RESUMO
The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.
Assuntos
Caricaceae , Transtorno Depressivo Maior , Humanos , Astrócitos , Depressão , Mitocôndrias , Neurônios , Fibroblastos , MitomicinaRESUMO
Inherited retinal diseases can result from various genetic defects and are one of the leading causes for blindness in the working-age population. The present study aims to provide a comprehensive description of changes in retinal structure associated with phenotypic disease entities and underlying genetic mutations. Full macular spectral domain optical coherence tomography scans were obtained and manually segmented in 16 patients with retinitis pigmentosa, 7 patients with cone−rod dystrophy, and 7 patients with Stargardt disease, as well as 23 age- and sex-matched controls without retinal disease, to assess retinal layer thicknesses. As indicated by generalized least squares models, all IRDs were associated with retinal thinning (p < 0.001), especially of the outer nuclear layer (ONL, p < 0.001). Except for the retinal nerve fiber layer, such thinning was associated with a reduced visual acuity (p < 0.001). These advances in our understanding of ultrastructural retinal changes are important for the development of gene-, cell-, and optogenetic therapy. Longitudinal studies are warranted to describe the temporal component of those changes.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Humanos , Tomografia de Coerência Óptica/métodos , Retina/diagnóstico por imagem , Retinose Pigmentar/genética , Doença de Stargardt/genéticaRESUMO
OBJECTIVE: We report on two German siblings diagnosed with congenital hypotrichosis and juvenile macular dystrophy, an extremely rare syndrome affecting both hair growth and visual functions. METHODS: A detailed ophthalmological examination was carried out including fundus examination, visual acuity assessment, visual field determination, color vision testing, and electrophysiology (electroretinography [ERG]). Additionally, fundus photography and autofluorescence imaging (FAF) was performed, along with optical coherence tomography (OCT) and adaptive optics (AO) fundus imaging. Targeted Sanger sequencing and next-generation gene panel sequencing were carried out. RESULTS: Macular dystrophy was evident in the fundus of both patients, as was a central scotoma in the static visual field. The kinetic visual field was normal. The ERG recordings were also normal, but the amplitudes of the multifocal ERG were reduced in the central 4-5° of the retina. The FAF images revealed a large central hypofluorescent area surrounded by a hyperfluorescent ring. The OCT images showed atrophy in the outer layers and tubulations. The AO images depicted a loss of central photoreceptors, as well as severe central atrophy in patient 1. A cone mosaic was observable in the peripheral AO fundus images of both patients. The disrupted cone mosaic on the AO images correlated with the hypofluorescent areas on autofluorescence. DNA testing identified the homozygous, likely pathogenic variant c.1508G>A/p.(Arg503His) (chr16:68719191) in the CDH3 gene. CONCLUSIONS: The two siblings revealed hypotrichosis and macular dystrophy in both eyes. The identification of a homozygous CDH3 mutation in each patient confirms the syndromic entity of hypotrichosis with juvenile macular degeneration.
Assuntos
Caderinas/genética , DNA/genética , Hipotricose/diagnóstico , Degeneração Macular/diagnóstico , Mutação , Células Fotorreceptoras Retinianas Cones/patologia , Acuidade Visual , Adolescente , Adulto , Caderinas/metabolismo , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Humanos , Hipotricose/congênito , Hipotricose/metabolismo , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Masculino , Irmãos , Tomografia de Coerência ÓpticaRESUMO
Autosomal recessive bestrophinopathy (ARB) has been reported as clinically heterogeneous. Eighteen patients (mean age: 22.5 years; 15 unrelated families) underwent ophthalmological examination, fundus photography, fundus autofluorescence, and optical coherence tomography (OCT). Molecular genetic testing of the BEST1 gene was conducted by the chain-terminating dideoxynucleotide Sanger methodology. Onset of symptoms (3 to 50 years of age) and best-corrected visual acuity (0.02-1.0) were highly variable. Ophthalmoscopic and retinal imaging defined five phenotypes. Phenotype I presented with single or confluent yellow lesions at the posterior pole and midperiphery, serous retinal detachment, and intraretinal cystoid spaces. In phenotype II fleck-like lesions were smaller and extended to the far periphery. Phenotype III showed a widespread continuous lesion with sharp peripheral demarcation. Single (phenotype IV) or multifocal (phenotype V) vitelliform macular dystrophy-like lesions were observed as well. Phenotypes varied within families and in two eyes of one patient. In addition, OCT detected hyperreflective foci (13/36 eyes) and choroidal excavation (11/36). Biallelic mutations were identified in each patient, six of which have not been reported so far [c.454C>T/p.(Pro152Ser), c.620T>A/p.(Leu207His), c.287_298del/p.(Gln96_Asn99del), c.199_200del/p.(Leu67Valfs*164), c.524del/p.(Ser175Thrfs*19), c.590_615del/p.(Leu197Profs*26)]. BEST1-associated ARB presents with a variable age of onset and clinical findings, that can be categorized in 5 clinical phenotypes. Hyperreflective foci and choroidal excavation frequently develop as secondary manifestations.
Assuntos
Bestrofinas/genética , Oftalmopatias Hereditárias/genética , Fenótipo , Doenças Retinianas/genética , Adulto , Alelos , Criança , Pré-Escolar , Oftalmopatias Hereditárias/diagnóstico por imagem , Oftalmopatias Hereditárias/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologiaRESUMO
PURPOSE: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Análise Mutacional de DNA/métodos , Íntrons , Sondas Moleculares , Mutação , Doença de Stargardt/diagnóstico , Doença de Stargardt/genética , Alelos , Biologia Computacional , Éxons , Estudos de Associação Genética , Predisposição Genética para Doença , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Anotação de Sequência Molecular , Linhagem , Splicing de RNARESUMO
Sorsby fundus dystrophy (SFD), an autosomal dominant, fully penetrant, degenerative disease of the macula, is manifested by symptoms of night blindness or sudden loss of visual acuity, usually in the third to fourth decades of life due to choroidal neovascularization (CNV). SFD is caused by specific mutations in the Tissue Inhibitor of Metalloproteinase-3, (TIMP3) gene. The predominant histo-pathological feature in the eyes of patients with SFD are confluent 20-30 m thick, amorphous deposits found between the basement membrane of the retinal pigment epithelium (RPE) and the inner collagenous layer of Bruch's membrane. SFD is a rare disease but it has generated significant interest because it closely resembles the exudative or "wet" form of the more common age-related macular degeneration (AMD). In addition, in both SFD and AMD donor eyes, sub-retinal deposits have been shown to accumulate TIMP3 protein. Understanding the molecular functions of wild-type and mutant TIMP3 will provide significant insights into the patho-physiology of SFD and perhaps AMD. This review summarizes the current knowledge on TIMP3 and how mutations in TIMP3 cause SFD to provide insights into how we can study this disease going forward. Findings from these studies could have potential therapeutic implications for both SFD and AMD.
Assuntos
Degeneração Macular/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Animais , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Retina/metabolismo , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Inibidor Tecidual de Metaloproteinase-3/metabolismoRESUMO
PURPOSE: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. METHODS: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. RESULTS: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. CONCLUSION: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Oligonucleotídeos Antissenso/genética , Isoformas de Proteínas/genética , Doença de Stargardt/genética , Adolescente , Adulto , Idoso , Criança , Éxons/genética , Células HEK293 , Humanos , Íntrons/genética , Pessoa de Meia-Idade , Mutação/genética , Oligonucleotídeos Antissenso/farmacologia , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Splicing de RNA/genética , Doença de Stargardt/patologia , Adulto JovemRESUMO
OBJECTIVE: CADASIL is a genetic paradigm of cerebral small vessel disease caused by NOTCH3 mutations that stereotypically lead to the extracellular deposition of NOTCH3 ectodomain (Notch3(ECD) ) on the vessels. TIMP3 and vitronectin are 2 extracellular matrix proteins that abnormally accumulate in Notch3(ECD) -containing deposits on brain vessels of mice and patients with CADASIL. Herein, we investigated whether increased levels of TIMP3 and vitronectin are responsible for aspects of CADASIL disease phenotypes. METHODS: Timp3 and vitronectin expression were genetically reduced in TgNotch3(R169C) mice, a well-established preclinical model of CADASIL. A mouse overexpressing human TIMP3 (TgBAC-TIMP3) was developed. Disease-related phenotypes, including cerebral blood flow (CBF) deficits, white matter lesions, and Notch3(ECD) deposition, were evaluated between 6 and 20 months of age. RESULTS: CBF responses to neural activity (functional hyperemia), topical application of vasodilators, and decreases in blood pressure (CBF autoregulation) were similarly reduced in TgNotch3(R169C) and TgBAC-TIMP3 mice, and myogenic responses of brain arteries were likewise attenuated. These defects were rescued in TgNotch3(R169C) mice by haploinsufficiency of Timp3, although the number of white matter lesions was unaffected. In contrast, haploinsufficiency or loss of vitronectin in TgNotch3(R169C) mice ameliorated white matter lesions, although CBF responses were unchanged. Amelioration of cerebrovascular reactivity or white matter lesions in these mice was not associated with reduced Notch3(ECD) deposition in brain vessels. INTERPRETATION: Elevated levels of TIMP3 and vitronectin, acting downstream of Notch3(ECD) deposition, play a role in CADASIL, producing divergent influences on early CBF deficits and later white matter lesions.
Assuntos
Encéfalo/patologia , CADASIL/patologia , CADASIL/fisiopatologia , Circulação Cerebrovascular , Inibidores Teciduais de Metaloproteinases/metabolismo , Vitronectina/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
PURPOSE: To investigate the association of reticular pseudodrusen (RPD) with Sorsby fundus dystrophy (SFD). DESIGN: Prospective, monocenter, cross-sectional case series. SUBJECTS: Sixteen patients of 4 unrelated families with SFD caused by mutations in TIMP3. METHODS: All subjects underwent multimodal imaging including near-infrared (NIR) reflectance and fundus autofluorescence with a confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography (SD OCT). MAIN OUTCOME MEASURES: Prevalence, topographic distribution, and phenotype of RPD. RESULTS: Mean age of the investigated patients was 56.8 years (range, 23-78 years). Reticular pseudodrusen were identified frequently in SFD patients in the sixth decade of life (5 of 7 [71%]) and were absent in younger (n = 3) or older (n = 6) patients. They were most abundant in the superior quadrant and spared the foveal region. Reticular pseudodrusen appeared as yellowish round to oval (dot subtype; n = 5) or confluent, wriggled (ribbon subtype; n = 3) lesions, sometimes forming irregular networks. Reticular pseudodrusen were hyporeflective on NIR reflectance and hypofluorescent on fundus autofluorescence imaging. They appeared as subretinal deposits on SD OCT imaging. Other lesions, such as peripheral pseudodrusen and soft drusen, were present less frequently. CONCLUSIONS: Reticular pseudodrusen are a frequent finding in patients with SFD. Although SFD patients with RPD are younger, distribution and phenotype of RPD are similar to those observed in patients with age-related macular degeneration. The association of RPD with SFD implicates a role of Bruch's membrane, the Bruch's membrane-retinal pigment epithelium interface, or both in the pathogenesis of RPD.
Assuntos
Braquidactilia/complicações , Lâmina Basilar da Corioide/patologia , Coloboma/complicações , Drusas Retinianas/etiologia , Epitélio Pigmentado da Retina/patologia , Adulto , Idoso , Braquidactilia/genética , Coloboma/genética , Estudos Transversais , Feminino , Angiofluoresceinografia , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Imagem Multimodal , Mutação , Oftalmoscopia , Imagem Óptica , Prevalência , Estudos Prospectivos , Drusas Retinianas/diagnóstico , Inibidor Tecidual de Metaloproteinase-3/genética , Tomografia de Coerência Óptica , Adulto JovemRESUMO
Loss-of-function mutations in the gene encoding FAM161A were recently discovered as the cause for RP28, an autosomal recessive form of retinitis pigmentosa. To initiate the characterization of the cellular role of FAM161A in the retina, we focused on its subcellular localization and conducted in vitro studies to identify FAM161A-interacting proteins and associated cellular structures. Immunohistochemistry revealed the presence of mouse FAM161A in the photoreceptor inner segments, the synaptic regions of the outer and inner plexiform layers and the ganglion cells. In mouse and human retinal sections from unfixed eyes, FAM161A localized to the ciliary region linking photoreceptor outer and inner segments. High-resolution immunofluorescence and immunoelectron microscopy mapped FAM161A to the connecting cilium, the basal body region and the adjacent centriole. Ectopic FAM161A was found in the centrosome and concentrated at the base of primary cilia in cultured cells. In addition, overexpressed FAM161A was clearly associated with microtubules during interphase and mitosis. The presence of FAM161A increased microtubule acetylation and stabilization. We further show that the evolutionarily conserved UPF0564 domain of FAM161A is crucial for its binding to microtubules and mediates homo- and heterotypic FAM161A and FAM161B interaction. In conclusion, our study shows that FAM161A is a microtubule-associated ciliary protein presumably involved in microtubule stabilization to maintain the microtubule tracks and/or in transport processes along microtubules in photoreceptors and other retinal cell types.
Assuntos
Proteínas do Olho , Microtúbulos , Células Fotorreceptoras , Retina , Retinose Pigmentar/genética , Animais , Centrossomo/metabolismo , Centrossomo/ultraestrutura , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Camundongos , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Mutação , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/ultraestrutura , Retina/metabolismo , Retina/ultraestrutura , Retinose Pigmentar/metabolismoRESUMO
PURPOSE: To describe the phenotypic variability in a consanguineous family with genetically confirmed X-linked retinoschisis. METHODS: Five patients, including one homozygous female, were characterized by clinical examination, optical coherence tomography, fundus autofluorescence, mapping of macular pigment optical density, electroretinography, and DNA testing. RESULTS: The 36-year-old male index patient showed a ring of enhanced autofluorescence and outer retinal atrophy on optical coherence tomography. Electroretinography testing revealed a reduced a/b ratio. His mother presented with a central atrophic retina with markedly reduced autofluorescence signal and a surrounding ring of enhanced autofluorescence. The 40-year-old brother of the index patient and his 2 sons showed characteristic signs for X-linked retinoschisis, including retinal schisis and a reduced a/b ratio. Genetic testing revealed a c.293C>A mutation in the RS1 gene in all affected family members while the mother of the index patient was homozygous for this mutation. CONCLUSION: X-linked retinoschisis can present with a wide phenotypic variability. Here, detailed family history and genetic testing established the diagnosis of X-linked retinoschisis despite striking differences in phenotypic presentation in affected subjects, homozygosity of one affected female, and seemingly dominant inheritance in three subsequent generations because of multiple consanguinity.
Assuntos
Consanguinidade , Proteínas do Olho/genética , Mutação , Retinosquise/genética , Adulto , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Angiofluoresceinografia , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Pigmentos da Retina/genética , Retinosquise/diagnóstico , Tomografia de Coerência Óptica , Adulto JovemRESUMO
Retinitis pigmentosa (RP) is an inherited disease of the retina leading to vision impairment due to progressive photoreceptor cell death. Homozygous and compound heterozygous null mutations in the CRX-regulated FAM161A gene of unknown function were identified as a cause for autosomal recessive RP (RP28) in patients from India, Germany, Israel, the Palestinian territories, and the USA. The FAM161A protein has been found to be localized to the connecting cilium, the basal body, and the adjacent centriole in mammalian photoreceptors and was also present in synaptic layers and ganglion cells of the retina. In addition, FAM161A was shown to be part of microtubule-organizing centers in cultured cells and associates with the intracellular microtubule network. Moreover, FAM161A directly binds to microtubules and increases the acetylation of α-tubulin. An evolutionary highly conserved, C-terminal protein domain (UPF0564) of FAM161A was shown to mediate microtubule association, homo- and heterotypic interaction among UPF0564-containing proteins and binding to several ciliopathy-associated proteins. In summary, FAM161A is a novel centrosomal-ciliary protein that likely is implicated in the regulation of microtubule-based cellular processes in the retina.
Assuntos
Centrossomo/fisiologia , Cílios/fisiologia , Proteínas do Olho/genética , Centro Organizador dos Microtúbulos/fisiologia , Retinose Pigmentar/genética , Proteínas do Olho/fisiologia , Humanos , Retinose Pigmentar/metabolismoRESUMO
Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.
Assuntos
Degeneração Macular , Humanos , Mutação , Penetrância , Linhagem , Degeneração Macular/genética , Retina , Fenótipo , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas do Olho , Proteínas Relacionadas a Caderinas , Proteínas do Tecido Nervoso/genéticaRESUMO
Mutations in the RS1 gene that encodes the discoidin domain containing retinoschisin cause X-linked juvenile retinoschisis (XLRS), a common macular degeneration in males. Disorganization of retinal layers and electroretinogram abnormalities are hallmarks of the disease and are also found in mice deficient for the orthologous murine protein, indicating that retinoschisin is important for the maintenance of retinal cell integrity. Upon secretion, retinoschisin associates with plasma membranes of photoreceptor and bipolar cells, although the components by which the protein is linked to membranes in vivo are still unclear. Here, we show that retinoschisin fails to bind to phospholipids or unilamellar lipid vesicles. A recent proteomic approach identified the Na/K-ATPase subunits ATP1A3 and ATP1B2 as binding partners of retinoschisin. We analyzed mice deficient for retinoschisin (Rs1h(-/Y)) and ATP1B2 (Atp1b2(-/-)) to characterize the role of Na/K-ATPase interaction in the organization of retinoschisin on cellular membranes. We demonstrate that both the Na/K-ATPase and retinoschisin are significantly reduced in Atp1b2(-/-) retinas, suggesting that retinoschisin membrane association is severely impaired in the absence of ATP1A3 and ATP1B2 subunits. Conversely, the presence of ATP1A3 and ATP1B2 are obligatory for binding of exogenously applied retinoschisin to crude membranes. Also, co-expression of ATP1A3 and ATP1B2 is required for retinoschisin binding to intact Hek293 cells. Taken together, our data support a predominant role of Na/K-ATPase in anchoring retinoschisin to retinal cell surfaces. Furthermore, altered localization of ATP1A3 and ATP1B2 is a notable consequence of retinoschisin deficiency and thus may be an important downstream aspect of cellular pathology in XLRS.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular/deficiência , Membrana Celular/metabolismo , Retinosquise/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Adenosina Trifosfatases/genética , Animais , Proteínas de Transporte de Cátions/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Linhagem Celular , Membrana Celular/genética , Proteínas do Olho/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/metabolismo , Ligação Proteica , Transporte Proteico , Retinosquise/genética , Retinosquise/metabolismo , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Retinitis pigmentosa (RP) is a degenerative disease of the retina leading to progressive loss of vision and, in many instances, to legal blindness at the end stage. The RP28 locus was assigned in 1999 to the short arm of chromosome 2 by homozygosity mapping in a large Indian family segregating autosomal-recessive RP (arRP). Following a combined approach of chromatin immunoprecipitation and parallel sequencing of genomic DNA, we identified a gene, FAM161A, which was shown to carry a homozygous nonsense mutation (p.Arg229X) in patients from the original RP28 pedigree. Another homozygous FAM161A stop mutation (p.Arg437X) was detected in three subjects from a cohort of 118 apparently unrelated German RP patients. Age at disease onset in these patients was in the second to third decade, with severe visual handicap in the fifth decade and legal blindness in the sixth to seventh decades. FAM161A is a phylogenetically conserved gene, expressed in the retina at relatively high levels and encoding a putative 76 kDa protein of unknown function. In the mouse retina, Fam161a mRNA is developmentally regulated and controlled by the transcription factor Crx, as demonstrated by chromatin immunoprecipitation and organotypic reporter assays on explanted retinas. Fam161a protein localizes to photoreceptor cells during development, and in adult animals it is present in the inner segment as well as the outer plexiform layer of the retina, the synaptic interface between photoreceptors and their efferent neurons. Taken together, our data indicate that null mutations in FAM161A are responsible for the RP28-associated arRP.
Assuntos
Códon sem Sentido/genética , Proteínas do Olho/genética , Genes Recessivos/genética , Loci Gênicos/genética , Retinose Pigmentar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Análise Mutacional de DNA , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/patologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
The ABCA4 gene is the most frequently mutated Mendelian retinopathy-associated gene. Biallelic variants lead to a variety of phenotypes, however, for thousands of cases the underlying variants remain unknown. Here, we aim to shed further light on the missing heritability of ABCA4-associated retinopathy by analyzing a large cohort of macular dystrophy probands. A total of 858 probands were collected from 26 centers, of whom 722 carried no or one pathogenic ABCA4 variant, while 136 cases carried two ABCA4 alleles, one of which was a frequent mild variant, suggesting that deep-intronic variants (DIVs) or other cis-modifiers might have been missed. After single molecule molecular inversion probes (smMIPs)-based sequencing of the complete 128-kb ABCA4 locus, the effect of putative splice variants was assessed in vitro by midigene splice assays in HEK293T cells. The breakpoints of copy number variants (CNVs) were determined by junction PCR and Sanger sequencing. ABCA4 sequence analysis solved 207 of 520 (39.8%) naive or unsolved cases and 70 of 202 (34.7%) monoallelic cases, while additional causal variants were identified in 54 of 136 (39.7%) probands carrying two variants. Seven novel DIVs and six novel non-canonical splice site variants were detected in a total of 35 alleles and characterized, including the c.6283-321C>G variant leading to a complex splicing defect. Additionally, four novel CNVs were identified and characterized in five alleles. These results confirm that smMIPs-based sequencing of the complete ABCA4 gene provides a cost-effective method to genetically solve retinopathy cases and that several rare structural and splice altering defects remain undiscovered in Stargardt disease cases.
Assuntos
Degeneração Macular , Distrofias Retinianas , Humanos , Células HEK293 , Mutação/genética , Degeneração Macular/genética , Distrofias Retinianas/genética , Análise de Sequência , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.
Assuntos
Degeneração Retiniana , Animais , Humanos , Camundongos , Centrossomo/metabolismo , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Neurogênese/genética , Retina/metabolismo , Degeneração Retiniana/metabolismoRESUMO
BACKGROUND: Inherited retinal diseases with cone dysfunction can be accompanied by severe visual loss and a marked loss of color vision despite relatively normal fundus appearance. Autosomal dominant occult macular dystrophy (RP1L1 gene) and Xchromosomal retinitis pigmentosa (RPGR gene, including heterozygous female carriers) are important examples. New examination techniques enable quantification of the extent of color vision disturbances. METHODS: After a thorough clinical examination, color discrimination and cone function were quantified. The Cambridge color test is a computer-based test that generates pseudo-isochromatic plates with Landolt C figures for quantifying color discrimination along several axes in color space. Examination of photorecepor-specific temporal contrast sensitivity is performed by subtle cyclic modulation of the spectral composition of a light stimulus. Molecular diagnostics were carried out by next generation sequencing (NGS)-based targeted gene panel analysis and Sanger sequencing. RESULTS: Markedly reduced color discrimination as well as reduced photoreceptor-specific temporal contrast sensitivity could be demonstrated in two patients with occult macular dystrophy and two heterozygous female carriers of RPGR mutations. CONCLUSION: The demonstration of dyschromatopsia is very helpful in the diagnosis of inherited retinal diseases, in addition to modern imaging techniques, such as optical coherence tomography (OCT) and fundus fluorescence. New functional techniques enable quantification of color vision disturbances and could be useful as outcome parameters in clinical trials of new gene and stem cell-based therapies.
Assuntos
Visão de Cores , Sensibilidades de Contraste , Eletrorretinografia , Proteínas do Olho/genética , Feminino , Humanos , Mutação/genética , Linhagem , Fenótipo , Tomografia de Coerência ÓpticaRESUMO
Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca(2+)-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca(2+)-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca(2+)-dependent chloride channel in the photoreceptor synapse.
Assuntos
Canais de Cloreto/fisiologia , Proteínas de Membrana/metabolismo , Neurônios/citologia , Células Fotorreceptoras/citologia , Terminações Pré-Sinápticas/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Anoctaminas , Cálcio/metabolismo , Linhagem Celular Transformada , Clonagem Molecular , Proteína 4 Homóloga a Disks-Large , Estimulação Elétrica , Olho/citologia , Expressão Gênica , Guanilato Quinases , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Domínios PDZ/fisiologia , Técnicas de Patch-Clamp , Retina/citologia , TransfecçãoRESUMO
BACKGROUND: The anoctamin family of transmembrane proteins are found in all eukaryotes and consists of 10 members in vertebrates. Ano1 and ano2 were observed to have Ca2+ activated Cl- channel activity. Recent findings however have revealed that ano6, and ano7 can also produce chloride currents, although with different properties. In contrast, ano9 and ano10 suppress baseline Cl- conductance when co-expressed with ano1 thus suggesting that different anoctamins can interfere with each other. In order to elucidate intrinsic functional diversity, and underlying evolutionary mechanism among anoctamins, we performed comprehensive bioinformatics analysis of anoctamin gene family. RESULTS: Our results show that anoctamin protein paralogs evolved from several gene duplication events followed by functional divergence of vertebrate anoctamins. Most of the amino acid replacements responsible for the functional divergence were fixed by adaptive evolution and this seem to be a common pattern in anoctamin gene family evolution. Strong purifying selection and the loss of many gene duplication products indicate rigid structure-function relationships among anoctamins. CONCLUSIONS: Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family.