RESUMEN
OBJECTIVE: Friedreich's ataxia patients are homozygous for expanded alleles of a GAA triplet-repeat sequence in the FXN gene. Patients develop progressive ataxia due to primary neurodegeneration involving the dorsal root ganglia (DRGs). The selective neurodegeneration is due to the sensitivity of DRGs to frataxin deficiency; however, the progressive nature of the disease remains unexplained. Our objective was to test whether the expanded GAA triplet-repeat sequence undergoes further expansion in DRGs as a possible mechanism underlying the progressive pathology seen in patients. METHODS: Small-pool polymerase chain reaction analysis, a sensitive technique that allows the measurement of repeat length in individual FXN genes, was used to analyze somatic instability of the expanded GAA triplet-repeat sequence in multiple tissues obtained from six autopsies of Friedreich's ataxia patients. RESULTS: DRGs showed a significantly greater frequency of large expansions (p < 0.001) and a relative paucity of large contractions compared with all other tissues. There was a significant age-dependent increase in the frequency of large expansions in DRGs, which ranged from 0.5% at 17 years to 13.9% at 47 years (r = 0.78; p = 0.028). INTERPRETATION: Progressive pathology involving the DRGs is likely due to age-dependent accumulation of large expansions of the GAA triplet-repeat sequence. Thus, somatic instability of the expanded GAA triplet-repeat sequence may contribute directly to disease pathogenesis and progression. Progressive repeat expansion in specific tissues is a common theme in the pathogenesis of triplet-repeat diseases.
Asunto(s)
Ataxia de Friedreich/genética , Ataxia de Friedreich/patología , Ganglios Espinales/metabolismo , Proteínas de Unión a Hierro/genética , Expansión de Repetición de Trinucleótido/genética , Adolescente , Adulto , Factores de Edad , Femenino , Ganglios Espinales/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , FrataxinaRESUMEN
The capsids of spherical viruses may contain from tens to hundreds of copies of the capsid protein(s). Despite their complexity, these particles assemble rapidly and with high fidelity. Subunit and capsid represent unique end states. However, the number of intermediate states in these reactions can be enormous-a situation analogous to the protein folding problem. Approaches to accurately model capsid assembly are still in their infancy. In this paper, we describe a sail-shaped reaction landscape, defined by the number of subunits in each species, the predicted prevalence of each species, and species stability. Prevalence can be calculated from the probability of synthesis of a given intermediate and correlates well with the appearance of intermediates in kinetics simulations. In these landscapes, we find that only those intermediates along the leading edge make a significant contribution to assembly. Although the total number of intermediates grows exponentially with capsid size, the number of leading-edge intermediates grows at a much slower rate. This result suggests that only a minute fraction of intermediates needs to be considered when describing capsid assembly.
Asunto(s)
Proteínas de la Cápside/química , Modelos Moleculares , Complejos Multiproteicos/química , Virus/químicaRESUMEN
Friedreich ataxia is caused by expansion of a GAA triplet repeat (GAA-TR) in the FRDA gene. Normal alleles contain <30 triplets, and disease-causing expansions (66-1700 triplets) arise via hyperexpansion of premutations (30-65 triplets). To gain insight into GAA-TR instability we analyzed all triplet repeats in the human genome. We identified 988 (GAA)(8+) repeats, 291 with >or=20 triplets, including 29 potential premutations (30-62 triplets). Most other triplet repeats were restricted to <20 triplets. We estimated the expected frequency of (GAA)(6+) repeats to be negligible, further indicating that GAA-TRs have undergone significant expansion. Eighty-nine percent of (GAA)(8+) sequences map within G/A islands, and 58% map within the poly(A) tails of Alu elements. Only two other (GAA)(8+) sequences shared the central Alu location seen at the FRDA locus. One showed allelic variation, including expansions analogous to short Friedreich ataxia mutations. Our data demonstrate that GAA-TRs have expanded throughout primate evolution with the generation of potential premutation alleles at multiple loci.
Asunto(s)
Elementos Alu/genética , Genoma Humano , Proteínas de Unión a Hierro/genética , Mutación , Expansión de Repetición de Trinucleótido , Algoritmos , Secuencia de Bases , Evolución Molecular , Ataxia de Friedreich/genética , Frecuencia de los Genes , Humanos , Modelos Genéticos , FrataxinaRESUMEN
The assembly of virus capsids or other spherical polymers--empty, closed structures composed of hundreds of protein subunits--is poorly understood. Assembly of a closed spherical polymer is unlike polymerization of a filament or crystal, examples of open-ended polymers. This must be considered to develop physically meaningful analyses. We have developed a model of capsid assembly, based on a cascade of low-order reactions, that allows us to calculate kinetic simulations. The behavior of this model resembles assembly kinetics observed in solution (Zlotnick, A., J. M. Johnson, P. W. Wingfield, S. J. Stahl, and D. Endres. 1999. Biochemistry. 38:14644-14652). We exhibit two examples of this general model describing assembly of dodecahedral and icosahedral capsids. Using simulations based on these examples, we demonstrate how to extract robust estimates of assembly parameters from accessible experimental data. These parameters, nucleus size, average nucleation rate, and average free energy of association can be determined from measurement of subunit and capsid as time and concentration vary. Mathematical derivations of the analyses, carried out for a general model, are provided in an Appendix. The understanding of capsid assembly developed in this paper is general; the examples provided can be readily modified to reflect different biological systems. This enhanced understanding of virus assembly will allow a more quantitative analysis of virus stability and biological or antiviral factors that affect assembly.