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Purpose: Pre-Descemet corneal dystrophy (PDCD) with X-linked ichthyosis (XLI) is associated with mutations in or deletions of the steroid sulfatase gene (STS). As only three cases of genetically confirmed PDCD associated with XLI have been reported, we sought to expand our understanding of the genetic basis of PDCD by screening STS in two previously unreported families. Materials and Methods: The affected individuals underwent cutaneous and slit-lamp examinations. Saliva samples collected from each affected individual served as a source of DNA for the amplification of the 10 coding exons of STS and flanking DNA markers. Results: The slit-lamp examination of three affected men (two of whom were brothers) from two families revealed bilateral punctate posterior corneal stromal opacities anterior to the Descemet membrane. Cutaneous examination demonstrated dry, coarse, scaly ichthyotic changes characteristic of XLI in all individuals. Genetic examination of the STS locus on the X chromosome in Case 1 revealed a deletion that spanned across DNA markers DXS1130-DXS237, which includes all the coding exons (exons 1-10) of STS. Genetic screening of Cases 2 and 3 revealed a partial deletion of the STS locus involving exons 1-7 and flanking DNA marker DXS1130 on the X chromosome. Conclusions: PDCD with XLI may be associated with either partial or complete deletion of STS. Despite the identification of point mutations, partial deletion, and complete deletion of STS in different affected families reported to date, there was no apparent difference in the affected phenotype between the families, suggesting that the identified variants likely all resulted in loss of function of steroid sulfatase.
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Distrofias Hereditarias de la Córnea , Ictiosis Ligada al Cromosoma X , Ictiosis , Masculino , Humanos , Esteril-Sulfatasa/genética , Marcadores Genéticos , Ictiosis Ligada al Cromosoma X/complicaciones , Ictiosis Ligada al Cromosoma X/genética , Ictiosis/genética , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/diagnóstico , Eliminación de GenRESUMEN
Mutations associated with posterior polymorphous corneal dystrophy (PPCD) have been identified in three genes: ZEB1 (zinc-finger E-box binding homeobox 1) associated with sub-type PPCD3; OVOL2 (ovol-like zinc finger 2) associated with sub-type PPCD1; and GRHL2 (grainyhead like transcription factor 2) associated with sub-type PPCD4. Each of these genes encodes a transcription factor that regulates cell-state transitions. While the discovery of these PPCD-associated genes has greatly expanded our knowledge of the genetic basis of PPCD, the molecular mechanisms via which mutations in these genes lead to indistinguishable disease phenotypes have yet to be elucidated. To characterize the gene expression profiles of the genetic sub-types of PPCD, RNA-seq was performed on corneal endothelium derived from an individual with PPCD1 who harbors a c.-307Tâ¯>â¯C OVOL2 promoter mutation. Transcriptomic analysis of this and previously-reported RNA-seq data from two individuals with PPCD (the first with PPCD3 associated with a ZEB1 truncating mutation (c.1381delinsGACGAT) and the second with genetically unresolved PPCD in which ZEB1 coding region, OVOL2 promoter and GRHL2 promoter, exon 1, and intron 1 mutations were excluded) revealed: OVOL2 expression increased in PPCD1 (259 fold), unchanged in PPCD3 and slightly increased in genetically unresolved PPCD (from 0 TPM to 0.86 TPM, undefined fold change); ZEB1 expression decreased in PPCD1 (-5.9 fold), PPCD3 (-3.95 fold) and genetically unresolved PPCD (-3.96 fold); and GRHL2 expression increased in PPCD1 (333.5 fold), slightly increased (from 0 TPM to 0.67 TPM, undefined fold change) in PPCD3 and increased in genetically unresolved PPCD (1853 fold). Additionally, as the majority of pedigrees affected with PPCD remain genetically unresolved, we screened the promoter, exon 1, and intron 1 regions of GRHL2 in 24 PPCD probands who do not harbor a ZEB1 or OVOL2 mutation. GRHL2 screening did not identify any novel or rare GRHL2 variant in these 24 individuals. As ZEB1 can act as an activator or repressor of downstream target gene expression depending on Wnt signaling pathway activation or deactivation, we also sought to determine whether or not Wnt signaling is active in PPCD by performing immunohistochemistry in corneal tissue sections derived from an individual affected with PPCD3 and from an individual with genetically unresolved PPCD. Immunohistochemistry results demonstrated corneal endothelial nuclear accumulation of S552 phos-ß-catenin and cytosolic localization of S33/37/T42 non-phosphorylated ß-catenin in PPCD, indicating aberrant activation of Wnt signaling, which was not observed in control corneal endothelium. These findings suggest that alterations in the ZEB1-OVOL2-GRHL2 axis (caused by PPCD-associated mutations) lead to changes in corneal endothelial cell state and molecular pathways, including the aberrant activation of the Wnt signaling pathway.
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Distrofias Hereditarias de la Córnea/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Mutación , Factores de Transcripción/genética , Vía de Señalización Wnt/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Anciano , Distrofias Hereditarias de la Córnea/metabolismo , Distrofias Hereditarias de la Córnea/patología , Exones/genética , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Intrones/genética , Masculino , Persona de Mediana Edad , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
PURPOSE: To investigate the functional role that the zinc e-box binding homeobox 1 (ZEB1) gene, which underlies the genetic basis of posterior polymorphous corneal dystrophy 3 (PPCD3), plays in corneal endothelial cell proliferation, apoptosis, migration, and barrier function. METHODS: A human corneal endothelial cell line (HCEnC-21T) was transfected with siRNA targeting ZEB1 mRNA. Cell proliferation, apoptosis, migration, and barrier assays were performed: Cell proliferation was assessed with cell counting using a hemocytometer; cell apoptosis, induced by either ultraviolet C (UVC) radiation or doxorubicin treatment, was quantified by measuring cleaved caspase 3 (cCASP3) protein levels; and cell migration and barrier function were monitored with electric cell-substrate impedance sensing (ECIS). RESULTS: ZEB1 knockdown in HCEnC-21T cells transfected with siRNA targeting ZEB1 did not result in a significant difference in cell proliferation when compared with control. Although knockdown of ZEB1 in HCEnC-21T cells sensitized the cells to UV-induced apoptosis, ZEB1 knockdown did not alter the cells' susceptibility to doxorubicin-induced apoptosis, as measured with cCASP3 protein levels, compared with controls. Similarly, no difference was observed in cell migration following ZEB1 knockdown. However, cell barrier function increased significantly following ZEB1 knockdown. CONCLUSIONS: The corneal endothelium in PPCD3 is characterized by morphologic, anatomic, and molecular features that are more consistent with an epithelial-like rather than an endothelial-like phenotype. Although these characteristics have been well documented, we demonstrate for the first time that susceptibility to UV-induced apoptosis and cell barrier function are significantly altered in the setting of reduced ZEB1. The significance of an altered cellular response to apoptotic stimuli and increased cell barrier function in the pathobiology of PPCD remains to be fully elucidated.
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Distrofias Hereditarias de la Córnea/fisiopatología , Endotelio Corneal/fisiología , Regulación de la Expresión Génica/fisiología , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Antibióticos Antineoplásicos/toxicidad , Apoptosis/fisiología , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Doxorrubicina/toxicidad , Impedancia Eléctrica , Endotelio Corneal/efectos de la radiación , Técnicas de Silenciamiento del Gen , Humanos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Transfección , Rayos Ultravioleta/efectos adversosRESUMEN
PURPOSE: The aim of this study was to report a novel heterozygous variant c.1712G>T (p.Gly571Val) in the nucleotide-binding domain, leucine-rich repeat family, pyrin domain-containing 3 gene ( NLRP3 ) in a previously unreported non-Finnish individual with keratitis fugax hereditaria (KFH). METHODS: Ophthalmologic examination of the proband was performed with slit-lamp biomicroscopy and anterior segment optical coherence tomography. Saliva was collected as a source of DNA, after which targeted exome sequencing of candidate genes was performed using a commercially available panel. Identified presumed pathogenic variants were confirmed by Sanger sequencing. RESULTS: Slit-lamp examination of the 52-year-old female proband revealed peripheral arcus-like degeneration and bilateral central corneal opacification, observed on anterior segment optical coherence tomography to involve the anterior half of the corneal stroma. Examination of the proband's parents revealed clear corneas in each eye. Genetic testing of the proband identified the presence of a novel heterozygous NLRP3 missense mutation (c.1712G>T, p.Gly571Val), which was confirmed by Sanger sequencing. This mutation was absent in the proband's parents. CONCLUSIONS: Although KFH has been reported only in individuals of Finnish descent and only in association with a missense mutation in exon 1 of NLRP3 , we report an individual of non-Finnish descent with KFH associated with a novel heterozygous variant in exon 2 of NLRP3 . Thus, ophthalmologists should be aware of the ethnic and genetic heterogeneity associated with KFH.
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Queratitis , Proteína con Dominio Pirina 3 de la Familia NLR , Femenino , Humanos , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Mutación , Queratitis/diagnóstico , Queratitis/genética , Mutación Missense , LinajeRESUMEN
Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.
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Proteínas de Transporte de Anión , Antiportadores , Distrofias Hereditarias de la Córnea , Distrofia Endotelial de Fuchs , Humanos , Proteínas de Transporte de Anión/genética , Antiportadores/genética , Trastornos de los Cromosomas , Distrofias Hereditarias de la Córnea/genética , Células Endoteliales , Distrofia Endotelial de Fuchs/genética , Mutación , Proteínas SLC4ARESUMEN
PURPOSE: To report the genetic etiology of Lisch epithelial corneal dystrophy (LECD). DESIGN: Multicenter cohort study. METHODS: A discovery cohort of 27 individuals with LECD from 17 families, including 7 affected members from the original LECD family, 6 patients from 2 new families and 14 simplex cases, was recruited. A cohort of 6 individuals carrying a pathogenic MCOLN1 (mucolipin 1) variant was reviewed for signs of LECD. Next-generation sequencing or targeted Sanger sequencing were used in all patients to identify pathogenic or likely pathogenic variants and penetrance of variants. RESULTS: Nine rare heterozygous MCOLN1 variants were identified in 23 of 27 affected individuals from 13 families. The truncating nature of 7 variants and functional testing of 1 missense variant indicated that they result in MCOLN1 haploinsufficiency. Importantly, in the homozygous and compound-heterozygous state, 4 of 9 LECD-associated variants cause the rare lysosomal storage disorder mucolipidosis IV (MLIV). Autosomal recessive MLIV is a systemic disease and comprises neurodegeneration as well as corneal opacity of infantile-onset with epithelial autofluorescent lysosomal inclusions. However, the 6 parents of 3 patients with MLIV confirmed to carry pathogenic MCOLN1 variants did not have the LECD phenotype, suggesting MCOLN1 haploinsufficiency may be associated with reduced penetrance and variable expressivity. CONCLUSIONS: MCOLN1 haploinsufficiency is the major cause of LECD. Based on the overlapping clinical features of corneal epithelial cells with autofluorescent inclusions reported in both LECD and MLIV, it is concluded that some carriers of MCOLN1 haploinsufficiency-causing variants present with LECD.
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Distrofias Hereditarias de la Córnea , Mucolipidosis , Canales de Potencial de Receptor Transitorio , Humanos , Canales de Potencial de Receptor Transitorio/genética , Estudios de Cohortes , Mucolipidosis/diagnóstico , Mucolipidosis/genética , Mucolipidosis/patología , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genéticaRESUMEN
The purpose of this experiment was to investigate the feasibility of treating arsenopyrite-containing excavated crushed rock (ECR) in cropland by examining the amounts of arsenic released from various sizes of ECR mixed with soils at different ratios under three water levels using a batch incubation experiment. A total of 4 particle sizes of ECR were mixed with soil from 0% to 100% in 25% increments under three mass water contents such as 15%, 27%, and saturation. The results showed that the amount of As released from ECR mixed with soil was in the order of 27% saturation and 15% for 180 days regardless of the ECR:soil ratios, and the increase in the amount of As released before 90 days was slightly greater than that after 90 days. The maximum and minimum contents of released As were observed at 350.3 mg·kg-1 (ECR:Soil = 100:0, ECR size = 0.0-0.053 mm, and Æm = 32.2%), indicating that the smaller the ECR particle size resulted in a higher extractable As concentration. The amount of As released was higher than the relevant standard (25 mg·kg-1), except for ECR with a mixing ratio (25:75) and particle size (4.75-10.0 mm). In conclusion, we assumed that the amount of As released from ECR was influenced by the higher surface area of smaller ECR particle sizes and mass water content, which determine the porosity of the soil. However, further studies are needed on the transport and adsorption of released As depending on the physical and hydrological properties of the soil to determine the size and incorporation rate of ECR into the soil in view of the government standard.
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PURPOSE: Congenital stromal corneal dystrophy (CSCD) is a rare congenital, dominantly inherited disorder characterized by diffuse stromal opacification associated with mutations in the decorin gene ( DCN ). As only 5 families with genetically confirmed CSCD have been reported, the identification of a novel pedigree provides the opportunity to better characterize the phenotype and genetic basis. METHODS: An Armenian family with individuals in 4 consecutive generations demonstrated clinical features consistent with CSCD. Consented individuals underwent slit lamp examination, optical coherence tomography, and confocal microscopy. Genomic DNA was collected from saliva and all coding and adjacent intronic regions of DCN were sequenced. In silico analysis was performed for identified mutation(s). Excised corneal tissue underwent light, electron microscopic, and immunohistochemical evaluation. RESULTS: Affected individuals demonstrated bilateral, diffuse, panstromal corneal opacification. Three of the 6 individuals diagnosed with CSCD underwent genetic analysis; all demonstrated a novel heterozygous frameshift deletion in exon 8 of DCN (p.His317Thrfs*11), predicted to cause a 33 amino acid truncation and to be damaging and disease causing by SIFT and MutationTaster. Light and electron microscopic examination of an excised cornea demonstrated increased corneal thickness, stromal scarring, keratocyte loss, and an irregularity of lamellar collagen spacing and fibril formation. Immunofluorescent examination demonstrated increased DCN immunostaining, predominantly in the widened interlamellar spaces. CONCLUSIONS: We report only the sixth pedigree with genetically confirmed CSCD, associated with a novel DCN frameshift mutation. The clinical evaluation, multimodal imaging, and histopathologic assessment in this family with CSCD broaden our understanding of this rare corneal disease.
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Distrofias Hereditarias de la Córnea , Humanos , Decorina/genética , Decorina/metabolismo , Armenia , Distrofias Hereditarias de la Córnea/genética , Mutación , Linaje , Análisis Mutacional de ADNRESUMEN
PURPOSE: To provide the initial confirmation of the c.1772C>T (p.Ser591Phe) mutation in the transforming growth factor-ß-induced (TGFBI) gene as being associated with variant lattice corneal dystrophy (LCD). METHODS: Ophthalmologic examination of the proband was performed with slit lamp biomicroscopy. Saliva was collected as a source of DNA for screening all 17 exons of TGFBI, after which three family members were selectively screened for variants in exon 13. Rosetta-based structure prediction was used to calculate changes in TGFBI protein (TGFBIp) stability secondary to the c.1772C>T (p.Ser591Phe) missense mutation. RESULTS: Slit lamp examination of the 38-year-old proband revealed a clear cornea right eye and unilateral, discrete, and branching lattice lines in the anterior and mid-stroma of the central cornea left eye. Screening of TGFBI in the proband revealed a heterozygous missense mutation in exon 13 (c.1772C>T (p.Ser591Phe)) that was also identified in her affected mother but not in her brother or maternal grandmother. Calculated energy change in Rosetta (ΔΔG) for the TGFBIp variant p.Ser591Phe was 23.5, indicating a thermodynamic destabilization resulting from energetic frustration. CONCLUSIONS: The p.Ser591Phe mutation in TGFBI is associated with an unilateral variant of LCD. Rosetta-predicted stability changes indicate that the p.Ser591Phe variant is destabilizing, which is consistent with other observations for LCD-causing mutations.
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Neuropatías Amiloides Familiares , Distrofias Hereditarias de la Córnea , Proteínas de la Matriz Extracelular , Factores de Crecimiento Transformadores , Adulto , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Masculino , Mutación , Mutación Missense , Linaje , Factor de Crecimiento Transformador beta , Factores de Crecimiento Transformadores/genéticaRESUMEN
PURPOSE: The aim of this study was to report the results of screening peroxiredoxin 3 (PRDX3) and PDZ domain-containing protein 8 (PDZD8) in a previously unreported pedigree with punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD) to confirm that the PRDX3 mutation c.568G>C is the genetic basis of PPPCD. METHODS: Ophthalmologic examination of the proband and her affected father was performed with slit lamp biomicroscopy. Saliva was collected from the proband as a source of DNA, after which screening for PRDX3 and PDZD8 was performed. RESULTS: Slit lamp examination of the proband revealed polychromatic deposits diffusely distributed at the pre-Descemet level in both corneas and anterior subcapsular in the crystalline lens of both eyes. The proband's father also demonstrated diffuse pre-Descemetic polychromatic deposits in both eyes but no lenticular deposits. Screening of PRDX3 in the proband demonstrated the c.568G>C (p.Asp190His) variant previously associated with PPPCD and failed to identify any variants in PDZD8. CONCLUSIONS: We report the initial confirmation of PRDX3 as the genetic basis of PPPCD in a previously unreported pedigree and expand the phenotype of PPPCD to include polychromatic lenticular deposits.
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Distrofias Hereditarias de la Córnea , Peroxiredoxina III , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Córnea/metabolismo , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Distrofias Hereditarias de la Córnea/metabolismo , Femenino , Humanos , Masculino , Mutación , Linaje , Peroxiredoxina III/genética , Peroxiredoxina III/metabolismoRESUMEN
BACKGROUND: Concomitant corneal ectasia and posterior lamellar corneal opacification is rare, and the genetic relationship between these two conditions is unclear. We report the genetic and clinical characterization of this phenotype in three unrelated individuals. MATERIALS AND METHODS: One previously reported affected individual and two unreported, unrelated, affected individuals were recruited for the study. Subjects and unaffected relatives underwent slit lamp examination, refraction, and multi-modal imaging. Saliva samples were obtained from two of the three affected individuals, from which DNA was extracted. Sanger sequencing was performed to identify mutations in genes associated with posterior amorphous corneal dystrophy (PACD), brittle cornea syndrome (BCS), and posterior polymorphous corneal dystrophy (PPCD), while copy number variation (CNV) analysis was used to identify CNV in the PACD locus. RESULTS: Affected individuals demonstrated bilateral corneal steepening, stromal thinning and lamellar posterior corneal opacification. Corneal topography and tomography revealed conical or globular corneal steepening and decreased thickness. Anterior segment optical coherence tomography demonstrated hyperreflectivity of the posterior stroma in each of the affected individuals. Genetic testing did not detect a heterozygous deletion involving the PACD locus on chromosome 12 or a pathogenic mutation in the genes associated with BCS or PPCD. CONCLUSIONS: Corneal ectasia may be associated with posterior lamellar stromal opacification that appears consistent with PACD. However, genetic testing for PACD as well as BCS and PPCD in affected individuals fails to reveal pathogenic deletions or mutations, indicating that other genetic factors are involved.
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Córnea/patología , Opacidad de la Córnea/diagnóstico , Sustancia Propia/patología , Queratocono/diagnóstico , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/genética , Topografía de la Córnea , Variaciones en el Número de Copia de ADN , Dilatación Patológica , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Femenino , Humanos , Recién Nacido , Inestabilidad de la Articulación/congénito , Inestabilidad de la Articulación/diagnóstico , Inestabilidad de la Articulación/genética , Masculino , Mutación , Anomalías Cutáneas/diagnóstico , Anomalías Cutáneas/genética , Microscopía con Lámpara de Hendidura , Adulto JovenRESUMEN
A study was conducted to compare the effects of phosphate (P) materials in reducing cadmium extractability. Seven P materials (commercial P fertilizers--fused phosphate (FP), 'fused and superphosphate' [FSP], and rock phosphate [RP]; P chemicals--Ca[H(2)PO(4)](2).H(2)O, [NH(4)](2)HPO(4), KH(2)PO(4), and K(2)HPO(4)) were selected for the test. The selected P source was mixed with Cd-contaminated soil at the rate of 0, 200, 400, 800, and 1,600 mg P kg(-1) under controlled moisture conditions at 70% of water holding capacity, then incubated for 8 weeks. FP, Ca(H(2)PO(4))(2) H(2)O, KH(2)PO(4), and K(2)HPO(4) significantly decreased NH(4)OAc-extractable Cd (plant-available form) concentrations with increasing application rates. Compared to other phosphate materials used, K(2)HPO(4) was found to be the most effective in reducing the plant-available Cd concentration in soil, mainly due to the negative charge increase caused by soil pH and phosphate adsorption. Contrary to the general information, FSP and (NH(4))(2)HPO(4) increased Cd extractability at low levels of P application (<400 mg kg(-1)), and thereafter Cd extractability decreased significantly with increasing application rate. RP scarcely had an effect on reducing Cd extractability. Ion activity products of CdHPO(4), Cd(OH)(2), and CdCO(3) analyzed by the MINTEQ program were significantly increased by K(2)HPO(4) addition, but the effect of Cd-P compound formation on reducing Cd extractability was negligible. Conclusively, the P-induced alleviation of Cd extractability can be attributed primarily to Cd immobilization due to the increase in soil pH and negative charge rather than Cd-P precipitation, and therefore, alkaline P materials such as K(2)HPO(4) are effective for immobilizing soil Cd.
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Cadmio/química , Fosfatos/química , Contaminantes del Suelo/química , Cadmio/análisis , Difosfatos/química , Fertilizantes , Compuestos de Potasio/química , Suelo , Contaminantes del Suelo/análisisRESUMEN
BACKGROUND: Most transforming growth factor beta-induced (TGFBI) corneal dystrophies are associated with a characteristic phenotype, clinical course, and a conserved mutation in the TGFBI gene. However, we report a novel TGFBI missense mutation associated with a late-onset, variant Bowman layer dystrophy. METHODS: Participants underwent slit-lamp examination and multimodal imaging. Polymerase chain reaction amplification and Sanger sequencing were performed on saliva-derived genomic DNA to screen TGFBI exons 4 and 12 as well as COL17A1 exon 46. PolyPhen-2 and SIFT were used to predict the functional impact of any identified variants. RESULTS: A 56-year-old Thai woman reported a four-year history of decreased vision and intermittent eye irritation, suggestive of recurrent epithelial erosions, in both eyes. Slit-lamp exam revealed bilateral, irregular, limbal-sparing Bowman layer opacities, which were also noted on anterior segment optical coherence tomography. Phototherapeutic keratectomy was performed in the right eye, improving the best-corrected visual acuity from 20/50 to 20/30. Sequencing of the TGFBI gene revealed a novel heterozygous, missense mutation in exon 12 (c.1571 C > G; p.Ser524Cys), which was present in an affected son and absent in an unaffected son, and was predicted to be damaging by PolyPhen-2 and SIFT. The patient was diagnosed with a variant Bowman layer dystrophy given the late onset of an atypical phenotype and the identification of a novel TGFBI mutation. CONCLUSIONS: A novel TGFBI missense mutation is associated with a late-onset Bowman layer dystrophy. Given the atypical clinical appearance and course, molecular genetic analysis was utilized to establish a definitive diagnosis.
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Lámina Limitante Anterior/patología , Distrofias Hereditarias de la Córnea/patología , Epitelio Corneal/patología , Proteínas de la Matriz Extracelular/genética , Mutación , Fenotipo , Factor de Crecimiento Transformador beta/genética , Edad de Inicio , Autoantígenos/genética , Lámina Limitante Anterior/metabolismo , Distrofias Hereditarias de la Córnea/genética , Epitelio Corneal/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Colágenos no Fibrilares/genética , Linaje , Recurrencia , Microscopía con Lámpara de Hendidura , Colágeno Tipo XVIIRESUMEN
Meesmann epithelial corneal dystrophy (MECD) is a rare dominantly inherited disorder that is characterized by corneal epithelial microcysts and is associated with mutations in the keratin 3 (KRT3) and keratin 12 (KRT12) genes. In this study, we report a novel mutation in the KRT12 gene in a Vietnamese pedigree with MECD. Slit-lamp examination was performed on each of the 7 recruited members of a Vietnamese family to identify characteristic features of MECD. After informed consent was obtained from each individual, genomic DNA was isolated from saliva samples and screening of KRT3and KRT12 genes was performed by Sanger sequencing. The proband, a 31-year-old man, complained of a 1-year history of eye irritation and photophobia. Slit-lamp examination revealed intraepithelial microcysts involving only the corneal periphery in each eye with clear central corneas and no stromal or endothelial involvement. Three family members demonstrated similar intraepithelial microcysts, but with diffuse involvement, extended from limbus to limbus. Sanger sequencing of KRT3 (exon 7) and KRT12 (exons 1 and 6) in the proband revealed a novel heterozygous KRT12 variant (c.1273G>A [p.Glu425Lys]) that was present in the three affected family members but was absent in the three family members with clear corneas. This study is the first report of a Vietnamese family affected with MECD, associated with an atypical peripheral corneal epithelial phenotype in the proband and a novel mutation in KRT12.
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Purpose: To elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4. Methods: Comparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and Slc4a11 (Slc4a11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations. Results: CTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and Slc4a11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in Slc4a11-/- MCEnC. Conclusions: Reduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.
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Adenosina Trifosfato/biosíntesis , Endotelio Corneal , Transporte Iónico/fisiología , Mitocondrias/metabolismo , Proteínas SLC4A/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Células Cultivadas , Distrofias Hereditarias de la Córnea/genética , Endotelio Corneal/metabolismo , Endotelio Corneal/patología , Endotelio Corneal/fisiopatología , Metabolismo Energético , Perfilación de la Expresión Génica , Humanos , Ratones , Mutación , Proteínas Quinasas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The advent of cell culture-based methods for the establishment and expansion of human corneal endothelial cells (CEnC) has provided a source of transplantable corneal endothelium, with a significant potential to challenge the one donor-one recipient paradigm. However, concerns over cell identity remain, and a comprehensive characterization of the cultured CEnC across serial passages has not been performed. To this end, we compared two established CEnC culture methods by assessing the transcriptomic changes that occur during in vitro expansion. In confluent monolayers, low mitogenic culture conditions preserved corneal endothelial cell state identity better than culture in high mitogenic conditions. Expansion by continuous passaging induced replicative cell senescence. Transcriptomic analysis of the senescent phenotype identified a cell senescence signature distinct for CEnC. We identified activation of both classic and new cell signaling pathways that may be targeted to prevent senescence, a significant barrier to realizing the potential clinical utility of in vitro expansion.
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Técnicas de Cultivo de Célula/métodos , Endotelio Corneal/citología , Adolescente , Adulto , Movimiento Celular , Proliferación Celular , Senescencia Celular , Niño , Preescolar , Biología Computacional , Trasplante de Córnea , Femenino , Humanos , Masculino , Fenotipo , Transducción de Señal , Transcriptoma , Adulto JovenRESUMEN
PURPOSE: This study reports the clinical features and genetic bases of 3 previously unreported families with punctiform and polychromatic pre-Descemet corneal dystrophy (PPPCD). DESIGN: Observational case series. METHODS: Full ophthalmic assessment was performed for members of 3 unreported families with PPPCD. Structural and biomechanical alterations of the cornea were screened. Whole exome sequencing (WES) was performed in the first family. Novel or rare variants that segregated with the affected status were screened in the other 2 families using Sanger sequencing. Identified variants that segregated with the affected status in all families were characterized by using in silico prediction tools and/or in vitro splice assays. Additionally, 2 previously reported PPPCD families were screened for variants identified in the 3 unreported PPPCD families. RESULTS: PPPCD was diagnosed in 12 of the 21 examined members of the 3 unreported families. The only refractive, topographic, or biomechanical abnormality associated with PPPCD was a significantly increased corneal stiffness. WES and Sanger sequencing identified 2 variants that segregated with the affected status in all 3 families: a rare intronic PDZD8 c.872+10A>T variant and a novel missense PRDX3 c.568G>C (p.Asp190His) variant. The same PRDX3 variant was identified in the previously reported PPPCD family expressing the common PPPCD phenotype and was predicted by in silico prediction tools to be damaging to protein function. CONCLUSIONS: PPPCD is associated with an alteration of corneal biomechanics and a novel missense variant in PRDX3. Screening of additional families will determine whether all families demonstrate a PRDX3 variant or whether locus heterogeneity may exist for PPPCD.
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Distrofias Hereditarias de la Córnea/genética , Mutación Missense/genética , Peroxiredoxina III/genética , Adolescente , Adulto , Anciano , Segmento Anterior del Ojo/diagnóstico por imagen , Fenómenos Biomecánicos/fisiología , Estudios de Casos y Controles , Niño , Córnea/fisiología , Distrofias Hereditarias de la Córnea/diagnóstico por imagen , Distrofias Hereditarias de la Córnea/fisiopatología , Femenino , Humanos , Masculino , Microscopía Confocal/métodos , Persona de Mediana Edad , Linaje , Tomografía de Coherencia Óptica/métodos , Secuenciación del Exoma/métodos , Adulto JovenRESUMEN
PURPOSE: To report an unusual case of bilateral crystalline keratopathy presenting several years after a laser in situ keratomileusis (LASIK) procedure. METHODS: Case report and review of the literature. RESULTS: A healthy 42-year-old woman with post-LASIK ectasia, treated with long-term corneoscleral contact lenses, developed new-onset crystalline keratopathy in both eyes. Anterior segment optical coherence tomography demonstrated bilaterally symmetric paracentral rings of hyperreflectivity, involving the LASIK corneal flaps. There was good clearance of the corneoscleral contact lenses with no corneal-lens touch in all quadrants. There was no evidence of infectious or systemic etiologies. Genetic testing for the UBIAD1 gene for Schnyder corneal dystrophy was negative. Continued clinical observations have exhibited progressive corneal crystalline deposition, yet the patient has remained visually asymptomatic. CONCLUSIONS: This is the first known reported case of significantly delayed progressive noninfectious crystalline keratopathy in post-LASIK ectasia. The pathophysiology of this condition remains elusive to date and highlights the challenges of noninvasive diagnostic techniques and yet the utility of molecular genetic analysis in elucidating the etiology of this unique clinical presentation.
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Lentes de Contacto/efectos adversos , Enfermedades de la Córnea/etiología , Dilatación Patológica/terapia , Queratomileusis por Láser In Situ/efectos adversos , Adulto , Femenino , HumanosRESUMEN
PURPOSE: To report a case of bilateral and repetitive corneal perforations after corneal cross-linking (CXL) for keratoconus in a woman harboring potentially pathogenic variants in the ZNF469 gene and to characterize the keratoconus phenotype in this woman and her daughter who shared the same ZNF469 mutations. METHODS: Clinical characterization of the proband and her daughter followed by sequencing of the genes associated with brittle cornea syndrome, ZNF469 and PRDM5, in both individuals. RESULTS: An Ashkenazi Jewish woman in her sixth decade presented with diffuse corneal thinning and progressive steepening consistent with keratoconus. After CXL, epithelium-off in the first eye and epithelium-on in the second, she developed spontaneous corneal perforations in each eye. Her daughter in her fourth decade demonstrated a similar pattern of diffuse corneal thinning and progressive corneal steepening but did not undergo CXL and did not develop corneal perforation. Screening of the ZNF469 and PRDM5 genes revealed 3 missense ZNF469 variants (c.2035G>A, c.10244G>C, and c.11119A>G) in cis arrangement on 1 allele of ZNF469 in both proband and her daughter. Although the 3 variants share low (<0.01) global minor allele frequencies, each has significantly higher minor allele frequencies (0.01-0.03) in the Ashkenazi Jewish population, leading to uncertainty regarding a pathogenic role for the identified variants. CONCLUSIONS: CXL may be associated with the development of corneal perforation in particular at-risk individuals with keratoconus. Identifying clinical and genetic risk factors, including screening of ZNF469 and PRDM5, may be useful in the prevention of significant complications after CXL.
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Perforación Corneal/etiología , Reactivos de Enlaces Cruzados/efectos adversos , Queratocono/genética , Mutación Missense , Fotoquimioterapia/efectos adversos , Factores de Transcripción/genética , Adulto , Colágeno/metabolismo , Perforación Corneal/diagnóstico , Sustancia Propia/metabolismo , Topografía de la Córnea , Proteínas de Unión al ADN/genética , Femenino , Humanos , Judíos/genética , Queratocono/tratamiento farmacológico , Queratocono/metabolismo , Persona de Mediana Edad , Fármacos Fotosensibilizantes/efectos adversos , Reacción en Cadena de la Polimerasa , Rayos UltravioletaRESUMEN
The zinc finger e-box binding homeobox 1 (ZEB1) transcription factor is a master regulator of the epithelial to mesenchymal transition (EMT), and of the reverse mesenchymal to epithelial transition (MET) processes. ZEB1 plays an integral role in mediating cell state transitions during cell lineage specification, wound healing and disease. EMT/MET are characterized by distinct changes in molecular and cellular phenotype that are generally context-independent. Posterior polymorphous corneal dystrophy (PPCD), associated with ZEB1 insufficiency, provides a new biological context in which to understand and evaluate the classic EMT/MET paradigm. PPCD is characterized by a cadherin-switch and transition to an epithelial-like transcriptomic and cellular phenotype, which we study in a cell-based model of PPCD generated using CRISPR-Cas9-mediated ZEB1 knockout in corneal endothelial cells (CEnCs). Transcriptomic and functional studies support the hypothesis that CEnC undergo a MET-like transition in PPCD, termed endothelial to epithelial transition (EnET), and lead to the conclusion that EnET may be considered a corollary to the classic EMT/MET paradigm.