RESUMEN
Autism is a common neurodevelopmental disorder with genetic and environmental components. Though unproven, genetic susceptibility to high mercury (Hg) body burden has been suggested as an autism risk factor in a subset of children. We hypothesized that exposure to "safe" Hg levels could be implicated in the etiology of autism if genetic susceptibility altered Hg's metabolism or intracellular compartmentalization. Genetic sequences of four genes implicated in the transport and response to Hg were screened for variation and association with autism. LAT1 and DMT1 function in Hg transport, and Hg exposure induces MTF1 and MT1a. We identified and characterized 74 variants in MT1a, DMT1, LAT1 and MTF1. Polymorphisms identified through screening 48 unrelated individuals from the general and autistic populations were evaluated for differences in allele frequencies using Fisher's exact test. Three variants with suggestive p-values <0.1 and four variants with significant p-values <0.05 were followed-up with TaqMan genotyping in a larger cohort of 204 patients and 323 control samples. The pedigree disequilibrium test was used to examine linkage and association. Analysis failed to show association with autism for any variant evaluated in both the initial screening set and the expanded cohort, suggesting that variations in the ability of the four genes studied to process and transport Hg may not play a significant role in the etiology of autism.
Asunto(s)
Trastorno Autístico/genética , Proteínas de Transporte de Catión/genética , Proteínas de Unión al ADN/genética , Transportador de Aminoácidos Neutros Grandes 1/genética , Mercurio/metabolismo , Metalotioneína/genética , Polimorfismo Genético , Factores de Transcripción/genética , Adolescente , Trastorno Autístico/metabolismo , Estudios de Casos y Controles , Proteínas de Transporte de Catión/metabolismo , Niño , Preescolar , Proteínas de Unión al ADN/metabolismo , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Desequilibrio de Ligamiento , Masculino , Metalotioneína/metabolismo , Fenotipo , Medición de Riesgo , Factores de Riesgo , Tennessee , Factores de Transcripción/metabolismo , Adulto Joven , Factor de Transcripción MTF-1RESUMEN
Glutamate cysteine ligase, the rate-limiting enzyme for the synthesis of glutathione, represents an important component of chemoprevention paradigms. GCLC and GCLM, the genes encoding glutamate cysteine ligase subunits, are induced by indoles, such as indomethacin. Novel functionalized indole analogues and other structurally related compounds were synthesized and used for a comparative structure analysis of GCLC induction. Use of mouse embryo fibroblasts null for Nrf2 (nuclear factor-erythroid 2p45-related transcription factor) and HepG2 cells overexpressing Keap1 demonstrated that indole analogue-mediated GCLC expression was regulated by Nrf2-Keap1 interactions. Indole analogues capable of inducing GCLC were found to increase NADPH oxidase activity. Indole analogues unable to induce GCLC did not increase oxidase activity. HepG2 cells transfected with FLAG/Keap1 were exposed to indomethacin, and the redox state of Keap1 cysteine residues was assessed. The data indicated that Keap1 exhibited several oxidation states that were sensitive to indomethacin treatment. These indomethacin-mediated changes in thiol oxidation states were suppressed by diphenyleneiodonium, a NADPH oxidase inhibitor. Diphenyleneiodonium also suppressed indole analogue-mediated increases in GCLC mRNA. In summary, the use of the indole analogues identified NADPH oxidase activity as a novel upstream activity regulating Nrf2/Keap1 signaling of GCLC, provided data supporting the hypothesis that Keap1 is a downstream effector for oxidase activity, and afforded in vivo data to support the hypothesis that Keap1 thiols can act as molecular sensors of reactive oxygen species. Finally, the comparative structure analysis suggests that 2-indol-3-yl-methylenequinuclidin-3-ols may represent a prototype for the development of novel chemopreventative agents able to activate Keap1/Nrf2 signaling.
Asunto(s)
Anticarcinógenos/farmacología , Glutamato-Cisteína Ligasa/genética , Indoles/farmacología , NADPH Oxidasas/metabolismo , Proteínas/fisiología , Quinuclidinas/farmacología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glutamato-Cisteína Ligasa/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The tripeptide glutathione is an important biomolecule that acts as a scavenger of free radicals and plays a role in a number of other cellular processes. A number of diseases, including Parkinson's disease, cancer, sickle cell anemia, and HIV infection, are thought to involve oxidative stress and depletion of glutathione. The heterodimeric enzyme glutamate cysteine ligase catalyzes the first, rate-limiting step in the de novo synthesis of glutathione. Functional polymorphisms within the gene encoding the subunits of glutamate cysteine ligase have the potential to affect the body's capacity to synthesize glutathione and thus, may affect those diseases in which oxidative stress and glutathione have roles. We undertook systematic screening for polymorphisms within the exons and intronic flanking sequences of the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC). We identified 11 polymorphisms in GCLC and established allele frequencies for those polymorphisms in a population fitting the demographics of the middle Tennessee area. The nonsynonymous polymorphism C1384T was found only in individuals of African descent. In addition, allele frequencies for three other polymorphisms differ between Caucasians and African-Americans. Understanding these polymorphisms may lead to better understanding of diseases where glutathione is important so that better treatments may be developed.
