RESUMEN
We have identified a novel fibroblast growth factor receptor 3 (FGFR3) missense mutation in four unrelated individuals with skeletal dysplasia that approaches the severity observed in thanatophoric dysplasia type I (TD1). However, three of the four individuals developed extensive areas of acanthosis nigricans beginning in early childhood, suffer from severe neurological impairments, and have survived past infancy without prolonged life-support measures. The FGFR3 mutation (A1949T: Lys650Met) occurs at the nucleotide adjacent to the TD type II (TD2) mutation (A1948G: Lys650Glu) and results in a different amino acid substitution at a highly conserved codon in the kinase domain activation loop. Transient transfection studies with FGFR3 mutant constructs show that the Lys650Met mutation causes a dramatic increase in constitutive receptor kinase activity, approximately three times greater than that observed with the Lys650Glu mutation. We refer to the phenotype caused by the Lys650Met mutation as "severe achondroplasia with developmental delay and acanthosis nigricans" (SADDAN) because it differs significantly from the phenotypes of other known FGFR3 mutations.
Asunto(s)
Acantosis Nigricans/genética , Huesos/anomalías , Discapacidades del Desarrollo/genética , Proteínas Tirosina Quinasas , Receptores de Factores de Crecimiento de Fibroblastos/genética , Acantosis Nigricans/complicaciones , Acondroplasia/complicaciones , Acondroplasia/genética , Huesos/diagnóstico por imagen , Craneosinostosis/genética , Discapacidades del Desarrollo/complicaciones , Humanos , Immunoblotting , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Mutación Missense , Fenotipo , Fosfotransferasas/análisis , Mutación Puntual , Pruebas de Precipitina , Radiografía , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Displasia Tanatofórica/complicaciones , Displasia Tanatofórica/diagnóstico por imagen , Displasia Tanatofórica/genéticaRESUMEN
Various mutations in the fibroblast growth factor receptor 3 (FGFR3) gene have recently been reported in thanatophoric dysplasia (TD). We examined the clinical, radiographic, and histologic findings in 91 cases from the International Skeletal Dysplasia Registry and correlated them with the specific FGFR3 mutation. Every case of TD examined had an identifiable FGFR3 mutation. Radiographically, all of the cases with the Lys650Glu substitution demonstrated straight femora with craniosynostosis, and frequently a cloverleaf skull (CS) was demonstrated. In all other cases, the femora were curved, and CS was infrequently present but was occasionally as severe as TD with the Lys650Glu substitution. Histopathologically, all of the cases shared similar abnormalities, but cases with the Lys650Glu substitution had better preservation of the growth plate. Cases with the Tyr373Cys substitution tended to have more severe radiographic manifestations than the Arg248Cys cases, but there was overlap in the phenotypic spectrum between them. One common classification of TD distinguishes affected infants based on the presence or absence of CS. In contrast, and as originally proposed by Langer et al. [1987: Am J Med Genet 3: 167-179], our data suggest that TD can be divided into at least two groups (TD1 and TD2) based on the presence of straight or curved femora. The variable presence of CS and severity of the radiologic and histologic findings in the other substitutions may be due to other genetic, environmental, or stochastic factors.
Asunto(s)
Placa de Crecimiento/anomalías , Proteínas Tirosina Quinasas , Receptores de Factores de Crecimiento de Fibroblastos/genética , Displasia Tanatofórica/genética , Condrocitos/patología , Craneosinostosis/diagnóstico por imagen , Craneosinostosis/genética , Cisteína/genética , Fémur/anomalías , Fémur/diagnóstico por imagen , Feto/anomalías , Genotipo , Edad Gestacional , Placa de Crecimiento/diagnóstico por imagen , Humanos , Recién Nacido , Mutación , Fenotipo , Mutación Puntual , Reacción en Cadena de la Polimerasa , Radiografía , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Sistema de Registros , Cráneo/anomalías , Cráneo/diagnóstico por imagen , Columna Vertebral/anomalías , Columna Vertebral/diagnóstico por imagen , Displasia Tanatofórica/clasificación , Displasia Tanatofórica/diagnósticoRESUMEN
The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein. Nine segments of particularly high similarity have been identified and are termed segments A through I. We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth. The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296). In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified. They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro. To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene. In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination. We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Genes Letales , Genes Recesivos , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Extensión de la Cadena Peptídica de Translación , Fenotipo , Homología de Secuencia de AminoácidoRESUMEN
Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves. As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues. We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E). With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit. Allele specific suppression is a genetic criterion for protein-protein interaction. Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation. Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide. We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Secuencia Conservada , ADN Bacteriano , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Supresión Genética , Transcripción GenéticaAsunto(s)
Mutación Puntual , Proteínas Tirosina Quinasas , Receptores de Factores de Crecimiento de Fibroblastos/genética , Displasia Tanatofórica/genética , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Sitios de Unión , Cisteína/genética , ADN , Humanos , Datos de Secuencia Molecular , Receptor Tipo 3 de Factor de Crecimiento de FibroblastosRESUMEN
Thanatophoric dysplasia (TD), the most common neonatal lethal skeletal dysplasia, affects one out of 20,000 live births. Affected individuals display features similar to those seen in homozygous achondroplasia. Mutations causing achondroplasia are in FGFR3, suggesting that mutations in this gene may cause TD. A sporadic mutation causing a Lys650Glu change in the tyrosine kinase domain of FGFR3 was found in 16 of 16 individuals with one type of TD. Of 39 individuals with a second type of TD, 22 had a mutation causing an Arg248Cys change and one had a Ser371Cys substitution, both in the extracellular region of the protein. None of these mutations were found in 50 controls showing that mutations affecting different functional domains of FGFR3 cause different forms of this lethal disorder.