RESUMEN
OBJECTIVE: To report a novel mutation in TGFBI (GenBank NM_000358), p.Met619Lys, associated with a variant of combined granular-lattice corneal dystrophy. METHODS: Slitlamp examination and DNA collection from the proband and affected and unaffected relatives. All 17 exons of TGFBI were amplified and sequenced in the proband. Exon 14 was amplified and sequenced in the proband's family members and in 100 controls. Histopathologic examination of the excised corneal buttons from the proband and 3 family members was also performed. RESULTS: Affected individuals demonstrated an age-dependent phenotype, with the progression from central subepithelial needlelike deposits in younger individuals to polymorphic anterior stromal opacities in older family members. Screening of TGFBI in the proband demonstrated a novel mutation, p.Met619Lys, which was also present in all affected family members. Histopathologic examination revealed stromal deposits that stained with the Congo red and Masson trichrome stains as well as an antibody to the protein product of TGFBI. CONCLUSIONS: We present a unique corneal dystrophy phenotype associated with the novel p.Met619Lys mutation in TGFBI. Clinical Relevance The atypical and variable phenotype and the demonstration of both hyaline and amyloid stromal deposits indicate that neither clinical nor histopathologic features may be relied on to accurately diagnose and classify the corneal dystrophies.
Asunto(s)
Amiloidosis/genética , Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Variación Genética , Mutación Missense , Factor de Crecimiento Transformador beta/genética , Adulto , Anciano , Amiloide/metabolismo , Amiloidosis/diagnóstico , Amiloidosis/metabolismo , Distrofias Hereditarias de la Córnea/diagnóstico , Distrofias Hereditarias de la Córnea/metabolismo , Sustancia Propia/metabolismo , Sustancia Propia/patología , Análisis Mutacional de ADN , Exones , Femenino , Amplificación de Genes , Humanos , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Sunitinib, a multitargeted vascular endothelial growth factor and receptor tyrosine kinase inhibitor, causes hypothyroidism in patients who take it for treatment of cancer. Although the pathophysiologic mechanism of the hypothyroidism is unclear, it has been claimed that it is due to inhibition of iodide uptake. METHODS: To evaluate the pathologic mechanism of induction of the hypothyroidism, we studied the effect of sunitinib on FRTL-5 rat thyroid cells. We measured the effect of sunitinib on cell growth, (125)I-iodide uptake and efflux, TSH receptor (TSH-R), and sodium-iodide symporter (NIS) message. RESULTS: At 48 hours, sunitinib caused a dose-related inhibition of growth with LC(50) of 14.6 muM, but there was no apparent inhibition of growth at 24 hours at concentrations of 0.1-25 microM. Preincubation with sunitinib did not impair the response to TSH, indicating that it did not affect the TSH-R. Incubation with sunitinib for 24 hours caused a dose-related increase of (125)I-iodide uptake and did not reduce iodide efflux or NIS mRNA expression. CONCLUSION: The data indicate that sunitinib is unlikely to cause hypothyroidism by inhibition of iodide uptake.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Indoles/farmacología , Pirroles/farmacología , Glándula Tiroides/crecimiento & desarrollo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Relación Dosis-Respuesta a Droga , Radioisótopos de Yodo/farmacocinética , ARN Mensajero/metabolismo , Ratas , Receptores de Tirotropina/efectos de los fármacos , Receptores de Tirotropina/metabolismo , Sunitinib , Simportadores/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Factores de TiempoRESUMEN
PURPOSE: To determine the genetic basis of autosomal dominant cornea plana (CNA1) through the performance of a genome-wide linkage analysis and screening of the decorin (DCN), dermatan sulfate proteoglycan 3 (DSPG3), forkhead box C1 (FOXC1), keratocan (KERA), lumican (LUM,) and paired-like homeodomain transcription factor 2 (PITX2) genes in members of an affected multigenerational family. METHODS: Cycloplegic refraction, slit lamp biomicroscopy, corneal pachymetry, and corneal topography were performed to determine each patient's affected status. DNA was obtained from affected and unaffected subjects for the performance of a genome-wide linkage analysis as well as PCR amplification and sequencing of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2. RESULTS: Five affected and three unaffected individuals were examined and provided a peripheral blood sample for DNA isolation. All affected individuals demonstrated an average corneal dioptric power less than 39 D, as well as one or more of the following anomalies: high hyperopia, strabismus, microcornea, posterior embryotoxon, iridocorneal adhesions, iris atrophy, and pupillary irregularities. A genome-wide linkage analysis did not indicate or exclude linkage to the region on chromosome 12 to which CNA1 has been previously mapped, and did not provide a single or multipoint LOD score greater than 2.0 for any of the 400 microsatellite markers. Screening of DCN, DSPG3, FOXC1, KERA, LUM, and PITX2 revealed 12 previously described single nucleotide polymorphisms, 2 previously described duplications, and 1 previously described insertion. None of the mutations previously associated with autosomal recessive cornea plana (CNA2) were identified. Seven novel sequence variants were described, including 5 single nucleotide substitutions, 1 insertion and 1 deletion. None of the identified sequence variants demonstrated complete segregation with the affected phenotype in the pedigree. CONCLUSION: Although missense and nonsense mutations in KERA are associated with CNA2, we did not identify any of the previously described mutations or novel mutations that segregated with the disease phenotype in a family with CNA1. In addition, no pathogenic sequence variations were found in DCN, DSPG3, LUM, PITX2 and FOXC1, which have also been implicated in corneal and anterior segment dysgenesis.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Enfermedades de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Factores de Transcripción Forkhead/genética , Proteínas de Homeodominio/genética , Sulfato de Queratano/genética , Mutación/genética , Proteoglicanos/genética , Factores de Transcripción/genética , Adulto , Niño , Preescolar , Córnea/anomalías , Córnea/patología , Enfermedades de la Córnea/patología , Decorina , Femenino , Genes Dominantes , Ligamiento Genético , Humanos , Lumican , Masculino , Repeticiones de Microsatélite , Linaje , Reacción en Cadena de la Polimerasa , Proteoglicanos Pequeños Ricos en Leucina , Proteína del Homeodomínio PITX2RESUMEN
PURPOSE: To determine the genetic basis of autosomal recessive congenital hereditary endothelial dystrophy (CHED2) in an American patient of Chinese ancestry. METHODS: Slit-lamp examination of the proband and his parents, as well as histopathologic examination of excised corneal specimens from the proband, were performed to confirm the diagnosis of autosomal recessive CHED. DNA was collected from the proband and his parents, and all 19 exons of the SLC4A11 gene were amplified and screened. RESULTS: The proband showed diffuse bilateral corneal edema, which was not present in either of his parents. After the performance of bilateral penetrating keratoplasties, histopathologic examination of the excised corneal specimens showed marked corneal stromal edema and an absence of corneal endothelial cells. Screening of SLC4A11 showed 2 heterozygous mutations: c.743G>A (Ser232Asn) and c.1033A>T (Arg329X). The proband's mother was found to be heterozygous for the Ser232Asn missense mutation, and his father was heterozygous for the Arg329X nonsense mutation. No other coding region sequence variants were identified in the proband or his parents, and neither of the identified mutations was identified in 100 control individuals. CONCLUSIONS: CHED2 is associated with mutations in SLC4A11, a member of the SLC4 family of base transporters. Although the majority of affected individuals reported to date have shown homozygous mutations, associated with consanguinity in the Burmese, Indian, and Pakistani populations, we report 2 novel, independently sorting SLC4A11 mutations in an affected individual of Chinese ancestry.
Asunto(s)
Proteínas de Transporte de Anión/genética , Antiportadores/genética , Distrofias Hereditarias de la Córnea/genética , Genes Recesivos , Heterocigoto , Mutación , Adolescente , Asiático/genética , Distrofias Hereditarias de la Córnea/cirugía , Edema Corneal/genética , Edema Corneal/cirugía , Exones/genética , Humanos , Queratoplastia Penetrante , Masculino , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: To evaluate the suggested role of the COL8A1 and COL8A2 genes in the pathogenesis of the corneal ectatic disorders keratoconus and keratoglobus through mutation screening in affected patients. METHODS: DNA extraction, polymerase chain reaction amplification, and sequencing of COL8A1 and COL8A2 were performed in 50 unrelated keratoconus and 2 unrelated keratoglobus patients. RESULTS: No sequence variations were identified in COL8A1 and COL8A2 in the 2 patients with keratoglobus. Screening of COL8A1 in keratoconus patients revealed a previously identified single nucleotide polymorphism (SNP; c.1850C>T; Pro535Pro), in 1 patient. Screening of COL8A2 in keratoconus patients revealed 7 previously described SNPs: c.14G>A (Gly3Arg); c.112G>A (Ala35Ala); c.1012C>G (Leu335Leu); c.1308G>A (Arg434His); c.1492G>A (Gly495Gly); c.1512C>T (Thr502Met); and c.1765C>T (Pro586Pro). Four novel sequence variants were also identified, each in 1 affected patient: c.38_40dupCTG (Leu11dup), also identified in an unaffected relative of the affected proband, c.667G>A (Gly220Gly), c.1588G>A (Pro527Pro), and c.2026C>T (Val673Val). None of the 3 novel synonymous substitutions identified in COL8A2 was predicted to produce a splice acceptor site. CONCLUSIONS: The absence of pathogenic mutations in COL8A1 and COL8A2 in patients with keratoconus indicates that other genetic factors are involved in the pathogenesis of this corneal ectatic disorder.
Asunto(s)
Colágeno Tipo VIII/genética , Queratocono/genética , Mutación , Adulto , Análisis Mutacional de ADN , Femenino , Amplificación de Genes , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADNRESUMEN
PURPOSE: To investigate the genetic basis of late-onset, familial Fuchs endothelial corneal dystrophy (FECD) through screening of the COL8A1 and COL8A2 genes, in which mutations have been associated with both early and late-onset, familial and sporadic FECD. METHODS: DNA extraction, PCR amplification, and direct sequencing of the COL8A1 and COL8A2 genes was performed in affected and unaffected members of 15 unrelated families with two or more members with late-onset FECD. RESULTS: Screening of the COL8A1 gene did not reveal sequence variants in any affected individuals from the 15 FECD families. In the COL8A2 gene, the previously identified mutations presumed to play a pathogenic role in cases of familial FECD (Arg155Gln, Leu450Trp, and Gln455Lys) were not discovered in any of the affected patients. A mutation previously considered causative of FECD (Arg434His) was shown not to segregate with the disease in the one family in which it was identified. Two previously identified single-nucleotide polymorphisms (SNPs), Pro575Leu and Pro586Pro, were identified in a single affected individual and three affected individuals (two families), respectively. CONCLUSIONS: The Arg434His mutation in the COL8A2 gene, previously associated with FECD, has been shown not to segregate with the disease phenotype, and thus may not be considered a disease-causing mutation. The absence of pathogenic mutations identified in the COL8A1 or COL8A2 genes in affected members of 15 pedigrees with familial FECD indicates that other genetic factors are involved in the development of this autosomal dominant corneal dystrophy.
Asunto(s)
Colágeno Tipo VIII/genética , Distrofia Endotelial de Fuchs/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , ADN/aislamiento & purificación , Análisis Mutacional de ADN , Femenino , Distrofia Endotelial de Fuchs/patología , Humanos , Masculino , Persona de Mediana Edad , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To determine whether mutations of the VSX1 gene play a pathogenetic role in the development of keratoconus (KTCN). METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1 gene were performed in 100 unrelated patients with diagnoses of clinical and topographic features of KTCN. RESULTS: Of the four previously identified presumed pathogenic mutations in the VSX1 gene (Leu17Pro, Asp144Glu, Leu159Met, and Arg166Trp), only Asp144Glu was identified in a single affected patient. Two novel single nucleotide polymorphisms (SNPs), both resulting in synonymous substitutions, were identified: c.53G>T (Ser6Ser) in four affected patients and c.209G>T (Pro58Pro) in two affected patients. Two previously reported SNPs were also identified: c.426C>A (Arg131Ser) in one affected patient and c.581A>G (Ala182Ala) in 51 of the 100 affected patients. CONCLUSIONS: Only one of the presumed pathogenic mutations in the VSX1 gene, Asp144Glu, was identified in a single member of the cohort of affected patients. However, as previously demonstrated, Asp144Glu is a non-disease-causing polymorphism. The absence of pathogenic mutations in the VSX1 gene in a large number of unrelated KTCN patients indicates that other genetic factors are involved in the development of this disorder.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Queratocono/genética , Mutación , Adolescente , Adulto , Anciano , Análisis Mutacional de ADN , Femenino , Humanos , Queratocono/cirugía , Queratoplastia Penetrante , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: To determine whether primary, polymorphic, corneal amyloid deposition is associated with a mutation of the TGFBI gene. METHODS: Interventional case series of 8 patients. Slit lamp examination of all patients and photodocumentation of 5 patients were performed. Genomic DNA was isolated from buccal mucosal swabs obtained from all patients and all 17 exons of the TGFBI gene were amplified and sequenced. RESULTS: Multiple polymorphic, refractile deposits were noted throughout the central corneal stroma in all patients. The deposits appeared gray-white on direct illumination and translucent on retroillumination, characteristic of amyloid. In 2 patients, linear, branching opacities, reminiscent of lattice corneal dystrophy, were identified. Histopathologic examination confirmed the presence of stromal amyloid in the cornea of 1 patient who required corneal transplantation for pseudophakic corneal edema. Screening of the entire coding region of the TGFBI gene revealed 4 previously described synonymous substitutions, Leu217Leu, Val327Val, Leu472Leu, and Phe540Phe. A previously unreported missense change, Asp299Asn, was identified in one affected patient but not in her affected sister. No pathogenic mutations, including the Ala546Asp missense mutation previously associated with polymorphic corneal amyloidosis, were identified in any of the patients. CONCLUSIONS: TGFBI gene mutations were not identified in a series of patients with polymorphic corneal amyloid deposition. As bilateral, discrete stromal amyloid deposits may be dystrophic or degenerative, differentiation between these phenotypically similar conditions is facilitated with the use of molecular genetic analysis.
Asunto(s)
Amiloide/metabolismo , Amiloidosis Familiar/genética , Enfermedades de la Córnea/genética , Sustancia Propia/metabolismo , Proteínas de la Matriz Extracelular/genética , Mutación , Factor de Crecimiento Transformador beta/genética , Anciano , Anciano de 80 o más Años , Amiloidosis Familiar/metabolismo , Amiloidosis Familiar/patología , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Sustancia Propia/patología , Análisis Mutacional de ADN , Femenino , Humanos , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Sunitinib malate (Sutent, Pfizer, Inc.; SU11248) is a selective, multitargeted inhibitor of receptor tyrosine kinases and has been shown to inhibit receptors for VEGF, PDGF, KIT, FLT3, and RET. The objective of this study was to determine the effects of sunitinib on signal transduction pathways and on gene expression of iodide-metabolizing proteins in papillary cancer cells with the RET/PTC1 rearrangement. METHODS: We investigated the effects of sunitinib on cell growth, signal transduction pathways, and thyroid-specific gene expression in papillary thyroid cancer (PTC) cell lines that had the RET/PTC1 rearrangement. RESULTS: Sunitinib inhibited proliferation of RET/PTC1 subclones in a time- and dose-related manner. The mean 50% lethal concentration in the RET/PTC1 subclones was 1.81 microM. Incubation of RET/PTC1 cells with 1 microM sunitinib inhibited their migration potential and transformed their morphology. Sunitinib inhibited RET autophosphorylation at Y1062 and the activation of signal transducer and activator of transcription 3 by blocking Y705 phosphorylation. Sunitinib caused cell cycle arrest in the G0/G1 phase and dephosphorylation of retinoblastoma protein, but did not induce apoptosis. Western blot analysis of the p38, MEK/ERK, and SAPK/JNK mitogen-activated protein kinase signal transduction pathways showed that sunitinib blocked ERK 1/2 and JNK phosphorylation in the cytoplasm. Sunitinib treatment of RET/PTC1 cell lines, in combination, with forskolin induced expression of the sodium (Na)/iodide (I) symporter (NIS) and the transcription factors that bind the NIS upstream enhancer. Mechanistically, the inhibition of both MEK/ERK and SAPK/JNK cytoplasmic pathways individually and in combination caused an increase in NIS gene expression. CONCLUSION: Sunitinib appears to target the cytosolic MEK/ERK and SAPK/JNK pathways in the RET/PTC1 cell lines, suggesting that blocking these pathways is at least part of the mechanism by which sunitinib inhibits cell proliferation and causes stimulation of NIS gene expression in RET/PTC1 cells.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Papilar/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Indoles/farmacología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Simportadores/metabolismo , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacos , Sunitinib , Simportadores/análisisRESUMEN
Mutations in the two-handed zinc-finger homeodomain transcription factor gene (TCF8) have been associated with posterior polymorphous corneal dystrophy (PPCD) and extraocular developmental abnormalities. We performed screening of TCF8 in 32 affected, unrelated probands, affected and unaffected family members of probands identified with a TCF8 mutation, and in 100 control individuals. Eight different pathogenic mutations were identified in eight probands: four frameshift (c.953_954insA, c.1506dupA, c.1592delA, and c.3012_3013delAG); three nonsense (Gln12X, Gln214X, Arg325X); and one missense (Met1Arg). Screening of TCF8 in affected and unaffected family members in six families demonstrated that each identified mutation segregated with the disease phenotype in each family; two probands did not have additional family members available for analysis. None of the eight TCF8 mutations was identified in 200 control chromosomes. The prevalence of hernias of the abdominal region in affected individuals with PPCD associated with TCF8 mutations was significantly higher than the prevalence in both individuals with PPCD not associated with a TCF8 mutation and in unaffected individuals. Therefore, PPCD is associated with TCF8 mutations in one quarter of affected families in this study, or about one third of all PPCD families that have been screened thus far. In these families, the presence of apparently causative TCF8 mutations is associated with abdominal and inguinal hernias.