Zebra , Albinismo Oculocutâneo/genética , Animais , Humanos , Oxirredutases Intramoleculares , Camundongos , Camundongos Endogâmicos C57BL , Mutação
4.
Am J Hum Genet ; 100(5): 706-724, 2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28413018

RESUMO

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.


Assuntos
Epilepsia/genética , Proteínas/genética , Espasmos Infantis/genética , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantis/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismo
5.
Am J Hum Genet ; 98(5): 981-992, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27108798

RESUMO

Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.


Assuntos
Aniridia/etiologia , Aniridia/patologia , Ataxia Cerebelar/etiologia , Ataxia Cerebelar/patologia , Genes Dominantes/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Deficiência Intelectual/etiologia , Deficiência Intelectual/patologia , Mutação/genética , Adolescente , Adulto , Animais , Células Cultivadas , Criança , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Camundongos , Microscopia Confocal , Pessoa de Meia-Idade , Linhagem , Conformação Proteica
6.
PLoS Genet ; 10(5): e1004359, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24809698

RESUMO

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.


Assuntos
Genes Dominantes , Genes Letais , Glaucoma/genética , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Alelos , Animais , Padronização Corporal , Dimerização , Heterozigoto , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto
7.
PLoS Genet ; 7(7): e1002114, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21750680

RESUMO

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.


Assuntos
Anoftalmia/genética , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Mutação , Osteonectina , Síndrome de Waardenburg/genética , Animais , Proteína Morfogenética Óssea 1/genética , Coloboma/genética , Análise Mutacional de DNA , Extremidades/crescimento & desenvolvimento , Olho/crescimento & desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Osteonectina/genética , Osteonectina/metabolismo , Linhagem , Sindactilia/genética , Xenopus laevis
8.
Nat Commun ; 15(1): 4316, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773095

RESUMO

As signalling organelles, cilia regulate their G protein-coupled receptor content by ectocytosis, a process requiring localised actin dynamics to alter membrane shape. Photoreceptor outer segments comprise an expanse of folded membranes (discs) at the tip of highly-specialised connecting cilia, into which photosensitive GPCRs are concentrated. Discs are shed and remade daily. Defects in this process, due to mutations, cause retinitis pigmentosa (RP). Whilst fundamental for vision, the mechanism of photoreceptor disc generation is poorly understood. Here, we show membrane deformation required for disc genesis is driven by dynamic actin changes in a process akin to ectocytosis. We show RPGR, a leading RP gene, regulates actin-binding protein activity central to this process. Actin dynamics, required for disc formation, are perturbed in Rpgr mouse models, leading to aborted membrane shedding as ectosome-like vesicles, photoreceptor death and visual loss. Actin manipulation partially rescues this, suggesting the pathway could be targeted therapeutically. These findings help define how actin-mediated dynamics control outer segment turnover.


Assuntos
Actinas , Proteínas do Olho , Retinose Pigmentar , Animais , Actinas/metabolismo , Camundongos , Retinose Pigmentar/metabolismo , Retinose Pigmentar/genética , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Cílios/metabolismo , Humanos , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Membrana Celular/metabolismo
9.
Science ; 384(6694): eadf5489, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38662826

RESUMO

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.


Assuntos
Axonema , Centríolos , Cílios , Transtornos da Motilidade Ciliar , Tubulina (Proteína) , Animais , Humanos , Camundongos , Axonema/metabolismo , Centríolos/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Mutação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Masculino , Feminino , Camundongos Knockout
10.
Hum Mol Genet ; 20(2): 223-34, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20943750

RESUMO

Renal-coloboma syndrome, also known as papillorenal syndrome, is an autosomal dominant human disorder in which optic disc coloboma is associated with kidney abnormalities. Mutations in the paired domain transcription factor PAX2 have been found to be the underlying cause of this disease. Disease severity varies between patients, and in some cases, renal hypoplasia has been found in the absence of any retinal defects. Here we report an N-ethyl-N-nitrosourea-induced mouse mutation, Opdc, which is an isoleucinetothreonine missense mutation, I40T, in the first α-helix of the Pax2 paired domain. The mutant protein binds target DNA sequences less strongly than the wild-type protein and acts poorly to transactivate target promoters in culture. The phenotypic consequence of this mutation on the development of the eye and ear is similar to that reported for null alleles of Pax2. However, in homozygotes, cerebellar development is normal on a genetic background in which loss of Pax2 results in failure of cerebellar formation. Moreover, there is a genetic background effect on the heterozygous phenotype such that on some strain backgrounds, kidney development is unaffected. Opdc is the first hypomorphic mutation reported for Pax2 that differs in phenotype from loss-of-function mutations. These results suggest that PAX2 is a strong candidate gene for cases in which human patients have optic disc coloboma not associated with renal dysplasia.


Assuntos
Coloboma/genética , Coloboma/patologia , Mutação de Sentido Incorreto , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Fenótipo , Insuficiência Renal/genética , Insuficiência Renal/patologia , Refluxo Vesicoureteral/genética , Refluxo Vesicoureteral/patologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Animais , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Modelos Animais , Mutação Puntual , Ativação Transcricional/genética
11.
Elife ; 122023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36790165

RESUMO

Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia, and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.


Assuntos
Centríolos , Cílios , Animais , Feminino , Humanos , Camundongos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas do Citoesqueleto/metabolismo
12.
PLoS Genet ; 5(11): e1000748, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19956733

RESUMO

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.


Assuntos
Aciltransferases/genética , Diferenciação Celular , Células Epidérmicas , Folículo Piloso/citologia , Homeostase , Lipoilação/fisiologia , Animais , Mutação da Fase de Leitura , Camundongos , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-fyn/metabolismo
13.
Genes (Basel) ; 13(7)2022 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-35885947

RESUMO

We have recently identified DCT encoding dopachrome tautomerase (DCT) as the eighth gene for oculocutaneous albinism (OCA). Patients with loss of function of DCT suffer from eye hypopigmentation and retinal dystrophy. Here we investigate the eye phenotype in Dct-/- mice. We show that their retinal pigmented epithelium (RPE) is severely hypopigmented from early stages, contrasting with the darker melanocytic tissues. Multimodal imaging reveals specific RPE cellular defects. Melanosomes are fewer with correct subcellular localization but disrupted melanization. RPE cell size is globally increased and heterogeneous. P-cadherin labeling of Dct-/- newborn RPE reveals a defect in adherens junctions similar to what has been described in tyrosinase-deficient Tyrc/c embryos. The first intermediate of melanin biosynthesis, dihydroxyphenylalanine (L-Dopa), which is thought to control retinogenesis, is detected in substantial yet significantly reduced amounts in Dct-/- postnatal mouse eyecups. L-Dopa synthesis in the RPE alone remains to be evaluated during the critical period of retinogenesis. The Dct-/- mouse should prove useful in understanding the molecular regulation of retinal development and aging of the hypopigmented eye. This may guide therapeutic strategies to prevent vision deficits in patients with albinism.


Assuntos
Albinismo Oculocutâneo , Albinismo , Albinismo/genética , Albinismo Oculocutâneo/genética , Animais , Modelos Animais de Doenças , Humanos , Oxirredutases Intramoleculares , Levodopa , Melanossomas , Camundongos , Monofenol Mono-Oxigenase/genética
14.
PLoS One ; 17(11): e0268149, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36413568

RESUMO

Classical aniridia is a congenital and progressive panocular disorder almost exclusively caused by heterozygous loss-of-function variants at the PAX6 locus. We report nine individuals from five families with severe aniridia and/or microphthalmia (with no detectable PAX6 mutation) with ultrarare monoallelic missense variants altering the Arg51 codon of MAB21L1. These mutations occurred de novo in 3/5 families, with the remaining families being compatible with autosomal dominant inheritance. Mice engineered to carry the p.Arg51Leu change showed a highly-penetrant optic disc anomaly in heterozygous animals with severe microphthalmia in homozygotes. Substitutions of the same codon (Arg51) in MAB21L2, a close homolog of MAB21L1, cause severe ocular and skeletal malformations in humans and mice. The predicted nucleotidyltransferase function of MAB21L1 could not be demonstrated using purified protein with a variety of nucleotide substrates and oligonucleotide activators. Induced expression of GFP-tagged wildtype and mutant MAB21L1 in human cells caused only modest transcriptional changes. Mass spectrometry of immunoprecipitated protein revealed that both mutant and wildtype MAB21L1 associate with transcription factors that are known regulators of PAX6 (MEIS1, MEIS2 and PBX1) and with poly(A) RNA binding proteins. Arg51 substitutions reduce the association of wild-type MAB21L1 with TBL1XR1, a component of the NCoR complex. We found limited evidence for mutation-specific interactions with MSI2/Musashi-2, an RNA-binding proteins with effects on many different developmental pathways. Given that biallelic loss-of-function variants in MAB21L1 result in a milder eye phenotype we suggest that Arg51-altering monoallelic variants most plausibly perturb eye development via a gain-of-function mechanism.


Assuntos
Aniridia , Microftalmia , Humanos , Animais , Camundongos , Microftalmia/genética , Fator de Transcrição PAX6/genética , Aniridia/genética , Mutação de Sentido Incorreto , Heterozigoto , Fatores de Transcrição/genética , Proteínas de Homeodomínio/genética , Proteínas de Ligação a RNA/genética , Proteínas do Olho/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética
15.
Dis Model Mech ; 14(9)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34368841

RESUMO

Brittle cornea syndrome (BCS) is a rare recessive condition characterised by extreme thinning of the cornea and sclera. BCS results from loss-of-function mutations in the poorly understood genes ZNF469 or PRDM5. In order to determine the function of ZNF469 and to elucidate pathogenic mechanisms, we used genome editing to recapitulate a human ZNF469 BCS mutation in the orthologous mouse gene Zfp469. Ophthalmic phenotyping showed that homozygous Zfp469 mutation causes significant central and peripheral corneal thinning arising from reduced stromal thickness. Expression of key components of the corneal stroma in primary keratocytes from Zfp469BCS/BCS mice is affected, including decreased Col1a1 and Col1a2 expression. This alters the collagen type I/collagen type V ratio and results in collagen fibrils with smaller diameter and increased fibril density in homozygous mutant corneas, correlating with decreased biomechanical strength in the cornea. Cell-derived matrices generated by primary keratocytes show reduced deposition of collagen type I, offering an in vitro model for stromal dysfunction. Work remains to determine whether modulating ZNF469 activity will have therapeutic benefit in BCS or in conditions such as keratoconus in which the cornea thins progressively. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas de Ligação a DNA , Anormalidades da Pele , Animais , Córnea , Proteínas de Ligação a DNA/genética , Anormalidades do Olho , Humanos , Instabilidade Articular/congênito , Camundongos , Mutação/genética , Anormalidades da Pele/genética , Fatores de Transcrição/genética , Dedos de Zinco
16.
Sci Rep ; 10(1): 437, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949211

RESUMO

Fam151b is a mammalian homologue of the C. elegans menorin gene, which is involved in neuronal branching. The International Mouse Phenotyping Consortium (IMPC) aims to knock out every gene in the mouse and comprehensively phenotype the mutant animals. This project identified Fam151b homozygous knock-out mice as having retinal degeneration. We show they have no photoreceptor function from eye opening, as demonstrated by a lack of electroretinograph (ERG) response. Histological analysis shows that during development of the eye the correct number of cells are produced and that the layers of the retina differentiate normally. However, after eye opening at P14, Fam151b mutant eyes exhibit signs of retinal stress and rapidly lose photoreceptor cells. We have mutated the second mammalian menorin homologue, Fam151a, and homozygous mutant mice have no discernible phenotype. Sequence analysis indicates that the FAM151 proteins are members of the PLC-like phosphodiesterase superfamily. However, the substrates and function of the proteins remains unknown.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Retina/fisiologia , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Contagem de Células , Técnicas de Inativação de Genes , Humanos , Camundongos , Modelos Moleculares , Mutação , Células Fotorreceptoras de Vertebrados/citologia , Conformação Proteica , Retina/citologia
17.
Invest Ophthalmol Vis Sci ; 60(8): 2875-2887, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31266059

RESUMO

Purpose: We previously found a dominant mutation, Rwhs, causing white spots on the retina accompanied by retinal folds. Here we identify the mutant gene to be Tmem98. In humans, mutations in the orthologous gene cause nanophthalmos. We modeled these mutations in mice and characterized the mutant eye phenotypes of these and Rwhs. Methods: The Rwhs mutation was identified to be a missense mutation in Tmem98 by genetic mapping and sequencing. The human TMEM98 nanophthalmos missense mutations were made in the mouse gene by CRISPR-Cas9. Eyes were examined by indirect ophthalmoscopy and the retinas imaged using a retinal camera. Electroretinography was used to study retinal function. Histology, immunohistochemistry, and electron microscopy techniques were used to study adult eyes. Results: An I135T mutation of Tmem98 causes the dominant Rwhs phenotype and is perinatally lethal when homozygous. Two dominant missense mutations of TMEM98, A193P and H196P, are associated with human nanophthalmos. In the mouse these mutations cause recessive retinal defects similar to the Rwhs phenotype, either alone or in combination with each other, but do not cause nanophthalmos. The retinal folds did not affect retinal function as assessed by electroretinography. Within the folds there was accumulation of disorganized outer segment material as demonstrated by immunohistochemistry and electron microscopy, and macrophages had infiltrated into these regions. Conclusions: Mutations in the mouse orthologue of the human nanophthalmos gene TMEM98 do not result in small eyes. Rather, there is localized disruption of the laminar structure of the photoreceptors.


Assuntos
Proteínas de Membrana/genética , Microftalmia/genética , Mutação de Sentido Incorreto , Células Fotorreceptoras de Vertebrados/patologia , Doenças Retinianas/genética , Animais , Comprimento Axial do Olho/patologia , Sistemas CRISPR-Cas , Eletrorretinografia , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microftalmia/patologia , Microscopia Eletrônica de Transmissão , Oftalmoscopia , Reação em Cadeia da Polimerase , Doenças Retinianas/patologia
18.
Dis Model Mech ; 11(12)2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30478029

RESUMO

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one ß and one γ subunit. Loss-of-function and missense mutations in both IDH3A and IDH3B have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Idh3a Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of Idh3a and found that homozygous mice do not survive past early embryogenesis. Idh3a-/E229K compound heterozygous mutants exhibit a more severe retinal degeneration compared with Idh3aE229K/E229K homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both Idh3aE229K/E229K and Idh3a-/E229K cells. Loss-of-function Idh3b mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in Idh3a mutant mice.This article has an associated First Person interview with the first author of the paper.


Assuntos
Isocitrato Desidrogenase/genética , Mitocôndrias/patologia , Mutação/genética , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Animais , Fibroblastos/metabolismo , Genótipo , Isocitrato Desidrogenase/metabolismo , Mutação com Perda de Função/genética , Camundongos , Mutação de Sentido Incorreto/genética , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Retina/fisiopatologia
19.
Invest Ophthalmol Vis Sci ; 46(9): 3443-50, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16123450

RESUMO

PURPOSE: To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa. METHODS: Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visual-tracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis. RESULTS: Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b(rd1) mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles. CONCLUSIONS: These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b(rd1) mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa.


Assuntos
Mutação , Diester Fosfórico Hidrolases/genética , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/genética , Alquilantes/toxicidade , Alelos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Análise Mutacional de DNA , Modelos Animais de Doenças , Progressão da Doença , Eletroforese Capilar , Etilnitrosoureia/toxicidade , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/enzimologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/enzimologia , Degeneração Retiniana/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Acuidade Visual
20.
Invest Ophthalmol Vis Sci ; 56(5): 3015-26, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25736793

RESUMO

PURPOSE: As part of a large scale systematic screen to determine the effects of gene knockout mutations in mice, a retinal phenotype was found in mice lacking the Slc9a8 gene, encoding the sodium/hydrogen ion exchange protein NHE8. We aimed to characterize the mutant phenotype and the role of sodium/hydrogen ion exchange in retinal function. METHODS: Detailed histology characterized the pathological consequences of Slc9a8 mutation, and retinal function was assessed by electroretinography (ERG). A conditional allele was used to identify the cells in which NHE8 function is critical for retinal function, and mutant cells analyzed for the effect of the mutation on endosomes. RESULTS: Histology of mutant retinas reveals a separation of photoreceptors from the RPE and infiltration by macrophages. There is a small reduction in photoreceptor length and a mislocalization of visual pigments. The ERG testing reveals a deficit in rod and cone pathway function. The RPE shows abnormal morphology, and mutation of Slc9a8 in only RPE cells recapitulates the mutant phenotype. The NHE8 protein localizes to endosomes, and mutant cells have much smaller recycling endosomes. CONCLUSIONS: The NHE8 protein is required in the RPE to maintain correct regulation of endosomal volume and/or pH which is essential for the cellular integrity and subsequent function of RPE.


Assuntos
Mutação , Doenças Retinianas/genética , Epitélio Pigmentado da Retina/patologia , Trocadores de Sódio-Hidrogênio/genética , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Eletrorretinografia , Imunofluorescência , Técnicas de Inativação de Genes , Inativação Gênica , Pressão Intraocular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Oftalmoscopia , Plasmídeos , Reação em Cadeia da Polimerase em Tempo Real , Doenças Retinianas/diagnóstico
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