RESUMO
Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometrybased quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cellspecific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from lowgenetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.
Assuntos
Cromossomos Humanos Par 10 , Cromossomos Humanos Par 1 , Degeneração Macular , Mastócitos , Peptídeo Hidrolases , Alelos , Corioide/enzimologia , Corioide/patologia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 10/genética , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Mastócitos/patologia , Peptídeo Hidrolases/genética , Proteômica , Risco , Triptases/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10-10; FHR-2, p = 6.0 × 10-10; FHR-3, p = 1.5 × 10-5; FHR-4, p = 1.3 × 10-3; FHR-5, p = 1.9 × 10-4) and FHL-1 (p = 4.9 × 10-4) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.
Assuntos
Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/genética , Predisposição Genética para Doença , Degeneração Macular/sangue , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Casos e Controles , Proteínas Inativadoras do Complemento C3b/genética , Feminino , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Masculino , Fatores de RiscoRESUMO
BACKGROUND: Aberrant sympathetic nerve activity exacerbates cardiovascular risk in hypertension and diabetes, which are common comorbidities, yet clinically sympathetic nerve activity remains poorly controlled. The hypertensive diabetic state is associated with increased reflex sensitivity and tonic drive from the peripheral chemoreceptors, the cause of which is unknown. We have previously shown hypertension to be critically dependent on the carotid body (CB) input in spontaneously hypertensive rat, a model that also exhibits a number of diabetic traits. CB overstimulation by insulin and leptin has been similarly implicated in the development of increased sympathetic nerve activity in metabolic syndrome and obesity. Thus, we hypothesized that in hypertensive diabetic state (spontaneously hypertensive rat), the CB is sensitized by altered metabolic signaling causing excessive sympathetic activity levels and dysfunctional reflex regulation. METHODS: Using a hypothesis-free RNA-seq approach, we investigated potential molecular targets implicated in energy metabolism mediating CB sensitization and its regulation of sympathetic outflow in experimental hypertension. Identified targets were characterized using molecular and functional techniques assessing peripheral chemoreflex sensitivity in situ and in vivo. RESULTS: We discovered GLP1R (glucagon-like peptide-1 receptor) expression in the CBs of rat and human and showed that its decreased expression is linked to sympathetic hyperactivity in rats with cardiometabolic disease. We demonstrate GLP1R to be localized to CB chemosensory cells, while targeted administration of GLP1R agonist to the CB lowered its basal discharge and attenuated chemoreflex-evoked blood pressure and sympathetic responses. Importantly, hyperglycemia-induced peripheral chemoreflex sensitization and associated basal sympathetic overactivity were abolished by GLP1R activation in the CB suggesting a role in a homeostatic response to high blood glucose. CONCLUSIONS: We show that GLP1 (glucagon-like peptide-1) modulates the peripheral chemoreflex acting on the CB, supporting this organ as a multimodal receptor. Our findings pinpoint CBs as potential targets for ameliorating excessive sympathetic activity using GLP1R agonists in the hypertensive-diabetic condition.
Assuntos
Corpo Carotídeo , Hipertensão , Animais , Pressão Sanguínea , Corpo Carotídeo/metabolismo , Glucose/metabolismo , Ratos , Ratos Endogâmicos SHRRESUMO
INTRODUCTION: The primate retina has evolved regional specialisations for specific visual functions. The macula is specialised towards high acuity vision and is an area that contains an increased density of cone photoreceptors and signal processing neurons. Different regions in the retina display unique susceptibility to pathology, with many retinal diseases primarily affecting the macula. OBJECTIVES: To better understand the properties of different retinal areas we studied the differential distribution of metabolites across the retina. METHODS: We conducted an untargeted metabolomics analysis on full-thickness punches from three different regions (macula, temporal peri-macula and periphery) of healthy primate retina. RESULTS: Nearly half of all metabolites identified showed differential abundance in at least one comparison between the three regions. Furthermore, mapping metabolomics results from macula-specific eye diseases onto our region-specific metabolite distributions revealed differential abundance defining systemic metabolic dysregulations that were region specific. CONCLUSIONS: The unique metabolic phenotype of different retinal regions is likely due to the differential distribution of different cell types in these regions reflecting the specific metabolic requirements of each cell type. Our results may help to better understand the pathobiology of retinal diseases with region specificity.
Assuntos
Macula Lutea , Doenças Retinianas , Animais , Metabolômica , Retina/metabolismo , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Macula Lutea/metabolismo , Neurônios/metabolismoRESUMO
Opticin is an extracellular glycoprotein present in the vitreous. Its antiangiogenic properties offer the potential for therapeutic intervention in conditions such as proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the hypothesis that intravitreal administration of recombinant human opticin can safely protect against the development of pathological angiogenesis and promote its regression. We generated and purified recombinant human opticin and investigated its impact on the development and regression of pathological retinal neovascularization following intravitreal administration in murine oxygen-induced retinopathy. We also investigated its effect on normal retinal vascular development and function, following intravitreal injection in neonatal mice, by histological examination and electroretinography. In oxygen-induced retinopathy, intravitreal administration of human recombinant opticin protected against the development of retinal neovascularization to similar extent as aflibercept, which targets VEGF. Opticin also accelerated regression of established retinal neovascularization, though the effect at 18 h was less than that of aflibercept. Intravitreal administration of human recombinant opticin in neonatal mice caused no detectable perturbation of subsequent retinal vascular development or function. In summary we found that intraocular administration of recombinant human opticin protects against the development of pathological angiogenesis in mice and promotes its regression.
Assuntos
Hiperóxia , Neovascularização Retiniana , Retinopatia da Prematuridade , Animais , Modelos Animais de Doenças , Humanos , Hiperóxia/complicações , Recém-Nascido , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica , Oxigênio/toxicidade , Neovascularização Retiniana/tratamento farmacológico , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/prevenção & controleRESUMO
Rare variants in the complement factor I (CFI) gene, associated with low serum factor I (FI) levels, are strong risk factors for developing the advanced stages of age-related macular degeneration (AMD). No studies have been undertaken on the prevalence of disease-causing CFI mutations in patients with geographic atrophy (GA) secondary to AMD. A multicenter, cross-sectional, noninterventional study was undertaken to identify the prevalence of pathogenic rare CFI gene variants in an unselected cohort of patients with GA and low FI levels. A genotype-phenotype study was performed. Four hundred and sixty-eight patients with GA secondary to AMD were recruited to the study, and 19.4% (n = 91) demonstrated a low serum FI concentration (below 15.6 µg/ml). CFI gene sequencing on these patients resulted in the detection of rare CFI variants in 4.7% (n = 22) of recruited patients. The prevalence of CFI variants in patients with low serum FI levels and GA was 25%. Of the total patients recruited, 3.2% (n = 15) expressed a CFI variant classified as pathogenic or likely pathogenic. The presence of reticular pseudodrusen was detected in all patients with pathogenic CFI gene variants. Patients with pathogenic CFI gene variants and low serum FI levels might be suitable for FI supplementation in therapeutic trials.
Assuntos
Fator I do Complemento , Atrofia Geográfica , Fator I do Complemento/genética , Estudos Transversais , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/epidemiologia , Atrofia Geográfica/genética , Humanos , Mutação , Fenótipo , PrevalênciaRESUMO
The t-soluble NSF-attachment protein receptor protein Syntaxin-1a (Stx-1a) is abundantly expressed at pre-synaptic terminals where it plays a critical role in the exocytosis of neurotransmitter-containing synaptic vesicles. Stx-1a is phosphorylated by Casein kinase 2α (CK2α) at Ser14, which has been proposed to regulate the interaction of Stx-1a and Munc-18 to control of synaptic vesicle priming. However, the role of CK2α in synaptic vesicle dynamics remains unclear. Here, we show that CK2α over-expression reduces evoked synaptic vesicle release. Furthermore, shRNA-mediated knockdown of CK2α in primary hippocampal neurons strongly enhanced vesicle exocytosis from the reserve pool, with no effect on the readily releasable pool of primed vesicles. In neurons in which endogenous Stx-1a was knocked down and replaced with a CK2α phosphorylation-deficient mutant, Stx-1a(D17A), vesicle exocytosis was also increased. These results reveal a previously unsuspected role of CK2α phosphorylation in specifically regulating the reserve synaptic vesicle pool, without changing the kinetics of release from the readily releasable pool.
Assuntos
Caseína Quinase II/metabolismo , Endocitose/fisiologia , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Sintaxina 1/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Fosforilação/fisiologia , Gravidez , Ratos , Ratos WistarRESUMO
The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.
Assuntos
Channelrhodopsins/genética , Variação Genética , Células Bipolares da Retina/metabolismo , Opsinas de Bastonetes/genética , Animais , Channelrhodopsins/metabolismo , Dependovirus/genética , Humanos , Injeções Intravítreas , Camundongos , Optogenética , Opsinas de Bastonetes/metabolismo , Transdução GenéticaRESUMO
Although the triggers causing angiogenesis in the context of neovascular age-related macular degeneration (nAMD) are not fully understood, oxidative stress is likely involved. Oxidative stress in the eye can occur through exposure of macular tissues to sunlight and local or systemic exposure to oxidative stressors associated with environmental or lifestyle factors. Because trace elements have been implicated as regulators of oxidative stress and cellular antioxidant defense mechanisms, we hypothesized that they may play a role as a risk factor, modifying the progression toward nAMD. Herein, we determined whether levels of human plasma trace elements are different in 236 individuals with nAMD compared to 236 age-matched controls without AMD. Plasma levels of 16 trace elements including arsenic, barium, calcium, cadmium, cobalt, chromium, copper, iron, magnesium, manganese, molybdenum, lead, antimony, selenium, vanadium and zinc were measured using inductively coupled plasma mass spectrometry. Associations of trace elements with demographic, environmental and lifestyle factors and AMD-associated genetic variants were assessed. Elevated levels of barium and cadmium and reduced levels of chromium were observed in nAMD patients compared to controls. Mean plasma concentrations of barium were 1.35 µg/L (standard deviation [SD] 0.71) in nAMD and 1.15 µg/L (SD 0.63) in controls (P = 0.001). Mean levels of chromium were 0.37 µg/L (SD 0.22) in nAMD and 0.46 µg/L (SD 0.34) in controls (P = 0.001). Median levels for cadmium, which were not normally distributed, were 0.016 µg/L (interquartile range [IQR] 0.001-0.026) in nAMD and 0.012 µg/L (IQR 0.001-0.022) in controls (P = 0.002). Comparison of the Spearman's correlation coefficients between nAMD patients and controls identified a difference in correlations for 8 trace elements. Cadmium levels were associated with the smoking status (P < 0.001), while barium levels showed a trend of association with the usage of antihypertensive drugs. None of the AMD-associated genetic variants were associated with any trace element levels. In conclusion, in this case-control study we detected elevated plasma levels of barium and cadmium and reduced plasma levels of chromium in nAMD patients. An imbalance in plasma trace elements, which is most likely driven by environmental and lifestyle factors, might have a role in the pathogenesis of AMD. These trace elements may be incorporated as biomarkers into models for prediction of disease risk and progression. Additionally, population-based preventive strategies to decrease Cd exposure, especially by the cessation of smoking, could potentially reduce the burden of nAMD. Future studies are warranted to investigate whether supplementation of Cr would have a beneficial effect on nAMD.
Assuntos
Plasma/metabolismo , Degeneração Macular Exsudativa/sangue , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Estudos Retrospectivos , Oligoelementos/sangueRESUMO
Opticin is an endogenous vitreous glycoprotein that may have therapeutic potential as it has been shown that supranormal concentrations suppress preretinal neovascularization. Herein we investigated the pharmacokinetics of opticin following intravitreal injection in rabbits. To measure simultaneously concentrations of human and rabbit opticin, a selected reaction monitoring mass spectrometry assay was developed. The mean concentration of endogenous rabbit opticin in 7 uninjected eyes was measured and found to be 19.2 nM or 0.62 µg/mL. When the vitreous was separated by centrifugation into a supernatant and collagen-containing pellet, 94% of the rabbit opticin was in the supernatant. Intravitreal injection of human opticin (40 µg) into both eyes of rabbits was followed by enucleation at 5, 24, and 72 h and 7, 14, and 28 days postinjection (n = 6 at each time point) and measurement of vitreous human and rabbit opticin concentrations in the supernatant and collagen-containing pellet following centrifugation. The volume of distribution of human opticin was calculated to be 3.31 mL, and the vitreous half-life was 4.2 days. Assuming that rabbit and human opticin are cleared from rabbit vitreous at the same rate, opticin is secreted into the vitreous at a rate of 0.14 µg/day. We conclude that intravitreally injected opticin has a vitreous half-life that is similar to currently available antiangiogenic therapeutics. While opticin was first identified bound to vitreous collagen fibrils, here we demonstrate that >90% of endogenous opticin is not bound to collagen. Endogenous opticin is secreted by the nonpigmented ciliary epithelium into the rabbit vitreous at a remarkably high rate, and the turnover in vitreous is approximately 15% per day.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacocinética , Proteínas da Matriz Extracelular/administração & dosagem , Proteínas da Matriz Extracelular/farmacocinética , Injeções Intravítreas/métodos , Proteoglicanas/administração & dosagem , Proteoglicanas/farmacocinética , Inibidores da Angiogênese/biossíntese , Animais , Colágeno/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/metabolismo , Meia-Vida , Humanos , Masculino , Espectrometria de Massas/métodos , Neovascularização Fisiológica/efeitos dos fármacos , Proteoglicanas/biossíntese , Proteoglicanas/metabolismo , Coelhos , Retina/metabolismo , Corpo Vítreo/metabolismoRESUMO
PURPOSE: To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. DESIGN: Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. PARTICIPANTS: Seven families with apparently autosomal dominant EOMD. METHODS: Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. MAIN OUTCOME MEASURES: Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. RESULTS: All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1GâT het; c.619+1GâA het, c.380GâA, p.(Arg127His) het; c.694CâT p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291TâA, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. CONCLUSIONS: Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.
Assuntos
Fator H do Complemento/genética , Mutação , Drusas Retinianas/genética , Idoso , Feminino , Variação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Drusas Retinianas/metabolismo , Estudos RetrospectivosRESUMO
BACKGROUND: Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. METHODS: We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). RESULTS: Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. CONCLUSIONS: Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.
RESUMO
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1-5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide array of alternative functions has been proposed. UCH-L1 is not essential for neuronal development but it is absolutely required for the maintenance of axonal integrity and UCH-L1 dysfunction is implicated in neurodegenerative disease. Here we review the properties of UCH-L1, and how understanding its complex structure can provide new insights into its roles in neuronal function and pathology.
Assuntos
Encéfalo/enzimologia , Ubiquitina Tiolesterase/metabolismo , Animais , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Ubiquitina Tiolesterase/químicaRESUMO
PURPOSE: To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). DESIGN: Case series. PARTICIPANTS: A total of 562 patients diagnosed with IRD. METHODS: We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. MAIN OUTCOME MEASURES: Diagnostic yield of genomic testing. RESULTS: Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. CONCLUSIONS: We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.
Assuntos
Oftalmopatias Hereditárias/genética , Genoma , Técnicas de Diagnóstico Molecular , Doenças Retinianas/genética , Análise de Sequência de DNA , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Time-dependent post-mortem biochemical changes have been demonstrated in donor cornea and vitreous, but there have been no published studies to date that objectively measure post-mortem changes in the retinal metabolome over time. The aim of the study was firstly, to investigate post-mortem, time-dependent changes in the rat retinal metabolome and secondly, to compare the metabolite composition of healthy rat ocular tissues. To study post-mortem changes in the rat retinal metabolome, globes were enucleated and stored at 4 °C and sampled at 0, 2, 4, 8, 24 and 48 h post-mortem. To study the metabolite composition of rat ocular tissues, eyes were dissected immediately after culling to isolate the cornea, lens, vitreous and retina, prior to storing at -80 °C. Tissue extracts were subjected to Gas Chromatograph Mass Spectrometry (GC-MS) and Ultra High Performance Liquid Chromatography Mass Spectrometry (UHPLC-MS). Generally, the metabolic composition of the retina was stable for 8 h post-mortem when eyes were stored at 4 °C, but showed increasing changes thereafter. However, some more rapid changes were observed such as increases in TCA cycle metabolites after 2 h post-mortem, whereas some metabolites such as fatty acids only showed decreases in concentration from 24 h. A total of 42 metabolites were identified across the ocular tissues by GC-MS (MSI level 1) and 2782 metabolites were annotated by UHPLC-MS (MSI level 2) according to MSI reporting standards. Many of the metabolites detected were common to all of the tissues but some metabolites showed partitioning between different ocular structures with 655, 297, 93 and 13 metabolites being uniquely detected in the retina, lens, cornea and vitreous respectively. Only a small percentage (1.6%) of metabolites found in the vitreous were only detected in the retina and not other tissues. In conclusion, mass spectrometry-based techniques have been used for the first time to compare the metabolic composition of different ocular tissues. The metabolite composition of the retina stored at 4 °C post-mortem is mostly stable for at least 8 h.
Assuntos
Córnea/metabolismo , Cristalino/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Retina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Córnea/patologia , Cromatografia Gasosa-Espectrometria de Massas , Cristalino/patologia , Masculino , Modelos Animais , Preservação de Órgãos , Ratos , Ratos Sprague-Dawley , Retina/patologia , Fatores de TempoRESUMO
The tight regulation of innate immunity on extracellular matrix (ECM) is a vital part of immune homeostasis throughout the human body, and disruption to this regulation in the eye is thought to contribute directly to the progression of age-related macular degeneration (AMD). The plasma complement regulator factor H (FH) is thought to be the main regulator that protects ECM against damaging complement activation. However, in the present study we demonstrate that a truncated form of FH, called FH-like protein 1 (FHL-1), is the main regulatory protein in the layer of ECM under human retina, called Bruch's membrane. Bruch's membrane is a major site of AMD disease pathogenesis and where drusen, the hallmark lesions of AMD, form. We show that FHL-1 can passively diffuse through Bruch's membrane, whereas the full sized, glycosylated, FH cannot. FHL-1 is largely bound to Bruch's membrane through interactions with heparan sulfate, and we show that the common Y402H polymorphism in the CFH gene, associated with an increased risk of AMD, reduces the binding of FHL-1 to this heparan sulfate. We also show that FHL-1 is retained in drusen whereas FH coats the periphery of the lesions, perhaps inhibiting their clearance. Our results identify a novel mechanism of complement regulation in the human eye, which highlights potential new avenues for therapeutic strategies.
Assuntos
Lâmina Basilar da Corioide/metabolismo , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Degeneração Macular/metabolismo , Retina/metabolismo , Drusas Retinianas/metabolismo , Lâmina Basilar da Corioide/imunologia , Lâmina Basilar da Corioide/patologia , Ativação do Complemento , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/imunologia , Fator H do Complemento/genética , Fator H do Complemento/imunologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Regulação da Expressão Gênica , Glicosilação , Heparitina Sulfato/imunologia , Heparitina Sulfato/metabolismo , Homeostase , Humanos , Imunidade Inata , Degeneração Macular/genética , Degeneração Macular/imunologia , Degeneração Macular/patologia , Ligação Proteica , Transporte Proteico , Retina/imunologia , Retina/patologia , Drusas Retinianas/genética , Drusas Retinianas/imunologia , Drusas Retinianas/patologia , Transdução de SinaisRESUMO
BACKGROUND: There are a number of different manifestations of pulmonary aspergillosis. This study aims to review the radiology, presentation, and histological features of lung nodules caused by Aspergillus spp. METHODS: Patients were identified from a cohort attending our specialist Chronic Pulmonary Aspergillosis clinic. Patients with cavitating lung lesions, with or without fibrosis and those with aspergillomas or a diagnosis of invasive aspergillosis were excluded. Demographic, laboratory, and clinical data and radiologic findings were recorded. RESULTS: Thirty-three patients with pulmonary nodules and diagnostic features of aspergillosis (histology and/or laboratory findings) were identified. Eighteen (54.5 %) were male, mean age 58 years (range 27-80 years). 19 (57.6 %) were former or current smokers. The median Charleston co-morbidity index was 3 (range 0-7). All complained of a least one of; dyspnoea, cough, haemoptysis, or weight loss. None reported fever. Ten patients (31 %) did not have an elevated Aspergillus IgG, and only 4 patients had elevated Aspergillus precipitins. Twelve patients (36 %) had a single nodule, six patients (18 %) had between 2 and 5 nodules, 2 (6 %) between 6 and 10 nodules and 13 (39 %) had more than 10 nodules. The mean size of the nodules was 21 mm, with a maximum size ranging between 5-50 mm. No nodules had cavitation radiographically. The upper lobes were most commonly involved. Histology was available for 18 patients and showed evidence of granulation tissue, fibrosis, and visualisation of fungal hyphae. CONCLUSION: Pulmonary nodules are a less common manifestation of aspergillosis in immunocompetent patients. Distinguishing these nodules from other lung pathology may be difficult on CT findings alone.
Assuntos
Aspergillus/isolamento & purificação , Aspergilose Pulmonar/diagnóstico por imagem , Aspergilose Pulmonar/patologia , Nódulo Pulmonar Solitário/diagnóstico por imagem , Nódulo Pulmonar Solitário/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Feminino , Humanos , Pulmão/microbiologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X , Reino UnidoRESUMO
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed in neurons. A possible role for UCH-L1 in neurodegeneration has been highlighted because of its presence in Lewy bodies associated with Parkinson disease and neurofibrillary tangles observed in Alzheimer disease. UCH-L1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-L1(C)) and a membrane-associated form (UCH-L1(M)). Alzheimer brains show reduced levels of soluble UCH-L1(C) correlating with the formation of UCH-L1-immunoreactive tau tangles, whereas UCH-L1(M) has been implicated in α-synuclein dysfunction. Given these reports of divergent roles, we investigated the properties of UCH-L1 membrane association. Surprisingly, our results indicate that UCH-L1 does not partition to the membrane in the cultured cell lines we tested. Furthermore, in primary cultured neurons, a proportion of UCH-L1(M) does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death, and an abnormal intracellular distribution, consistent with protein dysfunction and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared with clonal cell lines and that farnesylation does not account for the membrane association in neurons.
Assuntos
Neurônios/metabolismo , Prenilação de Proteína , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , Cultura Primária de Células , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , UbiquitinaçãoRESUMO
It is a longstanding puzzle why non-coding variants in the complement factor H (CFH) gene are more strongly associated with age-related macular degeneration (AMD) than functional coding variants that directly influence the alternative complement pathway. The situation is complicated by tight genetic associations across the region, including the adjacent CFH-related genes CFHR3 and CFHR1, which may themselves influence the alternative complement pathway and are contained within a common deletion (CNP147) which is associated with protection against AMD. It is unclear whether this association is mediated through a protective effect of low plasma CFHR1 concentrations, high plasma CFH or both. We examined the triangular relationships of CFH/CFHR3/CFHR1 genotype, plasma CFH or CFHR1 concentrations and AMD susceptibility in combined case-control (1256 cases, 1020 controls) and cross-sectional population (n = 1004) studies and carried out genome-wide association studies of plasma CFH and CFHR1 concentrations. A non-coding CFH SNP (rs6677604) and the CNP147 deletion were strongly correlated both with each other and with plasma CFH and CFHR1 concentrations. The plasma CFH-raising rs6677604 allele and raised plasma CFH concentration were each associated with AMD protection. In contrast, the protective association of the CNP147 deletion with AMD was not mediated by low plasma CFHR1, since AMD-free controls showed increased plasma CFHR1 compared with cases, but it may be mediated by the association of CNP147 with raised plasma CFH concentration. The results are most consistent with a regulatory locus within a 32 kb region of the CFH gene, with a major effect on plasma CFH concentration and AMD susceptibility.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Inativadoras do Complemento C3b/genética , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Alelos , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Fator H do Complemento/genética , Estudos Transversais , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Íntrons , Degeneração Macular/imunologia , Polimorfismo de Nucleotídeo Único , Deleção de SequênciaRESUMO
Ob/ob mice provide an animal model for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) in patients with obesity and type-2 diabetes. Low liver copper has been linked to hepatic lipid build-up (steatosis) in animals with systemic copper deficiency caused by low-copper diets. However, hepatic copper status in patients with NAFLD or NASH is uncertain, and a validated animal model useful for the study of hepatic copper regulation in common forms of metabolic liver disease is lacking. Here, we report parallel measurements of essential metal levels in whole-liver tissue and defatted-dried liver tissue from ob/ob and non-obese control mice. Measurements in whole-liver tissue from ob/ob mice at an age when they have developed NAFLD/NASH, provide compelling evidence for factitious lowering of copper and all other essential metals by steatosis, and so cannot be used to study hepatic metal regulation in this model. By marked contrast, metal measurements in defatted-dried liver samples reveal that most essential metals were actually normal and indicate specific lowering of copper in ob/ob mice, consistent with hepatic copper deficiency. Thus ob/ob mice can provide a model useful for the study of copper regulation in NAFLD and NASH, provided levels are measured in defatted-dried liver tissue.